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Dominant Effect of Human Immunodeficiency Virus Type 1 (HIV-1) gag-pol on Viral Fitness and Resistance to Protease Inhibitors

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Title:
Dominant Effect of Human Immunodeficiency Virus Type 1 (HIV-1) gag-pol on Viral Fitness and Resistance to Protease Inhibitors
Creator:
KOCH, SARAH JANE ( Author, Primary )
Copyright Date:
2008

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Subjects / Keywords:
AIDS ( jstor )
Amino acids ( jstor )
Genetic mutation ( jstor )
Genotypes ( jstor )
HIV ( jstor )
HIV 1 ( jstor )
Infections ( jstor )
Protease inhibitors ( jstor )
Virion ( jstor )
Viruses ( jstor )

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University of Florida
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University of Florida
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Copyright Sarah Jane Koch. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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12/31/2015

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DOMINANT EFFECT OF HUMAN IMMUNODE FICIENCY VIRUS TYPE 1 (HIV-1) GAG-POL ON VIRAL FITNESS AND RESISTANC E TO PROTEASE INHIBITORS By SARAH JANE KOCH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Sarah Jane Koch

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This dissertation is dedicated to my parent s Susan and Leslie Koch for their unwavering love and support.

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ACKNOWLEDGMENTS I would like to thank my mentor, Dr. Maureen Goodenow, for her constant advice and encouragement. Over the past four years, I have learned many invaluable lessons from Maureen, and I feel privileged to have been able to pursue my degree in her laboratory. I am also especially grateful to my other advisory committee members (Dr. John Sleasman, Dr. Ben Dunn, Dr. Maurice Swanson, and Dr. David Ostrov) for their guidance and contribution to my work. I would like to express my gratitude to past and present members of the Goodenow lab (particularly Dr. Stephanie Rose, Steve Pomeroy, Amanda Lowe, Luke Smith, Christina Gavegnano, Dr. Guity Ghaffari, and Rose Mills) for their support, both scientifically and personally. I would also like to acknowledge my friends, Kim Aiken, Melanie Powers, Christine Schaub, and Nilanjana Sengupta. We all took this journey together, and I will always have great memories of graduate school because of them. I am particularly grateful to Christine whose encouragement helped me get through the countless hours we spent together while writing our dissertations. Most importantly, I would like to thank my parents, my sisters, and my fianc for their never-ending love and support. They have all been behind me every step of the way, encouraging me to pursue my dreams, and I would not be where I am today without them. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv LIST OF TABLES .............................................................................................................ix LIST OF FIGURES .............................................................................................................x ABSTRACT .....................................................................................................................xiii CHAPTER 1 INTRODUCTION AND BACKGROUND.................................................................1 Human Immunodeficiency Virus.................................................................................1 HIV-1 Genome.............................................................................................................1 HIV-1 Life Cycle..........................................................................................................2 HIV-1 Target Cells.......................................................................................................2 HIV-1 Envelope............................................................................................................3 HIV-1 Gag-Pol..............................................................................................................4 Protease..................................................................................................................6 Cleavage Sites.......................................................................................................7 Matrix (p17 MA )......................................................................................................8 Capsid (p24 CA ).......................................................................................................9 p2.........................................................................................................................10 Nucleocapsid (p7 NC )............................................................................................10 p1.........................................................................................................................11 p6.........................................................................................................................11 p6 Pol ......................................................................................................................12 Reverse Transcriptase..........................................................................................13 Recombination............................................................................................................13 Antiretroviral Therapy................................................................................................14 Response to Therapy...........................................................................................14 Reverse Transcriptase Inhibitors.........................................................................15 Protease Inhibitors and Resistance to Therapy....................................................15 Fitness.........................................................................................................................16 Hypothesis..................................................................................................................17 Specific Aims..............................................................................................................18 Specific Aim 1: Analyze the Relationship Between gag-pol Genotype and Therapy Response............................................................................................18 v

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Specific Aim 2: Identify gag-pol Determinants of Replicative Fitness and Drug Sensitivity in a Protease Inhibitor-Resistant HIV-1 Variant in CXCR4 CD4 + T Lymphocytes........................................................................18 Specific Aim 3: Determine the Impact of Recombination and Reversion on HIV-1 Fitness in CXCR4 CD4 + T Lymphocytes............................................19 Specific Aim 4: Investigate the Impact of Protease Inhibitors on Fitness in CXCR4 and CCR5 CD4 + T Lymphocytes.......................................................19 Significance................................................................................................................19 2 IMPACT OF THERAPY ON GENETIC DETERMINANTS IN HIV-1 GAG-POL.............................................................................................................................28 Introduction.................................................................................................................28 Methods......................................................................................................................30 Study Design.......................................................................................................30 Viral and Immune Outcome................................................................................32 Sequencing..........................................................................................................34 Data Analysis.......................................................................................................37 Results.........................................................................................................................38 Protease Analysis.................................................................................................38 Pretherapy Primary and Secondary Polymorphisms....................................38 Posttherapy Primary and Secondary Mutations...........................................40 Pretherapy Supplemental Polymorphisms...................................................41 Posttherapy Supplemental Mutations...........................................................42 Cleavage Site Analysis........................................................................................43 Pretherapy.....................................................................................................43 Posttherapy...................................................................................................45 Nucleocapsid Analysis........................................................................................46 Pretherapy.....................................................................................................46 Posttherapy...................................................................................................47 p6 Analysis..........................................................................................................48 Pretherapy.....................................................................................................48 Posttherapy...................................................................................................49 p6 Pol Analysis.......................................................................................................50 Pretherapy.....................................................................................................50 Posttherapy...................................................................................................51 Covariation Analysis...........................................................................................53 Envelope Analysis...............................................................................................53 Pretherapy.....................................................................................................53 Posttherapy...................................................................................................54 Discussion...................................................................................................................55 3 GAG-POL DETERMINANTS OF FITNESS AND DRUG SENSITIVITY IN A PROTEASE INHIBITOR RESISTANT VARIANT IN CXCR4 CD4 + T LYMPHOCYTES.......................................................................................................85 Introduction.................................................................................................................85 vi

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Materials and Methods...............................................................................................87 Construction of Replication Competent gag-pol Recombinant Viruses.............87 Production of Virus Stocks..................................................................................87 PBMC Stimulation and Infections.......................................................................88 Site Directed Mutagenesis...................................................................................89 Gag Mutagenesis.................................................................................................90 Protease Mutagenesis..........................................................................................91 p6/p6 Pol Mutagenesis...........................................................................................92 Determination of IC 50 ..........................................................................................92 Statistical Analysis..............................................................................................93 Results.........................................................................................................................93 Replicative Fitness of Recombinant Viruses in X4 PBMC.................................93 Mutagenesis Strategy...........................................................................................94 Replicative Fitness of Gag and Protease Mutants...............................................95 Replicative Fitness of Recombinant Viruses in the Presence Indinavir..............97 Indinavir IC 50 Values for Recombinant Viruses.................................................97 Indinavir IC 50 Values for Mutant Viruses...........................................................97 Relationship between IDV IC 50 and Viral Replication.......................................98 Replicative Fitness of Recombinant Viruses in the Presence Ritonavir.............98 Ritonavir IC 50 Values for Recombinant Viruses.................................................99 Ritonavir IC 50 Values for Mutant Viruses...........................................................99 Relationship between RTV IC 50 and Viral Replication....................................100 Discussion.................................................................................................................100 4 RECOMBINATION AND REVERSION MODULATE HIV-1 FITNESS IN CXCR4 CD4 + T LYMPHOCYTES.........................................................................122 Introduction...............................................................................................................122 Materials and Methods.............................................................................................124 Competition Experiments..................................................................................124 Sequencing of Cell-Associated DNA................................................................125 Sequence Analysis.............................................................................................125 Mutagenesis of Position 54 in PR.....................................................................126 PBMC Infections with A54V and A54I Mutant Viruses..................................126 Results.......................................................................................................................127 Recombination Confers a Replicative Advantage to HIV-1.............................127 Reversion of the Posttherapy Virus in the Absence of Protease Inhibitors.......129 Amino Acid Position 54 in PR is an Important Modulator of Viral Fitness.....129 Discussion.................................................................................................................130 5 DISCUSSION...........................................................................................................141 APPENDIX A DIFFERENTIAL IMPACT OF PROTEASE INHIBITORS ON HIV-1 FITNESS IN CD4 + T LYMPHOCYTE SUBSETS..................................................................147 vii

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Introduction...............................................................................................................148 Methods....................................................................................................................149 Construction of R5 Recombinant Viruses.........................................................149 Replication of R5 Recombinant Viruses...........................................................149 R5 Competition Experiments............................................................................150 Sequencing of Cell-Associated DNA................................................................151 Sequence Analysis.............................................................................................151 Determination of IC 50 Values for R5 Recombinant Viruses.............................152 Determination of Intracellular PI Levels...........................................................152 Results.......................................................................................................................153 Parallel Infections in the Absence of Protease Inhibitors..................................153 Competition Experiments..................................................................................153 Replication of R5 Recombinant Viruses in the Presence of RTV and IDV......154 Determination of IC 50 Values for R5 Recombinant Viruses.............................155 Intracellular PI Levels.......................................................................................155 Discussion.................................................................................................................156 B ALIGNMENT OF PROTEASE SEQUENCES.......................................................169 C ALIGNMENT OF GAG SEQUENCES...................................................................196 D ALIGNMENT OF P6 POL SEQUENCES..................................................................213 E ALIGNMENT OF ENVELOPE SEQUENCES.......................................................228 LIST OF REFERENCES.................................................................................................245 BIOGRAPHICAL SKETCH...........................................................................................272 viii

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LIST OF TABLES Table page 2-1 Baseline Clinical Data for VSIS Subjects................................................................60 2-2 Baseline Clinical Data for VFIS Subjects................................................................60 2-3 Baseline Clinical Data for VFIF Subjects................................................................60 2-4 24 Week Clinical Data for VSIS Subjects...............................................................61 2-5 24 Week Clinical Data for VFIS Subjects...............................................................61 2-6 24 Week Clinical Data for VFIF Subjects...............................................................61 2-7 Summary of Baseline Clinical, Immune, and Viral Characteristics........................62 2-8 Summary of Clinical, Immune, and Viral Characteristics Following 24 Weeks of Therapy.....................................................................................................................62 2-9 Median Preand Posttherapy Supplemental Mutations in Protease........................73 2-10 Median Preand Posttherapy Mutations in p7 NC .....................................................78 ix

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LIST OF FIGURES Figure page 1-1 HIV-1 Proviral Genome...........................................................................................21 1-2 HIV-1 Life Cycle.....................................................................................................22 1-3 HIV-1 Virion Morphology.......................................................................................23 1-4 Structure of HIV-1 Protease.....................................................................................24 1-5 Gag-Pol Cleavage Sites............................................................................................25 1-6 Viral and Immune Response to Antiretroviral Therapy...........................................26 1-7 Resistance Associated Mutations in Protease..........................................................27 2-1 Therapy Outcome of VSIS, VFIS, and VFIF Response Groups..............................63 2-2 Immune and Viral Response to Therapy..................................................................64 2-3 Gag-Pol and Envelope Sequences from HIV HXB2 ......................................................65 2-4 Pretherapy Protease Genotypes................................................................................66 2-5 Proportion of Sensitive and Resistant Genotypes Prior to Therapy.........................67 2-6 Proportion of Therapy Specific and Therapy Non-Specific Polymorphisms in Protease Prior to Therapy.........................................................................................68 2-7 Posttherapy Protease Genotypes..............................................................................69 2-8 Median Number of Cell Associated Preand Posttherapy Mutations in Protease...70 2-9 Supplemental Polymorphisms in Protease Prior to Therapy....................................71 2-10 Supplemental Mutations in Protease after 24 Weeks of Therapy............................72 2-11 Pretherapy D Cleavage Site Polymorphisms............................................................74 2-12 Pretherapy C Cleavage Site Polymorphisms............................................................75 x

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2-13 Pretherapy E Cleavage Site Polymorphisms............................................................76 2-14 Preand Posttherapy Polymorphisms in p7 NC ..........................................................77 2-15 Pretherapy Insertions in p6.......................................................................................79 2-16 Pretherapy Polymorphisms in p6 Pol ..........................................................................80 2-17 Posttherapy Mutations in p6 Pol .................................................................................81 2-18 Covariation in Gag-Pol.............................................................................................82 2-19 Preand Posttherapy V3 Loop Charge.....................................................................83 2-20 Location of Amino Acids 36 and 77 on Protease.....................................................84 3-1 Viral and Immune Characteristics of Subject D1...................................................104 3-2 Replication Kinetics of Pre-and Posttherapy Variants in X4 PBMC.....................105 3-3 Sequence Alignment of Preand Posttherapy Variants........................................106 3-4 Mutagenesis Strategy.............................................................................................107 3-5 Mutagenesis Constructs..........................................................................................108 3-6 Replicative Capacity of C Cleavage Site and p7 NC Mutants..................................109 3-7 Replicative Capacity of Protease Mutants.............................................................110 3-8 Replicative Capacity of p6 and p6 Pol Mutants.......................................................111 3-9 Replication of Recombinant Viruses in the Presence of Indinavir........................112 3-10 IDV IC 50 of Recombinant Viruses.........................................................................113 3-11 IDV IC 50 Values and Curves for C site and p7 NC Mutant Viruses.........................114 3-12 IDV IC 50 Values and Curves for Protease Mutant Viruses....................................115 3-13 Linear Regression of IDV IC 50 and Viral Replication...........................................116 3-14 Replication of Recombinant Viruses in the Presence of Ritonavir........................117 3-15 RTV IC 50 of Recombinant Viruses........................................................................118 3-16 RTV IC 50 Values and Curves for C Site and p7 NC Mutant Viruses.......................119 3-17 RTV IC 50 Values and Curves for Protease Mutant Viruses...................................120 xi

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3-18 Linear Regression of RTV IC 50 and Viral Replication..........................................121 4-1 Replicative Capacity after 1 and 5 Weeks of Culture............................................133 4-2 Proportion of Virus Clones after 5 Weeks in Culture............................................134 4-3 Recombination Frequency in Protease and Gag after 1 and 5 Weeks in Culture..135 4-4 Proportion of Single and Multiple Crossovers in Gag and Protease......................136 4-5. Protease Recombinant Clones after 5 weeks in culture...........................................137 4-6 Gag Recombinant Clones after 5 weeks in Culture...............................................138 4-7 Replication and Sequences of the Posttherapy Virus at Passages 1 and 5.............139 4-8 Replication of A54I and A54V Mutants................................................................140 A-1 Replication Kinetics of Pre-and Posttherapy Variants in R5 PBMC.....................158 A-2 Replication of Single Virus Controls and Virus Competitions after 1 Week Culture in R5 PBMC..............................................................................................159 A-3 Proportion of Protease Virus Clones after 1 Week in Culture in R5 PBMC.........160 A-4 Proportion of Gag Virus Clones after 1 Week in Culture in R5 PBMC................161 A-5 Replication of R5 Recombinant Viruses in the Presence of Indinavir...................162 A-6 Replication of R5 Recombinant Viruses in the Presence of Ritonavir..................163 A-7 IC 50 Curves for R5 Recombinant Viruses..............................................................164 A-8 X4 and R5 Differences in Indinavir and Ritonavir IC 50 Values............................165 A-9 Intracellular Indinavir Concentrations...................................................................166 A-10 Intracellular Ritonavir Concentrations...................................................................167 A-11 Intracellular Nelfinavir Concentrations..................................................................168 xii

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DOMINANT EFFECT OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 (HIV-1) GAG-POL ON VIRAL FITNESS AND RESISTANCE TO PROTEASE INHIBITORS By Sarah Jane Koch December 2005 Chair: Maureen M. Goodenow Major Department: Pathology, Immunology, and Laboratory Medicine Human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS), currently affects almost 40 million people worldwide. Treatment of HIV-1 involves a combination of antiretroviral therapies, including protease inhibitors (PI), which act to prevent newly formed virions from becoming infectious. Initial mutations that accumulate in protease cause resistance to PI, usually associated with a reduction in viral replicative capacity. The accumulation of subsequent mutations in gag and protease may help restore the reduced replication of the virus. The hypothesis of this dissertation is that polymorphisms in HIV-1 gag-pol exert a dominant impact on viral fitness and resistance to protease inhibitors. Four specific aims were designed to examine this hypothesis: 1) to analyze the relationship between gag-pol genotype and therapy response, 2) to identify gag-pol determinants of replicative fitness and drug sensitivity in a PI-resistant HIV-1 variant in CXCR4 CD4 + T lymphocytes, 3) to xiii

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determine the impact of recombination and reversion on HIV-1 fitness in CXCR4 CD4 + T lymphocytes, and 4) to investigate the impact of protease inhibitors on fitness in CXCR4 and CCR5 CD4 + T lymphocytes. Following 24 weeks of antiretroviral therapy, the therapy response of pediatric patients was classified by viral and immune parameters. Polymorphisms in novel positions present in gag-pol prior to the start of therapy can be used to distinguish between viral and immune outcomes. A posttherapy variant that accumulated PI-specific mutations was less fit than the pretherapy variant in CXCR4 CD4 + T lymphocytes. Mutagenesis and replication studies in CXCR4 CD4 + T lymphocytes in the presence and absence of PI identified unique determinants of viral replicative capacity and PI-resistance in gag-pol, specifically the C cleavage site, p7 NC , p6/p6 Pol , and protease. Competition between two viruses with similar replicative capacities resulted in significant levels of recombination in Gag and protease that were associated with increases in replication, demonstrating a fitness advantage for recombinants. In conclusion, our data identify novel modulators of HIV-1 fitness and functional relationships between Gag and Pol that will significantly aid in discovering a new class of drugs that target interactions between protease and its gag substrates without the emergence of resistance. xiv

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CHAPTER 1 INTRODUCTION AND BACKGROUND Human Immunodeficiency Virus Human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS), has become a global epidemic since it was first identified in the 1980s. According to the World Health Organization, there were 5 million new infections of HIV-1 in 2004 alone, and by the end of that year, almost 40 million people worldwide were living with the disease, including 2.2 million children. HIV-1 is divided into groups and subgroups that are distributed according to geographic origin [231]. Group M (major), which contains subtypes A, B, C, D, F, G, H, J, and K is the most common, and groups N (new) and O (outlier) have been identified in Africa and eastern Europe [72]. The most prevalent subtype in North America, Europe, India, and parts of South America is subtype B. Subtype C is predominant in sub-Saharan Africa, while subtype E is most common in Southeast Asia. In addition, recombination between different subtypes resulted in several distinct circulating recombinant forms, or CRFs. HIV-1 Genome HIV-1 is a lentivirus which packages 2 copies of a positive-sense RNA strand genome of about 9 kilobases into each virion (Figure 1-1). The genome, which is flanked by two identical long terminal repeats (LTR), contains the three major genes gag, pol, and env, as well as the accessory proteins Vif, Vpr, Vpu, Tat, Rev, and Nef. The structural proteins, matrix (p17 MA ), capsid (p24 CA ), nucleocapsid (p7 NC ), and p6, that 1

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2 make up the virion are encoded in gag, while the enzymes protease (PR), reverse transcriptase (RT), integrase (IN), and RNaseH are encoded within the pol region [77]. HIV-1 Life Cycle The HIV-1 life cycle is depicted in Figure 1-2. An HIV-1 virion attaches to the cellular receptor CD4 and a coreceptor. The viral membrane fuses with the cell membrane and releases the viral core into the cytoplasm. The viral RNA is reverse transcribed into double-stranded DNA, which is then translocated into the nucleus and integrated into the host genome. Transcription of the RNA and the translation of gene products occur next, followed by assembly at the plasma membrane, budding of the newly formed virion, and protease processing to produce a mature virion [133]. HIV-1 Target Cells The main cellular targets for HIV-1 infection in vivo and in culture are CD4 + T lymphocytes and cells of the monocyte/macrophage lineage. In vitro, HIV-1 strains are typically classified based on their tropism for a particular cell type. Strains that can infect peripheral blood mononuclear cells (PBMC) and T-cell lines and use the CXCR4 (X4) coreceptor are classified as T-tropic. Strains that use the CCR5 (R5) coreceptor, and can infect PBMC and macrophages but not T-cell lines, are known as M-tropic. Dual-tropic strains of HIV-1 can infect PBMC, macrophages, and T-cell lines using the CXCR4 coreceptor alone (DX4) or CXCR4 in addition to CCR5 (DR5X4) [26,89]. R5 viruses tend to occur early in infection, while X4 and DX4 viruses emerge later during infection and are associated with pathogenesis and advanced disease [45]. Lymphocytes, thymocytes and cord blood cells predominantly express X4, while monocyte derived macrophages (MDM) predominantly express R5.

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3 Lymphocytes and macrophages provide two very different environments for HIV-1. CD4 + T lymphocytes are a heterogeneous population of cells. The majority of CD4 + cells express CXCR4, although a small subset of activated memory T lymphocytes expresses CCR5 [89]. Lymphocytes represent the majority of infected cells, are part of the adaptive immune response, and continuously circulate between the blood and the lymphoid organs until encountering antigen [113,230]. Once an antigen is recognized during infection, the lymphocytes begin to proliferate and differentiate into effector cells, which help control the infection. Macrophages, which constitute only a small portion of infected cells, are phagocytic cells that are part of the innate immune response [113,163]. Monocytes, the immature form of macrophages, circulate in the blood and terminally differentiate into macrophages upon migration into the tissues. Lymphocytes tend to have a relatively short half-life (fewer than 2 days), whereas macrophages can live for much longer periods of time (2 weeks) [4,6,191]. Furthermore, lymphocytes require lower levels of protease inhibitors than macrophages to inhibit viral replication [5,193,237]. HIV-1 Envelope HIV-1 envelope (gp160) is a 160 kilodalton protein that is expressed from singly spliced mRNA, and forms trimers on the surface of infected cells and virions [13]. Envelope is initially synthesized in the endoplasmic reticulum, and as it travels through the Golgi complex, it undergoes glycosylation, which is necessary for infectivity [30]. gp160 is cleaved by a cellular protease to produce gp120 and gp41. gp120 is located on the surface of an infected cell, and subsequently, on the surface of the virion, and is noncovalently attached to gp41, which spans the membrane. gp120 is organized into five hypervariable regions (known as V1-V5), as well as five conserved regions (C1-C5).

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4 One hypervariable region in particular, the V3 loop, interacts with the coreceptors CXCR4 and CCR5 and determines the cellular tropism of HIV-1 [26,105]. Viruses that use the CXCR4 coreceptor and replicate well in CD4 + T cells typically have a V3 loop with a high charge ( > +5). The V3 loops of viruses that replicate well in macrophages and use CCR5 are usually low charge ( < +4). The N-terminal domain of gp41 is necessary for mediating fusion of the viral and cellular membranes, the transmembrane region anchors envelope into the membrane, and the cytoplasmic domain interacts with matrix and is involved in incorporation into the membrane [49,63,269,275]. HIV-1 Gag-Pol The precursors p55 Gag and p160 Gag-Pol are translated from the gag open reading frame, and serve as the substrates for protease activity. p55 Gag encodes the structural proteins matrix (p17 MA ), capsid (p24 CA ), nucleocapsid (p7 NC ), and p6. p160 Gag-Pol encodes all of the structural proteins in addition to the enzymes protease, reverse transcriptase, and integrase. To maintain the correct ratio of structural proteins to enzymatic proteins, HIV-1 utilizes a ribosomal frameshifting mechanism to produce approximately 20 copies of p55 Gag for every one copy of p160 Gag-Pol [111]. The frameshift site contains a slippery sequence (UUUUUUA) followed by a stem loop. During translation, the ribosome reaches the stem loop and stalls on the slippery sequence approximately 5% of the time. The ribosome then slips back one nucleotide and translates pol [110,111,235]. Any change to the frameshifting site reduces the efficiency of frameshifting, thereby altering the Gag to Gag-Pol ratio [16]. In addition, the stability of the stem loop is crucial to viral replication as mutations which alter the stability of the stem loop cause the virions produced to be noninfectious [99]. Multiple groups have shown that the correct ratio of Gag to Gag-Pol is necessary for both virus assembly and

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5 replication [104,123,225]. The structure of a mature virion is closely linked to the ability of the virus to replicate, so the incorrect amount of structural proteins are produced, viral maturation and subsequent infectivity would be negatively affected. Similarly, efficient replication requires specific amounts of the viral enzymes, and insufficient levels would reduce or inhibit replication. The Gag precursor is synthesized on cytosolic ribosomes and a myristic acid moiety, which increases Gag’s affinity for membranes, is added through a cotranslational modification [28,91,251]. Gag molecules have been found associated with lipid rafts, which are discrete regions of the membrane that are enriched with cholesterol and sphingolipids [2,173,227]. N-terminal myristoylation is a common signal to target proteins to lipid rafts, and data demonstrate that lipid rafts provide a location for viral components to assemble before budding [164]. There are currently conflicting reports as to whether Gag multimerizes prior to or following membrane targeting. However, after association with the plasma membrane, particles begin to protrude from, and eventually bud from the cell surface, producing an immature virion. The process of maturation involves a series of ordered cleavages to produce a mature virion in which p17 MA lines the inner surface of the membrane, and the p7 NC /genomic RNA core is surrounded by a conical shell formed by p24 CA . Initial cleavage at the N-terminus of p7 NC releases p7 NC to allow condensation of the core [265]. Cleavage between p17 MA and p24 CA then allows p24 CA to separate from the membrane, and final cleavage at the C-terminus of p24 CA releases a spacer peptide and allows for capsid condensation [265]. Immature virus particles have a spherical morphology, with a translucent center (Figure 1-3a), while a fully mature virion contains a condensed conical core (Figure 1-3b).

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6 Protease HIV-1 protease is an aspartic protease which is active only as a homodimer (Figure 1-4) [66,150]. Each 99 amino acid monomer contains the consensus sequence Asp-Thr-Gly, which is characteristic of aspartic proteases [249]. Late in the viral life cycle, the two monomers dimerize to form the active site of the enzyme, which consists of two aspartic acids [172]. Interactions in the dimer interface, which contains a four-stranded antiparallel -sheet made up of residues 1-4 and 96-99 of each monomer, also help to maintain the mature protease dimer [94,262]. Once active, protease acts to cleave Gag and Gag-Pol into the various structural and enzymatic proteins. This cleavage is essential for virion maturation and infectivity [54,121,122,133]. Initial processing of the Gag-Pol polyprotein by the immature protease dimer occurs intramolecularly, and begins at the membrane of an infected cell at the time of budding [121,195]. The mature, free protease is significantly more sensitive to the protease inhibitor ritonavir than the immature, embedded protease, confirming that the initial cleavages are intramolecular [195]. Recent studies demonstrate that the location of the embedded protease within Gag-Pol limits the number of cleavages that can be made [194]. In fact, the proline located at the first position in protease is responsible for preventing protease from cleaving sites downstream from protease [194]. Significantly, these data demonstrate that the structure of Gag-Pol helps to determine the order of processing by protease. The first cleavage to release protease from the Gag-Pol polyprotein occurs at the N-terminus of protease, and occurs through an intramolecular mechanism [151,266]. Evidence suggests that this N-terminal cleavage helps to stabilize the dimer [152]. Cleavage at the C-terminus then occurs by an intermolecular mechanism [267]. Protease

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7 release from the polyprotein appears to be a relatively late event in virion assembly, most likely occurring after the virion has been released from the membrane of the host cell [196]. Cleavage Sites Multiple cleavage sites exist in Gag-Pol, and the processing of these sites by protease is not a random process. Protease recognizes specific substrates that must be at least 7 amino acids long. The residues on the N-terminal side of the scissile bond are designated P 1 , P 2 , etc., while the residues on the C-terminal side of the bond are termed P 1 ’, P 2 ’,etc. P 1 and P 1 ’ flank the scissile bond [221]. The cleavage sites have been categorized into three classes based on their biochemical composition, specifically the amino acid present at position P1’ and the preference for particular residues at P1 [200]. Cleavage sites defined as Type 1 contain a proline at P1’, with a preference for a phenylalanine or a tyrosine at P1. Type 2 sites include an alanine, leucine, or valine at P1’ and a preference for leucine in the P1 position. The amino acid at P1’ in Type 3 sites is different than what is present in either Type 1 or 2 sites, but the overall sequence composition is similar to Type 2 sites. Using alternative sequence sets, other groups have also classified the cleavage sites with additional preferences for amino acids at positions P2’ and P4 [97,186]. In all cleavage sites, the P1 position prefers an amino acid that is hydrophobic and does not contain beta-branched side chains [197]. Initial studies using synthetic substrates demonstrated that processing in vitro occurs in the order E (p6 Pol /PR), F (PR/RT), C (p2/p7 NC ), D (p1/p6), A (p17 MA /p24 CA ), D’ (p7 NC /p1), B (p24 CA /p2), while recent studies suggest that cleavage at the C site occurs via an intramolecular mechanism, occurring before cleavage at E and F (Figure 1-5) [137,151,195,199,250].

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8 Since accurate processing of Gag and Gag-Pol is necessary for the formation of infectious virions, it is not surprising that changes in gag occur to compensate for mutations in PR after exposure to protease inhibitors (PI) [51,64,85,156,278,280]. Mutations in the D and D’ cleavage sites confer a growth advantage to resistant viruses and increase resistance to protease inhibitors [51,64,154,278,280]. An association between the selection of a mutation at position 431 in the D’ cleavage site and mutations at positions 46 and 82 in protease was found [8,204]. In addition, heterogeneity in the C cleavage site and a functional linkage between regions in PR and gag has been observed [10,20,87,178]. Matrix (p17 MA ) Matrix is located at the N-terminus of Gag and is involved in targeting of Gag to the plasma membrane as well as incorporation of envelope glycoproteins into the new virus particles. After protease cleavage, the matrix region remains associated with the lipid envelope, while capsid and nucleocapsid condense around the viral genome to form a mature, infectious virion. The six residues located at the N-terminus of matrix are sufficient to signal the addition of the myristyl group [142]. In addition, a cluster of conserved basic residues located near the N-terminus also contributes to the targeting of matrix to the membrane [277,283]. Similar to other retroviruses, the matrix region of HIV-1 forms trimers with a positively charged face, which facilitates interaction with the acidic phospholipids of the membrane bilayer [32,46,160,162,209]. Evidence suggests that the myristyl moiety is exposed when matrix is still part of the Gag precursor, but is sequestered or hidden after Gag processing [98,180,236,283]. This myristoyl switch model would account for the association of the Gag precursor with the membrane during budding, but allow matrix in the mature viral particle to dissociate from the membrane

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9 after infection and enable the viral core to enter the cytoplasm of the newly infected cell. Matrix is necessary for the incorporation of the envelope glycoproteins during virus assembly, and a stable interaction between matrix and the cytoplasmic domain of envelope suggests that Gag and envelope interact prior to transport to the plasma membrane [49,63,269,275]. Capsid (p24 CA ) HIV-1 capsid consists of an N-terminal domain and a C-terminal domain separated by a flexible linker region [82,86,169]. The C-terminal domain is necessary for particle assembly as well as core formation, whereas the N-terminal domain is only required for core formation [22,61,155,161,211,212,228,284]. During virion maturation, capsid assembles into a closed conical shell that surrounds the p7 NC /RNA complex. Assembly of immature capsids is a multi-step process that requires the presence of the host protein HP68, which appears to act as a molecular chaperone [148,287]. In addition, the N-terminal domain interacts with the host protein cyclophilin A (CyPA), which leads to incorporation into the virion [75,153,248]. While it is known that incorporation of cyclophilin A is necessary for virion infectivity, the specific role of CyPA is still under investigation. One hypothesis suggests that CyPA serves to destabilize the core of the virion in order to facilitate uncoating, while another indicates that CyPA may act as a chaperone to help rearrange capsid during virus maturation [24,81,93]. The C-terminal domain of capsid mediates dimerization of Gag-Pol. Evidence suggests that the dimerization domain extends from capsid into the spacer peptide p2, forming an -helix, which appears to be necessary for assembly [1,82]. Small molecules targeted at the p24 CA /p2 junction can slow PR cleavage at that site, thereby delaying

PAGE 24

10 virion maturation and reducing viral infectivity [281,282]. The major homology region (MHR), which is a stretch of 20 amino acids in the C-terminal domain, is essential for viral replication [155]. Mutations in capsid can have effects on assembly, infectivity, and replication, indicating that capsid has both structural and nonstructural roles in the viral life cycle [83,258]. p2 The p2 domain is fourteen amino acids long in HIV-1 HXB2 and located between p24 CA and p7 NC [210]. The location of p2 in Gag is conserved among primate lentiviruses, but the sequence and length are not well conserved [97]. p2 has been suggested to play a role in the regulation of Gag processing. Pettit et al. demonstrated that cleavage at the B site (p24 CA /p2) was accelerated significantly when cleavage of the C site (p2/p7 NC ) was blocked, suggesting that p2 may function to slow the rate of cleavage at the B site during processing [198]. Further evidence by this group also indicates that p2 is necessary for the correct formation of the core of the virus, as virions produced from viruses with p2 deleted were not infectious and contained an improperly formed core [198]. Nucleocapsid (p7 NC ) Initial studies of nucleocapsid focused on the presence of the highly conserved zinc binding motifs, which bind to stem loop 3 of the -RNA recognition element on viral RNA [53]. Alteration of the zinc binding motifs resulted in decreased packaging of genomic RNA, as well as decreased viral infectivity [29,202]. Attempts to develop drugs that would knock the zinc out of the binding motifs proved unsuccessful because these drugs also removed zinc atoms from cellular proteins. Current studies are focusing on the identification of compounds that bind reversibly and are non-chelating [239]. The

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11 highly basic nature of nucleocapsid has become an area of interest recently, and studies focused on the importance of charged residues in RNA incorporation, particle formation, and particle morphology. Studies identified specific residues as being critical to RNA encapsidation, and others that are essential for the packaging of viral RNA [62,203]. Multiple groups have shown that replacing the basic resides in nucleocapsid with neutral amino acids results in the production of virions with altered morphology, indicating that perhaps there is a processing defect associated with the mutations [14,33,203]. Pettit et al. further demonstrated that the basic residues RKK located between the zinc fingers are needed to upregulate processing at the D and D’ cleavage sites upon interaction with RNA, while Goodenow et al. demonstrated that both charged and non-charged residues in p7 NC can impact PR processing [87,199]. p1 p1, located between p7 NC and p6, is 16 amino acids long in HIV HXB2 [210]. As with p2, the sequence and length of p1 are not very well conserved among lentiviruses, and not much is known about the function of p1 [97]. Because of its location, p1 overlaps with the slippery site and stem loop of the frameshift site. By making a series of mutations that altered the sequence of p1, while retaining stem loop stability, Hill et al. demonstrated reduced stability of genomic RNA dimers as well as noninfectious virions [99]. Furthermore, alteration of two highly conserved proline residues (at positions 7 and 13) reduced dimer stability and viral infectivity, demonstrating the importance of p1 for viral replication [99]. p6 Three functional domains have been mapped to p6, which is located at the C terminus of Gag. Demirov et al. demonstrated that the effects of p6 mutations on

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12 replication are cell-type dependent [57]. The P(T/S)AP motif located at the N-terminus binds the host cellular factor TSG101 and is required for efficient virus release [57,76,84,90,103,158,257,276]. A repeat of the P(T/S)AP sequence can occur, and a complete or partial duplication of P(T/S)AP appears to provide an advantage for survival of the virus in the presence of protease inhibitors [106]. Insertions such as the P(T/S)AP repeat or SRPE in p6 reduce the enzymatic activity of wild-type protease, while restoring Gag processing and subsequent replication of protease inhibitor-resistant variants [245]. A truncated p6 did not inhibit processing of p160Gag-Pol in COS-7 cells, while Demirov et al. showed that in certain T cell lines, mutations in the P(T/S)AP motif did cause processing defects [57,276]. An LXSLFG motif in the C-terminus binds Vpr, and mutations in this region diminish Vpr incorporation into virions, although impact on virus infectivity was not assayed [7,135]. The LXSLFG motif and the upstream LYP motif, which are necessary for the binding to the cellular factor AIP1, are involved in budding [241]. von Schwedler et al. showed that AIP1 directly binds to TSG101 and suggested a model in which Gag forms a ternary complex with both TSG101 and AIP1 during budding [259]. In uninfected cells, TSG101 and AIP1 are involved in a pathway that sorts cellular proteins into vesicles that bud into multivesicular bodies, and the interaction of HIV-1 p6 with these proteins suggests that in infected cells, HIV-1 uses this cellular network to promote budding of new virions. p6 Pol p6 Pol , located between p7 NC and PR in the Gag-Pol polyprotein, is generated by a -1 frameshift during translation. Deletion of p6 Pol improves protease processing and a recombinant purified p6 Pol inhibits mature PR activity, suggesting that p6 Pol is involved in the regulation of protease activity [184,185]. Mutations in p6 Pol can cause defects in PR

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13 autoprocessing, and alter the susceptibility of wild-type PR to protease inhibitors [264]. The presence of the p6 Pol region alone inhibits autoprocessing by PR, the addition of p7 NC causes a large increase in autoprocessing, suggesting that both p6 Pol and p7 NC influence PR activity, albeit in contrasting ways [288]. Reverse Transcriptase Reverse transcriptase is encoded within the pol gene, and acts as both an RNA and DNA dependent DNA polymerase to make a double-stranded DNA copy of the single-stranded viral RNA genome. The RNase H activity of reverse transcriptase degrades the RNA strand of a DNA/RNA hybrid, which then allows for synthesis of the complementary DNA strand. Reverse transcriptase lacks any proof-reading activity or 3’ 5’ exonuclease activity, and as a result is extremely error-prone [11]. The mutation rate is approximately 1 in 10 4 errors per base incorporated, meaning that one new mutation is introduced into each new copy of the genome, contributing to the large viral diversity seen in HIV-1 [39]. Recombination HIV-1 is similar to many other retroviruses in that it packages two copies of the complete viral RNA genome into one virion. During reverse transcription, pieces of both genomes can serve as a template for the growing strand of DNA, resulting in a recombinant viral genome [41,102]. HIV-1 recombines at a higher frequency than other retroviruses, so recombination plays a crucial role in generating a high level of viral diversity [213]. Multiple studies have shown that HIV-1 has a high recombination frequency, with an average rate of 2 to 3 crossovers per genome per replication cycle [115,213,214,276,279,286]. Recombination in HIV-1 can occur between different strains of the same subtype (intrasubtype recombination), between strains of different subtypes

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14 (intersubtype recombination), or between strains of different groups (intergroup recombination) [21,25,48,60,144,149,188,220,244,273,285]. In order for recombination to occur, virions with two different RNA genomes must be produced from a cell that was infected with two different viruses. Infection with two viruses is known as dual infection, which can occur through either coinfection or superinfection. Coinfection is when infection by two different strains occurs simultaneously, or within a short time of each other. In superinfection, also known as reinfection, the second infection occurs sometime after the first infection, and after an immune response has already been mounted to the first virus. Coinfection occurs frequently in HIV-1, and multiple cases of superinfection have been reported [3,52,118,132,208,233,234,246,272]. Antiretroviral Therapy Introduction in the mid 1990s of combination therapy containing protease inhibitors and reverse transcriptase inhibitors, termed highly active antiretroviral therapy (HAART), resulted in a dramatic decrease in the number of AIDS cases. However, resistant viruses emerged as a result of multiple factors including the error-prone nature of reverse transcriptase, patient non-compliance, differing bioavailablities of drugs, and anatomical reservoirs inaccessible to drugs. Response to Therapy Viral and immune parameters can be used to define a patient’s response to antiretroviral therapy (ART). Three distinct clinical responses have been described in pediatric patients (Figure 1-6) [192]. Approximately 35% of pediatric patients are defined as a viral success and immune success (VSIS), ~ 20% are considered viral failure and immune failure (VFIF), and ~ 45% are identified as a viral failure, but immune

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15 success (VFIS) [71,114,192]. VSIS patients experience an increase in CD4 + T cells and a decrease in viral load, while VFIF patients are marked by a decrease in CD4 + T cells and an increase in viral load after an initial transient drop in plasma viral RNA. The VFIS, or discordant response, group is characterized by an increase in CD4 + T cells while maintaining a high viral load. Reverse Transcriptase Inhibitors Reverse transcriptase inhibitors (RTI) were the first therapies designed to target HIV-1, and were first approved for use in the late 1980s. Reverse transcriptase inhibitors fall into two categories: nucleoside reverse transcriptase inhibitors (NRTI) and non-nucleoside reverse transcriptase inhibitors (NNRTI). NRTIs are competitive inhibitors that are incorporated into the growing viral DNA chain by reverse transcriptase, but since they lack a 3’ hydroxyl group, further elongation by the enzyme is blocked [79,215]. Zidovudine, abacavir, didanosine, lamivudine, and stavudine are examples of NRTIs. NNRTIs, such as efavirenz and nevirapine, bind near the active site of reverse transcriptase, and cause a conformational change in the active site of the enzyme resulting in inhibition of DNA polymerization [134,215]. Single mutations can cause high level resistance to some RTIs, such as lamivudine, while the sequential accumulation of several mutations is necessary to confer resistance to others, such as zidovudine [23,139]. Protease Inhibitors and Resistance to Therapy The necessity for an active protease in viral infectivity makes PR an attractive target for therapy. Protease inhibitors are molecules that mimic the natural substrates in the Gag-Pol polyprotein, and therefore compete for active-site binding. Once bound, the inhibitors cannot be cleaved and consequently inactivate the enzyme. Many of the PI

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16 resistance associated mutations appeared within the gag-protease (gag-pro) region [183]. Resistance, defined as the loss of virus susceptibility to a protease inhibitor, involves multiple gradual changes [70]. Resistance to PI can be attributed to several mechanisms. First, mutations known to reduce sensitivity to PI are found in therapy nave patients [27,140,192,218]. In addition, as a result of sub-optimal drug therapy, viruses begin to accumulate mutations in a stepwise fashion [43,168]. The first mutations selected are primary, or major, mutations, which cause resistance to the given inhibitor, as well as cross resistance to other similar drugs [50,170]. These mutations, however, also cause a decrease in the replicative capacity of the virus [51,261]. Secondary, or minor, mutations, which can be present before therapy or appear in response to therapy, help to restore the replicative capacity of the virus [10,64,192,280]. Mutations in protease that are associated with reduced sensitivity to protease inhibitors are shown in Figure 1-7. Fitness In the strict Darwinian sense of the word, fitness is the capacity of an organism to survive and transmit its genotype to reproductive offspring as compared to competing organisms. In the context of HIV, however, the definition is not so clear. In this dissertation, fitness is defined as the relative replicative capacity of an HIV-1 variant in a given environment [206]. In primary isolates, posttherapy PI resistant viruses replicate as well, if not better than pretherapy sensitive viruses in lymphocytes [147,190]. However, in vitro, the posttherapy viruses from discordant VFIS patients are qualitatively different than the pretherapy viruses. Multiple lines of evidence suggest that posttherapy viruses demonstrate reduced fitness compared to pretherapy viruses in lymphocytes cultured in the absence of protease inhibitors [51,159,219,240]. The situation in macrophages appears to be a little different. A study designed in the Goodenow laboratory

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17 investigated the number of infected CD4 + T cells posttherapy in all three response groups. The number of infected CD4 + T cells after therapy decreased for the VSIS and VFIS groups, while the VFIF group remained the same. Paradoxically, the VFIS group maintains a high viral load, despite the decrease in infected CD4 + T cells, indicating that there is an additional viral reservoir besides lymphocytes. One potential source of this virus is macrophages, since posttherapy recombinant viruses replicate as well, if not better, than pretherapy viruses in MDM [219]. Few studies have been done to examine the fitness of resistant viruses in the presence of PI. Many different methods are used to assess viral fitness in vitro, including replication kinetics assays, single cycle infection assays, growth competition assays, and enzymatic assays [37,206]. Viral fitness can be measured using primary isolates or recombinant viruses. The advantage of using recombinant viruses is the ability to study a specific region of the genome. Since the background is otherwise identical, any effects on fitness can be isolated to the particular region of interest. Limited information is available about the fitness of posttherapy viruses from discordant response group, so studying fitness, both in the absence and presence of PI, could help define factors that lead to a VFIS response. Hypothesis The hypothesis of this dissertation is that polymorphisms in HIV-1 gag-pol exert a dominant impact on viral fitness and resistance to protease inhibitors. Four specific aims were proposed to examine this hypothesis.

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18 Specific Aims Specific Aim 1: Analyze the Relationship Between gag-pol Genotype and Therapy Response Previous studies in the Goodenow laboratory have demonstrated that clinical variables and protease genotype can be used to predict the therapy outcome of approximately 80% of the pediatric HIV-1 patients studied [192]. In addition, polymorphisms in gag have been functionally linked to protease activity, and mutations in gag following protease inhibitor containing therapy have been shown to alter viral fitness [10,20,51,64,85,87,156,178,278,280]. The goal of this study was to determine if gag-pol genotype can be used as a predictive indicator of therapy response, and to elucidate any relationship between polymorphisms in gag and polymorphisms in protease. The results of Specific Aim 1 are presented in Chapter 2. Specific Aim 2: Identify gag-pol Determinants of Replicative Fitness and Drug Sensitivity in a Protease Inhibitor-Resistant HIV-1 Variant in CXCR4 CD4 + T Lymphocytes Treatment of HIV-1 infection with protease inhibitors results in the accumulation of mutations in both gag and protease [43,51,64,156,168,183,278,280]. The mutations in protease have been well characterized, and some have been found to alter sensitivity to protease inhibitors directly, while others have an impact on the replicative capacity of the virus [10,50,64,170,192,280]. The majority of studies that have examined the effect of gag mutations on viral fitness have focused on the cleavage sites. The goal of this study was to identify gag-pol determinants of viral fitness and drug resistance in a protease inhibitor-resistant variant. The results of Specific Aim 2 are presented in Chapter 3.

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19 Specific Aim 3: Determine the Impact of Recombination and Reversion on HIV-1 Fitness in CXCR4 CD4 + T Lymphocytes The high rate of HIV-1 replication and the error-prone nature of reverse transcriptase result in an elevated level of viral diversity [11]. The presence of a quasispecies of genetically diverse, but related viral variants provides the opportunity for recombination to occur and further contribute to the high genetic diversity of HIV-1. Furthermore, the presence of anatomical compartments unreachable by drugs or latent cellular reservoirs allows the emergence of reversion from a drug resistant phenotype to a drug sensitive one. The goal of this study was to examine the impact of recombination and reversion on viral fitness in lymphocytes that express CXCR4. The results of Specific Aim 3 are presented in Chapter 4. Specific Aim 4: Investigate the Impact of Protease Inhibitors on Fitness in CXCR4 and CCR5 CD4 + T Lymphocytes HIV-1 infects cells that express the CXCR4 or CCR5 coreceptor. Studies have examined the differential activity of protease inhibitors in lymphocytes versus macrophages, both of which express CCR5, and found that more drug is needed to inhibit viral replication in macrophages than in lymphocytes [6,193]. Currently, no studies have examined the impact of protease inhibitors on viral fitness in the two subsets of lymphocytes. The goal of this study was to investigate viral fitness and drug sensitivity in CXCR4 and CCR5 CD4 + T lymphocytes. The preliminary data for Specific Aim 4 are presented in Appendix A. Significance Resistance to antiretroviral therapy is currently one of the main problems facing treatment of HIV-1 infection. Since drug resistance is a critical factor that impacts viral fitness, additional knowledge about fitness is needed to design new and improved

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20 inhibitors. This work will contribute information about gag-pol factors that modulate viral fitness and drug resistance in CD4 + T lymphocytes.

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21 LTR Tat LTR Gag Pol Vif Vpr Vpu Env Nef Rev LTR Tat LTR Gag Pol Vif Vpr Vpu Env Nef Rev Figure 1-1. HIV-1 Proviral Genome. Two identical long terminal repeats (LTR) flank the viral genome, which includes the main genes gag, pol, and env (shown in blue), as well as the accessory genes vif, vpr, vpu, tat, rev, and nef (shown in red).

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22 RT INT PR 124 35 6 RT INT PR RT INT PR 124 35 igure 1-2. HIV-1 Life Cycle. HIV-1 attaches to CD4 and a coreceptor, either CXCR4 or CCR5 (1). The virion then fuses with the cell and releases the viral RNA into the cytoplasm (2), where it is reverse transcribed into double-stranded DNA (3). The viral DNA is translocated into the nucleus and integrated into the host genome (4). Transcription of the RNA and translation of gene products occurs next (5), followed by assembly at the plasma membrane, budding of the newly formed virion, and protease processing to produce a mature virion (6). The main enzymes are shown underlined in purple. 6 F

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23 A.B. A.B. Figure 1-3. HIV-1 Virion Morphology. A, Dougnut shaped morphology of an immature virion, with a translucent center. B, Morphology of a mature virion, with a condensed conical core. Adapted from Nisole et al. [176].

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24 igure 1-4. Structure of HIV-1 Protease. Protease is a homodimer made up of two identical monomers. The backbone of each monomer is shown as a green e F ribbon structure. Active site positions are shown in orange, and non-activesite positions are shown in blue. A protease inhibitor is depicted in pink in thactive site. (PDB 1OHR [119]).

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25 F p1 Gag (95%)Gag-Pol (5%) Polyproteins 7 p6Pol A BCD’DE p24 p7 p6 PRRT p2p1 F p1 7 p6Pol A BCD’DE p24 p7 p6 PRRT p2p1 Gag (95%)Gag-Pol (5%)Polyproteins Figure 1-5. Gag-Pol Cleavage Sites. Translation of p55 Gag and p160 Gag-Pol results in ed at specific sites, denoted by the oduces the structural proteins p17MA (matrix), NC (nucleocapsid), and p6. Cleavage of Gag-Pol produces proteins, including p6Pol, as well as the enzymatic proteins e), RT (reverse transcriptase), and integrase (IN, not shown). polyproteins which are subsequently cleavletters A-F. Cleavage of Gag prp24 CA (capsid), p7all of the structural PR (proteas

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26 50010001003005001000 01020304050 VSISVFIFVFIS 46 2 4 62 462 WeeksLog10 RNAcCD4 T cells opies per mlper ul 500 10001003005001000 01020304050 VSISVFIF VFIS 46 262 4 462 WeeksLog10 RNAcCD4 T cells e opies per mlper ul Figure 1-6. Viral and Immune Response to Antiretroviral Therapy. Top panel, viral success and immune success (VSIS); center panel, viral failure and immune failure (VFIF); bottom panel, viral failure and immune success (VFIS). Median log 10 viral RNA copies per milliliter (purple boxes) are shown on thleft axis. Median CD4 T-cell counts per microliter (red triangles) are shownon the right axis. Error bars represent the 25 th -75 th quartile range. Figure from Perez et al. [192].

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27 PR Inhibitor IdiaviRtoavirSauiaviNlfiavirAmpreavirLpiaviAtazaavirTiraavir nnrinqnrennonrnpn 843367546132848 377778890 1020203233644485053 ** ** Inhibitor *PR IdiaviRtoavirSauiaviNlfiavirAmpreavirLpiaviAtazaavirTiraavir nnrinqnrennonrnpn 843367546132848 377778890 1020203233644485053 PR Inhibitor IdiaviRtoavirSauiaviNlfiavirAmpreavirLpiaviAtazaavirTiraavir Inhibitor nnrinqnrennonrnpnPR IdiaviRtoavirSauiaviNlfiavirAmpreavirLpiaviAtazaavirTiraavir nnrinqnrennonrnpn 843367546132848 377778890 1020203233644485053 843367546132848 377778890 1020203233644485053 **Figure 1-7. Resistance Associated Mutations in Protease. Aino acid mutations commonlitors are listed to the left of the char. Nin protease. Black boxes represet maor mtatioent inorpolymsappe areotedwith n astrisk.Dataromohnsn et l. [1]. ** y seen in response to protease inhibitors mber reprsent mutatons. osic natal * m.The eight currently approved protease inhibtuseaino acid positions njuns and grey boxes represm mi Psition at whhurorphism ar n ae f Joa16

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CHAPTER 2 IMPACT OF THERAPY ON GENETIC DETER MINANTS IN HIV-1 GAG-POL In , CD4+ T cell counts and plasma virus levels are used as markers of HIV-to uld , the of the pan troduction Currently 1 disease progression, and serve as the foundation for the guidelines for the use of antiretroviral agents [182]. Unfortunately, clinical variables alone are not sufficient to correctly predict a patient’s response to therapy, and other markers are needed. Genotypic studies performed in the Goodenow laboratory analyzed HIV-1 gag-pro sequences from cell-associated virus prior to the start of protease inhibitor therapy evaluate whether natural polymorphisms in viral protease or reverse transcriptase coserve as prognostic variables to help predict a patient’s response to therapy [192]. No association was found between reverse transcriptase genotype and therapy outcome, but results demonstrated that pretherapy protease genotype could predict, to some degreeviral and immune outcomes of protease inhibitor-nave patients. Furthermore, when PR genotype was combined with the two best clinical variables, CD4 + T cell count and disease stage, the correct therapy response was predicted in 100% of VFIF patients, 78% of VSIS patients, and 75% of VFIS patients. While the combination of clinical variables and PR genotype could correctly predict the outcome of a majority of patients, 20% tients were misclassified, indicating that other variables also impact therapy response. One region of the genome that may influence therapy outcome in combinatio 28

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29 with PR is gag. Genetic variability in protease and in gag from the B site to p6 were analyzed, and the B, D, D’, E, and F sites were found to contain low heterogeneity [10].The C cleavage site was highly variable, although a direct relationship between C site polymorphisms and therapy response was not apparent. Additional studies in the Goodenow and Dunn laboratories demonstrated that positions in the C cleavage site, as well as in p7 d positi 19, 35 and 38, 37 and 41, 62 and 71, 63 and 64, 71 and 77, 71 and 93, and 7d aining therapy revealed that an alanin NC , modulate protease processing activity and subsequent viral replication, indicating a functional role for gag polymorphisms [87]. Multiple groups have reported the existence of covariation between amino aci ons in protease following protease inhibitor containing therapy [27,112,140,268,271]. Using a large set of sequences from the Stanford HIV RT and Protease Sequence Database, Hoffman et al. examined covariation in both untreated and PI-treated subjects [100,120]. Thirty-two pairs of sites were found to be linked followingPI treatment, while in PI-nave subjects, 9 pairs of sites in protease were found to covary:10 and 93, 12 and 7 and 93. Furthermore, some pairs of sites were found to covary in both the treateand untreated groups, while some pairs varied together only in the untreated, or only in the treated groups, indicating that the selective pressure of drug may be advantageous for some linkages, but detrimental to others. Currently, few studies have examined covariation in gag, or covariation between positions in gag and protease. Analysis of cleavage site and protease genotype following PI-cont e to valine change in the p2 position of the D’ site is associated with the selection of a mutation at positions 46 and 82 in protease [8,131]. Given the effect polymorphisms in gag, outside of the cleavage sites, can have on protease processing and viral

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30 replication, understanding covariation between gag and PR could help lead to understanding the evolution of resistance to protease inhibitors. This study was design ed to determine if pretherapy polymorphisms in gag-pol can nse, and to elucidate any connection between mutations that o d 8 ks of lfinavir impacts viral and immune outco ew lt, teen, were nave to protease inhibitor therapy, and had viral loads higher than 3.5 log10 copies per milliliter. Eight be used to predict therapy respo ccur in gag and protease prior to and following 24 weeks of combination antiretroviral therapy including a protease inhibitor. Methods Study Design The entire protease inhibitor cohort consisted of 50 subjects. Twenty-three (46%)were treated with nelfinavir (NFV), 19 (38%) were treated with ritonavir (RTV), anwere treated with indinavir (16%). The Perez cohort included 26 of 50 subjects, including 17 treated with RTV, 6 treated with IDV, and 3 treated with NFV [192]. Of the 23 NFV treated subjects, preand posttherapy samples were available for 7 of the subjects. Initial analysis of the NFV subjects demonstrated the absence of primary resistance associated polymorphisms prior to therapy. Surprisingly, following 24 weeof therapy, 86% (6/7) of the subjects failed to demonstrate the expected accumulationnelfinavir associated mutations, indicating that ne me differently than indinavir and ritonavir. For this reason, all NFV subjects wereexcluded from the present analysis. Three subjects included in the Perez cohort were excluded from this cohort due to either non-compliance or insufficient sample, and 4 nsubjects not included in the Perez cohort were included in the current study. As a resuthe cohort in this dissertation included 24 HIV-infected children and adolescents. Eligible patients were between the ages of one and eigh

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31 (33% least gh as d to 22% (25th to 75th quartile ranges from 10 to in ) received IDV as their first PI, while 16 (67%) received RTV. Previous exposureto reverse transcriptase inhibitors (RTI) was allowed if the patient was nave to atone RTI in their new regimen. The median age of the cohort was 9 years (range six to thirteen years). Thirteen (54%) patients were male and 11 (46%) patients were female. Thirteen (54%) children were African American, 9 (38%) were Caucasian, 1 (4%) was Asian, and 1 (4%) was Hispanic. A majority of the patients (18/24, 75%) were infected through maternal (vertical) transmission. Four patients (17%) were infected throucontaminated blood products, and two (8%) were infected by sexual transmission. Prior to therapy, median CD4 + T cell counts at baseline were 166 cells per microliter (25 th to 75 th quartile ranges from 47 to 334 c/l), median percent of CD4 + cells was 8% (25 th to 75 th quartile ranges from 5 to 19%), and median CD4 to CD8 ratio w0.18 (25 th to 75 th quartile ranges from 0.07 to 0.46). The baseline median HIV-1 plasma RNA level was 5.2 log 10 copies per milliliter (25 th to 75 th quartile ranges from 4.4 to 5.5 log 10 copies/ml). Following 24 weeks of combination therapy including a protease inhibitor, the median number of CD4 + T cells increased to 467 c/l (25 th to 75 th quartile ranges from 172 to 582 c/l), median CD4 percent increase 27%), and the median CD4/CD8 ratio increased to 0.47 (25 th to 75 th quartile ranges from 0.21 to 0.93). The viral load decreased from 5.2 log 10 copies/ml to 4.4 log 10 (25 th to75 th quartile ranges from 2.9 to 5.3 log 10 copies/ml). An additional 24 weeks of therapy (48 weeks total) resulted in a slight decrease the number of CD4 + T cells to 448 c/l (25 th to 75 th quartile ranges from 326 to 780 c/l). The percent of CD4 + T cells increased to 23% (25 th to 75 th quartile ranges from 13 to

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32 31%), and the CD4/CD8 ratio increased to 0.59 (25 th to 75 th quartile ranges from 0.31 to 0.99). The viral load decreased to 4.1 log 10 copies/ml (25 th to 75 th quartile ranges from 3.1 to 5.0 log 10 copies/ml). Viral and Immune Outcome A patient’s response to therapy was determined at 24 weeks based on both viral and immune parameters. Viral success was define s as less than 400 copies per milliliter s of therapy, whereas subjects with detectable viral loads following therapy were ing T cell after 24 week classified as viral failures. An immune success was characterized as havcounts greater than 200 or CD4% that increased by > 5% following therapy. Failure to achieve immune success was defined as immune failure. Two subjects were CDC immune stage 1 ( > 500 CD4 T cells and CD4% > 25%) at baseline, and were classifiimmune successes because CD4 T cell counts remained stable and CD4% increaweeks. Following 24 weeks of therapy, six out of 24 (25%) patients were viral successes and immune successes (VSIS) and five (21%) were viral and immune failures (VThirteen (54%) patients displayed a discordant response of viral failure, but immune success. Baseline and 24 week clinical data for all subjects are shown in Tables through 2-3, and 2-4 through 2-6, respectively. The median age of the VSIS group (8, 25 ed as sed at 24 FIF). 2-1 er 0.01). th to 75 th interquartile range 6-9) did not differ from the VFIS (8, 5-13) or VFIF (10, 9-14) groups (P = 0.482) (Table 2-7). All three groups displayed similar viral loads and CD4 percentages prior to therapy (P = 0.296 and 0.079, respectively). In contrast, the absolute number of CD4 + T cells pmicroliter in the VFIF group (25, 3-45) was statistically different from both the VSIS (225, 161-387) and VFIS (256, 57-410) groups (P =

PAGE 47

33 A summary of clinical characteristics following 24 weeks of therapy is shTable 2-8. Both the CD4 own in iffered nts demonstrated an 75th 0 uncha6 IS (Figure iral roup + T cell number and CD4% of the VFIF group dsignificantly from both immune success groups after therapy (P = 0.003 and P < 0.001, respectively). In contrast, the viral load of the VSIS group (2.6) was significantly lower than either of the viral failure groups (P < 0.001). The VSIS patieincrease in median CD4 + T cell counts from 225 c/l at baseline to 562 c/l (25 th toquartile ranges from 466 to 1063 c/l) after 24 weeks, and a significant 2.4 log 10 copies/ml drop in viral RNA (P = 0.002). The viral failure, immune failure group displayed no net change in viral load over 24 weeks of therapy, remaining at 5.3 log 1copies/ml (P = 0.274), and an insignificant increase in CD4 T cell number (P = 0.306). Even though the new viral load steady state of the VFIS patients remained virtually + nged from baseline (P = 0.165), the number of CD4 + T cells nearly doubled to 48c/l (25 th to 75 th quartile ranges from 451 to 576 c/l) (P = 0.046). In summary, the VSgroup demonstrated a sustained decrease in viral load and an increase in CD4%2-1A), the VFIS group demonstrated a transient suppression of viral load and an increasein CD4% (Figure 2-1B), and VFIF group demonstrated a transient suppression of vload, but no increase in CD4% (Figure 2-1C). Comparison of CD4% among the three response groups confirms that the immune response of the VSIS and VFIS groups did not differ from each other, but both differed from the VFIF group (Figure 2-2A). Comparison of viral load over time revealed that while all three groups displayed a reduction in viral load by 4 weeks, the magnitude of the decline differed between the groups (Figure 2-2B). The viral load of the VFIF gdecreased less than one log, while the viral loads of both the VFIS and VSIS groups

PAGE 48

34 decreased by greater than a log, with the VSIS group showing the greatest decline. Over the course of therapy, the VSIS group maintained a low viral load, while the viral loof the VFIS and VFIF groups rebounded to pretherapy levels by 24 weeks. The preaposttherapy viral loads of the two viral failure groups were similar, and since the VFIS group demonstrated a greater suppression of virus, the magnitude of rebound was highefor the VFIS group compared to the VFIF group. Sequencing Plasma samples and cells collected prior to and following combination antiretroviral therapy were used to evaluate gag, protease, and envelope genotype. Rwas extracted from plasma using the QIAamp Viral RNA Mini Kit (Qiagen, ValenCA), followed by cDNA preparation. Ten microliters of RNA was added to 1 l of primer and incubated at 7 ads nd r NA cia, 0C for 10 minutes, then placed on ice. Primer Pol 4 (Invit19 l of l, rogen, Carlsbad, CA, 5’-TCCTACATACAAATCATCC-3’, nucleotides 3101-31in HIV HXB2 ) was used for gag-PR preparation, and primer 194G (Invitrogen, 5’-CTTCTCCAATTGTCCCTCATA-3’) was used for envelope. After a quick spin, 8 the reverse transcriptase cocktail (10X PCR Buffer (Invitrogen), 25 mM MgCl 2 (Invitrogen), 10 mM dNTP (Invitrogen), 0.1 M DTT (Invitrogen), 4 mg/ml bovine serumalbumin (BSA, Invitrogen), RNase OUT (Invitrogen)) was added, and the mixture was incubated at 42C for 5 minutes. One microliter of SuperScript II RT enzyme (200 U/mInvitrogen) was added, followed by a 45 minute incubation at 42C and a 15 minute incubation at 70C. The resultant cDNA was stored at -20C until used for PCR amplification. DNA from cells was isolated using the QIAamp DNA Blood Mini Kit (Qiagen) and stored at -20C until used for PCR.

PAGE 49

35 The gag-P R region was amplified using two rounds of PCR. First round amplite, at 0 l of LB an g ), fication was performed with forward primer Gag 7 (Invitrogen, 5’-GTTAAAAGAGACCATCAAT-3’, nucleotides 1389-1407) and reverse primer Pol 4 (Invitrogen, 5’-TCCTACATACAAATCATCC-3’, nucleotides 3101-3119), followed by second round with nested primers G100 (Invitrogen, forward, 5’-TAGAAGAAATGATGACAG-3’, nucleotides 1817-1834) and Pol I (Invitrogen, reverse, 5’-ACTTTTGGGCCATCCATTCCTGGC-3’, nucleotides 2588-2611). Amplifications were performed in a 96-well GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA) and consisted of an initial denaturation at 94C for 10 minutes, 35 cycles of denaturation at 94C for 1 minute, annealing at 55C for 1 minuand extension at 72C for 2 minutes, with a final elongation for 10 minutes at 72C.One microliter of second round PCR product was cloned into pCR 2.1-TOPO vector using the TOPO TA Cloning Kit (Invitrogen). The reaction was incubatedroom temperature for 30 minutes, transformed into TOP10F’ competent cells (Invitrogen), plated on LB plates that contained 100 mg/ml ampicillin (Sigma), 520mg/ml X-GAL (Gibco), and 50 l of 100mM IPTG (Invitrogen), and incubated overnight at 37C. To screen for clones which contained an insert, white colonies were picked, used to inoculate a 96-well flat bottom tissue culture plate which contained 20 ml d 10 l of 50 mg/ml ampicillin (200 l per well), and then streaked onto a LB plate(hereafter referred to as slug plate) containing 100 mg/ml ampicillin (Sigma). The sluplate was incubated at 37C overnight, and the 96-well plate was incubated at 37C for 1 hour. After the hour incubation, a reaction containing PCR Supermix (Invitrogenforward primer P1 (Invitrogen, 5’-CAGAGCCAACAGCCCCACCAG-3’, nucleotides

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36 2147-2167), reverse primer P2 (Invitrogen, 5’-CTTTTGGGCCATCCATTCCTGGC-3’, 10), and 2 l of inoculated culture was set up, and gag-PR was ampli f 7 E mers CCTGTGTGGAA-3’) and 194G (Invitd of s. 13 nucleotides 2588-26 fied with an initial denaturation at 94C for 2 minutes, 35 cycles of 94C for 30seconds, 56C for 30 seconds, and 72C for 1 minute, followed by a final extension ominutes at 72C . Ten microliters of the PCR reaction was run on a 1% agarose gel to confirm a positive insert. For correct clones, the corresponding slug from the slug plate was picked and used to inoculate a 96-well flat bottom plate containing LB and ampicillin as described above. This plate was then sent to the Genome Sequencing Service Laboratory at the University of Florida. The sequences were prepared with DYEnamic ET dye terminator cycle sequencing kit for MegaBACE DNA Analysis Systems (GE Healthcare, Chalfont St. Giles, United Kingdom), and run on a MegaBAC1000 (GE Healthcare). The env region was amplified with two rounds of PCR. The first round pri were D1 (Invitrogen, 5’-CACAGTCTATTATGGGGTA rogen, 5’-CTTCTCCAATTGTCCCTCATA-3’), and the second round primers were Env5 (Invitrogen, 5’-GGGGATCCGGTAGAACAGATGCATGAGGAT-3’) an195C (Invitrogen, 5’-CTGGGTCCCCTCCTGAGG-3’). The amplification consistedan initial denaturation at 95C for 5 minutes, 35 cycles of 94C for 1 minute, 58C for 1 minute, and 72C for 2 minutes, followed by a final extension at 72C for 10 minuteThis region was cloned and sequenced as described above with the exception of the primers used to confirm the presence of an insert. In this case, universal primers MForward (Invitrogen, 5’-GTAAAACGACGGCCAG-3’) and M13 Reverse (Invitrogen, 5’-CAGGAAACAGCTATGAC-3’) were used.

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37 Data in sed to py sample to assess m cant. Analysis Approximately 10-15 clones were sequenced per subject (24) per time point (pre-and posttherapy) for two different regions of the genome (gag-pro and env), resultingthe analysis of over 1,000 sequences. Subjects were stratified into three groups based on both viral and immune outcomes (VSIS, VFIS, VFIF), as well as into two groups baon viral outcome alone (VS and VF) or immune outcome alone (IS and IF). Pretherapy amino acid sequences of Gag (from p2 through p6), p6 Pol , protease, and envelope V3 from 24 patients were compared to the amino acid sequence of HIV HXB2 (Figure 2-3), andany differences were counted as a polymorphism. Posttherapy samples were comparedHIV HXB2 to determine overall level of variability, as well as the prethera the accumulation of mutations following therapy. Cell-associated virus canrepresent virus from initial infection, currently replicating virus, or both, while virus frothe plasma represents only actively replicating virus. To determine any potential differences between cell-associated virus and plasma virus, posttherapy sequences from both cells and plasma were obtained for the VFIS and VFIF groups. Statistical significance of the differences between groups was assessed by Mann-Whitney Rank Sum Test or Kruskal-Wallis one-way analysis of variance (ANOVA) using SigmaStat 3.0 (Jandel Scientific Corp, San Rafael, CA). A P value of < 0.05 was considered signifiTo analyze envelope, the charge of the V3 loop of every sequence was calculated by counting arginines and lysines as positive and aspartic acids and glutamic acids as negative. Each subject was then scored as to whether the V3 loops of the viruses were all low charge ( < +4), all high charge ( > +5), or a mixture of high and low charge. Alignments of all amino acid sequences used in these analyses are located in Appendice s B-E.

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38 Results Protease Analysis To analyze polymorphisms in protease, amino acid positions were defined as primary, secondary, or supplemental. Mutations in primary and secondary positions increase resistance to protease inhibitors, while changes at supplemental positions have not previously been associated with reduced sensitivity to protease inhibitors. Mutations in the primary positions are the first mutations that a ccumulate in response to PI therapy, e. Mutations in secondary positions usualt the and istant positions (82, 84, and 90). However, the polym and are associated with an increase in drug resistanc ly occur following primary mutations, and have less impact on drug resistance, bumay act to increase replicative capacity. Polymorphisms at primary and secondary positions were then further categorized as therapy specific, if the new amino acid was associated with reduced sensitivity to the PI the subject received, or therapy non-specificif the amino acid has not been associated with resistance to the administered PI. Pretherapy Primary and Secondary Polymorphisms Pretherapy polymorphisms in the primary and secondary positions were restricted to positions 10, 36, 63, 71, 77 and 82. The most common pretherapy polymorphism occurred at amino acid 63 in 83% (20/24) of subjects. Eighty percent (16/20) ofsubjects with a polymorphism at 63 demonstrated a change from leucine to proline. Nineout of 24 (38%) subjects contained a polymorphism at position 36, and 6/24 (25%) contained a polymorphism at amino acid 77. The remaining three positions (10, 71, 82) were polymorphic in less than 15% of subjects. Only one subject displayed a polymorphism in any of the primary res orphism was not specific to the therapy the subject received.

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39 When the cohort was stratified into three response groups, the median number pretherapy polymorphisms was similar among all groups. The VSIS and VFIF groups both had a median of 1 pretherapy polymorphism (ranges 0-3 and 1-4, respectively), while the VFIS median was 2 (range 1-3). Positions 36, 63, and 77 were polymorphicall response groups. When stratified by immune outcome, no differences between immune success and immune failure subjects were found. However, when stratified by viral outcome, the number of subjects with 2 or more substitutions was higher in the viral failure group (11/18, 61%) than the viral success group (2/6, 33%), and therapy specifipolymorphisms at amino acid positions 10 and 71 were found only in the viral failure groups, demonstrating that protease genotype predicts viral outcome. To further analyze primary and secondary positions in protease, pretherapy genotypes were characterized as sensitive if none of the polymorphisms would reduce sensitivity to the PI the subject was given, or resistant if at least one of the substitutionwould confer reduced sensitivity to t of in c s he PI. Ten out of 24 (42%) subjects were classified d as resistant (Figure 2-4). Nine out of 10 (90%s ionine to isoleucine chang77, as sensitive and 14 of 24 (58%) were classifie ) sensitive genotypes contained one polymorphism, and none contained more than one substitution. In contrast, 12 out of 14 (86%) resistant genotypes contained 2 or more substitutions. In the resistant genotype group, therapy specific polymorphisms were restricted to positions 10, 36, 71, and 77. Furthermore, therapy specific polymorphismat positions 36 and 77 were mutually exclusive. If there was a meth e at 36, a valine was present at 77. If there was a valine to isoleucine change at there was a methionine at 36. The VSIS subjects were evenly distributed among sensitive and resistant genotypes, while a majority of the VFIS (62%) and VFIF (60%)

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40 groups were classified as resistant (Figure 2-5). All three response groups contained botherapy specific and therapy non-specific substitutions, but only the VSIS group contained a subject without any substitutions (Figure 2-6). The VSIS and VFIF gboth contained 2 subjects with therapy non-specific substitutions and 3 subjects witherapy specific substitutions. The discordant response group contained higher numbers of both types of polymorphisms with 5 subjects displaying non-specific substitutions and 8 subjects displaying therapy specific substitutions. Posttherapy Primary and Secondary Mutations Fo th roups th llowing 24 weeks of therapy, mutations accumulated in all of the primary and ception of amino acid 73 (Figure 2-7). The overa the most , ites 84 rapy specific mutations was the same in botge 0-8) as 5 r secondary protease positions, with the ex ll frequency of mutations at all of the resistant positions, with the exception of 77 which remained the same, increased following therapy. Position 63 remainedpolymorphic, with a mutation in 92% of subjects. The frequency of mutations at 10, 36, and 71 increased to 38%, 50%, and 21%, respectively. The proportion of subjects with mutations at amino acid 82, a primary resistance site, increased to 58%. Prior to therapyno polymorphisms were observed in either of the other two primary resistance sand 90, while after therapy, mutations accumulated in 4% and 17% of subjects, respectively. When subjects were grouped according to their pretherapy genotypes, the median number of total mutations as well as new the h the sensitive and resistant genotypes. However, the median number of total therapy specific polymorphisms was twice as high in the resistant group (4, ranin the sensitive group (2, range 0-10). Posttherapy cell associated sequences were obtained from 5/6 VSIS subjects, 4/VFIF subjects, and 12/13 VFIS subjects. Sequences from plasma RNA were obtained fo

PAGE 55

41 5 of the VFIF subjects and 12/13 of the VFIS subjects. The total median number of mutations following therapy did not differ between cell associated and plasma sequences for either the VFIS (5, range 1-11) or VFIF (4, range 2-5) groups. Similarly, the median number of mutations accumulated following therapy was identical between ceplasma for both VFIS (3, range 0-10) and VFIF (2, range 0-4). When grouped accorto viral response, differen lls and ding ces between the two groups became apparent. The viral failures accumumber of pretherapy polymorphisms and the median number of posttherapy ure 2-8). upplemental Polymorphisms to 92% ulated significantly more mutations than the viral successes following therapy (P = 0.004 for cells and P = 0.007 for plasma). The total number of posttherapy mutations was significantly higher in the viral failure group as compared to the viral successes (P = 0.003 for cells and P = 0.011 for plasma). When compared to the median number of pretherapy polymorphisms, the viral failures accumulated a significant number of mutations (P < 0.001), while the viral successes demonstrated no change between the median n mutations (P = 0.792) (Fig Pretherapy S Twenty-seven supplemental positions in protease contained polymorphisms priortherapy, but only 8 were polymorphic in greater than 20% of the cohort. Amino acid 37 was the most polymorphic, with a serine to an asparagine or aspartic acid change in (22/24) of subjects. Positions 13 (29%), 14 (25%), 15 (21%), 41 (25%), 57 (21%), 62 (25%), and 64 (25%) were also polymorphic in > 20% of subjects. When grouped into sensitive and resistant genotypes, the sensitive group had a median of 3 pretherapy polymorphisms, and the resistant group had a median of 4 (Figure 2-9). When the cohort was stratified according to response outcome, the median numberof pretherapy polymorphisms was similar among all three response groups. The VSIS

PAGE 56

42 and VFIF groups had a median number of 4 supplemental polymorphisms (ranges 2-7 and 1-5, respectively), while the VFIS group had a median of 3 (range 1-8). Positions41, 57, 62, and 64 were polymorphic in all response groups. The occurrence of polymorphisms at specific positions differed when stratified by immune and viral outcomes, suggesting that supplemental positions can help predict therapy response. For example, polymorphisms at supplemental position 14 were found only in immune success 37, patiene , cid 37 remained the most polymve r median of 4 to a median of 5, while the VFIS increased to a median of 4 from a median ts, and were absent from immune failures, while position 13 was found to be twicas polymorphic in the viral success group (50%) as compared to the viral failure group (22%). Posttherapy Supplemental Mutations The number of supplemental positions with mutations increased to 39 following therapy (Figure 2-10). Positions 22, 49, 51, 65, and 96 contained pretherapy polymorphisms, but after therapy, no mutations were detected, while 17 positions (9, 1723, 25, 27, 29, 30, 34, 45, 52, 53, 66, 79, 83, 92, 94, and 98) that were not polymorphic before therapy accumulated mutations after therapy. Amino a orphic, with 91% of subjects demonstrating a mutation in that position. Stratification by pretherapy genotype demonstrated that the resistant genotypes accumulated a median of 2 (range 1-4) new supplemental mutations, while the sensitigenotypes accumulated a median of 0 (range 0-1). Each of the three response groups demonstrated an increase in median number of supplemental mutations following therapy, but no difference in the number of posttherapy supplemental mutations between the cells and plasma was observed for eitheof the viral failure groups (Table 2-9). The VSIS and VFIF groups increased from a

PAGE 57

43 of 3. In both cells and plasma, the VSIS and VFIF groups each contained a median of 5 mutations following therapy. The VSIS group accumulated a median of 1 newhile the VFIF group accumulated a median of 1 new mutation in the cells, and no newmutations in the plasma. The VFIS group contained a median of 4 mutations posttherapy, and accumulated no new mutation w mutation, s in the cells and a median of 1 new plasma. . es. y ing at ber alysis of the location of the polymorphisms revea mutation in the The location of new mutations differed among viral and immune response subjectsMutations in position 93 were found only in the viral failure group, while, mutations at position 9 were found in a quarter of viral success subjects, but none of the viral failurIn addition, of the 22 positions that contained new supplemental mutations, 17 (77%) contained mutations only in the immune success group, 4 (18%) contained mutations only in the immune failures, and only 1 position (5%) accumulated new mutations in both groups. Cleavage Site Analysis Pretherapy The C and E cleavage sites were the most heterogeneous, with 100% of subjects showing variability from HIV HXB2 . The D’ cleavage site demonstrated low heterogeneitprior to therapy, with only 17% (4/24) of subjects showing polymorphisms. The D cleavage site demonstrated higher variability, with 54% (13/24) of subjects containleast one polymorphism. All three response groups contained the same average numof D site polymorphisms per subject. An led that position P2 was the only position on the non-prime side of the cleavage site that contained any polymorphisms. The prime side contained more heterogeneity, with polymorphisms in all three response groups at positions P1’, P3’, and P5’ (Figure 2-11A).

PAGE 58

44 Polymorphisms at P1’ and P3’ appeared less frequently in the VFIS group than either the VSIS or VFIF groups, although none of the polymorphisms were found in more than 50% of subjects. Stratification by immune outcome did not reveal any significant differences between the groups (Figure 2-11B). However, when stratified by viral outcome only, polymorphisms at position P3’ appeared in half of the viral successes, but only 2 and alf s, and and 26%, respectively) (Figure 2-12B). The viral failures also contained a eity at these two positions than the viral successes (50% for viral ared to 17% of viral successes) (Figure 2-12C). oups 8% of the viral failures (Figure 2-11C). In contrast to the D site, there was a greater level of variability on the non-prime side of the C cleavage site compared to the prime side (Figure 2-12A). Positions P3P3’ were variable in all three response groups. Position P2 was polymorphic in over h(7 out of 13, 54%) the VFIS subjects, but only 1 out of 6 (17%) of the VSIS subjectnone of the VFIF subjects. Strikingly, positions P4 and P5 are much more variable in the immune failure subjects (80% and 100%, respectively) compared to the immune success group (32% higher level of heterogen failures comp Similar to the C site, the non-prime side of the E cleavage site was considerably more variable than the prime side. Position P3’ was the only position on the prime side of the cleavage site that contained any polymorphisms, and was variable in 100% of subjects. HIV HXB2 contains a valine at this position, while HIV LAI contains an isoleucine,and all of the subjects demonstrated a valine to isoleucine polymorphism at this position. Position P2, and to a lesser extent, position P5, were variable in all three response gr(Figure 2-13A). Position P3 was more variable in the VSIS group (67%) than either the VFIS (31%) or the VFIF (40%) group. When stratified by immune outcome, positions

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45 P2, P3, P5, and P3’ were equally variable, while polymorphisms at P1 were found onthe immu ly in ne success group (Figure 2-13B). A difference in the frequency of polym/5 ma RNA were obtained for 5 bjects and 12/13 VFIS subjects. No differences in the appearance and frequage on bjects accumulated any mutations. The frequency of mutations at position P3’ remai orphisms at position P3 was observed when the subjects were stratified by viral outcome only. Polymorphisms at position P3 appeared in 2/3 of viral success subjects, but only 1/3 of viral failures (Figure 2-13C). Posttherapy Posttherapy cell-associated sequences were obtained from 5/6 VSIS subjects, 4VFIF subjects, and 13 VFIS subjects. Sequences from plas of the VFIF su ency of mutations between cells and plasma were observed in any of the cleavsites analyzed. Following therapy, the D cleavage site demonstrated a similar level of heterogeneity as pretherapy, with 48% (11/23) of subjects containing mutations. PositiP3’ remained the most polymorphic site, and contained mutations in 60% of the VSIS subjects, but only 23% of the VFIS subjects and 20% of the VFIF subjects. When stratified by viral outcome, the frequency of mutations at position P3’ remained higher inthe viral success group than the viral failure group. New mutations accumulated in only 3 out of 23 (13%) subjects, and all three were immune successes. None of the immunefailure su ned similar between the immune success and immune failure groups, while mutations were more frequent at positions P1’ and P5’ in the immune failure group compared to the immune success group. The C cleavage site remained highly polymorphic following 24 weeks of therapy. New mutations, which accumulated only in the VFIS group, occurred in 22% (5/23) of subjects. The accumulation of new mutations was restricted to positions P3, P4, P5, and

PAGE 60

46 P4’. The relative proportion of mutations at each position remained unchanged. P3 and P3’ remained polymorphic in all three response groups, and P2 was only variable in immune success group. Mutations at P4 and P5 were higher in the VFIF group (80% and 100%, respectively) than either the VSIS (20%) or VFIS (46% and 31%, respectively) groups. the ’ s tations at P5 appearing only in viral failure Analysis Prethes d ons 3, osition , The posttherapy E cleavage site was essentially identical to pretherapy. All subjects contained polymorphisms following therapy, but no subject accumulated any new mutations. The proportion of subjects with variability at positions P2 and P3remained invariant. The VSIS group continued to contain more mutations at P3 than either the VFIS or VFIF groups, and mutations at P1 appeared only in the immune success subjects. One polymorphism that was present at position P5 prior to therapy walost after 24 weeks of therapy, resulting in mu subjects. Nucleocapsid erapy Analysis of pretherapy p7 NC revealed the absence of polymorphisms in any of the conserved cysteine and histidine residues of either of the two zinc finger motifs (residu15, 18, 23, 28, 36, 39, 44, and 49). Twenty-five of 54 amino acids in p7 NC demonstratevariability (Figure 2-14B). The three most variable residues were located at positi12, and 26. Over half of the subjects (13/24, 54%) contained a polymorphism at p26, while residue 12 was variable in 46% (11/24) of subjects, and residue 3 in 42% (10/42) of subjects. A lower level of variability (< 30% of subjects) was observed in the following positions: 1, 4, 5, 6, 7, 8, 9, 11, 13, 20, 22, 27, 29, 31, 34, 38, 41, 46, 48, 50

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47 51, and 54. The remaining 30 positions in p7 NC contained no polymorphisms in any subject. When analyzed according to response group, the median number of pretherapy polymorphisms in all three response groups was 4. Positions 3, 7, 8, 13, 34, 41, and 48 were polymorphic in all three response groups. Residue 12 was more variable in the vir al failur, le c tations accumulated in 19 different positions in p7NC, 12 of which were polyme es. e (50%) and immune failure (60%) groups than either of the success groups (33% and 42%, respectively). In contrast, residue 26 was variable in 58% of the immune success group, but only 40% of the immune failure group. Posttherapy Following 24 weeks of therapy, the number of amino acid positions in p7 NC demonstrating variability from HIV HXB2 increased from 25 to 31. Similar to pretherapy, the three most variable positions were 3, 12, and 26, which showed mutations in 43%48%, and 57% of subjects, respectively. Amino acid position 12 remained more variabin the VFIF group than either VSIS or VFIS, and position 26 remained more polymorphiin the immune success group compared to the immune failures. In contrast, residues 41 and 48, which were equally polymorphic in all response groups prior to therapy, becamemore variable in the viral success group compared to the viral failure group following therapy. New mu orphic before therapy. Of the 7 positions that were not polymorphic prior to therapy, new mutations at 5 positions (25, 43, 45, 47, and 52) occurred only in the discordant VFIS group. In addition, new mutations at position 32 were found only in thimmune success subjects, and mutations at position 10 accumulated only in viral failurThe median number of new mutations in the cells was similar among the three response

PAGE 62

48 groups (VSIS = 0, VFIS = 0, VFIF = 1). No mutations accumulated in the cysteine and histidine residues of the zinc fingers. However, 10 out of the 19 residues with new mutations were located within the zinc fingers motifs (Figure 2-14C). Four out of t(29%) residues in the first zinc finger, and 6 out of 14 (43%) residue he 14 s in the second zinc fingerts, 4/5 ained at 4, the VFIS group increased to 5 in the cells,ells, and 0% of the he olymorphisms only in the subjects, while just 3 contained polymorphisms only in the immune ition 31 was more variable in the immune failures (60%) than the successes (42%n the the contained at least one new mutation following therapy. Posttherapy cell-associated sequences were obtained from 5/6 VSIS subjecVFIF subjects, and 13 VFIS subjects. Sequences from plasma RNA were obtained for 4/5 VFIF subjects and 12/13 VFIS subjects. The median number of posttherapy mutations in all three response groups was similar to the median number of pretherapy polymorphisms. The VSIS group rem but remained 4 in the plasma, and the VFIF group remained at 4 in the cdecreased to 3 in the plasma (Table 2-10). p6 Analysis Pretherapy The cohort contained highly polymorphic p6 regions, with variability in 7amino acid positions. Residues 17, 19, 30, and 39 were polymorphic in over 75% of tsubjects, and residues 25 and 42 were variable in > 50% of subjects. When analyzed according to response group, multiple positions were equally variable among all three response groups: 1, 5, 8, 12, 17, 19, 25, 30, 33, 34, 35, 39, 42, 48, and 50. When stratified by immune outcome, 10 positions contained p immune success failures. Pos ), while position 42 was more variable in the immune successes (68%) thafailures (40%). There were 15 positions in which polymorphisms were found only in

PAGE 63

49 viral failure group, and not in the success group. In contrast, amino acid 40 was the only position which contained polymorphisms in the viral success group, but was invariant in the viral failures. Two positions were found to be more variable in viral successes as compared to viral failures. Polymorphisms at amino acid positions 3 and 38 were fin 50% of viral success subjects, but only 28% and 6% of viral failures, respectively. The presence of three different insertions in p6 was found prior to therapy (Figur2-15). The first insertion was located between residues 7 and 8 and varied in length f3 to 15 amino acids. The second insertion, located between residues 20 and 21, ranged from 5 to 9 amino acids in length. The third insertion was a single amino acid insertion between positions 37 and 38, and was found in only 1 subject. The first insertion occurred in 9 out of 24 (38%) subjects while the second was found in only 8% (2/24) osubjects. The frequency of the presence of insertions did not appea ound e rom f r to be related to outcoed the most ted sequences were 5/6 VSIS subjects, 13 VFIS subjects, and 4/5 VFIF subjects, and plasma RNAhe me as the frequency was similar among all three response groups: 50% in VSIS, 46% in VFIS, and 40% in VFIF. The frequency of insertions between 7 and 8 occurrat similar frequencies in all response groups. However, insertions between residues 20 and 21 were found only in viral failures and in 20% of immune failures, but only 5% of immune successes. The insertion between residues 37 and 38 was also found only in viral failures. Posttherapy Similar to pretherapy, amino acid positions 17, 19, 25, 30, 39, and 42 wereheterogeneous after 24 weeks of therapy. Posttherapy cell-associa obtained from sequences were obtained from 12/13 VFIS subjects and 4/5 VFIF subjects. TVSIS group accumulated a median of 1 new mutation following therapy (range 0-3). In

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50 the cells, the VFIS subjects accumulated a median of 0 new mutations (range 0-7), while in the plasma, a median of 1 new mutation was acquired (range 0-5). The VFIF group did not accumulate any mutations in the cells, but accumulated a median of 2 new mutations in the plasma RNA (range 0-5). There were a total of 29 positions that did not accumulate any new mutations in p6. A majority of these were located in the C-terminus. More specifically, 8 of the unchanged positions spanned the AIP1 and VPR binding sites that are located within in the C-terminus. Furthermore, the location of the three insertions that were present before therapy remained unchanged after therapy, and no new mutations accumulated in any of the insertions. When separated by viral and immune outcomes , 15 positions remained polymf viral e found Pretherapy p6Pol sequences contained a high level of variability, and also demonstrated the presence of insertions. The most highly polymorphic positions, in orphic in only the viral failure subjects. Amino acid 40 remained polymorphic only in the viral successes, while position 38, which was found variable in both viral successes and viral failures prior to therapy, was found in polymorphic in 60% osuccesses and no viral failures posttherapy. Positions 3, 12, and 50 were more polymorphic in the viral success group compared to the viral failure group, while the opposite was true for position 25. A difference between cells and plasma was observed at amino acid 42. In the cells, mutations were found in 67% of immune success subjects and only 25% of immune failures. In contrast, in the plasma RNA, mutations werin 50% of both immune success and immune failures. p6 Pol Analysis Pretherapy

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51 which polymorphisms were found in greater than 50% of subjects, were residues 4, 8, 1135, 40, 50, and 54. Eleven out of 55 (20%) positions remained invarian , t in all subjects. tions were located at the N-terminus of p6Pol, while the remaining 3 werdues ues 52 and 53. The first t similar frequencies in all three response groups, whereas ee insertions were found only in the viral failures. Postt Eight of the eleven posi e located in the C-terminus. Differences in the frequency of polymorphisms between groups could be identified when the data was analyzed by viral and immune outcomes (Figure 2-16). Positions 16, 45, 46, and 53 were more polymorphic in viral successes, while polymorphisms at position 38 were found only in viral failures. In addition, changes at residues 4, 28, and 50 occurred more frequently in immune success subjects compared to immune failures, while positions 27 and 49 were found to be over twice as polymorphic in immune failures. Interestingly, amino acid position 11 was highly polymorphic in the VSIS group (83% of subjects) and the VFIF group (80% of subjects), but was only polymorphic in 38% of the VFIS subjects. Four insertions, which occurred at similar frequencies in the VSIS (50%), VFIS (46%), and VFIF (40%) groups, were identified in p6 Pol . The first, located between residues 22 and 23, had lengths ranging from 6 to 15 amino acids. Ten out of 24 subjects (41%) contained an insertion in this region. The remaining 3 insertions were each found in only one subject. The second insertion of 5 amino acids was located between resi35 and 36, the third insertion between amino acids 44 and 45 was 9 residues in length, and the fourth was a single amino acid insertion between resid insertion was found a remaining thr herapy Twelve out of 55 (22%) positions in p6 Pol contained no mutations following therapy. Eleven of those positions were located in the N-terminus, and the last was

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52 located at the far C-terminus. Similar to pretherapy, positions 4, 8, 11, 35, 40, 50, and 54 were the most variable, occurring in greater than half of the cohort. Six positions (4, 8, 11, 23, 50, and 54) were equally variable among the response groups. When analyzed by viral outcome, mutations at 2 positions (18 and 25) were found in 20% of the viral success group, but none of the viral failure group. There were 14 positions that contaa low leve ined l of variability in the viral failures and no variability in the viral successes. Anoths hat re ions after 24 weeks of therapy. In both the cells a, the discordant response group gained a median of 1 new mutation. The VFIFe re er 6 amino acids contained mutations in 60% of the viral success subjects, but lesthan 40% of the viral failures. In contrast, there were only two positions (35 and 40) twere more polymorphic in viral failures as compared to viral successes. When analyzed by immune outcome, mutations at position 33 occurred only in the immune failure subjects and none of the immune successes subjects. Cell-associated sequences were obtained for 5/6 VSIS subjects, 13 of the VFIS subjects, and 5 of the VFIF subjects after 24 weeks of therapy. Plasma sequences wealso obtained for 12/13 VFIS subjects and 4/5 VFIF subjects. The VSIS group accumulated a median of 2 new mutat and the plasm group gained a median of no new mutations in the cells, but 2 new mutations in thplasma. Amino acid 11 remained highly polymorphic in the cells of the VSIS and VFIF groups, with mutations occurring in 80% of subjects. The VFIS group contained mutations in only 38% of cell associated sequences, but over half (58%) of plasma RNA sequences. Mutations in 6 positions were found to a greater extent in the immune failugroup compared to the immune success group (Figure 2-17). All of these positions were

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53 found to be more variable in the immune failures prior to therapy, but the proportion of subjects with mutations at these positions increased following therapy. Cova ix 1, r therapy, such as position 13 in PR and position 50 in p6Pol of subject VFIS30. In total,sitions were found to covary (Figure 2-16) pol, and ts in group riation Analysis The preand posttherapy sequences from all 24 subjects were examined for covariation within protease alone, within gag alone, within pol alone, and between gag and pol. For this study, positions were considered linked if a polymorphism that occurredin one position also occurred in a second position in all sequences examined for that subject at that time point. Covariation was found in 83% (20/24) of subjects. Four of s(67%) VSIS subjects, 12 of 13 (92%) VFIS subjects, and 4 of 5 (80%) VFIF subjects demonstrated covariation. Some positions were found to covary only in pretherapy sequences, such as positions 14 and 62 in PR of subject VFIS22, while some covaried only in posttherapy sequences, such as positions 32, 33, and 54 of PR in subject VFIS1and some covaried both before and afte 40 sets of po . Eight covaried within protease, 12 covaried within gag, 9 covaried within11 covaried between gag and pol. In addition, insertions in p6 and p6 Pol were found to covary. If an insertion occurred between positions 7 and 8 in p6, then there was no insertion between positions 20 and 21. In p6 Pol , insertions between residues 22 and 23 and between residues 44 and 45 were mutually exclusive. Envelope Analysis Pretherapy Prior to therapy, envelope sequences were obtained for 22 out of the 24 subjecthe cohort: 5/6 VSIS, 12/13 VFIS, and 5 VFIF. Four out of five (80%) of the VSIS contained viruses with low charge V3 loops, and only 1 subject contained a mixture of

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54 viruses (Figure 2-19A). In contrast, 60% (3/5) of the VFIF group contained a mixture of high and low charge viruses, and 40% (2/5) contained viruses with only low charge V3 loops. Viruses with high charge V3 loops were only found in the discordant VFIS group prior to therapy. Three of 12 (25%) subjects contained only high charge viruses, 4 of 1(33%) contained only low charge viruses, and 5 of 12 (42%) contained a mixture of viruses. When analyzed by immune response, the proportion of subjects with low charge viruses was similar in the immune success (47%) and immune failure (40%) groups. High charge viruses were found only in the immune succ 2 ess subjects. A mix of high and low cntained only n prior e viruses after therapy. As a result, 100% of the VSISe loops. The proportion of subjects with only high charge viruses decreased to 16% (2/12), harge viruses was found in 60% of immune failures, but only 35% of immune successes. When divided by viral outcome, 80% of viral success subjects colow charge viruses, while only 35% of viral failures contained low charge viruses. Icontrast, the proportion of subjects with a mixture of viruses was higher in the viral failures (47%) compared to the viral successes (20%). High charge viruses alone were found only in the viral failure group. Posttherapy Following 24 weeks of therapy, the proportion of VFIF subjects with low charge viruses and a mixture of viruses remained unchanged (Figure 2-19B). Sixty percent (3/5)contained a mix of high and low charge viruses, and 40% (2/5) contained only low chargeviruses. In contrast, the one subject in the VSIS group who had a mixture of virusesto therapy, contained only low charg subjects contained viruses with only low charge V3 loops. Following therapy, thVFIS group was still the only response group that contained viruses with high charge V3

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55 while the proportion of subjects with only low charge viruses increased to 42% (5/1The number of subjects with a mixture of viruses (5/12) remained the same after theraFollowing therapy, the proportion of immune success subjects with low charge viruses increased slightly to 59%, the proportion with high charge viruses decreased from18% to 12%, and the proportion with a mixture of viruses decreased to 29%. There was no change in the immune failure group. Forty percent contained low charge viruses and60% contained a mixture of viruses. No high charge viruses were found in any oimmune failures after therapy. Viral success subjects contained a mixture of high and low charge viruses prior to therapy, but after therapy, contained only low charge virusesLess change was observed in the viral failure group following therapy. The proportion osubjects with a mixed population of viruses remained unchanged, while the proportion of subjects with only low charge viruses increased to 41%, and the proportion with only high charge viruses decreased to 12%. Discussion Mutations in protease that are known to reduce drug sensitivity have been identified in therapy nave subjects, and studies suggest that the existence of secondary mutations in protease may also contribute to the virological response of the subject [27,140,192,218,263]. In our cohort, pretherapy polymorphisms were found at positions 10, 36, 63, 71, 77, and 82. The presence of 2 or more substitutions in protease was associated with a resistant genotype. Furthermore, pretherapy polymorphisms at positions in protease (36 and 77) were found to be mutually excl 2). py. f the . f two usive, suggesting a negatmutations at both 36 and 77. However, examination of the sequences revealed that the ive correlation between them. M36 was associated with V77I, and M36I was associated with V77. Following therapy, only one subject (VFIS12) demonstrated

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56 majority of clones contained M36I and V77, and a minority of clones contained M36 anV77I. No single clone contained M36I and V77I. Given the close proximity of amino acid positions 36 and 77, and their location in and near the hinge of protease, mutationboth positions would most likely cause an alteration in the mobility of the flap (Figure 2-20). If the flaps are unable to open and close properly, the catalytic activity of proteasand subsequent viral replication will be inhibited. Hoffman et al. also found that the M36I and V77I double mutation is strongly suppressed, most likely due to van der Waals forces [100]. These data underscore the importance of the structure of protease, as well as that of gag-pol, in the d s at e regulation of viral replication. ects. s were associated with ds a ith Multiple groups have shown that mutations in Gag, specifically the cleavage sites, accumulate following therapy to help compensate for mutations in protease [51,64,85,156,278,280]. Few groups have examined the impact of pretherapy Gag mutations on viral and immune outcome. Cote et al. identified polymorphisms in Gag (specifically p7 and p6 Pol ) that were common before therapy [50]. Consistent with this report, our cohort demonstrated polymorphisms in p6 Pol in 79% and 71% of subjects at positions 4 and 8, and polymorphisms at position 3 in p7 were found in 42% of subjPretherapy analysis of Gag and p6 Pol revealed that in all regions examined (p7, p6, p6 Pol , C cleavage site, D cleavage site, and E cleavage site), specific position ifferences between viral and immune outcomes, demonstrating that Gag displaydominant impact on protease activity and subsequent viral replicative capacity andresponse to antiretroviral therapy. The presence of multiple insertions was found in both p6 and p6 Pol , and was not related to therapy outcome. However, the location of the insertions was associated w

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57 viral outcome, similar to a study by Kaufmann et al which found that subjects with insertions in the N-terminus of p6 were more likely to experience virological success [125]. In our cohort, insertions in the far N-terminus were found in half of viral sucsubjects and less than 40% of viral failures. The insertions between residues 7 and 8 op6 included the SRPE motif or a repeat of the P(T/S)AP motif, both of which have been shown to confer a replicative advantage for drug resistant variants, so insertions in this region appear to be beneficial for the virus in the presence of protease inhibitors [106,245]. In contrast, insertions in the C-terminus of both p6 and p6 cess f only IV-1 negative consequences for viral spread. The insertion in the far terminus of p6Pol is located within the E cleavage site. Cleavage at the E site is the first cleavage that occurs to release protease from Gag-Pol, and studies suggest that this cleavage is important for protease dimer stabilization [151,152,266]. Changes within the E cleavage site may alter the ability of protease to cleave itself out of the polyprotein, which could destabilize the dimer, prevent subsequent cleavage events, and inhibit replication. Mutations at position 36 in p6Pol have been shown to alter processing, and in our cohort, polymorphisms were found only in viral failures both prior to and following therapy, indicating that a substitution at this position is advantageous for the virus [178]. Covariation was found both within Gag and Pol, as well as between Gag and Pol. In agreement with other studies, two subjects demonstrated covariation between amino Pol were foundin viral failures. In p6, the insertion in the far C-terminus is located within the bindingsite for the cellular protein AIP-1. In an infected cell, a complex is formed between p6, AIP-1 and TSG101, and studies suggest that this interaction promotes budding of H[241,259]. Mutations that alter the binding site for AIP-1 would be expected to have

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58 acids 54 and 82 in protease [27,100,268]. An associa tion between a mutation in the p2 position of the D’ cleavage site (amino acid position 54 in p7NC) and the accumulation of mutations at positions 46 and 82 in protease was reported, but in our cohort, only one subject demonstrated an alanine to valine mutation following therapy [8,131]. Amino acid position 46 was invariant in this subject, while a mutation to alanine at position 82 did occur. However, the correlation was not perfect, as not all clones that contained the ulation of correlation coefficients is necessary to assign statistical significance to pairs of positions within Gag-Pol that appear Envelope V3 sequence predicts the cellular tropism of HIV-1. Low charge viruses are associated with an M-R5 phenotype whereas high charge viruses are associated with a T-X4 or dual tropic phenotype [26]. Prior to therapy, all response groups contained both high and low charge viruses, indicating that envelope V3 charge does not predict therapy outcome. However, only one VSIS subject contained a mixture of viruses, and all other e the CXCR4 coreceptor. Limited changes were observed following thapy, indicating limited genetic evolution in Until recently, drug resistance testing (both genotypic and phenotypic) was not used in the management of antiretroviral therapy. However, several studies have demonstrated the advantage of using drug resistance testing to select new therapies for patients who have failed their previous treatment [12,34,67,166,254]. Current genotypic drug resistance testing involves examining protease and reverse transcriptase for the D’ cleavage site mutation also contained V82A. Calc to covary in our cohort. VSIS subjects contained only low charge viruses, suggesting that VSIS subjects are less li kely than either VFIF of VFIS to contain viruses that us er envelope occurred during 24 weeks of antiretroviral therapy.

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59 presence or absence of known drug associated mutations, but do not involve analysis of gag. Phenotypic resistance testing is performed by amplifying the 3’ end of gag, protease and reverse transcriptase and creating a recombinant virus which is then tested for susceptibility to inhibitors. Several studies suggest that genetic variability in gag-pol impacts protease activity and contributes to changes in viral replication and response to therapy [51,64,70,88,156,278,280]. Our results identify novel positions in both Gag and Pol that are associated with the viral and immune outcomes of therapy nave patients. These data support a model of structural organization that is necessary for efficient protease processing and viral replication. The predictive value of Gag-Pol mutations Polgenotypic testing. A multivariate analysis including clinical variables and gag-pol genetic variables will help develop a m signifies the need for the inclusion of gag and p6 genotype, along with protease, in ore accurate model to predict therapy outcome.

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60 Table 2-1. Baseline Clinical Data for VSIS Subjects 5.9378VF AA6RTV14.713273VFC7RTV35.411176BPMC14RTV43.632425VMAA8IDV255.28156VFH9RTV284.0441445VFC4RTV27Log10Viral Load(copies/ml)CD4%CD4 T Cell #(cells/ul)Route ofInfectionSexEthnicityAgeDrugSubject 5.9378VF AA6RTV14.713273VFC7RTV35.411176BPMC14RTV43.632425VMAA8IDV255.28156VFH9RTV284.0441445VFC4RTV27Log10Viral Load(copies/ml)CD4%CD4 T Cell #(cells/ul)Route ofInfectionSexEthnicityAgeDrugSubject Table 2-2. Baseline Clinical Data for VFIS Subjects AA4.5357VMAA4.317256BPFAA5.78309BPMA5.15118VMC6.0341571VMC5.118302VMC5.9848BPMC4.421928VMAA4.222503VMAA4.118227VMAA5.218VFAA3.832410SFAALog10Viral Load(copies/ml)CD4%Cell #ls/ul)Route ofInfectionSexEthnicityAgeDrugSubject 5.5741VM 3RTV228RTV1713RTV213RTV129RTV152RTV103RTV1816IDV145IDV266IDV309IDV297RTV1117IDV24 CD4 T (cel 5.5741VMAA3RTV224.5357VMAA8RTV174.317256BPFAA13RTV25.78309BPMA13RTV125.15118VMC9RTV156.0341571VMC2RTV105.118302VMC3RTV185.9848BPMC16IDV144.421928VMAA5IDV264.222503VMAA6IDV304.118227VMAA9IDV295.218VFAA7RTV113.832410SFAA17IDV24Log10Viral Load(copies/ml)CD4%Cell #ls/ul)Route ofInfectionSexEthnicityAgeDrugSubject CD4 T (cel 5.1125VFC9RTV85.3747VMAA10RTV55.913SFAA14IDV65.3545VFC15IDV75.612VFAA7RTV9Log10Viral Load(copies/ml)CD4%4 T Cell #lls/ul)Route ofInfectionSexEthnicityAgeDrugSubject Table 2-3. Baseline Clinical Data for VFIF Subjects CD(ce 5.1125VFC9RTV85.3747VMAA10RTV55.913SFAA14IDV65.3545VFC15IDV75.612VFAA7RTV9Log10Viral Load(copies/ml)CD4%4 T Cell #lls/ul)Route ofInfectionSexEthnicityAgeDrugSubject CD(ce A, Asian; AA, African American; C, Caucasian; H, Hispanic; F, female; M, male; ission; BP, blood products; and S, sexual transmission. V, vertical transm

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61 Table 2-4. 24 Week Clinical Data for VSIS Subjects 2.6301218VF AA6RTV12.612446VFC7RTV32.624524BPMC14RTV42.636599VMAA8IDV252.625419VFH9RTV282.6421436VFC4RTV27Log10Viral Load(copies/ml)CD4%CD4 T Cell #(cells/ul)Route of InfectionSexEthnicityAgeDrugSubject 2.6301218VF AA6RTV12.612446VFC7RTV32.624524BPMC14RTV42.636599VMAA8IDV252.625419VFH9RTV282.6421436VFC4RTV27Log10Viral Load(copies/ml)CD4%CD4 T Cell #(cells/ul)Route of InfectionSexEthnicityAgeDrugSubject Table 2-5. 24 Week Clinical Data for VFIS Subjects 5.927576VMAA3RTV223.09146VMAA8RTV173.228451BPFAA13RTV24.813495BPMA13RTV125.210472VMC9RTV154.5353157VMC2RTV105.424738VMC3RTV184.916181BPMC16IDV143.6261275VMAA5IDV264.322523VMAA6IDV303.422244VMAA9IDV295.519462VFAA7RTV113.936486SFAA17IDV24Log10Viral Load(copies/ml)CD4%CD4 T Cell #(cells/ul)Route of InfectionSexEthnicityAgeDrugSubject 5.927576VMAA3RTV223.09146VMAA8RTV173.228451BPFAA13RTV24.813495BPMA13RTV125.210472VMC9RTV154.5353157VMC2RTV105.424738VMC3RTV184.916181BPMC16IDV143.6261275VMAA5IDV264.322523VMAA6IDV303.422244VMAA9IDV295.519462VFAA7RTV113.936486SFAA17IDV24Log10Viral Load(copies/ml)CD4%CD4 T Cell #(cells/ul)Route of InfectionSexEthnicityAgeDrugSubject Table 2-6. 24 Week Clinical Data for VFIF Subjects 5.313SFAA14IDV65.38141VFC15IDV74.014VFAA7RTV9Log10Viral Load(copies/ml)CD4%CD4 T Cell #(cells/ul)Route ofInfectionSexEthnicityAgeDrugSubject 5.3249VFC9RTV5.5975VMAA10RTV5 8 5.3249VFC9RTV5.5975VMAA10RTV5 85.313SFAA14IDV65.38141VFC15IDV74.014VFAA7RTV9Log10Viral Load(copies/ml)CD4%CD4 T Cell #(cells/ul)Route ofInfectionSexEthnicityAgeDrugSubject A, AV, vertical sian; AA, African American; C, Caucasian; H, Hispanic; F, female; M, male; transmission; BP, blood products; and S, sexual transmission.

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62 ary of Baseline Clinical, Immu ne, and Viral Characteristics VSISVFISVFIFResponse6135# of Subjects8 (6 -9)-13)-14)rs)*ile range) 8 (510 (9Median Age(Yea225 (161-387)256 (57-410)25 (3-45)CD4 T Cell #(cells/uL)*5.0 (4.2-5.4)5.1 (4.3-5.5)5.3 (5.3-5.6)Log10Plasma VL(copies/mL)*CD4 %*12 (9-27)17 (7-21)1 (1-5)* Median Value (25thto 75thinterquartP = 0.01 VSISVFISVFIFResponse6135# of Subjects8 (68 (510 (9Median Age(Yea225 (161-387)256 (57-410)25 (3-45)CD4 T Cell #(cells/uL)*5.0 (4.2-5.4)5.1 (4.3-5.5)5.3 (5.3-5.6)Log10Plasma VL(copies/mL)*CD4 %*12 (9-27)17 (7-21)1 (1-5) -9)-13)-14)rs)* VSISVFISVFIFResponseVSISVFISVFIFResponse6135# of Subjects8 (68 (510 (9Median Age(Yea225 (161-387)256 (57-410)25 (3-45)CD4 T Cell #(cells/uL)*5.0 (4.2-5.4)5.1 (4.3-5.5)5.3 (5.3-5.6)Log10Plasma VL(copies/mL)*CD4 %*12 (9-27)17 (7-21)1 (1-5)* Median Value (25thto 75thinterquartP = 0.01P = 0.01 Table 2-8. Summary of Clinical, Immune, and Viral Chas Following 24 Weeks of Therapy -9)-13)-14)rs)*ile range)racteristic range) * Median Value (25thto 75thinterquartile VSISVFISVFIFResponse6135# of Subjects8 (6-9)8 (5-13)10 (9-14Median A(Years)2 (466-1063)86 (451-576)49 (4-75)D4 T Cell #(cells/uL)*2.6 (2.6-2.6)4.5 (3.6-5.2)5.3 (5.3-5.3)Log10Plasma VL(copies/mL)*CD4 %*28 (24-35)22 (16-27)2 (1-8)P = 0.003P < 0.001* Median Value (25thto 75thinterquartil )ge*564Ce range) VSISVFISVFIFResponseVSISVFISVFIFResponse6135# of Subjects6135# of Subjects8 (6-9)8 (5-13)10 (9-14Median A(Years)8 (6-9)8 (5-13)10 (9-14Median A(Years)2 (466-1063)86 (451-576)49 (4-75)D4 T Cell #(cells/uL)*2.6 (2.6-2.6)4.5 (3.6-5.2)5.3 (5.3-5.3)Log10Plasma VL(copies/mL)*CD4 %*28 (24-35)22 (16-27)2 (1-8)P = 0.003P = 0.003P < 0.001P < 0.001 )ge*)ge*564C Table 2-7. Summ

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CD4% Log10 RNA ies/ml) (cop 020406080100 010203040 23456 VSIS (n = 6)A. 020406080100 010203040 23456 VFIS (n = 13)B.VFIF (n = 5)WEEKS 020406080100 010203040 23456 C.CD4% Log(cop 10RNA ies/ml) CD4% CD4% Log(cop 10RNA ies/ml) Log(cop 10RNA ies/ml) 020406080100 010203040 23456 VSIS (n = 6)A. 020406080100 010203040 23456 VFIS (n = 13)B.VFIF (n = 5)WEEKS 020406080100 010203040 23456 C. 020406080100 010203040 23456 VSIS (n = 6)A. 020406080100 010203040 23456 VFIS (n = 13)B.VFIF (n = 5)WEEKS 020406080100 010203040 23456 C. Figure 2-1. Therapy Outcome of VSIS, VFIS, and VFIF Response Groups. A, 6 subjects isplayed a viral success, immune success (VSIS) response. B, 13 isplayed viral failure and immune success (VFIS). C, 5 subjects onstrated viral failure and immune failure (VFIF). Mean viral load ean CD4% is shown with red represent standard error of the mean. (25%) d(54%) d(21%) demis shown with blue boxes on the left axis, and mtriangles on the right axis. Error bars 63

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64 CD4 % 020406080 100 0102030 A. CD4 % 020406080100 0102030 WEEKS WEEKS WEEKS CD4 % 020406080100 0102030 A. 020406080100 23456 WEEKSB.Log10RNA(copies/ml) 020406080100 23456 020406080100 23456 WEEKSB.Log10RNA(copies/ml) Figure 2-2. Immune and Viral Response to Therapy. A, Immune response to therapy. BViral response to therapy. Yellow squares represent VSIS, purple triangles , represent VFIS, and green circles represent VFIF. Error bars represent standard error of the mean.

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65NF********1020304050607080F. PRA. p2AEAMSQVTNSATIM*10C. p1FLGKIWPSYKGRPGNF*10G. Envelope V3 LoopCTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHC***102030MQRGNFRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQAN*****1020304050B. p7NCMQRGNFRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQAN*****1020304050MQRGNFRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQAN*****1020304050B. p7NCLQSRPEPTAPPEESFRSGVETTTPPQKQEPIDKELYPLTSLRSLFGNDPSSQ**1020*30*40*50D. p6LQSRPEPTAPPEESFRSGVETTTPPQKQEPIDKELYPLTSLRSLFGNDPSSQ**1020*30*40*50LQSRPEPTAPPEESFRSGVETTTPPQKQEPIDKELYPLTSLRSLFGNDPSSQ**1020*30*40*50*50D. p6FREDLAFLQGKAREFSSEQTRANSPTRRELQVWGRDNNSPSEAGADRQGTVSFNF*10*20*30*40*50E. p6PolFREDLAFLQGKAREFSSEQTRANSPTRRELQVWGRDNNSPSEAGADRQGTVSFNF*10*20*30*40*50FREDLAFLQGKAREFSSEQTRANSPTRRELQVWGRDNNSPSEAGADRQGTVSFNF*10*20*30*40*50*10*20*30*40*50*50E. p6PolPQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNNF********1020304050607080F. PRPQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNNF********1020304050607080PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNNF********1020304050607080F. PRA. p2AEAMSQVTNSATIM*10A. p2AEAMSQVTNSATIM*10AEAMSQVTNSATIM*10*10C. p1FLGKIWPSYKGRPGNF*10C. p1FLGKIWPSYKGRPGNF*10FLGKIWPSYKGRPGNF*10*10G. Envelope V3 LoopCTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHC***102030G. Envelope V3 LoopCTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHC***102030CTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHC***102030 Figure 2-3 Gag-Pol and Envelope Sequences from HIVHXB2. Amino acid sequences of p2 (A), p7NC (B), p1 (C), p6 (E), protease (F), and envelope V3 (G) are shown. Every 10th amino acid is marked with an asterisk and the position number. MQRGNFRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQAN*****1020304050B. p7NCLQSRPEPTAPPEESFRSGVETTTPPQKQEPIDKELYPLTSLRSLFGNDPSSQ**1020*30*40*50D. p6FREDLAFLQGKAREFSSEQTRANSPTRRELQVWGRDNNSPSEAGADRQGTVSFNF*10*20*30*40*50E. p6PolPQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNL LTQIGCTL*90LLTQIGCTL*90LLTQIGCTL*90LLTQIGCTL*90D), p6 Pol (

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66 VFIS RTV2 VFIS IDV14 VFIS IDV26 VFIS IDV30 VFIS RTV11 VFIF RTV5 VFIF IDV6 VSIS RTV1 VSISIDV25 VSIS RTV27908482777371635446363332242010OutcomePISubject Sensitive GenotypesA. VFIS RTV2 VFIS IDV14 VFIS IDV26 VFIS IDV30 VFIS RTV11 VFIF RTV5 VFIF IDV6 VSIS RTV1 VSISIDV25 VSIS RTV27908482777371635446363332242010OutcomePISubject908482777371635446363332242010OutcomePISubject Sensitive GenotypesA. VFIS RTV22 VFIS RTV17 VFIS RTV12 VFIS RTV15 VFIS RTV10 VFIS RTV18 VFIS IDV29 VFIS IDV24 VFIF RTV8 VFIF IDV7 VFIF RTV9 VSIS RTV3 VSIS RTV4 VSIS RTV28 908482777371635446363332242010OutcomePIjectResistant Genotypes SubB. Therapy Non-Specific PolymorphismTherapy Specific Polymorphism VFIS RTV22 VFIS RTV17 VFIS RTV12 VFIS RTV15 VFIS RTV10 VFIS RTV18 VFIS IDV29 VFIS IDV24 VFIF RTV8 VFIF IDV7 VFIF RTV9 VSIS RTV3 VSIS RTV4 VSIS RTV28 908482777371635446363332242010OutcomePIject908482777371635446363332242010OutcomePIjectResistant Genotypes SubSubB. Therapy Non-Specific PolymorphismTherapy Specific Polymorphism Figure 2-4. Pretherapy Protease Genotypes. A, Sensitive genotypes. B, Resistant genotypes. White boxes represent no substitution. RTV, ritonavir; IDV, indinavir. Therapy non-specific polymorphisms are shown with grey and therapy specific polymorphisms are shown with black boxes.

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67 1 02468 0SensitiveResistantNumber of Subje cts 024680SensitiveResistantNumber of Subje Figure 2-5. Proportion of Sensitive and Resistant Genotypes Prior to Therapy. VSIS is shown in yellow, VFIS in purple, and VFIF in green. 1 cts

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68 02468101214VSISVFISVFIFNumber of Subjects 02468101214VSISVFISVFIFNumber of Subjects Figure 2-6. Proportion of Therapy Specific and Therapy Non-Specific Polymorphisms in Protease Prior to Therapy. White bars represent no substitutions, grey bars represent therapy non-specific substitutions, and black bars represent therapy specific substitutions.

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69 VFIS RTV2 VFIS IDV14 VFIS IDV26 VFIS IDV30 VFIS RTV11 VFIF RTV5 VFIF IDV6 VSIS RTV1 VSIS RTV27 908482777371635446363332242010OutcomePISubjectA. VFIS RTV2 VFIS IDV14 VFIS IDV26 VFIS IDV30 VFIS RTV11 VFIF RTV5 VFIF IDV6 VSIS RTV1 VSIS RTV27 908482777371635446363332242010OutcomePISubject VFIS RTV2 VFIS IDV14 VFIS IDV26 VFIS IDV30 VFIS RTV11 VFIF RTV5 VFIF IDV6 VSIS RTV1 VSIS RTV27 908482777371635446363332242010OutcomePISubjectA. VFIS RTV22 VFIS RTV17 VFIS RTV 12 VFIS RTV 15 VFIS RTV 10 VFIS RTV 18 VFIS IDV29 VF IS IDV24 VFIF RTV8 VFIF IDV7 VFIF RTV9 VSIS RTV3 VSIS RTV4 VSIS RTV28 908482777371635446363332242010OutcomePISubjectB. Therapy Non-Specific PolymorphismTherapy Specific Polymorphism New Therapy Non-Specific MutationNew Therapy Specific Mutation VFIS RTV22 VFIS RTV17 VFIS RTV 12 VFIS RTV 15 VFIS RTV 10 VFIS RTV 18 VFIS IDV29 VF IS IDV24 VFIF RTV8 VFIF IDV7 VFIF RTV9 VSIS RTV3 VSIS RTV4 VSIS RTV28 908482777371635446363332242010OutcomePISubject VFIS RTV22 VFIS RTV17 VFIS RTV 12 VFIS RTV 15 VFIS RTV 10 VFIS RTV 18 VFIS IDV29 VF IS IDV24 VFIF RTV8 VFIF IDV7 VFIF RTV9 VSIS RTV3 VSIS RTV4 VSIS RTV28 908482777371635446363332242010OutcomePISubject908482777371635446363332242010OutcomePISubjectB. Therapy Non-Specific PolymorphismTherapy Specific Polymorphism Therapy Non-Specific PolymorphismTherapy Specific Polymorphism New Therapy Non-Specific MutationNew Therapy Specific Mutation New Therapy Non-Specific MutationNew Therapy Specific Mutation Figure 2-7. Posttherapy Protease Genotypes. A, Subjects with pretherapy sensitive genotypes. B, Subjects with pretherapy resistant genotypes. RTV, ritonavir; IDV, indinavir. White boxes represent no substitution. Pre-existing therapy non-specific polymorphisms are shown with grey and therapy specific polymorphisms are shown with black boxes. New therapy non-specific mutations are shown in blue and new therapy specific mutations are shown in red.

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70 Median Number of Mutations 0123 4 5PrePostViral SuccessPrePostViral Failure ** * Median Number of Mutations 01235PrePostViral SuccessPrePostViral Failure ** * ** 4 * Figure 2-8. Median Number of Cell Associated Preand Posttherapy Mutations in Protease. Pretherapy polymorphisms are shown in grey, and posttherapy mutations are shown in black. *P = 0.003, **P < 0.001, t-test.

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71 Sensitive Genotypes VFIS RTV VFIS IDV VFIS IDV VFIS IDV VFIS RTV VFIF RTV VFIF IDV VSIS RTV VSIS IDV VSIS RTV 2142630115612527121314151819223537394143495155576062646569707287919396SubjectPIOutcomeA.Sensitive Genotypes VFIS RTV VFIS IDV VFIS IDV VFIS IDV VFIS RTV VFIF RTV VFIF IDV VSIS RTV VSIS IDV VSIS RTV 2142630115612527121314151819223537394143495155576062646569707287919396SubjectPIOutcome121314151819223537394143495155576062646569707287919396121314151819223537394143495155576062646569707287919396SubjectPIOutcomeA. VFIS RTV VFIS RTV VFIS RTV VFIS RTV VFIS RTV VFIS RTV VFIS IDV VFIS IDV VFIF RTV VFIF IDV VFIF RTV VSIS RTV VSIS RTV VSIS RTV 121314151819223537394143495155576062646569707287919396SubjectPIOutcome22171215101829248793428Resistant GenotypesB. Pretherapy Polymorphism VFIS RTV VFIS RTV VFIS RTV VFIS RTV VFIS RTV VFIS RTV VFIS IDV VFIS IDV VFIF RTV VFIF IDV VFIF RTV VSIS RTV VSIS RTV VSIS RTV 121314151819223537394143495155576062646569707287919396SubjectPIOutcome22171215101829248793428 VFIS RTV VFIS RTV VFIS RTV VFIS RTV VFIS RTV VFIS RTV VFIS IDV VFIS IDV VFIF RTV VFIF IDV VFIF RTV VSIS RTV VSIS RTV VSIS RTV 121314151819223537394143495155576062646569707287919396SubjectPIOutcome121314151819223537394143495155576062646569707287919396121314151819223537394143495155576062646569707287919396SubjectPIOutcome22171215101829248793428Resistant GenotypesB. Pretherapy Polymorphism Pretherapy Polymorphism Figure 2-9. Supplemental Polymorphisms in Protease Prior to Therapy. A, Sensitive genotypes. B, Resistant genotypes. RTV, ritonavir; IDV, indinavir. White boxes represent no substitution, and grey boxes represent pretherapy polymorphisms.

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72 VFIS RTV VFIS IDV VFIS IDV VFIS IDV VFISRTV VFIF RTV VFIF VSIS IDV RTV VSIS RTV 1213141518192335373941434552555760626466697072879193PI9172527293034537983949298OutcomeA. Subject21426301156127 VFIS RTV VFIS IDV VFIS IDV VFIS IDV VFISRTV VFIF RTV VFIF VSIS IDV RTV VSIS RTV 1213141518192335373941434552555760626466697072879193PI9172527293034537983949298Outcome Subject21426301156127 VFIS RTV VFIS IDV VFIS IDV VFIS IDV VFISRTV VFIF RTV VFIF VSIS IDV RTV VSIS RTV 1213141518192335373941434552555760626466697072879193PI9172527293034537983949298Outcome Subject 1213141518192335373941434552555760626466697072879193PI9172527293034537983949298OutcomeA. Subject21426301156127 B. Pretherapy PolymorphismNew Posttherapy Mutation VFIS RTV VFIS RTV VFIS RTV VFIS RTV VFIS RTV VFIS RTV VFIS IDV IDV VFIF RTV IDV RTV VSIS RTV VSIS RTV VSIS RTV 221712151018292487934281213141518192335373941434552555760626466697072879193SubjectPI9172527293034537983949298Outcome VFIF VFIF VFIS B. Pretherapy PolymorphismNew Posttherapy Mutation B. Pretherapy PolymorphismNew Posttherapy Mutation Pretherapy PolymorphismNew Posttherapy Mutation VFIS RTV VFIS RTV VFIS RTV VFIS RTV VFIS RTV VFIS RTV VFIS IDV IDV VFIF RTV IDV RTV VSIS RTV VSIS RTV VSIS RTV 221712151018292487934281213141518192335373941434552555760626466697072879193SubjectPI9172527293034537983949298Outcome VFIF VFIF VFIS Figure 2-10. Supplemental Mutations in Protease after 24 Weeks of Therapy. A, Subjects with pretherapy sensitive genotypes. B, Subjects with pretherapy resistant genotypes. RTV, ritonavir; IDV, indinavir. White boxes represent no substitution. Pre-existing polymorphisms are shown with grey boxes and mutations that accumulated following therapy are shown with orange boxes.

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73 Table 2-9. Median Preand Posttherapy Supplemental Mutations in Protease VSISVFISVFIF VSISVFISVFIF PretherapyPosttherapy(Cells)Posttherapy(RNA) 45 (1)34 (0)4 (1)45 (1)5 (0) PretherapyPosttherapy(Cells)Posttherapy(RNA)45 (1)34 (0)4 (1)45 (1)5 (0) (#) Median number of new supplemental mutations following therapy

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74 R P G N F * L Q S R PP5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’ 0 0 0 0 0 17 0 50 0 170 0 0 11 0 11 0 28 6 11VSVF(n = 6)(n = 18)***C.B.R P G N F * L Q S R PP5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’ 0 0 0 11 0 11 0 32 5 11ISIF(n = 19)(n = 5)***0 0 0 0 0 20 0 40 0 10A.P5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’ 0 0 0 0 0 17 0 50 0 170 0 0 15 0 8 0 23 8 80 0 0 0 0 20 0 40 0 10VSISVFISVFIF(n = 6)(n = 13)(n = 5)****R P G N F * L Q S R PR P G N F * L Q S R PP5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’ 0 0 0 0 0 17 0 50 0 170 0 0 11 0 11 0 28 6 11VSVF(n = 6)(n = 18)***C.R P G N F * L Q S R PP5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’ 0 0 0 0 0 17 0 50 0 170 0 0 11 0 11 0 28 6 11VSVF(n = 6)(n = 18)***C.B.R P G N F * L Q S R PP5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’ 0 0 0 11 0 11 0 32 5 11ISIF(n = 19)(n = 5)***0 0 0 0 0 20 0 40 0 10B.R P G N F * L Q S R PP5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’ 0 0 0 11 0 11 0 32 5 11ISIF(n = 19)(n = 5)***0 0 0 0 0 20 0 40 0 10A.P5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’ 0 0 0 0 0 17 0 50 0 170 0 0 15 0 8 0 23 8 80 0 0 0 0 20 0 40 0 10VSISVFISVFIF(n = 6)(n = 13)(n = 5)****R P G N F * L Q S R PA.P5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’ 0 0 0 0 0 17 0 50 0 170 0 0 15 0 8 0 23 8 80 0 0 0 0 20 0 40 0 10VSISVFISVFIF(n = 6)(n = 13)(n = 5)****R P G N F * L Q S R P Figure 2-11. Pretherapy D Cleavage Site Polymorphisms. A, Subjects stratified by viral and immune outcome. B, Subjects stratified by immune outcome only. C, Subjects stratified by viral outcome only. Numbers represent the percentage of subjects who demonstrated polymorphisms at the specified position.

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75 S A T I M * M Q R G N P5 P4 1 1’ P2 P3 P2 PP’ P3’ P4’ P5’ 178 0 0 33 0 050 505039 0 11 0 44 0 6VSVF(n = 6)(n = 18)*C. 173170 ** ISIF(n = 19)(n = 5)S A T I M * M Q R G NP5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’42 0 5 0 42 100 0 20 0 40 0 20**B. 26 32 530 080 80 0 * VVVFIF(n = 5)S A T I M * M Q R G NP5’100 80 800 0 20 0 40 0 20*A.S A T I M * M Q R G N P3 P2 PP’ P3’ P4’ P5’ SISFIS(n = 6)(n = 13)P5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ 17 178317 0 0 0 330 031 38 38540 8 0 46 0 0*** VVVFIF(n = 5)S A T I M * M Q R G NP5’100 80 800 0 20 0 40 0 20*A. SISFIS(n = 6)(n = 13)P5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ 17 178317 0 0 0 330 031 38 38540 8 0 46 0 0*** P5 P4 1 1’ P2 178 0 0 33 0 050 505039 0 11 0 44 0 6VSVF(n = 6)(n = 18)*C.S A T I M * M Q R G N P3 P2 PP’ P3’ P4’ P5’ 173170 **P5 P4 1 1’ P2 178 0 0 33 0 050 505039 0 11 0 44 0 6VSVF(n = 6)(n = 18)*C. 173170 ** ISIF(n = 19)(n = 5)S A T I M * M Q R G NP5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’42 0 5 0 42 100 0 20 0 40 0 20**B. 26 32 530 080 80 0 * ISIF(n = 19)(n = 5)S A T I M * M Q R G NP5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’S A T I M * M Q R G NP5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’42 0 5 0 42 100 0 20 0 40 0 20**B. 26 32 530 080 80 0 * VVVFIF(n = 5)S A T I M * M Q R G NP5’100 80 800 0 20 0 40 0 20*S A T I M * M Q R G NP5’100 80 800 0 20 0 40 0 20*A. Figure 2-12. PretheraleSite Polymorphisms. A, Subjects stratified by viral and immune outcome. B, Subjects stratified by immune outcome only. C, Subjects stratified by viral outcome only. Numbers represent the percentage of subjects who demonstrated polymorphisms at the specified position. Values > 50% are shown in red. SISFIS(n = 6)(n = 13)P5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ 17 178317 0 0 0 330 031 38 38540 8 0 46 0 0***P5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ 17 178317 0 0 0 330 031 38 38540 8 0 46 0 0*** py C Cavage

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76 V S F N F * P Q V T L P5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’ 17 0 67100 33 0 0 100 0 023 0 31 9223 0 0 1000 020 0 40 1000 0 0 100 0 0VSISVFISVFIF(n = 6)(n = 13)(n = 5)****A.B.V S F N F * P Q V T LP5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’ 21 0 42 9521 0 0 1000 020 0 40 100 0 0 0 1000 0ISIF(n = 19)(n = 5)***C.V S F N F * P Q V T LP5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’ 17 0 6710017 0 0 1000 022 0 33 94 17 0 0 1000 0VSVF(n = 6)(n = 18)*** V S F N F * P Q V T L P5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’ 17 0 67100 33 0 0 100 0 023 0 31 9223 0 0 1000 020 0 40 1000 0 0 100 0 0VSISVFISVFIF(n = 6)(n = 13)(n = 5)****A.P5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’ V S F N F * P Q V T L V S F N F * P Q V T L 17 0 67100 33 0 0 100 0 023 0 31 9223 0 0 1000 020 0 40 1000 0 0 100 0 0VSISVFISVFIF(n = 6)(n = 13)(n = 5)****P5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’ 17 0 67100 33 0 0 100 0 023 0 31 9223 0 0 1000 020 0 40 1000 0 0 100 0 0VSISVFISVFIF(n = 6)(n = 13)(n = 5)****A.B.V S F N F * P Q V T LP5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’ 21 0 42 9521 0 0 1000 020 0 40 100 0 0 0 1000 0ISIF(n = 19)(n = 5)***B.V S F N F * P Q V T LP5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’ 21 0 42 9521 0 0 1000 020 0 40 100 0 0 0 1000 0ISIF(n = 19)(n = 5)***C.V S F N F * P Q V T LP5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’ 17 0 6710017 0 0 1000 022 0 33 94 17 0 0 1000 0VSVF(n = 6)(n = 18)***C.V S F N F * P Q V T LP5 P4 P3 P2 P1 P1’ P2’ P3’ P4’ P5’ 17 0 6710017 0 0 1000 022 0 33 94 17 0 0 1000 0VSVF(n = 6)(n = 18)*** Figure 2-13. Pretherapy E Cleavage Site Polymorphisms. A, Subjects stratified by viral and immune outcome. B, Subjects stratified by immune outcome only. C, Subjects stratified by viral outcome only. Numbers represent the percentage of subjects who demonstrated polymorphisms at the specified position. Values > 50% are shown in red.

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77 MQRGNFRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQA*****MQRGNFRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQA*****MQRGNFRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQA***** N 1020304050A. p7NCN1020304050N1020304050A. p7NC CCCHFNGKENGTARZn CCCHWKGKEDGQMKZn MQRGNFRNQRKIVKRAPRKKGTERQANB.10*20*30*40*50*CCCHFNGKENGTARZn CCCHWKGKEDGQMKZn MQRGNFRNQRKIVKRAPRKKGTERQANB.CCCHFNGKENGTARZn CCCHWKGKEDGQMKZn MQRGNFRNQRKIVKRAPRKKGTERQANCCCHFNGKENGTARZn CCCHFNGKENGTARCCCHFNGKENGTARZn CCCHWKGKEDGQMKZn CCCHWKGKEDGQMKCCCHWKGKEDGQMKZn MQRGNFRNQRKIVKRAPRKKGTERQANB.10*20*30*40*50*10*10*20*20*30*30*40*40*50*50* CCCHFNGKENGTARZn CCCHWKGKEDGQMKZn MQRGNFRNQRKIVKRAPRKKGTERQANC.10*20*30*40*50*CCCHFNGKENGTARZn CCCHWKGKEDGQMKZn MQRGNFRNQRKIVKRAPRKKGTERQANC.CCCHFNGKENGTARZn CCCHWKGKEDGQMKZn MQRGNFRNQRKIVKRAPRKKGTERQANCCCHFNGKENGTARZn CCCHFNGKENGTARCCCHFNGKENGTARZn CCCHWKGKEDGQMKZn CCCHWKGKEDGQMKCCCHWKGKEDGQMKZn MQRGNFRNQRKIVKRAPRKKGTERQANC.10*20*30*40*50*10*10*20*20*30*30*40*40*50*50* Figure 2-14. Preand Posttherapy Polymorphisms in p7NC. A, Amino acid sequence of p7NC from HIVHXB2. Every 10th residue is marked with an asterisk and position number. B, Pretherapy polymorphisms. Prior to therapy no polymorphisms appeared in the zinc binding residues, shown in red, or in any of the positions shown in black. Polymorphisms were present in over 40% of subjects at the positions shown in blue, and in less than 30% of subjects at positions shown in green. C, New posttherapy mutations. Positions that accumulated new mutations following therapy are shown in purple. No new mutations accumulated in the zinc binding residues, shown in red.

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78 Table 2-10. Median Preand Posttherapy Mutations in p7NC PretherapyPosttherapy(Cells)Posttherapy(RNA)VSIS44-VFIS45 4VFIF443 PretherapyPosttherapy(Cells)Posttherapy(RNA)VSIS44-VFIS45 4VFIF443

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79 LQSRPEP...TAPPEESFRSGVE...TTTPPQKQEPIDKELYP...LTSLRSLFGNDPSSQ*10*20*30*40*50VSIS50%0%0%VFIS38%8%8%VFIF40%20%0%VS50%0%0%VF39%11%6%IS42%5%5%IF40%20%0% LQSRPEP...TAPPEESFRSGVE...TTTPPQKQEPIDKELYP...LTSLRSLFGNDPSSQ*10*10*20*20*30*30*40*40*50*50VSIS50%0%0%VFIS38%8%8%VFIF40%20%0%VS50%0%0%VF39%11%6%IS42%5%5%IF40%20%0% Figure 2-15. Pretherapy Insertions in p6. The amino acid sequence of p6 (HIVHXB2) is shown on top, with every 10th residue marked with an asterisk and position number. The insertions are shown with (...) between residues 7 and 8, 20 and 21, and 37 and 38. The proportion of subjects in each response group that contained pretherapy insertions is shown below the corresponding insertion.

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80 41 12735 385053VSIS(n = 6)67%83%%0% 50%67% B. 17%50 FREDLAFLQGKAREFSSEQTRANSPTRRELQVWGRDNNSPSEAGADRQGTVSFNF*10*20*30*40*50A. p6Pol41385053VSIS(n = 6)67%83%%0% 50%67% 12735 VFIS(n = 13)85%38%15%62%54%62%31%VFIF(n = 5)40%80%60%80%20%40%40% ISIF4 1127 355079% 53%16% 58%58%40% 80%60%80%40% (n = 19)(n = 5)11 53VSVF83%67%50%33% C. (n = 6)(n = 18)D.VFIS(n = 13)85%38%15%62%54%62%31%VFIF(n = 5)40%80%60%80%20%40%40% 17%50 B.41385053VSIS(n = 6)67%83%%0% 50%67% 12735 17%50 41385053VSIS(n = 6)67%83%%0% 50%67% 12735 B. 17%50 VFIS(n = 13)85%38%15%62%54%62%31%VFIF(n = 5)40%80%60%80%20%40%40% VFIS(n = 13)85%38%15%62%54%62%31%VFIF(n = 5)40%80%60%80%20%40%40% ISIF4 1127 355079% 53%16% 58%58%40% 80%60%80%40% (n = 19)(n = 5)ISIF4 1127 355079% 53%16% 58%58%40% 80%60%80%40% C. (n = 19)(n = 5)ISIF4 1127 355079% 53%16% 58%58%40% 80%60%80%40% (n = 19)(n = 5)11 53VSVF83%67%50%33% C. (n = 6)(n = 18)D.11 53VSVF83%67%50%33% (n = 6)(n = 18)11 53VSVF83%67%50%33% (n = 6)(n = 18)D. FREDLAFLQGKAREFSSEQTRANSPTRRELQVWGRDNNSPSEAGADRQGTVSFNF*10*20*30*40*50A. p6PolFREDLAFLQGKAREFSSEQTRANSPTRRELQVWGRDNNSPSEAGADRQGTVSFNF*10*20*30*40*50FREDLAFLQGKAREFSSEQTRANSPTRRELQVWGRDNNSPSEAGADRQGTVSFNF*10*20*30*40*50*10*20*30*40*50*50A. p6Pol Pol from HXB2ber. by no Figure 2-16. Pretherapy Polymorphisms in p6 Pol . A, Amino acid sequence of p6 HIV. Every 10th residue is marked with an asterisk and position numB, Subjects stratified by viral and immune outcome. C, Subjects stratified immune outcome only. D, Subjects stratified by viral outcome only. Amiacid position is noted above the line, and the percent of subjects with pretherapy polymorphisms at that position is noted below the line, with values > 50% marked in red.

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81 04060801624272930% of Subjects with MutationsImmune Success (Cells) 2049Amino Acid Position Immune Failure (RImmune Failure (Cells) NA) 04060801624272930% of Subjects with MutationsImmune Success (Cells) 2049Amino Acid Position Immune Failure (RImmune Failure (Cells) NA) Figure 2-17. Posttherapy Mutations in p6. Amino acid positions 16, 24, 27, 29, 30, were more polymorphic in immune failure subjects as compared to immune success subjects after 24 weeks of therapy. Poland 49

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82 A.Protease A.Protease A.Protease Protease 13:6414:62 58:8236:54:82 C.Pol p6PolPR167735:6328:35:461323:28:40:46:47:541533:63:70:7152213504063 24:901414029 32:33:5432:71:8254:62:8210:34:46:62 Gag p2p1p7NCp6B.7:121298:4831112:20:2621308:23:25:32:42 714:2025 11:1225:3134:3935:52C.Pol p6PolPR167735:6328:35:461323:28:40:46:47:541533:63:70:7152213504063 24:901414029 C.Pol p6PolPR167735:6328:35:461323:28:40:46:47:541533:63:70:7152213504063 24:901414029 167735:6328:35:461323:28:40:46:47:541533:63:70:7152213504063 13:6414:62 58:8236:54:82 Gag-Pol D.p2p7 p1p6Pol10:11:13PR1032:63:70:71:82NC2111p6 17:35:37:4269876436191315:2063293223:5212:137143823:28:4935:521525:5025 24:901414029 Gag-Pol D.p2p7 p1p6Pol10:11:13PR1032:63:70:71:82NC2111p6 17:35:37:4269876436191315:2063293223:5212:137143823:28:4935:521525:5025 Gag-Pol D.Gag-Pol D.p2p7 p1p6Pol10:11:13PR1032:63:70:71:82NC2111p6 17:35:37:4269876436191315:2063293223:5212:137143823:28:4935:521525:5025 p1p6Pol10:11:13PR1032:63:70:71:82NC2111p6 17:35:37:4269876436191315:2063293223:5212:137143823:28:4935:521525:5025 p2p7 32:33:5432:71:8254:62:8210:34:46:62 13:6414:62 58:8236:54:82 13:6414:62 32:33:5432:71:8254:62:8210:34:46:62 58:8236:54:82 32:33:5432:71:8254:62:8210:34:46:62 Gag p2p1p7NCp6B.7:121298:4831112:20:2621308:23:25:32:42 714:2025 11:1225:3134:3935:52Gag p2p1p7NCp6B.7:121298:4831112:20:2621308:23:25:32:42 714:2025 11:1225:3134:3935:527:121298:4831112:20:2621308:23:25:32:42 714:2025 11:1225:3134:3935:52 covary, and multiple positions that covary within a region are separated by a Covariation within Pol. D, Covariation within Gag-Pol. Figure 2-18. Covariation in Gag-Pol. Each line represents a distinct set of positions that colon. A, Covariation within protease. B, Covariation within Gag. C,

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83 0268VSISVF 412ISVFIF# of SubjA. 10ects 0268VSISVF 412ISVFIF# of Subj 10ects 0268 412 10 0268VSISVF 412ISVFIF# of SubjA. 10ects 02481012VSISVFISVFIF# of Sbjects. 6uB 02481012VSISVFISVFIF# of Sbjects 6u 02481012 6 02481012VSISVFISVFIF# of Sbjects.V3 loop charges after 24 weeks of therapy. White bars indicate low ( 6uB Figure 2-19. Preand Posttherapy V3 Loop Charge. A, Pretherapy V3 loop charges. B, < +4) charge viruses, black bars indicate high (> +5) charge viruses, and grey bars indicate a mixture of high and low charge viruses.

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84 6371361010366371 77828277 6371361010366371 Figure 2-20 77828277 . Location of Amino Acids 36 and 77 on Protease. The two monomers of protease are shown as ribbons with a protease inhibitor bound in the active positions identified as pre-existing polymorphisms, site. Amino acids including 36 and 77, are noted in white. Coordinates for protease bound to the inhibitor from Kempf et al [127] and figure designed by Jose Clemente.

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CHAPTER 3 GAG-PROTE POL DETERMINANTS OF FITNESS AND DRUG SENSITIVITY IN A ASE INHIBITOR RESISTANT VARIANT IN CXCR4 CD4+ T LYMPHOCYTES -1 fitness. Genotype influences the basic ity , Introduction HIV-1 fitness can be defined as the ability of HIV-1 to replicate in a given environment. Multiple factors impact HIVreplicative capacity of the virus. Environmental factors, such as the host immune systemor the selective pressure of antiretroviral drugs, also impact the ability of the virus to replicate and produce infectious progeny. Viral fitness can be measured in multiple ways. Single cycle assays define the ability of the virus to enter a specific target cell, parallel infections with replication competent viruses demonstrate the replicative capacof the virus, competition experiments determine which of two viruses is more fitenzymatic assays determine the catalytic activity of protease and reverse transcriptase, and processing assays define the ability of protease to cleave a substrate. Previous studies in the Goodenow and Dunn laboratories used a bacterial expression system to examine proteolytic processing of the Gag-Pol polyprotein precursor, and demonstrated a functional linkage between Gag and protease [20,87]. Processing of chimeras constructed with Gag and PR regions isolated from patients demonstrated that optimal protease activity occurred when Gag and PR were isolated from the same individual. In contrast, heterologous Gag regions were processed less efficiently by protease. Natural variants isolated from HIV-1 infected patients in the absence of drug selection display multiple different processing phenotypes, categorized 85

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86 by the rate and completeness of processing. Type I variants demonstrate rapid and complete processing. Production of p24 occurs quickly, as early as 5 minutes post-induction, with essentially no accumulation of processing intermediates. An intermediatecomplete phenotype is observed for Type II var iants, with p24 production occurring d the appearance and accumulation of processing intermediates. Type a Type I gag-pol region ce s confirmed the impact of these two regions on PR processing and iants rated that in the presence of protease inhibitors, mutations in PR are slower than in Type I, an III variants are characterized by a slow, incomplete processing phenotype, never producing p24. Construction of replication competent recombinant viruses with patient derived gag-pol regions in a molecular clone backbone demonstrated that processing phenotype is reflected in viral replication. Viruses with produced the greatest levels of p24, followed by Type II, and finally, Type III. Sequenanalysis revealed differences between the variants in the C cleavage site and in p7 NC , andmutagenesis studieviral replication. The previous studies focused on the processing and replication of natural varprior to the initiation of antiretroviral therapy including a protease inhibitor. Manygroups demonstassociated with changes in Gag, specifically p7 NC and the cleavage sites [65,156,280]. Significant research has been dedicated to identifying mutations in protease that impact both drug resistance and viral fitness, while less has been done to examine the impact of mutations in gag. This study was designed to identify determinants in gag-pol that areresponsible for altering the viral replicative capacity of a drug resistant variant as well asthe sensitivity of the virus to protease inhibitors in CXCR4 CD4 + T lymphocytes.

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87 Materials and Methods Construction of Replication Compe tent gag-pol Recombinant Viruses etent recombinant viruses containing selected gag-pol alleles were s e py), and rum (FBS) (Gibco, Rockville, MD), ibco) , and streptomycin (100 g/ml) (Gibco) one day prior to transfsfected dded to (PBS Replication comp constructed as described previously [219]. Briefly, the molecular clone pLAI.4 waconstructed by removing a SpeI restriction site at nucleotide position 7 from thmolecular clone pLAI.2 [187]. pLAI.4 was then digested with the restriction enzymes SpeI (New England Biolabs, Inc, Beverly, MA, NEB) and Bstz17I (NEB), to remove theLAI gag-pol region, and a 1.7 kb patient fragment was inserted into the pLAI.4 backbone. Recombinant viruses were made with gag-pol alleles from a discordant response pediatric patient (D1) both prior to (referred to as D1.10 and pretherafollowing (referred to as 3188a02 and posttherapy), combination therapy. Production of Virus Stocks Virus stocks were generated by transfection of HEK293 cells [92]. 3.9 x 10 6 cells were seeded in a T-75 flask in 30 ml of Dulbecco’s modified Eagle media (DMEM) (Invitrogen) supplemented with 10% fetal bovine se penicillin (100 U/ml) (G ection. The cells were incubated overnight at 37C, 5% CO 2 , and were tranwhen confluency reached approximately 60%. Fifteen micrograms of DNA was a90 ul of 3 mg/ml SuperFect transfection reagent (Qiagen) in a total volume of 600 l of DMEM, mixed well, and incubated at room temperature for 10 minutes. During the incubation, the 293 cells were rinsed once with 15 ml of 1 X phosphate buffered saline ) (Gibco), and then 6 ml of supplemented DMEM was added to each flask. The transfection reagent mixture was added to the flask, and incubated at 37C, 5% CO 2 for 3 hours. Following incubation, the cells were rinsed twice with 15 ml of 1 X PBS, 6 ml of

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88 supplemented DMEM was added to each flask, and the cells were incubated at 37C, 5%CO s t n lues were calculated by the thod (Division of AIDS, 1997). PBMors g a in O) glutamine, 15% FBS, penicillin (100 U/momycin (100 g/ml), and IL-2 (30 DMSO. PBMC were resuspended in fresh m37C, 5% CO2. Additional PBMC media was then added to dilute the PHA to a final concentration of 2 g/ml and the cells were incubated for ~18 hours at 37C, 5% CO2. On day 1 the PHA was removed by transferring the cells to a 50 ml conical tube and centrifugation at 1000 RPM for 10 minutes. PBMC were resuspended in fresh media and 2 . Twenty four hours after transfection, the media was removed from the flasks, and 6ml of fresh supplemented DMEM was added. Forty eight hours later, the supernatantwere collected, syringe filtered (.45m Acrodisc), and stored at -80C. Virus stocks were titered in peripheral blood mononuclear cells (PBMC) thawere stimulated with phytohemaglutinin (PHA) (Sigma) and cultured in RPMI 1640 (Gibco) containing 15% FBS, Interleukin-2 (30 U/ml, Roche, Indianapolis, IN), penicilli(100 U/ml), and streptomycin (100 g/ml). TCID 50 va Spearman-Karber me C Stimulation and Infections PBMC were isolated from leukopack blood from normal, HIV-seronegative donby centrifugation over a Ficoll gradient [232]. The cells were then cyropreserved usinCryo 1C Freezing Container (Nalge Nunc International, Rochester, NY), and storedliquid nitrogen in 70% RPMI 1640, 20% FBS, and 10% dimethyl sulfoxide (DMS(Sigma). Three to four days prior to infection, PBMC were removed from liquidnitrogen, quick thawed at 37C, placed in 10 ml of PBMC media (RPMI 1640 + Ll), strept U/ml), and centrifuged for 10 minutes at 1000 RPM to remove edia and stimulated with 5 g/ml PHA (Sigma) for 15 minutes at

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89 split between the existing flask and a new flask. In a typical experiment 60 x 10 6 cells were stimulated, therefore each flask would contain 30 x 10 6 in 30 ml (final concentrationof 1 x 10 6 cells. ut in 50 ml conical tubes at 37C, 5% CO2, with gentle agitation once in 3188a e nd n 6 cells/ml). Cells were incubated at 37C, 5% CO 2 for an additional 2-3 days before infection. PBMC were infected with 1500 TCID 50 of virus stock per 1.2 x 10 Infections were carried o every 30 minutes for 2 hours. Following infection, the cells were washed by addition of 10 ml of 1 X PBS, centrifuged at 1000 RPM for 10 minutes, resuspendedPBMC media, and plated in 96 well round bottom tissue culture plates. Supernatants were harvested every 2 days for 10 days, concurrent with a 10% media change, and stored at -80C until supernatant p24 antigen levels were determined by the HIV-1 p24Antigen Assay ELISA (Beckman Coulter, Fullerton, CA) following the manufacturer’s protocol. Site Directed Mutagenesis To prepare for mutagenesis, the gag-pol fragment was digested out of the pLAI.402 gag-pol recombinant virus using the restriction enzymes SphI (NEB) and EcoRV (NEB). The pGEM5Zf+ cloning vector (Promega, Madison, WI) was digestedwith SphI and EcoRV, and the 3.0 kb backbone fragment was treated with calf alkalinphosphatase (CIAP) (Promega) to prevent plasmid religation. The 1.5 kb gag-pol fragment was ligated into the pGEM5Zf+ backbone using T4 DNA ligase (NEB), atransformed into DH5 cells (Invitrogen, Carlsbad, CA), plated on LB plates containing 100 mg/ml ampicillin (Sigma, St. Louis, MO), and incubated overnight at 37C. Ten clones were picked into Luria broth containing 100 ug/ml ampicillin (Sigma) and grow

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90 overnight at 37C. The plasmid DNA was extracted using QIAprep Miniprep Kit (Qiagen, Valencia, CA) following the manufacturer’s protocol. Insertion of the 1.5 kb gag-pol fragment was confirmed by restriction digest using SpeI (NEB) and BstZ17I (NEB). Gag Mutagenesis The QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene, CA) was used to introduce specific mutations into the gag-pol region fro La Jolla, m patient isolate GCAa , tamic n LB plates with ampicillin, and grown overnight. Ten colonies were picke , and 3188a02. Primer CSITE1 (Invitrogen, 5’CCAATTCACAGATCATAATGATGCAGAAAGGC-3’) was used to convert valine to an isoleucine in the C cleavage site (Construct 1). Primer NC1 (Invitrogen5’-TTGTGGCAAAGAGGGGCACATAGCC-3’) was used to mutate valine to gluacid in p7 NC (Construct 2). The mutagenic primers, reaction buffer, and QuikChangeMulti enzyme blend were added to the pGEM5Zf+/3188a02 template DNA following manufacturer’s protocol. The thermal cycling conditions consisted of an initial denaturation at 95C for 1 minute, followed by 30 cycles of denaturation at 95C for 1 minute, annealing at 55C for 1 minute, and extension at 65C for 9 minutes. Immediately following temperature cycling, the reactions were placed on ice for two minutes, 1 l of DpnI restriction enzyme (10 U/l) was added, and the reactions were incubated at 37C for 1 hour. One microliter of the reaction was transformed into DH5 cells, plated o d for each construct, DNA extracted with the QIAprep Miniprep Kit (Qiagen), andscreened by digestion with SpeI and Bstz17I. Correct clones were then sequenced to confirm mutagenesis using ABI Prism BigDye terminator cycle sequencing and ABI Prism 377XL automated sequence instrumentation (Perkin-Elmer, Wellesley, MA)

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91 the gag-pol regions were digested out of pGEM5Zf+ with SpeI and Bstz17I. To prepare for ligation, pLAI.4 was digested with SpeI and Bstz17I and treated with CIAP. Mutant gag-pol regions were ligated into the 10.2 kb pLAI.4 backbone, and the constructs weretransformed and prepped as described above. Protease Mutagenesis Using the construct that contained the mutation in the C cleavage site and the mutation in p7 to e Primer GATAted out mutagenesis reactions to produce constructs 4 and 5, respectively. Primers A and D were used in the same reaction to produce construct 6, NC (Construct 3) as a backbone, additional mutations were introduced intothe protease region. The gag-pol region from construct 3 was digested and ligated inpGEM5Zf+ as described above. Primer A (Invitrogen, 5’-CGACCCCTCGTCACAATAAAGATAGGGGGACAGC-3’) was used to mutatposition 10 from isoleucine to leucine and position 15 from valine to isoleucine. B (Invitrogen, 5’CAGTATTAGAAGAAATGACTTTGACAGGAAGATGGAAACC-3’) was designed to mutate position 34 from glutamine to glutamic acid, position 36 from isoleucine to methionine, and position 37 from asparagine to threonine. Primer C (Invitrogen, 5’-GGGGGAATTGGAGGTTTTATCAAAGTAAGACAGTATGATCAGG-3’) mutated position 54 from alanine to isoleucine and position 58 from glutamic acid to glutamine, and Primer D (Invitrogen, 5’-GTAGGACCTACACCTGTCAACATAATTGGAAG-3’) changed position 82 from alanine to valine. Mutagenesis was performed as described above, and following sequence confirmation, correct gag-pol regions were digesof pGEM5Zf+, ligated into the pLAI.4 backbone, transformed and prepped. Primes C and D were used alone in the

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92 primers B and C together produced construct 7, and construct 8 was produc ed by using primes E, F, and G. Primer TGGCCTTCCTACAAGGGG-3’) was uer a sine e same uct V) and Ritonavir (RTV) were obtained from the NIH AIDS cKesson BioServices Corporation, Germded in M. ted in rs A and D on construct 7. p6/p6 Pol Mutagenesis The construct which contained all 3 Gag mutations and all 8 protease mutations (Construct 8) was then used as the backbone for mutagenesis of p6 and p6 Pol . Mutagenesis was performed in the pGEM5Zf+ vector using primer E (Invitrogen, 5’-GCTAATTTTTTAGGGAAGATC sed to mutate the fifth amino acid of p6 Pol from asparagine to aspartic acid . PrimF (Invitrogen, 5’-GATAGGCAAGGAACTGTATCCCTTAGCTTCCC-3’) mutatedproline to a leucine in the AIP-1 binding site in p6, and Primer G (Invitrogen, 5’-CCCTTAGCTTCCCTCAAATCACTCTTTGGC-3’) changed an arginine to a lyin the Vpr binding site in p6. Primers E, F, and G were all used alone in single reactions to produce constructs 9, 10, and 11, respectively. Primers E and F were used in thmutagenesis reaction, followed by a reaction containing primer G to produce a constrwhich contained all 3 mutations (Construct 12). Determination of IC 50 Indinavir (ID Research and Reference Reagent Program (M antown, MD). Indinavir was reconstituted in water, while RTV was resuspenDMSO to obtain an initial stock concentration of 1.4 x 10 7 nM. Serial dilutions in water were then made to produce a range of concentrations between 140 nM to 1.4 x 10 6 n PBMC were PHA-stimulated and infected as described above. Following the 2 hour incubation period, the cells were rinsed, resuspended in PBMC media, and plaa 96-well round bottom plate. After 1 hour incubation at 37C, 5% CO 2 to allow the cells

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93 to settle, IDV or RTV was added to achieve the following range of final concentrations: 10 nM, 10 2 nM, 10 3 nM, 10 4 nM, and 105 nM. Since IDV was resuspended in dH20, and RTV e Replication of the gag-pol mutans tion antirepy was ses was resuspended in DMSO, controls were run with these two solvents. A 10% media change occurred every other day for 10 days, concurrent with supernatant collection. IDV or RTV was maintained in the cultures for the entire length of the infection. Replication was determined by p24 ELISA (Coulter) and the IC 50 values wercalculated with a nonlinear regression analysis using the GraphPad Prism 4.03 software package (GraphPad Software, Inc., San Diego, CA). Statistical Analysis Statistical analysis was performed using SigmaStat 3.0 software (Jandel Scientific Corp, San Rafael, CA). Differences between the replication of preand posttherapy viruses were tested on days 2, 4, 6, 8, and 10 with t-tests. ts was compared to the replication of the pretherapy virus and expressed as a percent of control (pretherapy). A Kruskal-Wallis one-way analysis of variance (ANOVA) was used to determine which viruses were capable of fully restoring replication to pretherapy levels. A P value of < 0.05 was considered significant. Results Replicative Fitness of Recombinant Viruses in X4 PBMC The gag-pol region from the pretherapy virus of discordant VFIS subject D1 wa obtained from a sample taken seven years prior to the initiation of combina troviral therapy including a protease inhibitor. The patient failed ritonavir theraafter 77 weeks, and was then placed on indinavir. The posttherapy gag-pol region isolated following 16 weeks of indinavir therapy. The viral and immune characteristics of subject D1 are shown in Figure 3-1. The preand posttherapy recombinant viru

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94 were constructed in the HIV LAI backbone, and so were targeted to PBMC that express CXCR4. Replication of the viruses was examined in seven independent donors over 10 days in culture in the absence of protease inhibitors (Figure 3-2). The pretherapy drugsensitive virus replicated to high levels, and produce d significantly more p24 on days 6, 8, andn . itonavir therapy [116]. Between the B cleavage site (p24CA/p2) and acid differences between the preand postthing n p6Pol. he their the 34, 36, and 37 are located on the hinge, amino acids 54 and 58 are on the, 10 of culture compared to the posttherapy drug-resistant virus. Mutagenesis Strategy In order to begin to map the gag-pol determinants of the fitness difference betweethe preand posttherapy variants, an amino acid alignment was performed to locate positions which varied between the two (Figure 3-3). Following ritonavir and indinavir therapy, mutations accumulated in protease at positions 10, 15, 34, 36, 37, 54, 58, and 82Changes at 4 of these positions (10, 36, 54, and 82) are known to confer resistance toboth indinavir and r the end of p1 in gag, there are only two amino erapy viruses, one in the C cleavage site, and one in p7 NC . Following PI-containtherapy, 7 amino acid mutations accumulated in p6, and 6 changes accumulated iTo attempt to restore the reduced replicative capacity of the posttherapy virus, tmutagenesis strategy was to revert the amino acid residues present in the posttherapy virus, either alone or in combination, back to the amino acids that were present in the pretherapy virus (Figure 3-4). The mutations in protease were grouped according tolocation in protease. Amino acids 10 and 15 are located near the active site of enzyme, amino acids flap, and amino acid 82 is located in the active site the enzyme. The amino acids that were mutated in p6 and p6 Pol were chosen because previous studies demonstrated functional activity for these regions. The mutation in p6 Pol is located at the N-terminus

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95 and the two mutations in p6 are located within the AIP1 and VPR binding sites. The first set of mutagenesis experiments focused on changing the residues in the C cleavage site and p7 NC . The virus that contained both the C site and p7 NC mutations was used as thebackbone to add the mutations in protease (Figure 3-4, Construct 3). Construct 8, whiccontained the C site and p7 h as tested by infection of PBMoved t . However, mutation of both the C site and p7NC together (Construct 3) resulted ination of replication to 42% of the prethedid uct flap NC mutations as well as all of the protease mutations, was thenused as the backbone to construct mutants with changes in p6 and p6 Pol . A representation of all of the mutants constructed is shown in Figure 3-5. Replicative Fitness of Gag and Protease Mutants Replicative capacity of the C site and p7 NC mutants w C in 6 independent donors (Figure 3-6). The posttherapy virus demonstrated 12-fold lower levels of p24 production compared to the pretherapy virus on day 8 post-infection. Mutation of a valine to an isoleucine in the C cleavage site imprreplication of the posttherapy virus from 13% of the pretherapy level to 23% (Construc1). Mutation of the valine in p7 NC alone was not capable of restoring any replicative ability to the posttherapy virus (Construct 2) partial restor rapy virus. The replication of the posttherapy virus and constructs 1, 2, and 3 not differ significantly from each other, but the replication of each differed significantly from the pretherapy virus (P < 0.001 for posttherapy and constructs 1 and 2, P = 0.001 forconstruct 3). Since full restoration of replicative capacity could not be achieved by mutation of positions in the C cleavage site and p7 NC , mutations in protease were added to constr3, and tested in 3 independent donors (Figure 3-7). Viruses with mutations in the nearactive site and active site together (Construct 6), or mutation of the hinge and

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96 together (Construct 7), were unable to significantly restore any of the replicative capacityof the posttherapy virus (P = 0.007 and P = 0.013, respectively). Reversion of all eigthe positions in protease to the amino acids present in the pretherapy virus was able topartially restore replication to 36% (Construct 8) of the pretherapy virus, although replication was significantly ht of less than that of pretherapy (P = 0.049). Mutation of active site a he % of the pretherapy virus (Figure 3-8). Replication of the virus in whls rly, lone from an alanine to a valine (Construct 5), or alteration of the alanine at aminoacid 54 to an isoleucine and the glutamic acid at position 58 to glutamine (Construct 4) was sufficient to fully restore the replicative capacity of the posttherapy virus. The replication of constructs 4 and 5 was not significantly different than the replication of tpretherapy virus (P = 0.637 and P = 0.067, respectively), although construct 5 only restored replication to 42%, while construct 4 restored replication to 85% of the pretherapy virus. Mutation of any of the amino acids in p6 or p6 Pol resulted in viruses that replicated to levels greater than 50 ich the proline in the AIP1 binding site of p6 was reverted to a leucine (Construct 10) was able to increase replication to 65% of pretherapy, although replication levewere statistically different than replication of the pretherapy virus (P = 0.002). Similacombining all three mutations in p6 and p6 Pol in addition to the C cleavage site, p7 NC , and protease mutations (Construct 12) was able to increase replication, but not to pretherapylevels (P = 0.03). In contrast, alteration of asparagine to aspartic acid in p6 Pol (Construct 9), or arginine to lysine in the VPR binding site of p6 (Construct 11) resulted in replicative capacities that were no different than the pretherapy virus (P = 0.181 and P = 0.169, respectively).

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97 Replicative Fitness of Recombinant Viruses in the Presence Indinavir Replication of the recombinant viruses LAI, pretherapy, and posttherapy wetested in three independent donors in the presence of increasing concentrations of indinavir. LAI was inhibited between 10 re the posttherapy virus was inhibie for eay f the C (Construct 2) resulted in a 2-fold increase in sensitivity, with an IC50 value of 110 nM. In 2 nM and 10 3 nM of IDV (Figure 3-9A). The pretherapy virus was completely inhibited by 10 2 nM, and ted in the presence of 10 3 nM IDV (Figure 3-9B and 3-9C). Indinavir IC 50 Values for Recombinant Viruses To determine the amount of drug needed to inhibit replication of the viruses by 50% (IC 50 ), the amount of p24 on day 8 of culture for each drug concentration was compared to controls that did not contain any drug. A representative IC 50 curve for indinavir is shown in Figure 3-10. More IDV is required to inhibit the posttherapy resistant virus than either of the sensitive viruses, LAI or pretherapy. A mean IC 50 valu ch virus was calculated from the individual IC 50 values of each of the three independent donors. There was a 3-fold difference between LAI and the pretherapvirus, with IC 50 values of 80 nM and 30 nM, respectively. The posttherapy virus requires over 6-fold more drug than the pretherapy virus to inhibit replication by 50%, with an IC 50 of 200 nM. Indinavir IC 50 Values for Mutant Viruses The impact of mutations in gag-pol on sensitivity to protease inhibitors was examined by determining the IC 50 values for mutant constructs 1 – 8. Mutation ocleavage site or p7 NC caused an increase in sensitivity to indinavir compared to the posttherapy virus (Figure 3-11). The C site mutant (Construct 1) had an IC 50 value of 40nM, which represented a 5-fold increase in sensitivity, while mutation of just p7 NC

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98 contrast, the double mutant (Construct 3) was as resistant to indinavir as the postthevirus, with an IC rapy r is 50 nt to uct 8, which contained all of the Gag mutations and all of the PR mutatus. R2 ug IDV of us dropped off between 103 nM and 104 nM of drug (Figure 3-14A and 3-14B). In contrast, the posttherapy virus 50 value of 260 nM. Replication of the protease mutants (Constructs 4-8) in the presence of indinavishown in Figure 3-12. Alteration of just the active site (Construct 5) caused a 3-folddecrease in IC 50 value (80 nM) compared to the posttherapy virus. Addition of the near active site mutations (Construct 6) caused a further decrease in resistance, with an ICvalue of 30 nM. Construct 4, with mutations in the flap (54 and 58), was as resistaindinavir as the posttherapy virus (IC 50 of 220 nM). However, addition of the 3 mutations in the hinge (Construct 7) was able to increase sensitivity 7-fold, with an IC 50 value of 30 nM. Constr ions (IC 50 value of 20 nM), was as sensitive to indinavir as the pretherapy virRelationship between IDV IC 50 and Viral Replication A linear regression was performed to determine if a correlation existed between the IC 50 of a virus and viral replication in the absence of indinavir (Figure 3-13). With anvalue of 0.022, no relationship was found between viral replication in the absence of drand the amount of drug required to inhibit replication by 50%. When compared to the preand posttherapy viruses, constructs could be resistant to IDV and replicate to either high (construct 4) or low (construct 3) levels. All constructs that were sensitive to replicated to levels that were less than 50% of the replication of the pretherapy virus Replicative Fitness of Recombinant Viruses in the Presence Ritonavir Replication of the recombinant viruses LAI, pretherapy, and posttherapy weretested in three independent donors in the presence of increasing concentrations ritonavir. Replication of both LAI and the pretherapy vir

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99 continued to replicate in the presence of 104 nM RTV, and was not inhibited until 105 nM of drug was added to the culture (Figure 3-14C). When compared to indinavir, all three viruses were more resistant to RTV, requiring higher levels of drug to inhibit replication. Ritonavir IC50 Values for Recombinant Viruses IC50 values on day 8 were calculated for ritonavir, and a representative example of the curve is shown in Figure 3-15. All three viruses were 25-30 fold more sensitive to indinavir than ritonavir, but the relative IC50 values between viruses remained similar. More drug was required to inhibit the posttherapy virus than either LAI or the pretherapy virus. In the presence of ritonavir, LAI has an IC50 of 1980 nM, and the pretherapy virus has an IC50 of 870 nM, a 2-fold difference. The posttherapy virus, with an IC50 of 5150 nM, requires 6-fold more ritonavir to inhibit replication by 50% as compared to the pretherapyRitonavir es In the presence of ritonavir, mutation of the C cleavage site alone (Construct 1) resulted in an IC50 value of 130 nM, which represents a 40-fold increase in drug sensitivity compared to the posttherapy virus (Figure 3-16). In contrast to indinavir, mutation of the amino acid in p7NC alone (Construct 2) caused an even greater 86-fold increase in sensitivity to ritonavir, with an IC50 value of 60 nM. Combination of the C site and p7NC mutations (Construct 3) resulted in a virus that was more resistant to ritonavir than either single mutant, but was still 3-fold more sensitive than the posttherapy virus. Replication of the protease mutants (Constructs 4-8) was also tested in the presence of ritonavir (Figure 3-17). Mutation of the active site alone (Construct 5) caused a 2-fold decrease in the IC50 value of compared to the posttherapy virus. Addition of the near virus. IC 50 Values for Mutant Virus

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100 active site mutations at positions 10 and 15 toe site mutation at 82 (Construct 6) caused a 9-fold increase in sensitivity, with an IC50 value of 590 nM. Mutation of amino acids 54 anincrease inacid positions 34, 36, and 37) to those in the flap (Construct 7) caused the sensitivity to ritonavir to increase further to 450 nM. Construct 8, which contained all of the protease mutations along with the C cleavage site and p7NC, had an IC50 value of 170 nM, making it 5-fold more sensitive to ritonavir than the pretherapy virus. Relationship between RTV IC50 and Viral Replication As was the case with indinavir, no correlation was found between the RTV IC50 and replication of the virus in the absence of ritonavir (Figure 3-18). In relation to the preand posttherapy viruses, two constructs (3 and 5) were able to replicate to intermediate levels in the absence of RTV, and had intermediate levels of resistance. Unlike construct 4 with indinavir, no viruses were able to replicate to high levels in the absence of RTV and have high IC50 values. Discussion One of the major problems facing HIV-1 treatment is the rapid emergence of mutations which alter the replicative capacity or the drug sensitivity of the virus. There are several methods of assessing HIV-1 viral fitness, but assaying viral replication is the most direct measure of fitness, as it takes in to account the entire life cycle of the virus [255]. The results in this chapter were all obtained using replication competent viruses, both in the presence and absence of protease inhibitors, and as a result, represent an accurate measure of viral fitness. Multiple studies have suggested that ex vivo HIV-1 fitness is directly related to, and is a strong predictor of disease progression the activ d 58 in the flap (Construct 4) resulted in an IC 50 value of 690 nM, a 7-fold sensitivity to ritonavir, while addition of the mutations in the hinge (amino

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101 [9,17,18,207,253]. Furthermore, Trkola et al. demonstrated th at fitness also impacts the extent of viral rebound and the viral set point in chronically infected patients [252]. Identifying genetic determinants of viral rep lication and drug resistance is critica to understanding how fitness impacts the course of disease so that better antiretroviral therapies can be designed. Multiple groups have demonstrated that in the absence of antiretrovirals, v with primary drug resistance mutations are le ss fit than wild-type viruses [40,96,174]. This is consistent with our data in which the PI-resistant posttherapy virus displayed a significant reduction in replica tion compared to the pretherapy sensitive virus. W mutation of the C cleavage site alone, or the position in p7NC alone from the am present in the posttherapy virus to those present in the pretherapy virus resulted in little to no restoration of viral replic ation, combining the two muta tions was able to cause an increase replication, indicating that positions in gag can act in concert with one another to improve viral fitness. Mutation of the two positions in gag was not sufficient to fully restore r to pretherapy levels, suggesting th at changes in protease and/or p6/p6Pol were also necessary. Addition of a mutation in the active site (amino acid 82) or mutations at positions 54 and 58, located on the flap of protease, to the C site and p7NC mu sufficient to completely restore replication. However, addition of the hinge m (34, 36, and 37) to the flap mutations caused a decrease in the repl icative capacity. One potential explanation for this could be that mutations in these positions alter the conformation of protease, inhibiting replicati on, and that additional mutations els are needed to restore activity. A similar si tuation was seen with the active site and near l iruses hile ino acids eplication tations was utations ewhere

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102 active site mutations. Mutation of just the active site allowed partial restoration of replication, while addition of the near active site mutations at 10 and 15 caused a reduction ior during thersome of th Studies demonstrated that reduced replication of wild-type virus could be explained partially by mutations in p6, particularly at residues involved in binding to cellular proteins such as AIP1 [143]. In agreement with those studies, mutation of just the AIP1 binding site from those present in the posttherapy virus to those present in the pretherapy virus was capable of increasing replication to 65% of pretherapy levels. Our data further demonstrate the dominant impact of residues that interact with viral proteins, such as VPR, as well as residues in the frameshift region of p6Pol, on viral replication. Reversion of the C cleavage site alone or p7NC alone resulted in little to no increase in replication of the viruses compared to the posttherapy virus, but caused a large decrease in resistance. These data indicate that therapy-associated mutations in the C site and p7NC will cause a large increase in resistance at a small fitness cost, demonstrating the dominant effect of gag on resistance to protease inhibitors. Combining the C site and p7NC mutations caused an increase in both replication and resistance to PI. So in the absence of any protease mutations, multiple therapy-related mutations in gag could cause a decrease in replication and resistance. Since decreases in both replication and resistance are detrimental for the virus, it is likely that gag mutations and mutations in protease act in concert to produce a fit virus. In fact, replication of the virus containing mutations in the C site, p7NC, and the active site of protease is the same as the replication n replication. The mutations present in the posttherapy virus were selected fapy, and it is likely that the conformation of protease is not ideal when only e amino acids are in the “correct” orientation.

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103 of the double gag mutant (construct 3) alone. However, addition of the active site mutation to the construct 3 reduces the susceptibility of the virus to indinavir. A similar pattern was observed with constructs 6, 7, and 8. The protease mutations in all three constructs reduced replication compared to construct 3, but also caused increases in sensitivity to both indinavir and ritonavir. Addition of mutations in the flap of protease to the gag mutations (construct 4) caused a significant increase in replication, and also increased drug sensitivity, although to a lesser extent than constructs 6, 7, and 8. Together, these data demonstrate how gag-pol functions as one connected unit. Specific combinations of mutations in gag and protease can have differential effects on replication and drug resistance. The overall goal of the mutagenesis experiments was to restore the reduced replicative capacity of the posttherapy PI-resistant virus. The positions chosen were based on previous functional data, and in the case of protease, amino acids were grouped according to their location on the enzyme. Consequently, amino acids were changed from those present after therapy to those present before therapy, beginning with the C cleavage site and p7NC, followed by protease, and finally p6/p6Pol. As a result, all of the protease mutants constructed contained a pretherapy C site and p7NC region. Testing of a virus that contains only the active site reverted back to pretherapy and a virus that contains all of the protease mutations, but none of the gag mutations will contribute to understanding the differential impact of gag and PR mutations on viral fitness. In addition, testing the p6/p6Pol mutations with posttherapy C site, p7NC, and protease contributio regions in the presence and absence of protease inhibitors will determine their n to replicative capacity and drug resistance.

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104 -60-40-20020406080100 012 3456 024681012141618 Log10(copies/ml) RNA CD4 %StartIDVe) WEEKSStartRTV Pr(-334 Post -60-40-20020406080100 012 3456 024681012141618 -60-40-20020406080100 012 3456 024681012141618 Log10(copies/ml) RNA Log10(copies/ml) RNA CD4 %WEEKSStartRTVStartIDVe) Pr(-334 Post Figure 3-1. Viral and Immune Characteristics of Subject D1. Log10 viral RNA copies r (blue circles) are shown on the left axis, and CD4 percents (red le time points and therapy start time per millilitebars) are shown on the right axis. Samppoints are shown with arrows.

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105 Daysp24 (ng/ml) 246 8 10 020040060080010001200 **Daysp24 (ng/ml) 2468 * 020040060080010001200 ** 10 2468 10 * 020040060080010001200020040060080010001200 ** Figure 3-2. Replication Kinetics of Pre-and Posttherapy Variants in X4 PBparallel cultures, the posttherapy virus (open circles) displayed a signreduction in p24 production relative to the pretherapy virus (filled circlesData represent mean and SEM of 7 independent infections (*P < 0.001 on day 6, P = 0.007 on day 8, P = 0.006 on day 10, t-test). *MC. In ificant ).

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A. Gag-Pol Cleavage SitesC. PolFFREDLAFLQGEAREFSSEQTREQTRANSPTRGELQVWGGDNNSLSEAGADRQGTVSLSF ----n--------------------s-----sr-----r----------d---------E Sitep6*PQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMTLTGRWKPKMIGGIGGFIKVRQYDQVPIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF ---------i----v------------------q-in----------------a---e-----------------------a----------------E SiteProtease F SiteB. GagKARVLAEAMSQATNSQIIMMQKGNFRNQRKIVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQAN-----------------v---------------------v---------------------------------C Sitep7 (Nucleocapsid) B Site D’ SitePrePostp2FLGKIWPSYKGRPGNFLQSKPESRPEPTAPPEESFRFGEETTTPSQKQEPIDKELYPLASLKSLFGNDPSSQ -------------------r-----a-----a-----------------t-g--p------r---------D Site D' Sitep6p1 F p17 p6* ABCD’DE p24 p7 p6 PRRT p2p1 PrePostPrePostPrePostA. Gag-Pol Cleavage SitesC. PolFFREDLAFLQGEAREFSSEQTREQTRANSPTRGELQVWGGDNNSLSEAGADRQGTVSLSF ----n--------------------s-----sr-----r----------d---------E Site E Sitep6*PQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMTLTGRWKPKMIGGIGGFIKVRQYDQVPIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF ---------i----v------------------q-in----------------a---e-----------------------a----------------E Site E SiteProtease F Site F SiteB. GagKARVLAEAMSQATNSQIIMMQKGNFRNQRKIVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQAN-----------------v---------------------v---------------------------------C Site C Sitep7 (Nucleocapsid) B Site B Site D’ Site D’ SitePrePostPrePostp2FLGKIWPSYKGRPGNFLQSKPESRPEPTAPPEESFRFGEETTTPSQKQEPIDKELYPLASLKSLFGNDPSSQ -------------------r-----a-----a-----------------t-g--p------r---------D Site D Site D' Site D' Sitep6p1 F p17 p6* ABCD’DE p24 p7 p6 PRRT p2p1 F p17 p6* ABCD’DE p24 p7 p6 PRRT p2p1 PrePostPrePostPrePostPrePostPrePostPrePost Figure 3-3. Sequence Alignment of Preand Posttherapy Variants. A, Schematic representation of Gag-Pol and the cleavage sites, denoted by the letters A through F. B, Sequence alignment of Gag. C, Sequence alignment of Pol. 106

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107 p6AIP1p6VPRCNC1015NearActive343637Hinge5854Flap82ActiveSiteProtease Gag p6PolPost Pre Mutagenesis of the C cleavage site and p7NCMutagenesis of p6 and p6PolMutagenesis of Protease38p6AIP1p6VPRCNC1015NearActive343637Hinge5854Flap82ActiveSiteProtease Protease Gag Gag p6PolPost Pre Mutagenesis of the C cleavage site and p7NCMutagenesis of p6 and p6PolMutagenesis of Protease38 Figure 3-4. Mutagenesis Strategy. The posttherapy virus was used as the backbone to uct e noted above the constructs. Red circles represent amino acids present in the posttherapy variant, while blue circles represent residues in the pretherapy variant. make mutations in the C cleavage site and p7 NC (Construct 3). Construct 3 was then used as the backbone to introduce mutations in protease (Constr8), which then became the backbone for the final mutations in p6 and p6 Pol . Positions and their locations ar

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108 CNC1015NearActive343637Hinge5854Flap82ActiveSitep6Polp6AIP1p6VPRProtease Gag 3 2 1 Post 8 4 5 7 6 9 10 11 12 Pre CNC1015NearActive343637Hinge5854Flap82ActiveSitep6Polp6AIP1p6VPRProtease Protease Gag Gag 3 3 2 2 1 1 Post Post 8 8 4 4 5 5 7 7 6 6 9 9 10 10 11 11 12 12 Pre Pre Figure 3-5ate resides that are identical to the posttherapy virus and blue circles represent amino acids identical to the pretherapy virus. . Mutagenesis Constructs. Amino acid positions and locations are noted above the constructs. Red circles indic

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109 Virus Replication CNC1015NearActive 343637ngero Hi5854Flap82ActiveSitePtease Gag p6AIP1p6VPRp6Pol 050100 Pre Post 1 2 3 ****Virus Replication Virus Replication CNC10151015NearActiveHi58545854Flap82ActiveSitePtease 343637343637ngero Ptease ro Gag Gag p6AIP1p6VPRp6Pol 050100 Pre Pre Pre Post Post Post 1 1 1 2 2 2 3 3 **** Figure 3-6. Replicative Capacity of C Cleavage Site Mutants. Representations of viral constructs are shown on the left. Red circles indicate resides that are identicosttherapy virus and blue circles represent amino acids identical to the pretherapy virus. Viral replication on day essed as a percent of the pretherapy virus, is shown in the bar graph on the right. Error bars represent mean and SEM ofdependent experiments. * P < 0.001, ANOVA. and p7 NC al to the p8, as expr 6 in

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110 54FlapAcSip6Polp6AIP1p6VPRProtease CNC1015NearActive343637Hinge 5882tivete Gag Virus Replication 050100 P re Post 4 5 6 7 8 ****CNC1015NearActive343637Hinge54FlapAcSip6Polp6AIP1p6VPRProtease 5882tivete Protease Gag Gag Virus Replication Virus Replication 050100 050100 P re P re P re Post Post Post 4 4 4 5 5 5 6 6 6 7 7 7 8 8 8 **** Figure 3-7. Replicative Capacity of Protease Mutants. Representations ofshown on the left. Red circles indicate resides that are identical to the posttherapy virus and blue circles represent amino acids identical to the pretherapy virus. Viral replication on day 8, as expressed as a percent of the pretherapy virus, is shown in the bar graph on the right. Error bars represent mean and SEM of 3 independent experime viral constructs are nts. * P = 0.028, ANOVA.

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111 CNC1015NearActive343637Hinge5854Flap82ActiveSitep6Polp6AIP1p6VPRProtease Gag Virus Replicat ion 05100 Post 9 10 11 12 Pre **CNC1015NearActive343637Hinge5854Flap82ActiveSitep6Polp6AIP1p6VPRProtease 0 * Gag Virus Replicat ion Protease Protease Gag Gag Virus Replicat ion Virus Replicat ion 05100 0 05100 0 05100 0 Post 9 10 11 12 Pre ** Figure 3-8. Replicative Capacity of p6 and p6Pol Mutants. Representations of viral constructs are shown on the left. Red circles indicate resides that are identical to the posttherapy virus and blue circles represent amino acids identical to the pretherapy virus. Viral replication on day 8, as expressed as a percent of the pretherapy virus, is shown in the bar graph on the right. Error bars represent mean and SEM of triplicate values from one experiment. * P < 0.001, ANOVA. *

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112 040080012001600246810p24 (ng/ml)DaysA. 040080012001600246810 040080012001600246810p24 (ng/ml)DaysA. B. 040080012001600246810p24 (ng/ml)DaysB. 040080012001600246810 040080012001600246810p24 (ng/ml)Days C. 0400800p24 (n 12001600246810g/ml)DaysC. 0400800p24 (n 12001600246810g/ml)Days 04 105 nM. Figure 3-9. Replication of Recombinant Viruses in the Presence of Indinavir. A, LAI. B, Pretherapy. C, Posttherapy. Blue diamonds represents no drug, pink squares 10 nM, yellow triangles 10 2 nM, aqua crosses 10 3 nM, purple stars 1nM, and red circles,

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113 0255075100101102103104105[IDV] nMReplication (% of Control) 0255075100101102103104105[IDV] nMReplication (% of Control) Figure 3-10. IDV IC50 of Recombinant Viruses. Blue diamonds represents LAI, pink squares represents the pretherapy virus, and yellow triangles represents the posttherapy virus.

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114 CNC1015Active343637Hinge5854Flap82SiteProtease Near Active Gag p6p6p6PolIC50(nM) AIP1VPR Post 200 1 40 2 110 3 260 Pre 30 02575100Replication(%ontrol) 50 of C [IDV] nM Pre Post 1 2 3NearActive 101102103104105CNC1015Active343637Hinge5854Flap82SiteProtease Gag p6p6p6PolIC50(nM) AIP1VPR Post 200 1 40 2 110 3 260 Pre 30NearActive CNC10151015Active343637343637Hinge58545854Flap82SiteProtease Gag p6p6p6PolIC50(nM) AIP1VPR IC50(nM) Post 200Post Post 200 1 40 1 1 40 2 110 2 2 110 3 2603 3 260 Pre 30Pre Pre 30 02575100Replication(%ontrol) 50 of C [IDV] nM Pre Post 1 2 3 101102103104105 02575100Replication(%ontrol) Pre Post 1 50 of C 2 3 Pre Post 1 2 [IDV] nM 3 50 101102103104105101102103104105 Figure 3-11. IDV IC Values and Curves for C site and p7 NC Mutant Viruses. Replication curves of the gag mutant viruses in the presence of indinavir are shown in the top panel. Virus construct representations and the corresponding IC 50 values are shown in the bottom panel.

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115 CNCActive343637Hingep6Polp6p6Protease 1015Near 5854Flap82ActiveSite AIP1VPR Gag IC50(nM) 4 220 5 80 6 307 308 20t Pos 200 Pre 30 025lica Ct) 5075100[IDV] nMReption(% ofonrol Pre Pos t 8 4 5 6 71015Near5854Flap82ActiveSite 101102103104105CNCActive343637Hingep6Polp6p6Protease AIP1VPR Gag IC50(nM) 4 220 5 80 6 307 308 20t Pos 200 Pre 301015Near5854Flap82ActiveSite CNCActive343637Hingep6Polp6p6Protease AIP1VPR Gag IC50(nM) IC50(nM) 4 2204 4 220 5 805 5 80 6 306 6 6 307 307 7 308 208 8 20t Pos 200t Pos Pos t 200 Pre 30Pre Pre 30 025lica Ct) 5075100[IDV] nMReption(% ofonrol Pre Pos t 8 4 5 6 7 101102103104105 025lica Ct) 5075100[IDV] nMReption(% ofonrol Pre Pos t 8 4 5 6 7 Pre Pre Pos t Pos t 8 8 4 4 5 5 6 6 7 7 101102103104105101102103104105 Figure 3-12. IDV IC 50 Values and Curves for Protease Mutant Viruses. Replication curves of the PR mutant viruses in the presence of indinavir are shown in thetop panel. Virus construct representations and the corresponding IC 50 valuesare shown in the bottom panel.

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116 IC50(nM) 050 100150200250300 02060100 80120 40 R2= 0.022ion(%ntrolPre6 P = 0.682Replicat of Co)4 Post123587 IC50(nM) 050 100150200250300 02060100 80120 40 R2= 0.022ion(%ntrolPre6 Figure 3-13. Linear Regression of IDV IC and Viral Replication. IDV IC values are P = 0.682Replicat of Co)4 Post123587 50 50 expressed on the x-axis, and viral replication in the absence of IDV is expressed on the y-axis. The virus construct number is shown in red to the right of each point.

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117 A.ng/ml) 80012001600A.ng/ml) 80012001600 80012001600 p24 ( 0246810Days 400p24 ( 0246810 400 400 0246810Days 4 (ng/m B.p2 l) 040080012001600DaysB.l) 246810p24 (ng/m 040080012001600 246810 040080012001600Days 246810C. 1600C. 1600 1600 p24 (ng/ ml) 0400800Daysp24 (ng/ 1200246810 ml) 0400800 1200246810 0400800Days B, Pretherapy. C, Posttherapy. Blue diamonds represents no drug, pink nM, and red circles, 10 1200246810 Figure 3-14. Replication of Recombinant Viruses in the Presence of Ritonavir. A, LAI. squares 10 nM, yellow triangles 102 nM, aqua crosses 103 nM, purple stars 104 5 nM.

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118 100 0255075[RTV] nMReplication Control10110210310 (% of )4105 100 0255075[RTV] nMReplication Control10110210310 Figure 3-15. RTV IC (% of )4105 nk he 50 of Recombinant Viruses. Blue diamonds represents LAI, pisquares represents the pretherapy virus, and yellow triangles represents tposttherapy virus.

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119 CNC 1015NearActive343637Hinge5854Flap82ActiveSiteProtease Gag p6AIP1p6VPRIC50(nM) p6Pol Post 51501 1302 603 1650Pre 870 0255075R(%f C 100rol) eplication oont Pre [RTV] nM Po st 1 2 3101102103104105 CNC 1015NearActive343637Hinge5854Flap82ActiveSiteProtease Gag p6AIP1p6VPRIC50(nM) p6Pol Post 51501 1302 603 1650Pre 87010151015NearActive343637343637Hinge58545854Flap82ActiveSiteProtease CNC Gag p6AIP1p6VPRIC50(nM) p6Pol IC50(nM) Post 5150Post Post 51501 1301 1 1302 602 2 603 16503 3 1650Pre 870Pre Pre 870 0255075R(%f C 100rol) eplication oont Pre [RTV] nM Po st 1 2 3101102103104105 0255075R(%f C 100rol) eplication oont Pre Po st 1 [RTV] nM 2 3 Pre Po st 1 2 3101102103104105101102103104105 NC Figure 3-16. RTV IC 50 Values and Curves for C Site and p7 Mutant Viruses. Replication curves of the gag mutant viruses in the presence of indinavir are shown in the top panel. Virus construct representations and the corresponding IC 50 values are shown in the bottom panel.

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120 CNCActiveHingeFlapSitep6Polp6AIP1p6VPRProtease 1015Near 343637585482Active Gag IC50(n M) 4 690 5 31506 5907 4508 170Post 5150 Pre 870 Pre Post 8 4 5 6 7 0255075100[RTV] nMReplication(% ofonrol101102103104105CNCActiveHingeFlapSitep6Polp6AIP1p6VPRProtease Ct)1015Near343637585482Active Gag IC50(n M) 4 690 5 31506 5907 4508 170Post 5150 Pre 8701015Near343637585482Active CNCActiveHingeFlapSitep6Polp6AIP1p6VPRProtease Gag IC50(n M) IC50(n M) 4 6904 4 690 5 31505 5 31506 5906 6 6 5907 4507 7 4508 1708 8 170Post 5150Post Post 5150 Pre 870Pre Pre 870 Pre Post 8 4 5 6 7 0255075100[RTV] nMReplication(% ofonrol101102103104105 Ct) Pre Post 8 4 5 6 7 Pre Pre Post Post 8 8 4 4 5 5 6 6 7 7 0255075100[RTV] nMReplication(% ofonrol101102103104105101102103104105 Figure 3-17. RTV IC Values and Curves for Protease Mutant Viruses. Replication curves of the PR mutant viruses in the presence of indinavir are shown in the top panel. Virus construct representations and the corresponding IC values are shown in the bottom panel. Ct)5050

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121 120 0100020003000400050006000 04080 2060100 R2= 0.0118ion orol)Pre6 120 P = 0.77 IC50(nM)Replicat(%f ContPost1234587 0100020003000400050006000 04080 2060100 R2= 0.0118ion orol) 120 P = 0.77 IC50(nM)Replicat(%f Cont 0100020003000400050006000 04080 2060100 R2= 0.0118ion orol)Pre6 Figure 3-18. Linear Regression of RTV IC50 and Viral Replication. RTV IC50 values are expressed on the y-axis. The virus construct number is shown in red to the P = 0.77 IC50(nM)Replicat(%f ContPost1234587expressed on the x-axis, and viral replication in the absence of IDV is right of each point.

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CHAPTER 4 RECOMBINATION AND REVERSION MODULATE HIV-1 FITNESS IN CXCR4 CD4+ T LYMPHOCYTES Introduction HI V-1 recombination has been shown to occur during negative strand DNA synthesis by a mechanism known as the copy choice model [78,1 to an e ror prone nature of reverspy. 02,115,175,189,242,276]. The copy choice model states that the low processivity ofreverse transcriptase causes the enzyme to dissociate from the template, allowing the short DNA-RNA hybrid to be disrupted so that the growing DNA strand is displaced the other RNA template [42,270]. In addition to creating viral diversity, recombinationcan also act as a repair mechanism for the HIV-1 genome by using one intact copy to fix detrimental mutations in the other copy [36,238]. HIV-1 recombination occurs throughout the entire genome, but some regions have been shown to have higher rates of recombination than others, indicating that there may be hotspots for recombination [36,80,107,115,138,146,175,206,213,286]. For example, the crossover rate in Pol is higher than the crossover rate in Env [107,115]. Furthermore,within envelope, recombination in the C2 region is 2-10 fold more frequent thsurrounding regions, suggesting that the structure of the genomic RNA may influencrecombination [80,206]. The frequency of recombination can be influenced by the er e transcriptase as well as mutations that accumulate in response to antiviral theraReverse transcriptase does not posses a 3’ to 5’ exonuclease proofreading activity, and as 122

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123 a result, has a base misincorporation rate of 1 in 10 4 , which is roughly equivalent tmisincorporated base per genome [39]. Multiple studies have demonstrated that base misincorporations by reverse transcriptase enhance strand transfer during transcription [59,181]. In addition, mutations that are associated with resistance to nucleoside analohave also been shown to increase the template switching frequency of reverse transcriptase [175]. An increase in template switching will lead to an increase in recombination. Conflicting reports have emerged regarding the relative frequency of recombination in different cell types. Levy et al. found that HIV-1 recombines 3-5more frequently in macrophages than T ce o one gs fold lls, but Chen et al. reported similar rates of recomf f y, amount of detectable drug resistant virus, and an apparent reversion to wild-f back tive bination between the two cell types [31,146]. The selective pressure of antiretroviral therapy (ART) results in the emergence oviruses that are resistant to treatment, but less fit than wild-type virus in the absence odrugs [51,278]. Consequently, an interruption in therapy should lead to the re-emergenceof the more fit wild-type virus, which can then be controlled by ART. This strategknown as structured treatment interruption (STI), is one approach to managing HIV-1 infection. Multiple studies have shown that following treatment interruption, there is a reduction in the type [55,56,58,108,167,256]. Unfortunately, the emergence of wild-type virus in the absence of therapy leads to an increase in viral load, and a rapid depletion of CD4 + T cells [55,274]. Many groups have suggested that the rebounding virus is an outgrowth oa less mutated, or wild-type virus, rather than a drug resistant virus that is revertingto wild-type in the absence of selective pressure [55,56,95]. In fact, using more sensireal-time PCR methods, multiple groups found that minority resistant populations of

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124 virus persist in patients, even though conventional sequencing methods detected only wild-type virus [95,109]. The rebounding wild-type virus could potentially be coming from two different sources. The first possibility is that wild-type virus could continue to replicate, even in the presence of drugs, and would be able to grow quickly when drug was removed. A second possibility is that there is a latent cellular reservoir of wild-type virus that reactivates after the removal of therapy. This study was designed to examine the role recombination and reversion play in HIV-1 fitness. HIV-1 variants were tested in direct competition experiments and a PI-resistant variant w as passaged in the absence of drugs to evaluate reversion. y of ere infected with a total of 4000 TCID50 of virus. PBMC were infected with either a single virus at 4000 TCID50, or two viruses, each at 2000 TCID50 for 2 hours, with gentle agitation every 20-30 minutes. The cells were then rinsed by addition of 10 ml of 1 X PBS (Gibco), centrifuged at 1000 RPM for 10 minutes, and resuspended in 5 ml PBMC media. The cell suspensions were placed in T-25 flasks and incubated upright at 37C, 5% CO2. On day 4 post-infection, 3.5 ml of supernatant was carefully removed, and 3.0 ml of fresh media was added back, and incubated for a further 3 days. Seven days post-infection, 20l of the cell suspension was removed and used to count the cells using dye exclusion with Trypan Blue (StemCell Technologies, Vancouver, BC, Canada), and the rest was placed in a 15 ml conical tube and centrifuged Materials and Methods Competition Experiments Competition experiments consisted of 5 one-week passages, beginning with infection on day 0. PBMC were stimulated with PHA (Sigma) three days prior to infection as described in the materials and methods section of Chapter 3. On the dainfection, 5 x 10 6 cells w

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125 at 1000 RPM for 10 minutes. The supernatants were syringe filtered (.45m Acrodisc) and 1 ml was used to infect a new batch of PHA-stimulated PBMC from the same donor as described previously. A small amount of supernatant (200 l) was used to determine p24 antigen levels by ELISA (Beckman Coulter), and the rest was frozen at -80C. Sequencing of Cell-Associated DNA The cells from day 7 of each passage were rinsed twice with 1.5 ml of 1 X PBS (Gibco) and spun at 12,000 RPM for 4 minutes. Following the second wash, the cells were lysed in the appropriate amount of 20 mg/ml Proteinase K (Fisher Scientific, Pittsburgh, PA) and K buffer (50 mM KCl, 10 mM Tris/Cl pH 8.3, 2.5 mM MgCl2, 0.1mg/ml gelatin, 0.45% Nonidet P40, 0.45% Tween 20) (5 l of Proteinase K per 1 ml of K following day, the Proteinase K was heat inactivated at 95C for 5 minutes. To prepare for sequencing, the DNA was cleaned up with the QiaAmp DNA Blood Mini Kit (Qiagen), and eluted in 100 l of dH20. The gag-PR region was amplified, cloned and sequenced as described in the high-throughput sequencing section of Chapter 2. Sequence Analysis Gag (p2 through p6) and PR sequences were aligned at both the nucleotide and amino acid level using the BioEdit Sequence Alignment Editor (Ibis Therapeutics, Carlsbad, CA) and analysis of recombination between LAI and the pretherapy virus was performed. The number of crossovers in Gag and PR was determined by using nucleotide differences that exist between LAI and the pretherapy virus as markers. In PR, there are 11 nucleotide differences between LAI and pretherapy. Only four of those differences translate into changes at the amino acid level at positions 37, 39, 62, and 63. There are 22 nucleotide differences between the two viruses in Gag that correspond to 10 buffer, and 1 ml K buffer for every 5 x 106 cells), and incubated overnight at 37C. The

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126 amino acid changes (three in p2, two in p7NC, and five in p6). In addition, LAI contains a 24 base pair insertion in p6 that is not present in the pretherapy virus. Mutagenesis of Position 54 in PR The QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene) was used to mutate the alanine at amino acid 54 in PR of the posttherapy virus into both a valine and an isoleucine. The mutagenesis was performed on the pGEM5Zf+/3188a02 gag-pol shuttle vector described in Chapter 3. Primer A54I (Invitrogen, 5’-GGGGGAATTGGAGGTTTTATCAAAGTCAGAGAGTATGATCAGG-3’) was used to mutate alanine to isoleucine, and primer A54V (Invitrogen, 5’-GGGGGAATTGGAGGTTTTGTCAAAGTAAGAGAGTATGATCAGG-3’) was used urer’s protocol deagments were ligated into the pLAI.4 backbone. PBMC Infections with A54V and A54I Mutant Viruses titered, as d1500 TCID hours, with gently shaking every 20-30 minutes. The cells were subsequently washed with 10 ml 1 X PBS, centrifuged for 10 minutes at 1000 RPM, nd resuspended in 1.2 ml of PBMC media. 200 l of the cell suspension was then liquotted into each well of a 96-well round bottom plate, and the cultures were incubated at 37C, 5% CO2. Supernatant collection and 10% media changes occurred on days 2, 4, 6, 8, and 10 post-infection. p24 antigen levels were determined by ELISA (Coulter). to mutate alanine to valine. Mutagenesis was performed according to the manufactscribed in Chapter 3, and after sequence confirmation, mutated gag-pol fr Once the mutagenesis of position 54 was complete, viral stocks were made andescribed in Chapter 3. PHA-stimulated PBMC (1.2 x 10 6 ) were infected with 50 of virus for 2 a a

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127 Results Recombination Confers a Replicative Advantage to HIV-1 To asses the effect of recombination on HIV-1 fitness, replication competent viruses were tested in direct competition experiments. Viruses were constructed using gag-pol alleles from discordant response patient D1 prior to (Pre) and following (Post) protease inhibitor containing therapy in an LAI backbone as described previously [219]. Each of the three viruses (LAI, Pre, and Post) was passaged alone in PBMC, as well as in combination with each of the other viruses for 5 weeks. Replication was assessed by p24 production at passages one and five (Figure 4-1). At the end of passage one, all viruses and virus combinations, with the exception of the posttherapy virus, showed similar levels of replication. The posttherapy virus displayed greater than a culture, allTo asses was sequenced from each culture after the first and fifth passages. All single infection ontrols (LAI, Pre, and Post) contained 100% of the expected clone at the end of 5 weeks in culture (Figure 4-2A). By the end of the first passage and continuing through passage indicating competed tpretherapys after 5 weeks (Figure 4-2B). Nine ut of 22 (41%) of clones were LAI and 4 of 22 (18%) were pretherapy. The remaining 9 lones appeared to be recombinants between LAI and the pretherapy virus. 6-fold reduction in replication compared to the pretherapy virus. After 5 weeks in viruses demonstrated a 2-7 fold increase in replication compared to baseline. hich virus clones were present in the cultures over time, cell-associated DNA w c 5, the presence of the posttherapy virus was not detected in the mixed infections, that both the pretherapy virus (Pre-Post) and LAI (LAI-Post) entirely out he posttherapy virus (Figure 4-2B). Competition between LAI and the virus resulted in a mixture of virus clone o c

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128 Further analysis of the LAI-pretherapy competition at the nucleotide level revealed the frequency of recombination in Gag and PRlevel of recombination was seen at passage 1. Five out of 24 (21%) clones were recombinants in the protease region and 4 of 30 (13%) clones in Gag. However, by the end of passage 5, the frequency of recombinanased to 41% (9/22) in PR, and to 66% (16/24) in Gag, representing 2 and 5 fold increases, respectively. The presence of single and multiple crossovers was detected in both PR and Gag. The proportion of single and multiple crossovers present at the end of passages 1 and 5 are shown in Figure 4-4. At passage 1 in protease, 60% (3/5) of the recombinant clones contained 1 crossover, and 40% (2/5) contained 2 crossovers. In Gag, 100% (4/4) of the recomant clones contained only 1 crossover. By passage 5, double and triple crossovers were evident in both Gag and PR. In PR, the number of single, double, and triple crossovers was equivalent. Three of nine clones contained 1, 2, and 3 crossovers. In contrast, a majority (64%) of Gag recombinants only had one crossover. 18% (3/16) of the recombinants contained a double crossover, and 18% (3/16) contained a triple crossover. Representative examples of the recombinantsres 4-5 (Protease) and 4-6sites mappclones conOver half owards the end of protease. In Gag, 63% (10/16) of the recombinants contained a rossover event which occured between the start of the second zinc finger in p7NC and the at passages 1 and 5 (Figure 4-3). A low ts increbin are shown in Figu (Gag). Inspection of the location of the crossovers reveals that many of the crossover ed to distinct regions. For example, in protease, 7 out of 9 (78%) recombinant tained a crossover in the flap region between amino acid positions 37 and 63. f the recombinants contained a crossover between amino acids 74 and 96, to c

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129 end of p1. Since the sequences of LAI and the pretherapy virus are identical in the ReversionTo aabsence ofe without drug. Figure 4-7A shows the sequence alignment of the preand posttherapy PR regions. Mutations in multiple positions associated with reduced sensitivity to protease inhibitors (10, 36, 54, and 82) accumulated over the course of therapy. Replication of the posttherapy virus and the sequences of the viruses present after 1 and 5 weeks in culture were evaluated. At the end of passage 1, there was a low level of replication (Figure 4-7B). By passage 5, the posttherapy virus demonstrated a 6-fold increase in p24 production. At the end of passage 1, 100% of the clones contained an alanine at position 54 in protease, which is the amino acid present in the posttherapy variant (Figure 4-7C). However, by the end of passage 5, a large majority (82%) of the clones contained a valine at position 54, while only 18% contained an alanine. All other positions in PR and Gag remained constant. Figure 4-8D demonstrates the amino acids that are commonly found at position 54 in PR, and the nucleotide differences between them. There is only one nucleotide difference between the alanine found in the posttherapy PI-resistant virus, andl difference bitive viruses. t Modulator of Viral Fitness o more directly assess the role of position 54 in PR in viral fitness, the alanine that is present at that position in the posttherapy PI-resistant virus was mutated to either the valine found after 5 weeks in culture, or the isoleucine found in PI-sensitive strains of crossover regions, it is impossible to know exactly where the crossovers occurred. of the Posttherapy Virus in the Absence of Protease Inhibitors ssess the impact of reversion on the fitness of a PI-resistant virus in the protease inhibitors, the posttherapy virus was passaged for 5 weeks in cultur the valine that appeared after 5 weeks in culture. In addition, there is only one additionaetween the valine and the isoleucine that is seen in PI-sens Amino Acid Position 54 in PR is an Importan T

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130 virus. The replicative capacities of the A54V and A54I mutants were then evaluated by infection of PBMC. Both viruses replicated to greater than 300-fold higher levels than the posttherapy virus (Figure 4-8). Thity of the mutants was similar to that of the pretherapy virus, with A54lication to 72% of the pretherapy virus,ime, on R quencing on culture. The frequency and extent of recomime, e replicative capac V increasing rep and the A54I mutant increasing replication even higher to 85% of the pretherapy virus. Discussion The replicative capacity of all viruses and virus combinations increased over tconsistent with previous reports that viral populations gain fitness with each successive passage in cell culture [35,177]. Previous studies have focused on recombination in envelope, or between gag-pol and envelope, and this study focused on recombinatiwithin the gag-PR region of the genome [107,138]. To assess recombination in gag-Pand its effect on viral fitness, two viruses with similar replicative capacities in parallel infections were used to co-infect PBMC from a healthy donor, and analysis of seand replication was performed over five weeks in culture. A low level of recombinatiwas seen in both gag and protease after one week in bination between two viruses with similar replicative capacity increase over tconcurrent with an increase in replication, suggesting a fitness advantage for recombinants. In addition, a higher proportion of the clones in the LAI-Pretherapy competition were LAI after five weeks of passaging, indicating a slightly higher replicative capacity for LAI as compared to the pretherapy virus. One potential explanation for the recombination observed between LAI and the pretherapy virus could be PCR mediated recombination. To assess this possibility, equalamounts of DNA from the single infections of LAI and the pretherapy virus were mixed

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131 together, used to amplify the gag-pol region, and sequenced as described above. Analysis at the amino acid level revealed no evidence of recombination, while anathe nucleotide level re lysis at vealed a low level of recombination. Furthermore, the recombinant clonewas n a is a rsisted d emerged when the drug pressure was r oir. s contained only one crossover. No evidence of double or triple crossovers observed, consistent with previous reports that PCR induced recombination results ilow level of recombination with only single crossover sites [101,165]. The high level of recombination in gag and protease, particularly after 5 weeks in culture, combined with the presence of double and triple crossover events rules out the possibility that the observed recombination was due to PCR. The idea of reversion of a drug resistant HIV-1 variant to a drug-sensitive variant controversial. Many studies indicate that in HIV-1 infected patients who go throughstructured treatment interruption, the drug-sensitive virus that appears following the interruption is actually a minority population of wild-type virus that somehow peduring treatment [95,109]. These data suggest that wild-type virus, whether in an anatomical compartment unavailable to drugs, or in a latent cellular reservoir, was still present in the patient during antiretroviral therapy, an emoved. In our system, however, a PI-resistant clone was used to infect lymphocytes froman uninfected, healthy donor. This eliminates the possibility of wild-type, or less mutated forms of the virus, being present in an anatomical compartment or a cellular reservOver five weeks in culture in the absence of selective drug pressure, the alanine in position 54 of protease mutated to a valine. The amino acid found in drug-sensitive strains of the virus is typically an isoleucine. A mutation to valine is the most common

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132 change at this position following protease inhibitor treatment, although mutation to an alanine is also associated with drug resistance [224]. Given the nucleotide differences between the three amino acids, our data suggest that the alanine reverted to a valine, andgiven more time in culture would revert to isoleucine. Reversion from alanine to valine occur of the f f red concomitantly with a significant increase in p24 production, and mutationalanine at position 54 to either a valine or an isoleucine was capable of restoring replication to levels similar to the pretherapy virus, demonstrating that in the absence oprotease inhibitors, reversion at a single amino acid in protease results in increased replicative fitness. Recombination and reversion both play a role in increasing the viral diversity of HIV-1, as well as impacting the replicative fitness of the virus itself. The high level oHIV-1 variability caused by these two mechanisms impacts both the immune system as well as antiviral therapy. Increased variability of HIV-1 quasi species makes it more difficult for the immune system to control and allows for drug-resistant variants to emerge easily [40,145].

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133 500700 p24 (ng/ml) 0100300PostPostPre LAIPrePostPre-LAI-LAIp24 (ng/ml) 0100300PostPostPre 500700 LAIPrePostPre-LAI-LAIFigure 4-1. Replicative Capacity after 1 and 5 Weeks of Culture. Grey bars, Passage 1; Black bars, Passage 5.

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134 0 20406080Proportion of Virus 100LAIPrePost ClonesA. 0100LAIPrePost 20406080 0100LAIPrePost ClonesA. 20406080Proportion of Virus 020 4060100Proportion of Vis Cl 80LAI-Pre-LAI-B.ruones PrePostPost 02080LAI-Pre-LAI4060100 PrePostPost 02080LAI-Pre-LAI-B.ruones Infections. B, Virus Mixtures. Black bars: LAI; Striped bars: Pretherapy; 4060100Proportion of Vis Cl PrePostPostFigure 4-2. Proportion of Virus Clones after 5 Weeks in Culture. A, Single Virus Dotted bar: Posttherapy; White bar: Recombinants.

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135 01020304050 6070ts ProteaseGag% Recombinan 01020304050ProteaseGag% Recombinan Figure 4-3. Recombination Frequency in Protease and Gag after 1 and 5 Weeks in Culture. The proportion of clones that demonstrated recombination at passages 1 (Grey bars) and 5 (Black bars) are shown for both the Protease and Gag regions. 6070ts

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136 A.Protease[21% (5/24)]Gag[13% (4/30)]B.Protease[41% (9/22)]Gag[66% (16/24)] 60%40% 33%33%34% 64%18%18% 100%A.Protease[21% (5/24)]Protease[21% (5/24)]Gag[13% (4/30)]Gag[13% (4/30)]B.Protease[41% (9/22)]Protease[41% (9/22)]Gag[66% (16/24)]Gag[66% (16/24)] 60% 60%40% 33%33%34% 33%33%34% 64%18%18% 64%18%18% 100% 100% Figure 4-4. Proportion of Single and Multiple Crossovers in Gag and Protease. A, Recombination at passage 1; B, Recombination at passage 5. The proportion and number of recombinants present in each region are noted in brackets. Single crossovers are shown in grey, double crossovers are shown in white, and triple crossovers are in black.

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137 P Q I T L W Q R P L V T I K I G G Q L K E A L L D T G A D D CCT CAGATC ACT CTT TGG CAA CGA CCC CTC GTC ACA ATA AAG ATA GGG GGGCAACTAAAGGAA GCT CTA TTA GAT ACA GGA GCA GAT GATCCT CA GATACA GTA TTA GAA GAAATG AGT TTG CCA GGA AGA TGG AAA CCA AAA ATG ATA GGG GGA ATT GGA GGT TTT ATC AAA GTA AGA CAG TAT GAT Q Y D L TTG AATC ACT CTT TGG CAA CGA CCC CTC GTC ACA ATA AAG ATA GGG GGGCAGCTGAAAGAA GCT CTA TTA GAT ACA GGA GCA GATACA GTA TTA GAA GAAATG ACT TTG AAA GGA AGA TGG AAA CCA AAA ATG ATA GGG GGA ATT GGA GGT TTT ATC AAA GTA AGA CAG TAT GATT V L E E M SL PG R W K P K M I G G I G G F I K V RQ I LI E I C G H K A I G T V L V G P T P V N I I G R N L CAG ATA CTC ATA GAA ATC TGT GGACAT AAA GCT ATA GGT ACAGTA TTA GTA GGA CCT ACA CCT GTC AAC ATA ATT GGA AGA AAT CTGCAG GTA CCC ATA GAA ATC TGT GGGCAT AAA GCT ATA GGT ACGGTA TTA GTA GGA CCT ACA CCT GTC AAC ATA ATT GGA AGA AAT CTG TTGTTVPT Q I G C T L N F ACT CAG ATT GGT TGC ACTTTA AAT TTTACT CAG ATT GGT TGC ACCTTA AAT TTT 16 16 16 16 22 22 22 22 01 01 01 01 09 09 09 09 19 19 19 19 08 08 08 08 P Q I T L W Q R P L V T I L K E A L L D T G A D D CCT CAGATC ACT CTT TGG CAA CGA CCC CTC GTC ACA ATA AAG ATA GGG GGGCAACTAAAGGAA GCT CTA TTA GAT ACA GGA GCA GAT GATCCT CA GATP Q I T L W Q R P L V T I L K E A L L D T G A D D CCT CAGATC ACT CTT TGG CAA CGA CCC CTC GTC ACA ATA AAG ATA GGG GGGCAACTAAAGGAA GCT CTA TTA GAT ACA GGA GCA GAT GATCCT CA GATACA GTA TTA GAA GAAATG AGT TTG CCA GGA AGA TGG AAA CCA AAA ATG ATA GGG GGA ATT GGA GGT TTT ATC AAA GTA AGA CAG TAT GAT Q Y D ACA GTA TTA GAA GAAATG AGT TTG CCA GGA AGA TGG AAA CCA AAA ATG ATA GGG GGA ATT GGA GGT TTT ATC AAA GTA AGA CAG TAT GAT Q Y D L TTG K I G G Q K I G G Q AATC ACT CTT TGG CAA CGA CCC CTC GTC ACA ATA AAG ATA GGG GGGCAGCTGAAAGAA GCT CTA TTA GAT ACA GGA GCA GATAATC ACT CTT TGG CAA CGA CCC CTC GTC ACA ATA AAG ATA GGG GGGCAGCTGAAAGAA GCT CTA TTA GAT ACA GGA GCA GATACA GTA TTA GAA GAAATG ACT TTG AAA GGA AGA TGG AAA CCA AAA ATG ATA GGG GGA ATT GGA GGT TTT ATC AAA GTA AGA CAG TAT GATT V L E E M SL PG R W K P K M I G G I G G F I K V RACA GTA TTA GAA GAAATG ACT TTG AAA GGA AGA TGG AAA CCA AAA ATG ATA GGG GGA ATT GGA GGT TTT ATC AAA GTA AGA CAG TAT GATT V L E E M SL PG R W K P K M I G G I G G F I K V RQ I LI E I C G H K A I G T V L V G P T P V N I I G R N L CAG ATA CTC ATA GAA ATC TGT GGACAT AAA GCT ATA GGT ACAGTA TTA GTA GGA CCT ACA CCT GTC AAC ATA ATT GGA AGA AAT CTGCAG GTA CCC ATA GAA ATC TGT GGGCAT AAA GCT ATA GGT ACGGTA TTA GTA GGA CCT ACA CCT GTC AAC ATA ATT GGA AGA AAT CTG TTGTTTTVPVPT Q I G C T L N F ACT CAG ATT GGT TGC ACTTTA AAT TTTACT CAG ATT GGT TGC ACCTTA AAT TTTT Q I G C T L N F ACT CAG ATT GGT TGC ACTTTA AAT TTTACT CAG ATT GGT TGC ACCTTA AAT TTT 16 16 16 16 16 16 16 16 16 16 16 16 22 22 22 22 22 22 22 22 01 22 22 22 22 01 01 01 01 01 01 01 01 01 01 01 09 09 09 09 09 09 09 09 09 09 09 09 19 19 19 19 19 19 19 19 19 19 19 19 08 08 08 08 08 08 08 08 08 Figure 4-5. Protease Recombinant Clones after 5 weeks in culture. The amino acid sequence of protease is located on the top line, the nucleic acid sequence of LAI on the middle line, and the nucleic acid sequence of the pretherapy virus on the third line, with differences between the two noted in color (blue for LAI and red for pretherapy). Regions of PR that were identical between LAI and pretherapy are shown in grey, and regions at which a crossover has occurred are denoted with grey stripes.

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138 A E A M S Q VT N S A TI M M Q RG N F R N Q R K I V K C FGCT GAGGCA ATG AGC CAA GCA ACA AAT TCGCAGATC ATA ATG ATGCAGAAA GGC AAT TTT AGAAAC CAA AGA AAG ATT GTT AAG TGT TTC GCT GAAGCA ATG AGC CAA GTA ACA AAT TCAGCTACC ATA ATG ATGCAAAGA GGC AAT TTT AGGAAC CAA AGA AAG ATT GTT AAG TGT TTC 3AQIK 9 19 22 8 05E P T A P P F L QS R P E P T A P P E E S F R SG VE T T TGAG CCA ACA GCC CCA CCATTT CTT CAGAGC AGA CCA GAG CCA ACA GCC CCA CCAGAAGAG AGC TTC AGG TCT GGG GTAGAG AGAG ... ... ... ... ... ... ... ...AGC AGA CCA GAG CCA ACA GCC CCA CCAGAGGAGAGC TTC AGG TTT GGG GAGGAGACA ACA CA ACAACT ACT 3 FE 9 19 22 8 05P S Q K Q E P I D K E L Y P L TS L RS L F G N D P S S QCCC TCCCC TC T CAG AAG CAG GAG CCG ATA GAC AAG GAA CTG TAT CCTTTA ACT TCC CTC AGA TCA CTC TTT GGC AAC GAC CCC TCG TCA CAAT CAG AAG CAG GAG CCG ATA GAC AAG GAA CTG TAT CCCTTA GCT TCC CTC AAA TCA CTC TTT GGC AAC GAC CCC TCG TCA CAA 3 AK 9 19 22 8 05 KN C G K E G H I A RN C R A P R K K G C W K C G K E G H Q MAAT TGT GGC AAA GAGGGG CAC ATA GCC AAA AAT TGC AGG GCC CCT AGAAAA AAG GGC TGT TGG AAA TGT GGA AAG GAA GGA AAT TGT GGC AAA GAAGGG CAC ATA GCC AGA AAT TGC AGG GCC CCT AGGAAA AAG GGC TGT TGG AAA TGT GGA AAG GAA GGA CAC CAA ATG CAC CAA ATG 3 9 19 22 A E A M S Q VT N S A TI M M Q RG N F R N Q R K I V K C FGCT GAGGCA ATG AGC CAA GCA ACA AAT TCGCAGATC ATA ATG ATGCAGAAA GGC AAT TTT AGAAAC CAA AGA AAG ATT GTT AAG TGT TTC GCT GAAGCA ATG AGC CAA GTA ACA AAT TCAGCTACC ATA ATG ATGCAAAGA GGC AAT TTT AGGAAC CAA AGA AAG ATT GTT AAG TGT TTC 3 3AQIK 9 9 19 19 22 22 8 8 8 05K D C T E R Q A N F L G K I W P S Y K G R P G N F L Q S R PAAA GAT TGT ACT GAG AGA CAG GCT AAT TTT TTA GGG AAG ATC TGG CCT TCC TAC AAG GGGAGG CCA GGG AAT TTT CTT CAG AGC AK AAA GAT TGT ACT GAG AGA CAG GCT AAT TTT TTA GGG AAG ATC TGG CCT TCC TAC AAG GGAAGG CCA GGG AAT TTT CTT CAG AGC AGA CCA AA CCA 3 9 19 22 8 05 05 05E P T A P P F L QS R P E P T A P P E E S F R SG VE T T TGAG CCA ACA GCC CCA CCATTT CTT CAGAGC AGA CCA GAG CCA ACA GCC CCA CCAGAAGAG AGC TTC AGG TCT GGG GTAGAG AGAG ... ... ... ... ... ... ... ...AGC AGA CCA GAG CCA ACA GCC CCA CCAGAGGAGAGC TTC AGG TTT GGG GAGGAGACA ACA CA ACAACT ACT 3 KN C G K E G H I A RN C R A P R K K G C W K C G K E G H Q MAAT TGT GGC AAA GAGGGG CAC ATA GCC AAA AAT TGC AGG GCC CCT AGAAAA AAG GGC TGT TGG AAA TGT GGA AAG GAA GGA AAT TGT GGC AAA GAAGGG CAC ATA GCC AGA AAT TGC AGG GCC CCT AGGAAA AAG GGC TGT TGG AAA TGT GGA AAG GAA GGA CAC CAA ATG CAC CAA ATG 3 9 19 22 8 05KN C G K E G H I A RN C R A P R K K G C W K C G K E G H Q MAAT TGT GGC AAA GAGGGG CAC ATA GCC AAA AAT TGC AGG GCC CCT AGAAAA AAG GGC TGT TGG AAA TGT GGA AAG GAA GGA AAT TGT GGC AAA GAAGGG CAC ATA GCC AGA AAT TGC AGG GCC CCT AGGAAA AAG GGC TGT TGG AAA TGT GGA AAG GAA GGA CAC CAA ATG CAC CAA ATG 3 9 19 22 8 05N C G K E G H I A RN C R A P R K K G C W K C G K E G H Q MAAT TGT GGC AAA GAGGGG CAC ATA GCC AAA AAT TGC AGG GCC CCT AGAAAA AAG GGC TGT TGG AAA TGT GGA AAG GAA GGA AAT TGT GGC AAA GAAGGG CAC ATA GCC AGA AAT TGC AGG GCC CCT AGGAAA AAG GGC TGT TGG AAA TGT GGA AAG GAA GGA CAC CAA ATG CAC CAA ATG 3 3 9 9 19 19 22 22 8 8 FE 9 19 22 8 05CA ACAACT ACT E P T A P P F L QS R P E P T A P P E E S F R SG VE T T TGAG CCA ACA GCC CCA CCATTT CTT CAGAGC AGA CCA GAG CCA ACA GCC CCA CCAGAAGAG AGC TTC AGG TCT GGG GTAGAG AGAG ... ... ... ... ... ... ... ...AGC AGA CCA GAG CCA ACA GCC CCA CCAGAGGAGAGC TTC AGG TTT GGG GAGGAGACA ACA 3 3 05 05K D C T E R Q A N F L G K I W P S Y K G R P G N F L Q S R PAAA GAT TGT ACT GAG AGA CAG GCT AAT TTT TTA GGG AAG ATC TGG CCT TCC TAC AAG GGGAGG CCA GGG AAT TTT CTT CAG AGC AK AAA GAT TGT ACT GAG AGA CAG GCT AAT TTT TTA GGG AAG ATC TGG CCT TCC TAC AAG GGAAGG CCA GGG AAT TTT CTT CAG AGC AGA CCA AA CCA 3 9 19 22 8 05 K D C T E R Q A N F L G K I W P S Y K G R P G N F L Q S R PAAA GAT TGT ACT GAG AGA CAG GCT AAT TTT TTA GGG AAG ATC TGG CCT TCC TAC AAG GGGAGG CCA GGG AAT TTT CTT CAG AGC AK AAA GAT TGT ACT GAG AGA CAG GCT AAT TTT TTA GGG AAG ATC TGG CCT TCC TAC AAG GGAAGG CCA GGG AAT TTT CTT CAG AGC AGA CCA AA CCA 3 3 9 9 19 19 22 22 8 8 FE 9 9 19 19 22 22 8 8 05 05P S Q K Q E P I D K E L Y P L TS L RS L F G N D P S S QCCC TCCCC TC T CAG AAG CAG GAG CCG ATA GAC AAG GAA CTG TAT CCTTTA ACT TCC CTC AGA TCA CTC TTT GGC AAC GAC CCC TCG TCA CAAT CAG AAG CAG GAG CCG ATA GAC AAG GAA CTG TAT CCCTTA GCT TCC CTC AAA TCA CTC TTT GGC AAC GAC CCC TCG TCA CAA 3 AK 9 19 22 8 05T CAG AAG CAG GAG CCG ATA GAC AAG GAA CTG TAT CCTTTA ACT TCC CTC AGA TCA CTC TTT GGC AAC GAC CCC TCG TCA CAAT CAG AAG CAG GAG CCG ATA GAC AAG GAA CTG TAT CCCTTA GCT TCC CTC AAA TCA CTC TTT GGC AAC GAC CCC TCG TCA CAA P S Q K Q E P I D K E L Y P L TS L RS L F G N D P S S QCCC TCCCC TC 3 3 AK 9 9 19 19 22 22 8 8 05 05 05 05 uence d Figure 4-6. Gag Recombinant Clones after 5 weeks in Culture. The amino acid seqof protease is located on the top line, the nucleic acid sequence of LAI on themiddle line, and the nucleic acid sequence of the pretherapy virus on the thirdline, with differences between the two noted in color (blue for LAI and red for pretherapy). Regions of Gag that were identical between LAI and pretherapy are shown in grey, and regions at which a crossover has occurred are denotewith grey stripes.

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139 ATC IVA GTCGCC D.1 0100150250350 50 200300 5p24 (l)156012060120 Passage NumberPassage Number02040801000204080100 pg/mB. Proportion o Virus lone fCsC.Pre PQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMTLTGRWKPKMIGGIGGFIKVRQYDQVPIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF --Post ---------i----v------------------q-in----------------a---e-----------------------a--------------*54*63*82*36*10A. ATCGTCGCC IVA D. ATCGTCGCC IVA ATCATCGTCGTCGCCGCC IIVVAA D.1 0100150250350 50 200300 5p24 (l)1 Passage Number pg/mB. 0100150250350 50 200300 5p24 (l)1 Passage Number pg/m 0100150250350 50 200300 5p24 (l)156012060120 Passage NumberPassage Number02040801000204080100 pg/mB. Proportion o Virus lonePassage Number02040801000204080100 fCsC.156012060120 Proportion o Virus lonePassage Number0204080100020408010002040801000204080100 fCs1560120601206012060120 Proportion o Virus lone protease alignment of the preand posttherapy viruses. Amino acid positions B, supernatant p24 production at passages 1 and 5. C, proportion of virus amino acid position 54 in PR after 1 and 5 weeks in culture. D, amino acids fCsC.Pre PQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMTLTGRWKPKMIGGIGGFIKVRQYDQVPIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF ---Pre PQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMTLTGRWKPKMIGGIGGFIKVRQYDQVPIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF ---Pre PQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMTLTGRWKPKMIGGIGGFIKVRQYDQVPIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF --Figure 4-7. Replication and Sequences of the Posttherapy Virus at Passages 1 and 5. A, with an asterisk are associated with reduced sensitivity to protease inhibitors. clones that contained either an alanine (black bars) or a valine (grey bars) at (top line) and nucleotides (bottom line) that are found at position 54 in PR. Post ---------i----v------------------q-in----------------a---e-----------------------a--------------*54*63*82*36*10A.Post ---------i----v------------------q-in----------------a---e-----------------------a--------------*54*63*82*36*10Post ---------i----v------------------q-in----------------a---e-----------------------a--------------*54*54*63*63*82*82*36*36*10*10A.

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140 0400ng/ 200246810Daysp24 (ml 600 600 ) 0400ng/ Figure 4-8. Replication of A54I and A54V Mutants. The pretherapy virus is shown in purple diamonds, the posttherapy virus in green squares, the A54I mutant in yellow circles, and the A54V mutant in red triangles. 200246810Daysp24 (ml )

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CHAPTER 5 ]. ing antiretroviral therapy has been demop6 the virus. For example, the P(T/S)AP motif in p6 recruits the cellular factors TSG101 DISCUSSION HIV-1 fitness is a balance between viral replication and resistance to inhibitors. Several cellular and viral factors influence the fitness of HIV-1. The genotype of thevirus defines its basic replicative capacity. The activity of the viral enzymes protease and reverse transcriptase and the ability of the virus to mature and form fully infectious virions impact replicative capacity, while the availability of target cells affects entry of the virus. The host immune system and the selective pressure of antiretroviral therapy then add additional pressures that select for the most fit virus. Many studies have examined the impact of mutations in protease and reverse transcriptase on replicative capacity and resistance to inhibitors [10,23,50,51,64,126,139,170,192,217,223,261,280 The accumulation of mutations in gag follow nstrated, but few studies examined the impact of those mutations on replicative fitness or drug sensitivity [51,64,85,156,278,280]. Genotypic studies on therapy-nave subjects prior to antiretroviral treatment identified novel positions in gag-pol that could predict viral and immune outcome. Positions were not solely located in the cleavage sites, but were also found in p7 NC , p6, and p6 Pol . Several polymorphisms, including the presence of particular insertions in and p6 Pol , were found only in viral failure subjects, indicating a dominant impact on viral replication. The proteins within Gag-Pol are crucial to the viral life cycle, and it is not surprising to find a relationship between polymorphisms in this region and the fitness of 141

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142 and AIP1, which are involved in budding, and p7 NC binds viral RNA and promotes Gamultimerization. Variation in any of these proteins could significantl g y alter viral replicative capacity. , while others have identiin f g-Pol, n only treated subjects, it is possible that optimal Gag-Pol organization may be absence of inhibitors. ation vels Multiple groups have identified covariation in protease fied positions in the cleavage sites which appear in concert with mutations in protease [8,100,125,131,154,243,268]. Since Gag and Pol are connected as a polyproteprior to protease activity, it is probable that the cleavage sites are not the only regions oGag that interact with protease. This study examined covariation within all of Gaand identified novel relationships within regions of Gag, outside the cleavage sites, as well as between Gag and Pol. Covariation of residues within Gag-Pol suggests that analteration in one region could cause a conformational change elsewhere that would modulate interactions between the active site of protease and its substrate. These data support a model of Gag-Pol organization that is necessary for protease activity. Furthermore, given that some positions covary only in untreated subjects, and some covary i different in the presence and Multiple lines of evidence demonstrate that amino acid residue 54 in protease is an important modulator of fitness. The posttherapy variant, which contains an alanine at position 54, replicates to significantly lower levels than the pretherapy variant. Mutof amino acid 54 alone to either the valine present in the pretherapy variant or the isoleucine present in wild-type variants was capable of restoring replication to lesimilar to the pretherapy virus. Furthermore, mutation of gag residues in the C cleavagesite and p7 NC from amino acids that were present in the posttherapy PI-resistant virus to

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143 amino se protease, and mutations at this position could bility of the fl the lter sttherapy acids present in the pretherapy PI-sensitive virus had little effect on increasing viral replication. Additional reversion of positions 54 and 58, however, was sufficient to fully restore the reduced replication of the posttherapy virus, demonstrating a dominant impact on replicative capacity. An impact on drug sensitivity is also evident, particularly in the presence of ritonavir. Reversion of positions 54 and 58, in combination with the reversion of positions in the C site and p7 NC , caused a greater than seven-fold decrease in the IC 50 value for ritonavir (compared to the posttherapy virus), suggesting that PI-associated mutations at position 54 that accumulate in subjects receiving ritonavir would increase resistance to RTV. In support of this, a study by Clemente et al. demonstrated that I54Vcauses a decrease in inhibitor binding [38]. The location of position 54 in proteasuggests a mechanism by which replication and drug sensitivity can be altered. Amino acid 54 is located near the tip of the flap of have profound effects on the opening and closing of the flaps, as well as the sta aps in either the open or closed conformation. Efficient opening and closing of the flaps is crucial to viral replication as substrates need to gain access to the active site for cleavage by protease. Changes in the opening and closing of the flaps could also alterbinding efficiency of protease inhibitors, effectively modulating drug sensitivity. The importance of an amino acid located outside the active site underscores the significant role of non-active site mutations in fitness. Not only can these positions areplication, but our study and others also demonstrate the significant role non-active site mutations play in resistance to protease inhibitors [38,170,179]. Reversion of positions in the hinge (34, 36, and 37) and flap (54 and 58) from those present in the po

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144 resistant virus to those present in the pretherapy sensitive virus, resulted in a virus that was spact in data, in combination with data that demonstrates the impact of C site anthat onserved pathway, while the tance is not so ordered [43,44,168]. In our study, subject D1 receiv ignificantly more sensitive to both indinavir and ritonavir, and in the case of indinavir, the IC 50 of the mutant virus was identical to the pretherapy virus. In addition to demonstrating that non-active site mutations in protease can impact fitness, studies in this dissertation have identified novel positions in gag that modulate fitness. Mutation of amino acids in the C cleavage site and p7 NC had a significant imon sensitivity to both indinavir and ritonavir. Reversion of either site alone resulteddecreases in IC 50 values that ranged from 2-fold to 86-fold compared to the posttherapy resistant virus. These d p7 NC mutations on both protease processing and viral replication, support our model of Gag-Pol organization and indicate the necessity for design of new therapies target interactions between Gag and Pol [87]. Interestingly, all of the viruses tested were more resistant to ritonavir than to indinavir. A similar trend was seen in a study by Mammano et al. which found that mutations associated with ritonavir and indinavir therapy conferred a greater advantage toviruses in the presence of ritonavir as compared to indinavir [157]. Although most of the mutations that accumulate in response to one drug also occur in response to the other, resistance to ritonavir has been shown to follow a fairly c pathway to indinavir resis ed ritonavir therapy for 77 weeks, but only 16 weeks of indinavir therapy. It is possible that given more time on indinavir, resistance could increase. An alternative explanation is that in CXCR4 CD4 + T lymphocytes, there is a difference in the

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145 intracellular levels of ritonavir and indinavir which could account for the observedifferences in IC d ral diversity, recombination in gag and proontribute to combining positions whichave [55,56 no rstanding 50 values. Serial passaging of single or dual infections reveals two additional modulators of fitness: recombination and reversion. Significant, high levels of recombination were found in both gag and protease. The frequency and extent of recombination increasedover time, indicating a fitness advantage for recombinants. In addition to increasing vi tease could c confer either a replicative advantage or an increase in drug resistance. The significant impact recombination has on creating viral diversity emphasizes the need for new antiretroviral therapies. Passage of a drug-resistant virus in the absence of inhibitors resulted in the reversion of amino acid position 54 in protease associated with a large increase in viral replication, and a similar study by Bleiber et al. demonstrated reversion in reverse transcriptase that also correlated with increased replication [19]. Several studies hindicated that wild-type virus that emerges following treatment interruption is due to an outgrowth of a more sensitive minority population of virus, rather than true reversion ,95]. However, our studies involved infection with a molecular clone, and somixture of virus was present, demonstrating that reversion does occur and impacts viral replication. The work in this dissertation clearly demonstrates the dominant impact of mutations in gag-pol on both replication and protease inhibitor resistance. Undethe complex connections between Gag and Pol will significantly aid in discovering a new

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146 class of drugs that target interactions between protease and its gag substrates without the emergence of resistance.

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APPENDIX A ENTIAL IMPACT OF PROTEASE INHIBITORS ON HIV-1 FITNESS IN CD4 DIFFER + T LYMPHOCYTE SUBSETS

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Introduction The efficacy of protease inhibitors and subsequent therapeutic outcome are commonly associated, in part, with drug concentrations in the plasma. However, the major target of protease inhibitors is within infected cells. While few studies demonstrated a correlation between antiviral activity and intracellular PI concentration, limited information is known about the clinical relevance of intracellular drug concentrations [15,171]. Several factors, such as oral bioavailability, physiochemical properties, plasma and cellular protein binding, and influx and efflux transport affect intracellular protease inhibitor levels [74]. Most PIs, with the exception of indinavir, are lipophilic, and can therefore penetrate the phospholipid bilayers of cell membranes [124]. PIs are substrates for transport proteins, such as P-glycoprotein (P-gp) [129,141]. P-gp is plasma membrane protein encoded by the MDR1 gene that acts to lower the intracellulaDifferential expression of P-gp on various lineages of lymphocytes and modulation of P-gp function by protease inhibitors both potentially alter intracellular PI concentrations [130,205,226]. Although determination of intracellular PI levels is highly dependent on experimental conditions, several studies examined the relative intracellular accumulation of PIs both in vitro and in vivo, and found a rank order of accumulation: NFV > SQV > RTV > IDV [73,117,128]. Previous studies examined the activity of antiretroviral drugs in different cell types. Aquaro et al. demonstrated that NRTIs have greater activity in macrophages than in lymphocytes, most likely due to the differential dNTP levels in the two types of cells [6]. Since NNRTIs are not dependent on intracellular dNTP levels, NNRTIs were equally effective in both macrophages and lymphocytes. In contrast, protease inhibitors (PI) a r concentration of a broad range of potentially toxic substrates [68]. 148

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149 were found to be more active in lymphocytes than in macrophages, meaning that more PI was needed to inhibit replication in macrophages. The results of this study suggest that there may be certain cell types in which antiretroviral drugs have lower antiviral activity, and as a result could allow continued replication of HIV-1, leading to the emergence of resistant viruses. Importantly, this study only examined the differences in drug sensitivities between cells that express the coreceptor CCR5. Cells that express CXCR4 are also significant targets for HIV-1 infection. While, both X4 and R5 viruses can be transmitted, R5 viruses tend to predominate during early infection [47,260,284]. A switch from R5 to X4 occurs later in infection and correlates with accelerated disease progression [45,136,216,222,247]. This study was designed to examine the differential impact of protease inhibitors on subsets of peripheral blood mononuclear cells. Methods Replherapy gag-pol alleles in an HIVLAI backbone were constructed as described in Chapter 3. HIVLAI is a CXCR4 utilizing virus, and to be able to target viruses to PBMC that express CCR5, it was necessary to alter the envelope region of the X4 recombinant viruses. The R5 recombinant viruses were designed and made as described previously [219]. Briefly, a shuttle vector system was employed to insert a 1.3 kb fragment encompassing the V1-V5 envelope region of HIVJRFL into the HIVLAI backbone. The viral constructs were then transfected into 293 cells and titered on PBMC as described in Chapter 3. Replication of R5 Recombinant Viruses The replicative capacity of the R5 recombinant viruses was tested in PBMC isolated fro Construction of R5 Recombinant Viruses ication competent recombinant viruses containing preand postt m healthy donors. Three to four days prior to infection, the PBMC were

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150 stimulated TCID50 of virus was used to infect 1.2 x 10 cells for 2 hours. The cells were then rinsed with 10 ml of 1 X PBS (Gibco), resuspended in PBMC media, and plated in a 96 well round bottom tissue culture plate at 200,000 cells per well. The cultures were maintained for 10 days, with a 10% media change and supernatant collection occurring every 2 days. The supernatant was assayed for p24 production with an HIV-1 p24 ELISA (Beckman Coulter). R5 Competition Experiments R5 competition experiments consisted o 5 one-week passages, beginning with infection on day 0. PBMC were stimulated with PHA (Sigma) three days prior to infection as described in the materials and methods section of Chapter 3. On the day of infection, 2 x 106 cells were infected with a total of 8000 TCID50 of virus. PBMC were infected with either a single virus at 8000 TCID50, or two viruses, each at 4000 TCID50 for 2 hours, with gentle agitation every 20-30 minutes. The cells were then rinsed by addition of 10 ml of 1 X PBS (Gibco), centrifuged at 1000 RPM for 10 minutes, and resuspended in 2 ml PBMC media. One milliliter of the cell suspension was placed in 4 post-infe was added back, and incubated for a further 3 days. Seven days post-infection, 250 l of cell suspension was removed, and 5 x 105 fresh PHA stimulated PBMC from the same donor were added back to the culture, which was further incubated at 37C, 5% CO2. Twenty microliters of the removed cell suspension was used to count the cells using dye exclusion with Trypan Blue (StemCell Technologies, Vancouver, BC, Canada), and the rest was placed in a screw top tube and lysed for sequencing as described below. A small with PHA (Sigma) as described in Chapter 3. On the day of infection, 1500 6 f each of duplicate wells in a 24 well culture plate and incubated at 37C, 5% CO 2 . On dayction, 500 l of supernatant was carefully removed, and 500 l of fresh media

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151 amount of supernatant (200 l) was used to determine p24 antigen levels by ELISA (Beckman Coulter). Sequencing of Cell-Associated DNA The cells from day 7 of the first passage were rinsed twice with 1 ml of 1 X PBS (Gibco) and spun at 12,000 RPM for 4 minutes. Following the second wash, the cells were lysed in the appropriate amount of 20 mg/ml Proteinase K (Fisher Scientific, Pittsburgh, PA) and K buffer (50 mM KCl, 10 mM Tris/Cl pH 8.3, 2.5 mM MgCl2, 0.1 mg/ml gelatin, 0.45% Nonidet P40, 0.45% Tween 20) (5 l of Proteinase K per 1 ml of K buffer, and 1 ml K buffer for every 5 x 106 cells), and incubated overnight at 37C. The following day, the Proteinase K was heat inactivated at 95C for 5 minutes. To prepare for sequencing, the DNA was cleaned up with the QiaAmp DNA Blood Mini Kit sequenced Sequence Analysis ag (p2 through p6) and PR sequences were aligned at both the nucleotide and Carlsbad, Cin Gag and PR was determined by using nucleotide differences that exist between two viruses. In protease, there are 4 amino acid differences and 11 nucleotide differences between LAI and the pretherapy virus; 11 amino acid and 20 nucleotide differences between LAI and the posttherapy virus; and 8 amino acid and 14 nucleotide differences between the preand posttherapy viruses. In gag, there are 10 amino acid differences and 24 nucleotide differences between LAI and the pretherapy virus; 15 amino acid and 29 nucleotide differences between LAI and the posttherapy virus; and 9 amino acid and 15 (Qiagen), and eluted in 100 l of dH 2 0. The gag-PR region was amplified, cloned and as described in the high-throughput sequencing section of Chapter 2. G amino acid level using the BioEdit Sequence Alignment Editor (Ibis Therapeutics, A) and analysis of recombination was performed. The number of crossovers

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152 nucleotide differences between the preand posttherapy viruses. In addition, LAI contains an 8 amino acid insertion that is not present in either the preor posttherapy virus. Determination of IC50 Values for R5 Recombinant Viruses The determination of IC50 values for the R5 recombinant viruses in the presence of ritonavir and indinavir (NIH AIDS Research and Reference Reagent Program) were carried out as described for the X4 viruses in Chapter 3. Briefly, PBMC were stimulated and infected as usual, and a range of drug concentrations (10 nM, 102 nM, 103 nM, 104 nM, and 105 nM) were added to the cultures following the 2 hour infection time. DMSO entire 10 dre determined by ELISA and the IC50 values were calculated with a nonlinear regression analysis using the GraphPad Prism 4.03 software package (GraphPad Software, Inc.). Determination of Intracellular PI Levels PBMC were isolated and stimulated with PHA as described previously. Three days post-stimulation, 15 x 106 cells were incubated with 103, 104, and 105 nM of the protease inhibitors indinavir, ritonavir, and nelfinavir in duplicate in T-25 flasks at 37C, 5% CO2. At 3, 18, and 48 hours post-treatment, 5 x 106 cells were removed from each flask for drug extraction. The cells were washed 3 times in 5 ml of ice cold 1 X PBS and spun at 700 x g for 6 minutes at 4C. Following the third wash, the cells were counted and extracted in 60% methanol overnight at 4C rotating, with a final concentration of 1 x 106 c/ml. The following day, the samples were spun at 700 x g for 6 minutes at 4C, and the supernatant fraction containing the intracellular drug, was transferred to a 1.8 ml cryovial (Sigma) and water controls were also run. Drug was maintained in the culture for the ays, and supernatants were collected every 2 days. p24 antigen levels we

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153 and stored at -20C until shipped to collaborators for mass spectroscopy analysis. To minimize drug loss, all steps were performed within an hour at 4C. Results Parallel Infections in the Absence of Protease Inhibitors The replicative capacity of the preand posttherapy R5 viruses was examined in six independent donors over 8 days in culture in the absence of protease inhibitors (Figure A-1). In R5 expressing PBMC, both viruses produce about a log fold less p24 than in PBMC that express X4. In contrast to what is seen in X4 PBMC, the pretherapy drug-sensitive virus replicated to similar levels as the posttherapy drug-resistant virus. Differences between the replication of LAI and the preand posttherapy viruses were tested on days 2, 4, 6, and 8 with one way ANOVAs using SigmaStat 3.0 software replicate toproduced ay 6, and P = 0.066 day 8). Competition Experiments competitioes. lture, LAI and the pretherapy virus single infections produced similar levels of p24, and the posttherapy virus produced about 3-fold less (Figure A-2). The LAI-pretherapy competition produced levels of p24 similar to the LAI and pretherapy single infections, while the LAI-posttherapy and the pretherapy-posttherapy competitions both produced p24 amounts more similar to the posttherapy single infection. (Jandel Scientific Corp, San Rafael, CA). Although the posttherapy virus appeared to lower levels, there was no statistical difference in the amount of p24 t each time (P = 0.907 day 2, P = 0.741 day 4, P = 0.240 da Since the R5 viruses replicated to equivalent levels in parallel infections, n experiments were set up to asses the comparative fitness of the R5 virus After one week in cu

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154 Cell-associated sequencing after the first passage revealed that in protease all single infection controls contained 100% of the appropriate virus (Figure A-3A). In the mixed infections, the pretherapy virus was more fit,mpeting LAI after one week (Figure A-3B). A 50rapy competition, confirming analogous replicative capacities. In the LAI-posttherapy competition, 54% (8/15) of the clones were posttherapy, 33% (5/15) were LAI, and 13% (2/15) were recombinants, each containing only one crossover. A similar situation was seen in gag, as all single infections contained 100% of the proper virus after one week (Figure A-4A). Nine of 10 clones in the LAI-pretherapy mixture were pretherapy, none were LAI, and one was a recombinant with one crossover (Figure A-4B). The pretherapy-posttherapy mixture resulted in 53% (8/15) pretherapy and 47% (7/15) posttherapy, and the LAI-posttherapy competition resulted in 40% (6/15) LAI and 60% (9/15) posttherapy, again confirming comparable fitness. Replication of R5 Recombinant Viruses in the Presence of RTV and IDV The replication of the R5 recombinant viruses (LAI, pretherapy, and posttherapy) in the presence of indinavir and ritonavir was tested in three independent donors. In the presence of indinavir, replication of both LAI and the pretherapy virus was inhibited between 103 nM and 104 nM (Figures A-5A and A-5B). Replication of the posttherapy virus was not completely inhibited, even in the presence of 105 nM (Figure A-5C). In the presence of ritonavir, replication of both LAI and the pretherapy virus was inhibited between 104 nM and 105 nM (Figures A-6A and A-6B). In contrast, the posttherapy virus replicates to relatively high levels even in the presence of 105 nM of ritonavir. All three viruses appear to be more sensitive to indinavir than ritonavir in R5 PBMC. entirely out co /50 mixture resulted from the pretherapy-postthe

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155 Determination of IC50 Values for R5 Recombinant Viruses For e noir ere calculated for all three viruses on day 8. The m curves are shown in Figure A-7. In the presence 50 of 140 nM, which is 207-fold lower than the IC t posttherapy R5 ritonav50 of 2,300 nM, 50 of the 5 nM, a A com ore resistant t than its X4 ore resistant than the X4 lly sensitive, with IC itonavir than the X4 A pilot expe f indinavir, ritonavir 4 nM of indinavir, old increase in increasing slightly ac p r PI Levels ir in non h of th re e do ent was set up to test the in a ent with 10 p rs, indinav an ttherapy virus has a value of 2.1 x 10 shown in Figure A-8. Surprisingly, in the e posttherapy R5 viruses were m the X4 and R5 pretherapy viruses were found g/ m d r t (41 hen treated with 10 l ri esen s over 200-fold m ) and consistent at 3, 18, and 48 hours ton ir, LAI-R5 has an IC trac a ce of indinavir. The IC m fo d in a 30-60 f vir o ore resis ld). ellular levels o I C 5 0 values w the resistan o ean IC 50 of indinavir, the IC an IC virus (2.9 x 10 which is 10-fold higher than the IC pretherapy virus is 900 nM, while the pos 233-fold difference. the presence of indinavir and ritonavir are presence of indinavir, both the preand th than the X4 viruses. Th counterpart, while the posttherapy R5 virus wa posttherapy virus. In the case of ritonavir, to be equa case with indinavir, the R5 pos posttherapy virus, although to a Intracellula and nelfinav intracellular levels rem (Figure A-9). Treatm intracellular levels com 50rim value of LAI-R5 is 230 nM. The sensitive pretherapy R5 virus has -sep 50ld m of 4arison of the IC nM). In the pres e preth arated ined low (< 1 ar ence of 50 in the p R5 virus was 5 f apy virus was r exten 0 50 values for the preand posttherapy X4 and R5 viruses in erapy re resistan tant to r 50tther le PBMC. W values of 870 nM and 900 nM, respectively. As was the sse nM IDV resulte 3 or 10 n ent with 10 5 ed to treatm 3 nM, with levels

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156 over 48 hours. Low intracellula r levels of ritonavir that declined over time were observed when the PBMC were treated with 104 nM RTV (Figure A-10A). When trea ed over 300-fold and a large spike at 103, 104, or 105 nM of nelfinavir er IDV or RTV (Figure e over time. In contrast to what was seen in PBMC ixtures ixt ure of viruses at the end of t pass s l, so analysis after more bination was observed in both protease and ilar to the competition ent in X4 PBMC. Analysis o allow a comparison of BMC and R5 PBMC. Replica resence of both re more sensitive avir than ritonav pared, the IC50 values for the s demonstrated the e inhibitor-containing lication in R5 PBMC 3 or 10 e p s in fitness. In addition, a low level of s was tested in the p ltip ted with 10 e in cultu an X 5 nM RTV, intracellular levels increas ilar to rito tion of C 18 hours was observed (Figure Aproduced higher levels of in A-11), but sim replicated to levels sim Results from the com involving the posttherapy vi the firs tim recom experim the frequency and extent of recom indinavir and ritonavir. As to indin posttherapy viruses in the two subset R5 viruses were highe preferential express HAART, and our finding that m th 1 llu were carried b b re drug is re in 0 la ac tial change ination inant viruse th B) r drug com ellu e f cl s of PBMC were com is . T la Discussion nts conf ses. Mu tr reatm that express CXCR4, the posttherapy virus that occurs in X4 P ng initiation of proteas sient suppression of X4 viruses [69,201,229]. nt ared to eith e ogag with d equivalent fitness, as all m r 5 weeks tota le group onstrated a decreas rus in CCR5 expressing PBMC. , sim ree viruses we trace rus contained a close to 50/50 m was true in X4 PBMC, all th ex navir, intr r as LAI and the pretherapy vi ir. Unexpectedly, when the IC f X4 viruses followi lp r levels dem ones after 5 weeks will an ila etition experim r than the X4 viru he p the R5 recom ion o ld irm out f quired to inhibit rep age. The infection re will reveal any poten M 50 values of the preand o pla 4 P B c ou

PAGE 171

157 One potential explanation for the observed differences in IC50 values between X4 and R5 essing t th ls of thegs ar the two cell types. The data from ability to determine intraceels, tudies regarding the hierar ulatio te PBMC into X4 and R5 populations and ine inIch subs et to elucidifferences between the two. xpre PB n. The next step is to separa trac M C ellu i ent with prev s tha pilot study verified our lar P e in tr ious s ls in ea acellular leve dru ate d e d chy of intracellular if fe re nt in llular PI lev and was in agreem accum determ leve

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158 246 8 05101520 25 p24 (ng/ml) Days 246 8 05101520 25 p24 (ng/ml) riants in R5 PBMC. In s (purple boxes) and ilar levels of p24 ean and SEM of 6 Figure A-1. Replication Kine tics of Daysis data represents m Pre-and Posttherapy Va parallel cultures, LAI (black circles)the posttherapy virus (grey trianglesproduction over 8 days in culture. Tindependent infections. , the pretherapy viru) displayed simh

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p24 (ng/ml) 159 0 2468LAI-PostPre-Post LAI Pre Pos t LAI-Pre 02468LAI-PostPre-Post petitions after 1 Week LAI p24 (ng/ml)Figure A-2. Replica tion of Single Virus Controls Culture in R5 PBMC. Pre Pos t and Virus Com LAI-Pre

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160 0204060800 10 LA I Pre Pos t Proportion of Virus Clones A. 0204060800 10 LA I LA I Pre Pos t Proportion of Virus Clones A. 020406080 100 LAI-Pre Pre-Post LAPost IProportion of Virus Clones B. 020406080 100 LAI-Pre Pre-Post LAPost IProportion of Virus Clones 020406080 100 LAI-Pre LAI-Pre Pre-Post Pre-Post LAPost ILAPostrs: L I-A Proportion of Virus Clones B.ars: Figure A-3. Proportion of Prot ease Virus Clones after 1 W A, Single Virus InfectionPretherapy; Grey bars: P e ek in Culture in R5 PBMC. s. B, Virus Mixtures. Black baostther apy; W h ite bar: Recom b inants. I; Purple b

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161 0204060800 10 LA I Pre Pos t Proportion of Virus Clones A. 0204060800 10 LA I LA I Pre Pos t Proportion of Virus Clones A. LAIPre Pre-Pos t LAI-Pos t 020406080 100 Proportion of Virus Clones B. Proportion of Virus Cloneset LAIPre Pre-Pos t LAI-Pos t LAIPre LAIPre Pre-Pos t Pre-Pos t LAI-Pos t LAI-Posa t 020406080; G 100py Figure A-4. Proportion of Gag B. Virus Clones after 1 War Single Virus Infections. B, Virus MixtPr he ra r ey b s: Po st th er ap y; eek in Culture in R5 PBMC. A, b ures. Black bars: LAI; Purple bars: h W ite ar : R e co mb in nts .

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162 Figure A-5. Replication of R5 05101520 24 6 8 1 0 Days p24 (ng/ml) A. 05101520 24 6 8 1 0 Days p24 (ng/ml) A. 05101520 24 6 8 1 0 Da ys p24 (ng/ml) B. 05101520 24 6 8 1 0 Da ys p24 (ng/ml) B. 05101520 24 6 8 1 0 Days p24 (ng/ml)C. 0510152005101520 24 24 66 88 11 00 DaysDays p24 (ng/ml)C.p24 (ng/ml)C. Recombinant Viruses in th diamrepresnM, and red circles represent 10 onds represent no drug, pink boxes ent 10 2 e Presence of Indinavir. Blue represents 10 nM, yellow triangles nM, green crosses represent 10 5 nM. 3 nM, purple stars represent 10 4

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163 Figure A-6. Replication of R5 24 6 810A.6 Days p24 (ng/ml) 012345 24 6 810A.6 Days p24 (ng/ml) 012345 0123456 2 468 10 Days p24 (ng/ml)B. 0123456 2 468 10 Days p24 (ng/ml)B. 24 6 810 Days p24 (ng/ml) C. 0123456 24 6 810 e Presence of Ritonavir. Blue 3 nM, purple stars represent 104 Days p24 (ng/ml) C. 0123456 Recombinant Viruses in th diamrepresnM, and red circles represent 10 onds represent no drug, pink boxes ent 10 2 represents 10 nM, yellow triangles nM, green crosses represent 10 5 nM.

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164 0 255075100 10 1 10 2[IDV] 10 3 n 10 4 105ReplicationA. M (% of Control) 0 255075100 10 1 10 2[IDV][RTV] 10 3 n 10 4 105ReplicationA. M (% of Control) nM B.105Replication 10 1 10 2[RTV] 10 3 10 4 0 255075100 (% of Control) nM B.105Replication ce of resent 10erap 1y R5 virus. 10inant Viruses. A, IC 2r 10 3 10 4 0e postth 255075100ent th (% of Control)indinavir. BLAI-R5, blue squares represent the prrepres Figure A-7. IC 50 Curves for R5 Rec , IC 50 om in the p curves b 505 virus, and green circles curves in the presen esence of ritonavir. Red triangles rep etherapy R

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165 Post-R52.1 x 105nMR5411Post-X45,150 nMX4Ritonavir Pre-900 nMPre-870 nM PosPos130 nMv tt -R54-X4 2.9 x 10a nM Pre-140 uM5Pre-30 nM R5X4I 226r n d in i Post-R52.1 x 105nMR5411Post-X45,150 nMX4RitonavirPost-R52.1 x 105nMR5411Post-X45,150 nMX4Ritonavir Pre-900 nMPre-870 nMPre-900 nMPre-870 nM PosPos130 nMvPosPos130 nMv tttt -R54-X4-R54-X4 2.9 x 10a2.9 x 10a nMnM Pre-140 uM5Pre-30 nMPre-140 uM5Pre-30 nM R5X4IR5X4I 226r226r nn dd inin ii 50 Values. The IC50 columns. The fold change row. Figure A-8. X4 and R5 Differen ces in Indinavir and Ritonavir IC values for preand posttherapy X4 and the lefbetween X4 and R5 viruses are show t two colum n s and riton R5 viruses are shown for indinavir in e right two avir in th n in the bottom

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166 0102030 40050n(ng/m Intracel Conce 0 1 02 0 3H 04ours) Tim e ( lultra ario tl) n 0102030onds), 10 40050n(ng/m eated with 103 nM Intracel 0Indinavir Concentrations. 1centration w 02 0s m 3H5 04ours) Tima e ( lultra(blue diamand the intracellular con ario ConceFigure A-9. Intra tl) ncellula r 4 PBMC were treasured at 3, 18, and 48 hours. nM (pink squares), or 10 nM (yellow triangles) of IDV,

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167 050304050Intracel(ng/mA. 1015n 0 10 2Time (Hours) lulal ario Conce ntr t) 050304050Intracel(ng/m 1015n 0 10 2Time (Hours) lulal ario Conce ntr t) 050304050 1015 0 10 2 050304050Intracel(ng/mA. Intracel(ng/m ConceConc B. 1015n 0 10 2Time (Hours) lulallulal) ariorn ntrtr t)atio en 00 1000200030004000 1 02 0304050me (Hours)Intracel(ng/m Conc Ti lulal) atio rn en tr 00itonavir Concentrations. A, 1000200030004000 1 02 0304050me (Hours) PBMC were treated with 103 onds), 104 les) of RTV. B.Figure A-10. Intracellular R nM (blue diamconcentration was mnM (pink squares), or 10 Tiquares) of RTV, and the intracellular and 48 hours. B, PBMC treated with 10w triang 4 nM (pink s easured at 3, 18, 5 nM (ye llo

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168 000 1002003004005 600014050acentrg/m Intr(n Conce 0 2Time (H 0 3 0ours) llulatil aro n ) 000onds), 10 1002003004005 600014050acentrg/m ted with 103 nM Intr(n 0 2Time (Hations. PBMC were treaa 0s m 35 0ours) llulatil. Intra(blue diamand the intracellular con ConceFigure A-11 ) aro ncellular Nelf inavir Con 4 centr nM (pink squares), or 10 centration w nM (yellow triangles) of NFV, easured at 3, 18, and 48 hours.

PAGE 183

T OF P APPENDIX B ALIGNME N R OTEASE S EQUENCES

PAGE 184

* * * * * * * * * HXB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF 170 VSIS 01 Pretherapy 2272.C01 --I---------V-V-------------------------K---------------------P-----K-----------------------------2272.C03 --I---------V-V-------------------------K---------------------P-----K-----------------------------2272.C04 --I---------V-V-------------------------K---------------------P-----K-----------------------------2272.C05 --I---------VR--------------------------K---------------------P-----K-------------------------2272.C06 --I---------V---------------------------K------E--R--------N--P----RK--------------------V---R----2272.C07 --I---------VR-----N--------------------K---------------------P-----K-----------------------------2272.C08 --I---------VR--------------------------K---------------------P-----K----------------------------2272.R01 --I---------VR--------------------------K---------------------P-----K-----------------------2272.R02 --I---------V-V-------------------------K---------------------P-----K-----------------------------2272.R04 --I---------VR--------------------------K---------------------P-----K---------------------------2272.R05 --I----------R--------------------------K---------------------P-----K-----------------------272.R06 --I---------V-V-------------------------K---------------------P-----K--------------------------2 Posttherapy 2774.A01 --I---------V-V-------------------------K---------------------P-----K-----------------------------2774.A02 --I---------V-V-------------------------K---------------------P-----K-----------------------------2774.A03 --I---------V-V-------------------------K---------------------P--V--K-----------------------------2774.A04 --I---------V-V-------------------------K---------------------P-----K-----------------------------2774.A05 --I---------V-V-------------------------K---------------------P-----K-----------------------------2774.O01 --I---------V-V-------------------------K---------------------P-----K-----------------------------2774.O02 --I---------V-V-------------------------K---------------------P-----K-----------------------------2774.O03 --I---------V-V-----------E-------------K---------------------P-----K-----------------------------2774.O04 --I---------V-V-------------------G-----K---------------------P-----K-----------------------------2774.O05 --I-------A-VR--------------------------K---------------------P-----K-----------------------------

PAGE 185

* * * * * * * * * HXB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF VSIS 03 171 --I---------------------------------N-T-K------------------E--PV------------I----I------------Pretherapy 2000.R01 --I---------------------------------N-T-K------------------E--PV------------I----I------2000.R02 --I---------------------------------N-T-K------------------E--PV------------I----I-------------2000.R03 --I---------------------------------N-T-K------------------E--PV-----------------I----------2000.R04 --I---------------------------------N-T-K------------------E--P-------------I----I-------------2000.R05 --I---------------------------------N-T-K------------------E--PV----------------------------2000.R06 --I---------------------------------N-T-K------------------E--PV------------I----I-------------000.R07 --I---------------------------------N-T-K------------------E--PV------------I----I----------2 2000.R08 Posttherapy 2590.A01 --I---------------------------------N-T-K------------------E--PV------------I----I----K-----------2590.A02 --I---------------------------------T-T-K---------------------P-----R-------------------G---2590.A03 --I---------------------------------N-T-K------------------E--PV-----------------ID-------NR----2590.A05 --I---------------------------------T-T-K---------------------P-----R---------L---D-----W 2590.O01 --I-------------------I-------------N-T-K------------------E--P-------------------D----2590.O02 --I---------------------------------T-T-K---R--------------E--P-------------------D--------------2590.O03 --II--------------------------------T-T-K---------------------P-----R---------L---D----2590.O04 --II-G---F-----------V--------------N-T-K------------------E--P-----------------------------------2590.O05 --I---------------------------------T-T-K------------------E--PV--------------L---D------2590.O06 --I-------------------I-------------N-T-K---------------------P-------------------D------2590.O07 --I---------------------------------N-T-K------------------E--PV-----------------ID----

PAGE 186

* * * * * * * * * HXB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF VSIS 04 Pretherapy 1669.01 --I--------------------------------IN------------------------VP--------------------------------P--1669.03 --I--------------------------------IN------------------------VP-----------------------K--------P--172----V--------------------IN------------------------VP-----------------------------------* * * * * * * * * XB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF SIS 25 retherapy 473.48.01 --I---------------------------------N-------------------------------------------------------------473.48.02 --I---------------------------------N-----R-------------------------------------------------------473.48.06 --I----------E----------------------N-------------------------------------------------------------473.48.08 --I---------------------------------N---------------------------------V---------------------------473.48.12 --I---------------------------------N-------------------------------------------------------------1669.05 --I--------------------------------IN------------------------VP-------------------H------------P--669.04 --I--------------------------------IN------------------------VP-------------------H--------P------1 1669.02 --I------Posttherapy 1779.01 --I--------------------------------IN------------------------VP--------------R-------------------I 1779.02 --I--------------------------------IN------------------------VP---------------------------------IPN 1779.03 --I---------V----------------------IN------------------------VP-----------------------K-----------1779.05 --I---------------------------------N-------------------K----VP-----------------------K----------IS H V P 2 2 2 2 2

PAGE 187

* * * * * * * * * HXB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF VSIS 27 173 Pretherapy 5761.48.02 --I----------R---E------------------N-------------------K-----P--------V--------------------------5761.48.03 --I---------V-----------------------NC----T-------------K-----P---G----VD-----L-L---M--Y----------5761.48.04 --I----------R---E------------------N-------------------K-----P--------V--------------------------5761.48.06 --I----------R---E------------------N-------------------K-----P--------V--------------------------5761.48.07 --I----------R----------------------N-------------------K-----P--------V--------------------------5761.48.08 --I----------R---E----------------D-N-------------------K-----P--------V--------------------------5761.48.09 --I----------R--------------------D-N-------------------K-----P--------V--------------------------5761.48.10 --I---------V-----------------------N-------------------K-----P--------V--------------------------5761.48.11 --I--------------------------------IN-------------------K-----P--------V--------------------------5761.48.12 --I----------R---E----------------D-NW----N-------------K-----P--------V--------------------------Posttherapy 6063.00.03 --I-------------------------------D-N-------------------K-----P--------V--------------------------6063.00.05 --I----------R--------------------D-N-------------------K-----P--------V--------------------------6063.00.06 --I-------------------------------D-N-------------------K-----P--------V--------------------------6063.00.07 --I-------------------------------D-N-------------------K-----P---R----V--------------------------6063.00.09 --II--------V-----------------------N-------------------K-----P--------V--------------------V-----6063.00.10 --I-------------------------------D-N-------------------K-----P--------V----------------------R---6063.00.11 --I-------------------------------D-N-------------------K-----P--------V--------------------------

PAGE 188

* * * * * * * * * HXB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF VSIS 28 174 Pretherapy 5332.48.02 --I--------------------------------IN--------------------------V----------------------------------5332.48.03 --I--------------------------------IN-------------------------------------------------------------5332.48.04 --I--------------------------------IN------------------------L-V----------------------------------5332.48.05 --I--------------------------------IN--------------------------V----------------------------------5332.48.06 --I--------------------------------IN--------------------------V----------------------------------5332.48.08 --I--------------------------------VN--------------------------V----------------------------------5332.48.09 --I---------V----------------------IN--------------------------V----------------------------------5332.48.10 --I--------------------------------IN--------------------------V----------------------------------5332.48.11 --I--------------------------------IN--------------------------V----------------------------------332.48.12 --I---------V----------------------IN--------------------------V----------------------------------5 Posttherapy 5715.00.01 --I-----T--------------------------IN-------------------------------------------------------------5715.00.02 --I--------------------------------IN-------------------------------------------------------------5715.00.03 --I-----T-A--------R---------------IN-------------------------------------------------------------5715.00.04 --I-F---T--------------------------IN--E----------------------------------------------------------5715.00.05 --I--------------------------------IN-------------------------------------------------------------5715.00.06 --I--------------------------------IN-----------------------------------------------------------F-5715.00.07 --I--------------------------------IN--------------------------V----------------------------------5715.00.08 --I-----T--------------------------IN-------------------------------------------------------------5715.00.09 --I--------------------------------IN-------------------------------------------------------------5715.00.10 --I--------------------------------IN-------------------------------------------------------------5715.00.11 --I--------------------------------IN-------------------------------------------------------------5715.00.12 --I--------------------------------IN-------------------------------------------------------------

PAGE 189

* * * * * * * * * HXB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF VFIS 02 Pretherapy 1850.48.01 --I-------------------------------D-N-------------------K-----P-----------------------------------1850.48.02 --I-------------------------------D-N-------------------K-----P-----------------------------------1850.48.03 --I-------------------------------D-N-------------------K-----P-----------------------------------1850.48.05 --I-------------------------------D-N-------------------K----VP-----------------------------------1850.48.06 --I-------------------------------D-N-------------------K-----P-----------------------------------1850.48.07 --I--------------K----------------D-N-------------------K-----PV----------------------------S-----1850.48.08 --I-------------------------------D-N-------------------K-----P-----------------------------------1850.48.09 --I----------------RK-------------D-N--------------V----K-----P-----------------------------------1850.48.10 --I-------------------------------D-N-------------------K-----P-----------------------------------1850.48.11 --I-------------------------------D-N-------------------K-----P-----------------------------------850.48.12 --I-------------------------------D-N-------------------K----VP-----------------------------------1 Posttherapy 2088.48.01 --I-------------------------------D-N----------------V--K----VP------------------A----------------2088.48.02 --I-------------------------------D-N----------------V--K----VP------------------A----------------2088.48.03 --I-------------------------------D-N----------------V--K----VP------------------A----------------175 2088.48.04 --I-------------------------------D-N----------------V--K----VP------------------A----------------2088.48.05 --I-------------------------------D-N----------------V--K----VP------------------A----------------2088.48.06 --I--------------R----------------D-N----------------V--K----VP------------------A----------------2088.48.07 --I-------------------------------D-N----------------V--K----VP------------------A----------------2088.48.08 --I-------------------------------D-N----------------V--K----VP------------------A----------------2088.48.09 --I-------------------------------D-N----------------V--K----VP------------------A----------------2088.48.10 --I----------------------------A--D-N----------------V--K----VP------------------A----------------2088.48.11 --I-------------------------------D-N----------------V--K----VP------------------A----------------2088.48.12 --I-------------------------------D-N----------------V--K----VP------------------A----------------2088a01 --I-------------------------------D-N-------------------K-----P-----------------------------------2088a02 --I-------------------------------D-N-------------------------P-----------------------------------2088a03 --I-------------------------------D-N-------------------K-----P-----------------------------------2088o01 --I-------------------------------D-N-------------------------P-----------------------------------2088o02 --I----------------E-------------KD-N----------------V--K----VP------------------A-V--------------2088o04 --I--------A--------D----A-------TD-Y----------------V--K----VP------------------A----------------2088o05 --I-------------------------------D-N-------------------------P-----------------------------------088o06 --I-------------------------------D-N-------------------K-----P-----------------------------------2

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* * * * * * * * * HXB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF VFIS 10 Pretherapy 1470c01 --I---------------I-----------------N-----R-------------------C-------------I------KF-------L-----1470C02 --I---------------I-----------------N-------------------------C-------------I---------------L-----1470U01 --I---------------I-----------------N-------------------------C-----------------------------L-----176--I---------------I-----------------N-------------------------C-G---------------------------L-----470.48.06 --I---------------I-----------------N-------------------------C-------------I---------------L-----470.48.07 --I---------------I-----------------N-------------------------C-------------I---------------L-----470.48.08 --I---------------I-----------------N-------------------------C-------------I---------------L-----470.48.09 --I---------------I-----------------N--------------------HN---C-------------I---------------L-----470.48.11 --I---------------I-----------------N-------------------------C-------------I---------------L-----470.48.12 --I---------------I-----------------N-------------------------C-------V-----I---------------L-----470.48.13 --I---------------I-K---------------N-------------------------C-------------I---------------L-----470.48.14 --I---------------I-----------------N-------------------------C-------------I----I----------L-----470.48.15 --I---------------I-----------------N-------------------------C-------------I---------------L-----470.48.16 --I---------------I-----------------N-------------------------C-------------I---------------L-----470R317 --IN--------------I-----------------N-------------------------C-------------I---------------L-----470R311 --I---------------I-----------------N-------------------------C-------------I---------------L-----470R312 --I---------------I-----------------N-------------------------C-------V-----I---------------L-----470R313 --I---------------I-K---------------N-------------------------C-------------I---------------L-----470R314 --I---------------I-----------------N-------------------------C-------------I----I----------L-----470R315 --I---------------I-----------------N-------------------------C-------------I---------------L-----1470R316 --I---------------I-----------------N-------------------------C-------------I---------------L-----1470U02 --I---------------I-----------------N-------------------------C-----------------------------L-----470U03 --I---------------I-----------------N-------------------------C-----------------------------L-----1 1470U04 1470U05 --I-------------E-I-----------------N-------------------------C-----------------------------L-----1470A01 --I---------------I-----------------N-------------------------C-------------I---------------L-----1470A02 --I---------------I-----------------N-------------------------C-----------------------------L-----1470A03 --I---------------I-----------------N-------------------------C-------------I---------------L-----1470A04 --I---------------I-----------------N-------------------------C-------------I-------F-------L-----1470O02 --I---------------I-----------------N-------------------------C-------------I---------------L-----1470O05 --I---------------I-----------------N-------------------------C-------------I---------------L-----1470O06 --I---------------I-----------------N-------------------------C-------------I---------------L-----1470PR --I---------------I-----------------N-------------------------C-------------I---------------L-----1470.48.01 --IN--------------I-----------------N-------------------------C-------------I---------------L-----1470.48.02 --I---------------I-----------------N-------------------------C-------------I---------------L-----1470.48.03 --I---------------I-----------------N-------------------------C-------------I---------------L-----1470.48.04 --I---------------I-----------------N-------------------------C-------------I---------------L-----470.48.05 --I---------------I-----------------N-------------------------C-------------I---------------L-----1 11 11 11 11 1 11 11 11 1

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* * * * * * * * * HXB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF 177Continued) --I------I--------I-----------------N----------------V--------C-------V-----I----A-------M--L-----664.48.20 --I------I--------I-----------------N----------------V--------C----R--V-----I----A-------M--LA----664.48.22 --I------I--------I-----------------N----------------V--------C-------V-----I----A-------M--L-R---664.48.23 --I------I--------I-----------------N-----------V--A-V--------C-------V-----I----A-------M--L-----664.48.24 --I------I--------I-----------------N----------------V--------C-------V-----I----A-------M--L-----664.48.25 --I------I--------I-----------------N------L---------V--------C-------V-----I----A-------M--L-----664.48.26 --I------I--------I-----------------N----------------V--------C--V----V-----I----A----------L-----664.48.27 --I------I--------I-----------------N----------------V--------C-------V-----I----A-------M--L-----664.48.28 --I------I--------I-----------------N----------------V--------C-------V-----I----A-------M--L-----VFIS 10 ( Pretherapy 1470R325 --I---------------I-----------------N-------------------------C-------------I---------------L-----1470R337 --I---------------I-----------------N-------------------------C-------------I---------------L-----1470R347 --I---------------I-----------------N-------------------------C-------------I---------------L-----1470R357 --I---------------I-----------------N-------------------------C-------------I---------------L-----1470R366 --I---------------I-----------------N-------------------------C-------------I---------------L-----1470R376 --I---------------I-----------------N-------------------------C-------------I---------------L-----1470R386 --I---------------I-----------------N-------------------------C-------------I---------------L-----1470R395 --I---------------I-----------------N--------------------HN---C-------------I---------------L-----Posttherapy 1664.48.01 -LI------I--------I-----------------N----------------V--------C-------V-----I----A-------M--L--A--664.48.02 --I------I--------I-----------------N----------------V--------C-------V-----I----A-------M--L-----1 1664.48.03 1664.48.04 --I------I--------I-----------------N----------------V--------C-------V-----I----A---E---M--L-----1664.48.05 --I------I--------I--------------K--N----------------V--------C-------V-----I----A-------M--L-----1664.48.06 --I------I--------I-----------------N----------------V--------C-------V-----I----A-------M--L-----1664.48.07 --I------I--------I-----------------N----------------V--------C-------V-----I----A-------M--L-----1664.48.08 --I------IA-------I-----------------N----------------V--------C-------V-----I----A-------M--L-----1664.48.09 --I------I----V---I-----------------N----------------V--------C-------V-----I----A-------M--L-----1664.48.10 --I------I--------I-----------------N----------------V--------C-------V-----I----A-------M--L-----1664.48.11 --I------I--------I-----------------N----------------V--------C-------V-----I----A-------M--L-----1664.48.12 --I------I--------IE----------------N-----T----------V--------C--L----V-----I-TK-A-------M--L-----1664.48.13 --I------I--------I-----------------N----------------V--------C-------V-----I----A-------M--L-----1664.48.14 --I------I--------I-----------------N----------------V--------C------QV-----I----A-------M--L-----1664.48.15 --I------I----V---I-----------------N----------------V--------C-------V-----I----A----------L-----1664.48.16 --I------I--------I-----------------N----------------V--------C-------V-----I----A----------L-----1664.48.17 --I------I--------I--------------K--N----------------V--------C-------V-----I----A--N----M--L-----1664.48.18 --I------I--------I-----------------N----------------V--------C-------V-----I----A-------M--L-----664.48.19 --I------I--------I-----------------N----------------V--------C-------V-----I----A-------M--L-----1 1 1 1 1 1 1 11

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* * * * * * * * * HXB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF 178Continued) -----I---------------I-----------------N-------------------------C-----------------------------L----K 664U02 --I---------------I-----------------N----------------V--------C-------V-----I----A----------L-----664U03 --I---------------I-----------------N-------------------------C-------------I---------------L-----664U04 --I---------------I-----------------N-------------------------C-------------I---------------L-----664U05 --I---------------I-----------------N-------------------------C-----------------------------L-----664U06 --I------I--------I-----------------N-------------------------C-------------I----A-V--------L-----VFIS 10 ( Posttherapy 1664A01 ---------------I-----------------N--------------S----------C-------------I---------------L-----1664A03 ---------------I-----------------N---------V---------------C-------------I---------------L-----1664A04 ------------K--I-----------------N----------R--RS----------C-------------I---------------L-----1664A05 ---------------I-----------------N---------T------V--------C-------V-----I----A-------M--L--------1664A07 --I--C------------I-----------------N-------------------------C-----------------------------L664A08 --IN--------------I-----------------N-------------------------C-------------I---------------L 1 1664A09 1664A10 --I---------------I-----------------N-------------------------C-------------I---------------L----K 1664O19 --I------I--------I-----------------N----------------V--------CV------V-----I-------------N-P----K 1664R02 --I------I--------I-----N-E---------N----------------V--------C-------V-----I----A-------M--L-----1664R03 --I------I--------I-----N-----------N----------------V--------C-------V-----I----A-------M--L-----1664R07 --I------I--------I-----N-----------N----------------V--------C-------V-----I----A-------M--L-----1664R09 --I------I--------T-----N-----------N----------------V--------C-------V-----I----A----------L-----664U01 --I---------------I-----------------N-------------------------C-----------------------------L-----1 1 1 1 1 1

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* * * * * * * * * HXB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF VFIS 11 Pretherapy 1446U01 --I-----------------------------------------------------K-----P-----------------------K-----------1446U02 --I------------------------T----------------------------K-----P-----------------------K-----------1446U03 --I-----------------------------------------------------K-----P-----------------------K-----------1446U04 --I-----------------------------------------------------K-----P-----------------------K-----------1446U05 --I-----------------------------------------------------K-----P-----------------------K-----------Posttherapy 1656U01 --I--------------------I---------------------I----------------P------------------A----------------1656U02 --I--------------------I---------------------I----------------P------------------A----------------1656U04 --I--------------------I---------------------I----------------P------------------A----------------1656U05 --I--------------------I---------------------I----------------P------------------A----------------1656U06 --I----------------R-----------IF--I--------------------------P-------V----------A-------M--------1656U07 --I----------------R---------------I-----------------V--------P-------V----------A-------M--------1656U08 --I----------------R-----------IF--I---------I----------------P-------V----------A-------M--------179656.48.09 --I------I---------R---------------I---------------A-V--------P-------V----------A-------M--------656.48.10 --I----------------------------IF--I---------I----------------P-------V----------A-------M--------656.48.11 --I------I---------R---------------I-----------------V--------P-------V----------A-------M--------656.48.12 --I----------------R---------------I-----------------V--------P-------V----------A-------M--------1656U09 --I----------------R---------------I-----------------V--------P-------V----------A-------M--------1656U11 --I------I---------R---------------I-----------------V--------P-------V----------A-------M--------1656.48.01 --I----------------R---------------I-----------------V--------P-------V----------A-------M--------1656.48.02 --I----------------------------IF--I---------I----------------P-------V----------A-------M--------1656.48.04 --I----------------------------IF--I---------I----------------P-------V----------A-------M--------1656.48.05 --I----------------R-----------IF--I---------I----------------P-------V----------A-------M--------1656.48.06 --I------I---------R---------------I-----------------V--------P-------V----------A-------M--------1656.48.07 --I----------------R---------------I-----------------V--------P-------V----------A-------M--------656.48.08 --I----------------------------IF--I---------I----------------P-------V----------A--T----M--------1 1 1 1 1

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* * * * * * * * * HXB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF VFIS 12 180-----------------------------------V------------D-N-------------------------P-K-----------I---------------------Pretherapy 1738.48.01 --I-------------------------------D-N-------------------------P--------------------------1738.48.07 --I-----------V-------------------D-N-------------------------P--------------------------------1738.48.08 --I-------------------------------D-N-------------------------P-------------I------------1738.48.09 --I-----------V-------------------D-N-------------------------P-----------------------------------1738.48.10 --I-----------V-------------------D-N--------------S----------P-------------I-------------1738.48.11 --I-------------------------------D-N-------------------------P-------------I---------------------1738.48.12 --I-----------V-------------------D-N-------------------------P-----------------------------------1738.48.13 --I-------------------------------DIN-------------------------P-----------------------------------1738.48.14 --I-----------V-------------------D-N-------------------------P-----------------------------------1738.48.15 --I-------------------------------D-N-------------E-----------P-----------------------------------1738.48.16 --I-----------V-------------------D-N------S------------------P------------------------------------1738A02 --I-------------------------------D-N-------------------------P---------------------------IR----V-1738A03 --II----------V------V------------D-N------------L------------S-----------------------------------1738A04 --I-----------V-------------------D-N-------------------------P---------------------------N-------1738A05 --I-----------V-------------------D-N-------------------------P-------------I---------------------738A06 --I-----------V-------------------D-N-------------------------P-------------I---------------------1 1738A07 --II-----1738O01 --I-------------------------------D-N-------------------------P-----------------------------------1738O03 --I-----------V-------------------D-N-------------------------P---------------------------N-------1738O04 --IA----------V------V------------D-N-------------------------P---------------------------I-------

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181 * * * * * * * * * HXB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF VFIS 12 (Continued) Posttherapy 1943.48.01 --I------I------------------------DIN--------------V-V--------P------------------A----------------1943.48.03 --I------I------------------------DIN--------------V-V--------P------------------A----------------1943.48.04 --I------I------------------------DIN----------------V--------P-------V----------A-V--------------1943.48.05 --I------I------------------------DIN----------------V--------P-------V----------A-V---D----------1943.48.06 --I-----------V----R--------------DIN----------------V--------P------------------A-----------V----1943.48.07 --I------I------------------------DIN----------------V--------P-------V----------A-V--------------1943.48.08 --I------I------------------------DIN----------------V--------P-------V----------A-V--------------1943.48.10 --I------I------------------------DIN----------------V--------P-------V----------A-V--------------1943.48.11 --I------I------------------------DIN----------------V--------P------------------A----------------1943.48.12 --I------I------------------------DIN----------------V--------P-------V----------A-V--------------1943A01 --I-----------V------V------------D-N-------------------------P-------------I---------------------1943A03 --I-----------V-------------------D-N-------------------------P-------------------K-------N-V----K1943A05 --I-------------------------------D-N-------------------------P-------------I-----K-------N-V---I-1943A06 --I-----------V-------------------D-N-----R-------------------P---------------------------N------K1943O01 --I----Q-I------------------------DIN----------------V--------P------------------A---------------I1943O11 --I-------------------------------DIN---K------------V--------P------------------A----------------1943O12 --I-------------------------------DIN---K------------V--------P------------------A----------------1943O13 --I-------------------------------DIN---K------------V--------P------------------A----------------1943O14 --I-----------V-------------------DVN----------------V--------P-------T----------A----------------1943O15 --I-----------V-------------------DVN----------------V--------P------------------A----------------1943O16 --I-----------V-------------------DVN----------------V--------P------------------A----------------1943R01 --I-----------V-------------------DVN----------------V--------P-------T----------A----------------1943U01 --I-----------V--------------------IN-------------------------------------------------------------1943U04 --I-----------V--------------------IN-------------------------------------------------------------

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* * * * * * * * * HXB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF VFIS 14 Pretherapy 1329pr --I-------------------------------------K----------------------V---------------------------------1329r13 RLI---------------------------------N---K----------------------V------------------------------A1 329r23 --I---------------------------------N---K---------------------PV---------------------------1329r24 --I---------------------------------N---K---------------------PV--------------------------N-GVE 1329u01 --I-------------------------------------K----------------------V----------------------------------1329u02 --I--------------------------------IN---K----------------------V----------------------------------Posttherapy 1576.48.02 --I---------------------------------N---K---------------------PV----------------------------L-----1576.48.03 --I---------------------------------N---K---------------------PV----------------------------L-----182576.48.05 --I---------------------------------N---K---------------------PV----------------------------L-----576.48.06 --I---------------------------------N---K---------------------PV----------------------------L-------8.08 --I---------------------------------N---K---------------------PV----------------------------L-----10 --I------------------------V--------N---K---------------------PV----------------------------L---------1 576.48.04 --I---------------------------------N---K---------------------PV----------------------------L-----1 1 1576.48.07 --I---------------------------------N---K---------------------PV----------------------------L--1576.4 1576.48. 1576.48.11 --I--------R----------V-------------N---K---------------------PV----------------------------L--1576.48.12 --I---------------------------------N---K---------------------PV----------------------------L--

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183 QIGCTLNF ------------------------------------------------D209R07 --I------I-----------------------Q-IN-T--------------V-------VP------------------A-----I---------------MI------I0 --I------I-----------------------Q-VN-T--------------V-------VP------------------A---------------I----------R------------------------209.48.11 --I--R---I----V------------------Q-IN-T-G------------V-------VP------------------A----------------209.48.12 --I------------------------------Q-IN-T--------------V-------VP------------------A---------------------3 --I---------------------------------N-T----------------------VP------------------F------------------I------------------------------Q-IN-T--------------V-------VP------------------A-------V---------------P-I* * * * * * * * * XB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLT H VFIS 15 Pretherapy 1916R01 --I--------------------------------IN-T----------------------VPL--------------------------1916R02 --I--------------------------------IN-T----------------------VPL--------------------------1916R03 --I--------------------------------IN-T-----------------------P---------------------------1916R04 --I--------------------------------IN-T----------------------VP----------------------------916R05 --I-----------V---------------------N---K---------------------S------R-----------I--------1 1916R06 --I--------------------------------IN-T----------------------VPL---E----------------------Posttherapy 209R06 --I------I-----------------------Q-IN-T--------------V-------VP------------------A--------2 2 2 209R08 --I------I-----------------------Q-VN-T--------------V-------VP------------------A-----S---209R09 --I------I-----------------------Q-VN-T--------------V-------VP------------------A--------2 2209R1 2209.48 .04 --I------I----V------------------Q-IN-T--------------V-------VP------------------A--------2209.48.06 --I------I----V------------------Q-IN-T--------------V-------VP------------------A--------2209.48.07 --I------I----V------------------Q-IN-T--------------V-------VP------------------A--------2209.48.08 --I------I----V------------------Q-IN-T--------------V-------VP------------------A--------2209.48.10 --I------I----V------------------Q-IN-T--------------V-------VP------------------A--------2 2 2209A02 --I------------------------------Q-IN-T--------------V-------VP------------------A----------2 209A0 2209A07 2209O02 --I------I-----------------------Q-VN-T--------------V-------VP------------------AH---K--V--2209O03 --I------I-----------------------Q-IN-T--------------V-------VP------------------A----------

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184 QIGCTLNF -----------------------------------7 --I--------R------I-----------------D-----R-----------R-------P-------------I----------------------------------IS ---------------------------D434.00.06 --I---------V-----------------------D-----R-----------R-------P-------T-----I---------------------434.00.07 --I---------V-----------------------D-----R-----------R-------P-------T-----I---------------------434.00.08 --I---------V-----------------------D-----R-----------R-------P-------T-----I---------------------434.00.09 --I--------P-R----------------------D-----R-----------R----E--P-------------I---------------------434.00.10 --I---------V-----------------------D-----R-----------R-------P-------T-----I---------------------434.00.11 --I---------V-----------------------D-----R-----------R-------P-------T-----I---------------------434.00.12 --I---------V-----------------------D-----R-----------R-------P-------T-----I---------------------* * * * * * * * * XB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLT H VFIS 17 Pretherapy 1875.48.01 --I------I--V-----------------------D-----R-----------R-------P-------T-----I-------------1875.48.02 --I--------P-R------------------P---D-----R-----------R-------P-------------I-------------1875.48.03 --I--------P-R----------------------D-----R-----------R-------P-------------I-------------1875.48.04 --I--------P-R----------------------D-S---R-----------R-------P-------T-----I-------------1875.48.05 --I--------P-R----------------------D-----R-----------R-------P-------------I-------------1875.48.0 1875.48.08 --I--------P-R----------------------D-----R-----------R-------P-------T-----I-------------1875.48.09 --I--------R------I-----------------D-----R-----------R-------P-------------I-------------1875.48.10 --I--------P-R----------------------D-----R-----------R-------P-------T-----I-------------1875.48.11 --I--------R------I-----------------D-----R-----------R-------P---------S---I-------------Posttherapy 2434.00.03 --I---------V-----------------------D-----R-----------R-------P-------T-----I-------------2 434.00.05 --I---------V-----------------------N-----R-----------R-------P-------T-----I-------------2 2 2 2 2 2 2

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185* LTQIGCTLNF ---L--------L--------L---S----L--------L--------L--------L--------L--------L-------I------I--V----------------------IN------------------------VNV----------------------------L--------L--------L--------L--------L--------L--------L--------L--------L-----M--L-----M--L-----M--L--------L--------L-----930.48.08 --I------I--V-------------------F---N----------------V-------VNV-----------------A-------M--L-----930.48.09 --I------I--V-------------------F--IN----------------V-------VNV-----------------A----------L-----930.48.10 --I------I--V-------------------F--IN----------------V-------VNV-----------------A----------L-----930.48.11 --I------I--VR------------------F--IN----------------V-------VNV-----------------A----------L-----930.48.12 --I------I--V-------------------F--IN----------------V-------VNV-----------------A----------L-----* * * * * * * * XB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNL H VFIS 18 Pretherapy 2550u02 --I------II-V-----------------------N------------------------VNV------------------------2550u03 --I------I--V-----------------------N------------------------VNV------------------------2550u04 --I------I--V-----------------------N---------T-E-E----------VNVK-----------------------2550u05 --I------I--V-----------------------N------------------------VNV------------------------2550u06 --I------I--V-----------------------N------------------------VNV------------------------2550u07 --I------I--V-----------------------N-----------E-E----------VNVK-----------------------2550u08 --I------I--V-----------------------N------------------------VN-------------------------2550u09 --I------I--V-----------------------N------------------------VNV------------------------2550u10 --I------I--V-----------------------N------------------------VN-------------------------2550u11 2550u12 --I------I--V-----------------------N------------------------VNV------------------------Posttherapy 2930C01 --I------I--V-------------------F-DIN----------------V-------VNV-----------------A------2930C02 --I------I--V-------------------F-DIN----------------V-------VNV-----------------A------2930C03 --I------I--V----L--------------F-DINF---------------V-------VNV-----------------A------2930C04 --I---R--I--V-------------------F--IN----------------V-------VNV-----------------A------2930C06 --I------I--V-------------------F-DIN----------------V-------VNV-----------------A------2930.48.01 --I------I--V-------------------FQ--N----------------V-------VNV-----------------A------2930.48.02 --I------I--V-------------------F--IN----------------V-------VNV-----------------A------2930.48.03 --I------I--V-------------------F---N----------------V-------VNV-----------------A------2930.48.04 --I------I--V-------------------F--IN----------------V-------VNV---------------S-A------2930.48.05 --I------I--V-------------------F--IN----------------V-------VNV-----------------A------2930.48.06 --I------I--V-------------------F--IN----------------V-------VNV-----------------A------2930.48.07 --I------I--V-------------------F---N----------------V-------VNV-----------------A------2 2 2 2 2

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186TLNF ------------------------------8 --I------I---R-------------------K-IN---K------------V--------P------------------A------------------------------------------S---* * * * * * * * * HXB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGC VFIS 22 Pretherapy 2041R11 --I----------R---------------------IN---K---------------------P-------------------------------2041R12 --I----------R-E-------------------IN---K---------------------P-------------------------------2041R13 --I----------R---------------------IN---K---------------------P-------------------------------2041R14 --I----------R---------------------IN---K---------------------P-------------------------------2041R15 --I--------------------------------IN---K--------------------VP-------------------------------2041R16 --I--------------------------------IN---K--------------------VP-------------------------------Posttherapy 2411.48.03 --I----------R---------------------IN---K----I-------V-------VP------------------A------------2411.48.04 --I------I---R-----N-------------K-IN---K------------V--------P------------------A------------R 2411.48.05 --I------I---R-------------------K-IN---K------------V--------P------------------A------------2 411.48.07 --I------I---R-------------------K-IN---K------------V--------P------------------A------------2411.48.0 2411.48.09 --I----------R---------------------IN---K----I-----V-V-------VP---------------L--A----G-------2411.48.10 --I----------R---------------------IN---K----I-------V-------VP------------------A----G-------2411.48.11 --I------I---R---------------G---K-IN---K------------V--------P------------------A------------2411.48.12 --I------I---R-------------------K-IN---K------------V--------P------------------A------------2411C01 --I----------R---------------------IN---K----I-------V-------VP------------------A------------2411C02 --I----------R---------------------IN---K----I-------V-------VP------------------A---W--------2411C03 --I----------R---------------------IN---K----I-------V-------VP------------------A------------2411C04 --I----------R---------------------IN---K----I-------V-------VP------------------A------------2411C05 --I----------R---------------------IN---K----I-------V-------VP------------------A------------2411C06 --I----------R---------------------IN---K----I-------V-------V-------------------A------------

PAGE 201

187 QIGCTLNF ----------------I----------R---------------------ID-------------------------P--------------------------------------------------------------------------------------------------* * * * * * * * * HXB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLT VFIS 24 Pretherapy 2098U01 --I----------R---------------------ID-------------------------P---------------------------2098U03 --I----------R---------------------ID-------------------------P---------------------------2098U04 Posttherapy 2375.48.04 --I--------------------------------IN------T------------------P---------------------------2375.48.06 --I--------------------------------ID-------------------------P--------T------------------2375.48.08 --I----------------N---------------ID-------------------------P------Q--------------------2375.48.10 --I----------R-----N--Q--K---------ID---------------L---------P----R---------------M------2375U03 --I----------R---------------------IN-------------------------P---------------------------2375U01 --I----------R---------------------IN-------------------------P-------------------D-------2375U02 --I----------R---------------------ID-------------------------P---------------------------2375U04 --I----------R--------------G------ID-------------------------P---------------------------2375U05 --I----------R---------------------ID------------------------VP----------------------R-S--

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* * * * * * * * * HXB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF VFIS 26 Pretherapy 2612.48.01 --I-----------V---------------------N-------------------------P------E----------------------------2612.48.02 --------------V-------V-------------N-------------------------P------E----------------------------2612.48.04 --I-----------V---------------------N-------------------------P------E----------------------------1882612.48.06 --I-----------V---------------------N-------------------------P------E--------------------------------------058.00.11 --I---------V-V---------------------N-------------------------P------E----------------------------2612.48.05 --I-----------V---------------------N-------------------------P------E----------------------------2612.48.08 --I-----------V---------------------N-------------------------P------E----------------------------2612.48.07 --I-----------L---------------------N--------------------------------E----------------------------2612.48.10 --I-----------V---------------------N--------------------------------E----------------------------2612.48.11 --I-----------V---------------------N--------------------------------E----------------------------2612.48.12 --I-----------V---------------------N--------------------------------E----------------------------Posttherapy 3058.00.01 --I-----------V----------------I----N--------V----------------P-------V----------A----------------3058.00.02 --I-----------V----------------I----N-------------------------P-------V----------A----------------3058.00.03 --I-----------V----------------I----N-------------------------P-------V----------A----------------3058.00.07 --I-----------V----------------I----N-------------------------P-------V--------------------------T3058.00.09 --I-----------V----------------I----N-------------------------P-------V----------A----------------3058.00.12 --I-----------V----------------I----N-------------------------P-------V----------A----------------3058.00.04 --I-----------V---------------------N---------------L----------------E-------------T--------T-----058.00.05 --I-----------V---------------------N--------------------------------E----------------------------3 3058.00.10 --I-----------VV--------------------N--------------------------------E-----------------3

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* * * * * * * * * HXB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF 189Pretherapy 3122.48.08 --I---------------------------------N-------------------------P-------------I---------------------501.00.06 --I---------------------------------N--------L----------------P--L----------I---------------L-----501.00.07 --I---------------------------------N-------------------------------------------------------------501.00.08 --I---------------------------------N----------R--------------P-----------------------------------501.00.09 --I---------------------------------N--------------A----------P-----------------------------------501.00.10 --I---------------------------------N-------------------------P-----------------------------------L 501.00.11 --I-------------R----T--------------N--------L----------------P-GL----------I---------------L-----501.00.12 --I---------------------------------N-------------------------P-----------------------------------501.48.01 --I---------------------------------N-------------------------P-------T---------------------L-----501.48.02 --I---------------------------------N-------------------------P-------------I---------------L-----501.48.03 --I---------------------------------N-------------------------P-------------I---------------L-----501.48.04 --I-----------------------E---------N-------------------------P-------T----------A----------------501.48.06 --I---------------------------------N-------------------------P-------------I---------------L-----501.48.07 --I---------------------------------N-------------------------P-------------I---------------L-----501.48.08 --I---------------------------------N-------------------G-----P-------------I---------------L-----501.48.09 --I---------------------------------N-------------------------P-------------I---------------L-----501.48.10 --I-----------------------E---------N-------------------------P-------T----------A----------------501.48.11 --I-----------------------E---------N-------------------------P-------T----------A----------------501.48.12 --I---------------------------------N------------------------NT-------L----------A----------------VFIS 29 3122.48.01 --I---------------------------------N-------------------------P-----------------------------------3122.48.02 --I---------------------------------N-------------------------P-----------------------------------3122.48.03 --I---------M---------------------K-N-------------------------P-----------------------------------3122.48.04 --I---------------------------------N-------------------------P-------------I---------------------3122.48.09 --I---------------------------------N-------------------------P-----------------------------------3122.48.10 --I---------------------------------N-------------------------P-------------I---------------------3122.48.11 --I---------------------------------N-------------------------S-----------------------------------3122.48.12 --I---------------------------------N-------------------------P-------------I---------------------Posttherapy 3501.00.01 --I-------------R-------------------N--------L----------------P--L----------I---------------L-----3501.00.02 --I---------------------------------N-------------------------P-----------------L-----------------3501.00.03 --I-------------R-------------------N-------------------------P-----------------------------------3501.00.04 --I---------------------------------N-------------------------P-----------------------------------3501.00.05 --I---------------------------------N--------L----------------P--L----------I---------------L-----3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

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190* LTQIGCTLNF ------------------S-----------------------------------------------------------------------------------------------------------------------------037.00.09 --I-----------V---------------------N-------------------------Q-----------------------------------037.00.11 --I----------------R----------------N-------------------------Q-----------------------------------037.00.12 --I-----------V-------------------V-N-----------VT------------Q------------------------Y----------037.48.01 --I-----------V----------A----------N-------------------------Q-----------------------------------037.48.02 --I-----------V---------------------N-------------------------Q-----------------------------------037.48.03 --I-----------V---------------------N-------------------------Q-----------------------------------037.48.04 --I-----------V---------------------N-------------------------Q-----------------------------------037.48.05 --I-----------V---------------------N-------------------------Q-----------------------------------037.48.07 --I-----------V---------------------N-------------------------Q-----------------------------------037.48.09 --I-----------V---------------------N-------------------------Q-----------------------------------037.48.10 --I-----------V---------------------N-------------------------Q-----------------------------------037.48.11 --I-----------V---------------------N-------------------------Q-----------------------------------* * * * * * * * HXB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNL VFIS 30 Pretherapy 2649.48.01 --I---------LR----------------------N-------------------------Q-------------------------2649.48.03 --I----------R----------------------N-------------------------Q-------------------------2649.48.04 --I---------------------------------N-------------------------Q-------------------------2649.48.05 --I---------LR----------------------N-------------------------Q-------------------------2 649.48.06 --I----------R----------------------N-----R-------------------R-D-----------I-----------2649.48.07 --I----------R----------------------N-------------------------Q-------------------------2649.48.08 --I----------R----------------------N-------------------------Q-------------------------2649.48.09 --I----------R----------------------N-------------------------Q-------------------------2649.48.10 --I----------R----------------------N-------------------------Q-------------------------2649.48.12 --I---------LR----------------------N-------------------------Q-------------------------Posttherapy 3037.00.01 --I----------R----------------------N-------------------------Q-------------------------3037.00.02 --I-----------V---------------------N-------------------------Q-------------------------3037.00.03 --I-----------V---------------------N-------------------------Q-------------------------3037.00.04 --I----------------R----------------N--------V----------------Q-------------------------3037.00.05 --I-----------V---------------------N-------------------------Q-------------------------3037.00.06 --I----------R----------------------N-------------------------Q-------------------------3037.00.07 --I----------------R----------------N-------------------------Q-----------------------------------3037.00.08 --I-----------V---------------------N-------------------------Q-----------------------------------3 3 3 3 3 3 3 3 3 3 3 3

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191* LTQIGCTLNF --------------------------------------I------------------------------Q--N-------------------K-----P-----------L------S-------------------------------------------------------------------------------------------------190.48. --I----------------R---------------IN----------------V--------P------------------A----------------* * * * * * * * HXB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNL VFIF 05 Pretherapy 1877u01 --I---------------------------------N-------------------------P-------------------------1877u03 --I---------------------------------N---------------------------------------------------1877u04 --I---------------------------------N---------------------------------------------------1877u05 --I---------------------------------N-------------------------P----------I--------------P osttherapy 2104c04 2104c06 --I------------------------------Q--N-------------------K-----P------------------S------2190.48.01 --I-----------V---------------------N-------------------------P-------------------------2190.48.02 --I----------------R---------------IN----------------V--------P------------------A------2190.48.03 --I----------------R---------------IN----------------V--------P------------------A------2190.48.04 --I------------------------------Q-IN-------------------------P------------------A------2190.48.05 --I----------------R---------------IN----------------V---R----P------------------A------2190.48.06 --I----------------R---------------IN----------------V--------P------------------A------2190.48.08 --I----------------R---------------IN----------------V--------P------------------A------2190.48.09 --I----------------R---------------IN----------------V--------P------------------A------2190.48.10 --I----------------R---------------IN----------------V--------P------------------A----------------2190.48.11 --I----------------R---------------IN----------------V--------P------------------A----------------2 12

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192 TQIGCTLNF --------2 --I---------------------------------N-------------------K--E--P---------------------------------------------------------------------------------------------------------------------------292.48.06 --I---------------------------------N-------------------K--E--P-----------------------------------292.48.07 --I------------E--------------------N-------------------K--E--P-------------I-----------------------------------------10 --I---------------------------------N-------------------K--E--P------------------------------------------------------------------M----------------------------* * * * * * * * *XB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLL H VFIF 06 Pretherapy 1944.48.01 --I-------------------------------D-N-------E-----------K--E--P--------------------------1944.48.0 1944.48.03 --I-------------------------------D-N-------------------K--E--P--------------------------1944.48.04 --I---------------------------------N-------------------K--E--P--------------------------1944.48.05 --I---------------------------------N-------------------K--E--P--------------------------1944.48.06 --I---------------------------------N-------------------K--E--P--------------------------1944.48.08 --I---------------------------------N-------------------K--E--P--------------------------1944.48.09 --I---------------------------------N-------------------K--E--P--------------------------1944.48.10 --I---------------------------------N-------------------K--E--P--------------------------1944.48.11 --I---------------------------------N-------------------K--E--P--------------------------1944.48.12 --I---------------------------------N-------------------K--E--P--------------------------Posttherapy 2292.48.01 --I------I----V-------------------D-N-------------------K--E--P--------------------------2292.48.03 --I-------------------------------D-N-------------------K--E--P--------------------------2292.48.04 --I---------------------------------N-------------------K--E--P-------------I---------------------2292.48.05 --I---------------------------------N-------------------K--E--P-----------------------------------2 2 2292.48.08 --I-------------------------------D-N-------------------K--E--P-------------I-----------292.48.09 --I---------------------------------N-------------------K--E--P-------------I----------2 2292.48. 2292.48.12 --I--R---F--------------------------N-------------------K--E--P-------T----------------2292u01 --I-P------------------------------IN-------------------K--E--P------------------------2292u02 --I--------------------------------IN-------------------K--E--P------------------------2292u03 --I------I------------------------D-N-------------------K--E--P------------------------2292u04 --I---------------------------------N-------------------K--E--P----------------A-------2292u05 --I--------------------------------IN-------------------K--E--P------------------------

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193 * LLTQIGCTLNF ----L---------L-------N-L---------L-------N----L---------L---------L---------L---------L---------L-----* * * * * * * * XB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRN H VFIF 07 Pretherapy 79458.2 ----I--------------------------N---K-R------------------VT-------T-----I----------79458.3 ----I--------------------------N---K-R------------------VT-------T-----I----------79458.4 --I------I--------------------------N---K-R------------------VT-------T-----I----------79458.11 ----I--------------------------N---K-R------------------VT-------T-----I-----------9458.14 --I------I--------------------------N---K-R------------------VT-------T-----I----------7 Posttherapy 1678U02 --I------I-A----------------G-------N---K-R------------------VT-------T-----I----------1678U03 --I------I---------R----------------N---K-R------V-S---------VT-------T-----I----------1678U04 --I------I--------------------------N---K-R------------------VT-------T-----I----------1678U05 --I------I--------------------------N---K-R------------------VT-------T-----I----------1678U06 --I------I-P----------------G-------N---K-R------------------VT-------T-----I----------1678U07 --I------I--------------------------N---K-R------------------VP-------T-----I----I-----

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194 * LLTQIGCTLNF ------------------------L-------------------------------R-----------------------------------------------------------------------------------------------------------------234U04 --I-----------V---I----------N-----IN------------------------VP-----------------------------------234U06 --I-----------V---I----------------IN------------------------VP-----------------------------------234U08 --I-----------V---I----------------IN------------------------VP-----------------------------------* * * * * * * * XB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRN H VFIF 08 Pretherapy 1934U01 --I-----------V--------------------IN--------------------------------------------------1934U03 --I-----------V--------------------IN--------------------------------------------------1934U04 --I------I--------I-----------------N---------------------------------------I----------1 934U06 --I-----------V---I----------------IN--------------------------------------------------1934U08 --I-----------V--------------------IN--------------------------------------------------Posttherapy 2234.48.02 --I-----------V---I----------N-----IN------------------------VP------------------------2234.48.03 --I-----------V---I----------N-----IN------------------------VP------------------------2234.48.05 --I--------------------------N-----IN------------------------VP------------------------2234.48.06 --I----------NV---IM---------N-----IN----S--Q----------------VP------------------------2234.48.07 --I-----------V---IT---------N-----IN-----E------------------VP------------------------2234.48.08 --I-----------V---IT---------N-----IN------------------------VP------------------------2234.48.09 --I-----------V---I----------K-----IN----R-------------------VP------------------------2234.48.10 --I-----------V---I----------N-----IN------------------------VP------------------------2234.48.11 --I-----------V---IT---------N-----IN------------------------VP------------------------2234.48.12 --I-----------V---IT---------N-----IN------------------------VP------------------------2234U02 --I-----------V---I----------N-----IN------------------------VP-------------------------234U03 --I-----------V---I----------------IN------------------------VP------------------------2 2 2 2

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195 * LLTQIGCTLNF --I------V--V-V---------------------N---K----------------------V--------------------------------------------------------------208.48.12 --I------V--V-V--------------------IN---K----------------------V-----------------------------------208.48.02 --I------V--V-V---------------------N--------------------------V---R------------------------------208.48.04 --I------V--V-V---------------------N--------------------------V----------------------------------208.48.08 --I------V--V-V---------------------N---K----------------------V----------------------------------208.48.09 --I------V--V-V---------------------N---K----------------------VD---------------------------------8.02 --I------V--V-L----R---------------ID---K----------------------V-----------------F------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------* * * * * * * * HXB2 PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRN VFIF 09 Pretherapy 2208.r02 --I------V--V-V--------------------IN---K----------------------V-----------------------2 208.r03 2208.r04 --I------V--V-V--------------------IN---K----------------------V-----------------------2208.r05 --I------V--V-V--------------------IN---K----------------------V-----------------------2208.r06 --I------V--V-V--------------------IN---K----------------------V-----------------------2208.48.01 --I------V--V-V--------------------IN---K----------------------V-----------------------2208.48.03 --I------V--V-V--------------------IN---K----------------------V-----------------------2208.48.05 --I------V--V-V--------------------IN---K----------------------V-----------------------2208.48.06 --I------I--V-V--------------------IN---K----------------------V----------------------------------208.48.07 --I------V--V-V--------------------IN---K----------------------V---------------------------------2 2 2 2 2 2 Posttherapy 2686.4 2686.48 .03 --I------V--V-L----------------I---ID--RK----I-----------------V---------A-------A-----2686.48.04 --I------V--V-V--------------------ID---K----------------------V-----------------F-----2686.48.05 --I--R---V--V-L----R---------------IN---K------------V-------T-V-----------------A-----2686.48.06 --I------V--V-V----R---------------ID---K----------------------V-----------------A-----2686.48.07 --I------V--V-V--------------------ID---K------------V---------V-----------------A-----2686.48.08 --I------V--V-V--------------------IN---K------------V---------V-----------------A-----2686.48.09 --I------V--V-V--------------------ID---K----------------------V-----------------F-----2686.48.11 --I------V--V-V----R-----------I---ID---K----I-----------------V-----------------A------686.48.12 --I------VI---V----R---------------ID---K----------------------VD----------------F-----2 2686.C01 --I------V--V-V----R---------------ID---K----------------------V-----------------A-----2686.C02 --I------V--V-V----R---------------ID---K----------------------V-----------------A-----2686.C03 --I------V--V-V----R---------------ID---K----------------------V-----------------A-----2686.C08 --I------V--V-V----R---------------ID---K----------------------V-----------------A-----2686.C04 --I------V--V-V----R---------------ID---K----------------------V-----------------A-----2686.U21 --I------V----V----R---------------IN---K----------------------------------------A-----2686.U02 --I------V----V----R---------------IN---K----------------------------------------A-----2686.U31 --I------V----V----R---------------IN---K----------------------------------------A-----

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APPENDIX C LIGNMENT OF GAG SEQUENCES A

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HXB2 KARVLAEAMSQVTNSATIMMQRGNFRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSYKGRPGNFLQSRPEPTAPPEESFRSG..............VETTTPPQKQEPIDKELYPLTSLRSLFGNDPSSQ VSIS 01 Pretherapy 2272C01 -----------A.---A----K---KG---------------I----------------Q------E--G------------H---------N-------------F-...............-E---S----K-------TAA-K---------2272C03 -----------A.---A----K---KG----I----------I----------------Q---------G------------H---------N-------------V-RTEPTAPPEESFRFG-E---SP---KV-------A--K---------2272C04 -----------A.---A----K---KG---------------I----------------Q---------G------------H---------N-------............TAPPEESFRFG-E--------KV-------A--K---------2272C05 -----------A.---A----KD--KG---------------I-------K--------Q------E--G------------H---------N-------------F-RPEPTAPPEETFRFG-E---S----KV-------A--K---------2272C06 --------A.---A----K---KG---------------I----------------Q------E--R------------H---------N-------------V-RTEPTAPPEESFRFG-E--------KV-------A--K---------2272C07 ---------A.---A----K---KG---------------I----------------Q------E--G------------H---------NK------------V-RTEPTAPPEESFRFG-E---S----KV------TAA-K---------Posttherapy 2774A01 -----------A.---A----K---KG---------------I----------------Q------E--G------------H---------N-------------V-RAEPTAPPEESFRFG-E---S----KV-------A--K---------2774A02 -----------A.---A----K---KG---------------I----------------Q------E--G------------H---------N----I--------V-RAEPTAPPEESFRFG-E---S----KV-------A--K---------2774A03 -----------A.---A----K---KG---------------I----------------Q------E--G------------H---------N-------------V-RAEPTAPPEESFRFG-E---S----KV-------A--K---------2774A04 -----------A.---A----K---KG---------------I----------------R------E--G------------H---------N-------------V-RAEPTAPPEESFRFG-E---S----KV-------A--K---------2774A05 -----------A.---A----K---KG---------------I----------------Q------E--G------------H---------N-------------V-RAEPTAPPEESFRFG-E---S----KV-------A--K---------1972774O03 -----------A.--------K---KG---------------I----------------Q------E--G------------H---------N-------------V-RTEPTAPPEESFRFG-E---S----K--------A--K---------90A02 --KI-------A---HIT------------------------I-K-----K--------Q------E-----------------------F-----........-----A-R--F-E-------------------A--K---------2590A03 --KI-------A---HIT------------------------I-K---------------------------------------------------QSRPE...-----A----F-E-------------------A--K---------2590A05 --KI-------A---HITV-----------------------I-K-----K--------Q------E-----------------------F-----........-----A-R--F-E-------------------A--K---------2590O01 --KI-------A---HIT------------------------I-K--------------Q------------------------------------.....PTA-----A----F-E-----L-------------A--K---------2590O02 --KI-------A---SIM------------------------I-K---------------------E-----------------------------........-----A----F-E-------------------A--K---------2590O03 --KI-------A---HIT------------------------I-K-----K--------Q------E-----------------------F-----.....PTA-----A-R-KF-EA---------T--E-----A--K-F-----L-2590O04 --KI-------A---SIM------------------------I-K--------------Q------------------------------F-----.....PTA-----A----F-E-----L-------------A--K---------2590O05 --NI-------A---HIT------------------------I-K---------------------E-----------------------------........-----A----F-E-----------T-------A--K---------2590O06 --KI-------A---SIM------------------------I-K---------------------------------------------------QSRPE...-----A----F-E--A----------------A--K---------2590O07 --KI-------A---SIM------------------------I-K---------------------------------------------------.....PTA-----A---KF-E----------TV-------S--K---------2774O01 -----------A.---A----K---KG---------------I----------------Q------E--G------------H---------N-------------V-RTEPTAPPEESFRFG-E---S----K--------A--K---------774O02 -----------A.--------K---KG---------------I----------------Q------E--G------------H---------N-------------V-RTEPTAPPEESFRFG-E---S----K--------A--K---------2 2774O04 -----------A.---A----K---KG---------------I----------------Q---------G------------H---------N-------------V-RPEPTAPPAESFRFG-E--------K-------TAA-K---------2774O05 -----------A.--------K---KG---------------I----------------Q------E--G------------H---------D-------------V-RPEPTAPPAESFRFG-E--------K--------A--K--------PHXB2 KARVLAEAMSQVTNSATIMMQRGNFRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSYKGRPGNFLQSRPE........PTAPPEESFRSGVETTTPPQKQEPIDKELYPLTSLRSLFGNDPSSQ VSIS 03 Pretherapy 1794U03 --KF-------A---SIM------------------------I-K-----------------------I---------------------------QSRPE...-----A----F-E----------T--------A--K---------1794U04 --KF-------A---SIM------------------------I-KD--------------------------------------------------QSRPE...-----A----F-E-------------------A--K---------1794U05 --KF-------AP--SIM------------------------I-K--------------Q---------Q--------------------------QSRPE...-S---A----F-E--A----------------A--K---------1794U06 --KF-------AP--SIM------------------------I-K--------------Q---------G--------------------------QSRPEPTA-----A----FRE----------T--------A--K---------1794U07 --KF-------AP--SIM------------------------I-K--------------Q---------Q--------------------------QSRPE...-----A----F-E-------------------A--K---------1794U08 --KF-------AP--SIM------------------------I-K--------------Q---------Q---D----------------------QSRPE...-S---A----F-E----------T--------A--K---------1794U09 --KI-------AP--SIM-------------G----------I-K---------------------------------------------------QSRPE...-S---A----F-E--AI---------------A--K---------1794U11 --KI-------AP--SIM-------------G----------I-K-------------R-------------------------------------QSRPE...-S---A----F-E--S----------------A--K---------Posttherapy 25

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198 PPQKQEPIDKELYPLTSLRSLFGNDPSSQ -------T--G----A--------------------------SA----------L---------------SA----------L--------T-QG----A-------------------T-QG----A-------------------T-QG----A--------------P----T-QG-F--A------CTT--.. -------T-QG----A-------Y-----------T-QG----A-------Y-----------R------SA-------------------R------SA-------------------R------SA----------L1779u05 ----------TTN-------------A-----------I-K-------------------------------------H-----------------------F-E--A--------T---R---A---------HXB2 KARVLAEAMSQVTNSATIMMQRGNFRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSYKGRPGNFLQSRPEPTAPPEESFRSGVETTTPPQKQEPIDKELYPLTSLRSLFGNDP VSIS 25 Pretherapy 2473.48.01 ------------A-------L----R------------I-K---------------------E---------------HQ------I---T-----------F-E-----S----L----M---A--K-------2473.48.02 ------------A-------L----R------------I-K---------------------E---------------HQ------I---T-----------F-E-----S----L----M---AF-K-------2473.48.03 ------------A-------L----R------------I-K---------------------E---------------HQ------I---T-----------F-E-----S----L----M---A--K-----E-2473.48.06 ------------A-------L----R------------I-K---------------------E---------------HQ------I---T-----------F-E-----S----L----M---A--K-------2471 -A--L-------E----------MK-2473.48.12 ------------A-------L----R------------I-K---------------------E----------------Q------I---T-----------F-E-----S----L----M---A--K------HXB2 KARVLAEAMSQVTNSATIMMQRGNFRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSYKGRPGNFLQSRPEPTAPP...EESFRSGVETTTPPQKQEPIDKELYPLTSLRSLFGNDPSSQ VSIS 27 Pretherapy 5761.48.02 -----------------K----G---------------I---------------------------------------H---------N--------APP-----F-E----------LK-------A--K-------L-5761.48.03 -----------------K----D---------------I---------------------------------------H---------N--------APP-----F-E----------MK-------A--K-------L-5761.48.04 -----------------K----G---------------I---------------------------------------H---------N--------APP-----F-E----------LK-------A--K-------L-5761.48.06 -----------------K----G---------------I---------------------------------------H---------N--------APP-----F-E----------MK-------A--K-------L-5761.48.07 -----------------K----G---------------I---------------------------------------H---------N--------APP-----F-E----------LK-------A--K-------L-5761.48.08 -----------------K----G---------------I--------------R------------------------H---------N--------APP-----F-E-A---H----VK-------A--K-------FTI 5761.48.10 -----------------K----G---------------I---------------------------------------H---------N--------APP-----F-E----------MK-------A--K-------L-5761.48.11 -----------------K----G---------------I---------------------------------------H---------N--------APP-----F-E----------MK-------A--K-------L-5761.48.12 -----------------K----G---------------I-------------------------P-------------H------------------APP-----F-E----------LK-------A--K-------L-Posttherapy 6063.00.02 KARV-----------------K--------------------I-----------------------E---------------H---------N--------APP-----F-E---N------LK-------A--K---------6063.00.03 KARV------H----------K--------N-----------I-----------------------E---------------H---------N--------APP----KF-E----------LKE------A--K-------L-6063.00.04 KARV------------A----K--------------------I--------E------------------------------H------------------APP-----F-E----------LK-------A--K-------L-6063.00.05 KARV-----------------K----G---------------I---------------------------------------H------------------APP-----F-E----------LK-------A--K-------L-6063.00.06 KARV----V------------K--------------------I-----------------------E---------------H---------N-----G--APP-----F-EG---------LK-------A--K-------L-6063.00.07 KARV----V-H----------K--------N-----------I-----------------------E---------------H---------N-----G--APP-----F-E----------LRE------A--K-------L-6063.00.09 KARV-----------------K--------------------I-----------------------E---------------H---------N--------APP-----F-E----------LKE------A--K-F-------6063.00.11 KARV------H----------K--------T-----------I----------------------QE---------------H---------N--------APP-----L-E---N------LK-------A--K---------6063.00.12 KARV---G-------------K--------------------I-----------------------E---------------H---------N--------APP-----F-E---N------LK-------A--K------------SSQ -----------------E ---A--L---HXB2 KARVLAEAMSQVTNSATIMMQRGNFRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSYKGRPGNFLQSRPEPTAPPEESFRSGVETTT VSIS 04 Pretherapy 1669u01 ----------P-N-------------N-----------I----K-----------R----------------------H-----------------------F-E----1669u02 ----------P-N-------------T-----------I-K--------------G----------------------H-----------------------F-E----1669u03 ----------P-N-------------T-----------I-K-----I--------G----------------------H------Y----------------F-E----1669u05 ----------P-N-------------T---------E-I-K---------Y----G----------------------H-----E-P---------------F-E--A-1669u04 ------------N-------------T---------E-I-K--------------G----------------------H-----------------------F-E--A-1691u01 ---------T-N-------------T-----------I-Q-----FI-------G----------------------H--------------------N--FWE----1691u03 -I---A-T-N--------K----T-----------I-Q-----W--------G----------------R-----H----R---------------N--F-E----1691u05 ----------T-N-------------T-----------I-Q-----M--------G------G---------------H-----------------------F-E----1691u06 ----------T-N-------------T-E-------R-I-Q-----L--------G------------------L---HT----------------------F-E---Posttherapy 1779u01 ----------P-N-------------T-----------I-K--------------G----------------------H-----K---N-------------F-E----1779u02 ----------P-N-------------T-----------I-K--------------G----------------------H---------N-------------F-E----1779u03 ----------P-N-------------T-----------I-K--------------G----------------------H-----------------------F-E--A---S---F-E --G-T-I---T HL------------------------------I-K ---------R -------------3.48.1

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199 HXB2 SIS 2ether32.4832.4832.4832.4832.4832.4832.4832.4832.4832.48stthe15.0015.0015.0015.0015.0015.0015.0015.0015.0015.0015.00 8 ap.0.0.0.0.0.0.0.1.1.1ra.0.0.0.0.0.0.0.0.0.1.1 y 1 2 3 5 6 8 9 0 1 2 py 1 2 3 4 5 6 7 8 9 1 2 K K K K K K K K K K K KAR VL AEA-----------------------------------I----I----I----I----I----I----I----I----I----I----I---MS------------------------------------V----QVT--------------------------------------------------------------NS-----------------------------------------ATI-A--A--A--G-TA---A---A---A---A---A---A---A---A---A---A---A---A---A---A---A---AVMM---------QRG-------NF RNQL--P-L--P-L--P-L--P-L--P-L--P-L--P-L--P-L--P-L--P-L--P-L--P-L--P-L--P-L--P-L--P-L--P-L--P-L--P-L--P-L--PRK IVK CFN CG KEG--------------------------------------------------------------------------------------------------------HT-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I ARN-KH-KH-KH-KH-KH-KH-KH-KH-KH-KH-KH-KH-KH-KH-KH-KH-KH-KH-KH-KH-KH CR-----------------------------------------APR--------------------------------------------------------------KK-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R-R GCW--------------------------------------------------------------KC-----------------------------------------GKE--------------------------------------------------------------GH---------------------------------------------------------QMK--------------DC TER--------------------------------------------------------------------------------------------------------QA-----------------------------------------NFL------------------------------------------D-------------------GKI--------------------------------------------------------------WP-----------------------------------------SYK-H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--HGR-----------------------------------------PGN--------------------------------------------------------------FL---------QSR-N--NPE PTA---S----S----S----S----S----S----S----S----S----S----S----S----S----S----S----S----S----S----S----S----SPP EES FR SGV ET-E---E---E---E---E-I-E---E---E---E---E---E---E---E---E---E---E---E---E---E---E---E-TTP--------------------------------------------------------------PQS-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-SKQE------------------------------------------------R-------------PI-----------------------------------------DKE--------------------------------------------------------------LY-----------------------------------------PLT-SA-SA-SA-SA-SV-SA-SA-SA-SA-SA--A--A--A--A--A--A-SA--A--A--A--A SL-----------------------------------------RSLK--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K-FG-----------NDP SS---P--------E---------------E---E-------E-------E--PE---------------E--------------Q VPr53--.-----------F53--.-----------F53-----.----N--------F53-----.----N--------Y53-------.-------N--------F---53 -------.---------N--------F---53 -------.---------N--------F--K53 -------.---------N-------KF---53 -------.---------N--------F---53 -------.---------N--------F--Po57AK-------.---------N--------F---57AK-------.---------N--------F---57AK-------.---------N--------F---57AK-------.---------N--------F---57AK-------.---------N--------F---57AK-------.---------N--------F---57AK--K----.---------N--------F---57AK-------.---------N--------F---57AK-------.---------N--------F---57AK-------.---------N--------F---57AK-------.---------N--------F--

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200 HXB2 VFIPret1722172217221722172218501850185018501850185018501850185018501850 Post2088208820882088208820882088208820882088208820882088208820882088208820882088208820882088 S 0her001002005a02a03.48.48.48.48.48.48.48.48.48.48.48the.48.48.48.48.48.48.48.48.48.48.48.48001002004006007a01a02a03a04o05 2 ap .0.0.0.0.0.0.0.1.0.1.1ra.0.0.0.0.0.0.0.0.0.1.1.1 y 1 2 3 4 5 7 8 0 9 1 2 py 1 2 3 4 5 6 7 8 9 0 1 2 LAE-----------------------------------------------------------------------------------------------------------------AM-----------------------------------------------R---------------------------SQV-H-----H-----H--H--------H--H--H--H--------H--H-----------------------------------------H--------H---------------TN---------------------------------------------------------------------------SAT-------------------------------------------------------------------------------------------------------------T---IMV-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-VMQR--------------------------------------------------------------------------------------------------------------------------------------------------GN--------------FRN QR--------------------------------------------------H----------------------------------------------------------------------------------------------------KIV-T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--TKC-----------------------R---------------------------------------------------FNC-----------------------------------------------------------------------------------------------------------------GKE----------------------------------------------------------------T------------------------------------------------GH---------------------------------------------------------------------------TARI--I--I--I--I--I-KI--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I-NC---------------------------------------------------------------------------RAP-------------------------------------------------------------------------D---------------------------------------RK-------------------------------------------------------------------------------------------------------------KGC-----------WK-----CGK EG-R----R----R----R----R----R----R----R----R---------R---------R----R----R----R----R----R----R----R----R----R----R----R----R----R----R----R----R----R----R----R----R----R----R----R----R----R--HQM KD CTE RQ ANF---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------LG---------------------------------------------------------------------------KIW-----------------------------------------------------------------------------------------------------------------PSY--H--H-PH--H--H--H--H--H--H--H--H--HA-H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--R--H--H--R--H--H--R--R--H--R KG---------------------------------------------------------------------------RPG-------------------------------------A---------------------------------------------------------------------------NF---------------------------------------------------------------------------LQS--------------------------------------N--------------------------------------------------------------------------RP---------------------------------------------------------------------------EPT-----A-----------------------------------------------------------------------------------------------------------AP--------------PEE SF-------------------------------------------------------------------------------------------------------------------------------------------------------RSG-F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--FVEE-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E.TT.--.--.--.--.--.--.--.--.--K--.--.--.--.--.--.--.--.--.--.--.--.--.--.--.--.--.--.--.--.--.--.--.--.--.--.--.--.-TP---------------------------------------------------------------------------PQKS--S--S--S--S--S--S--S--S-----S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S-QE---------------------------------------------------------------------------PID-M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--MKE-D-D-D-D-D--D--D--D--D--D--D--D--D--D--D--D--D--D--D--D--D--D--D--D--D--D--D--D--D--D--D--D--D--D--D--D--D--DLYP-----------LT-A--ASLR SL-K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K----K--FGN DP SSQ -----------------------------------------------------------A-----------------------A----------------------------A----------------------------A----------------------------A----------------------------A----------------------------A----------------------------A----------------------------A---------------P------------A----------------------------A----------------------------A----------------------------AP--E------------------------A----------------------------A----------------------------A----------------------------A----------------------------A----------------------------A----------------------------A----------------------------A----------------------------A----------------------------A----------------------------A----------------------------A----------------------------A----------------------------A----------------------------A----------------------------A----------------------------A----------------------------A----------------------------A----------------------------A----------------------------A----------------------------A-------

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201 HXB2 KAR VL AEA MS QV. TN SAT IM MQR GN FRN QR KIV KCF NC GKE GH TAR NC RAP RK KGC WK CGK EG HQM KD CTE-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------RQ-----------------------------------------------------------------------ANF---------------------------------------------------------------------------P---------------------------D---LGK-----------------------------------------------------------------R--R-----R-----R--R--R--------------------IW-----------------------------------------------------------------------PSY--H-PH--H--H--H--H--H--H--H--H--H--H--N--H--H--H--H--H--H--H--N--N--N--H--N--H--N--N--N--H--H--H--N--N--N--N KG-----------E------E----------------------------------------------------RPG------------------------------------------------------------G--------------S-------------------------------NF-----------------------------------------------------------------------LQS---------------------------------------------------------------------------R-------------------------------RP------------------------K---------------K-K-K---K---K-K-K-------K---K-KEP.--RG-R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R ..PEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPEPE .TAP--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P-VP--P--P--P--P--P--P--P--P--P--P--P-PP--------------------------------T--------------T-----------------------EES-----------------------------------------------------------------------------------------------------------FR-----------------------------------------------------------------------SGVF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-E ET-----I-----------------------------------------------------------------TTPA--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--AN-A--A--A--A--A--A-LA--A--A--A--A--A-PQS-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-L-S-S-SKQEQ-GQRGQ-GQ-GQ-GQ-GQ-GQ-GQ-GQ-GQ-GQ-GQ-GQ-GQ-GQ-GQ-GQ-GQ-GR-GQ-GQ-GQ-GQ-GQ-GQ-GQ-GQ-GQ-GQ-GQ-GQ-GQ-GQRGQ-GQ-G PIT--T--T--T--TDKE LY-G---G---G---G---G---G---G---G---G---G---G---G---G---G---G---G---G---G---G---G---G---G---G---G---G---G---S---G---G---G---GP--G---G---G---G---G-PLT--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A SL-----------------------------------------------------------------------RSLK--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K-FG-----------------------------------------------------------------------NDP-----------------------------------------------------------------L----E------------------------------------SS----------------------------------------------------------------L------Q VFIS 10 Pretherapy 1470A01 ..............--V--------------T-----------I----------------------1470A02 ..............--V--------------T-----------I----------------------1470A03 ..............--V--------------TA----------I----------------------1470A04 ..............--VN--------G----T-----------I----------------------1470C02 ...I-------A.---VN-------------T-----------I-----------------------1470C03 ---I-------A.---VN-------------T-----------I----------------------T--1470C04 ---I-------A.---VN-------------T-----------I----------------------T--1470C05 ..-I-------A.---VN-------------T-----------I----------------------T--1470C06 ---I-------A.---VN-------------T-----------I----------------------T--1470C07 -SKI-------A.---VN-------------T-----------I--------E-------------T--1470C08 --KI-------A.---VN-------------T-----------I--------------------P-T--1470O01 -----------A.-.NSN-----S-------TA----------I----------------------T--1470O02 -----------A.Q--VS-------------T-----------I---------------------LT--1470O05 ..K--------APP--VN-------------T-----------I---------------------LT--1470O06 ...........APP--VN-------------TA----------I----K-----------------T--1470U01 -----------A..TNSN-------------T----D------I----------------------T--1470U02 -----------A..TNSN-------------T-----------I----------------------T--1470U03 -----------A..TNSN-------------T-----------I----------------------T--1470U04 -----------A..TNSN-------------T-----------I----------------------T--1470U05 -----------A..TNSN-------------T-----------I----------------------T-Posttherapy 1664.48.17 ----------EA.Q--VG-------------T-----------I---------------------LT--1664.48.18 -----------AT---VN-------------T-----------I---------------------LT--1664.48.19 -----------AT---VN-------------T-----------I---------------------LT--1664.48.21 -----------A.Q--VG-------------T-----------I---------------------LT--1664.48.22 -----------AT---VN-------------T-----------I-----------S---------LT-N1664.48.23 -----------AT---VN-------------T-----------I--D--G---------------LT--1664.48.24 -----------A.Q--VG-------------T-----------I---------------------LT--1664.48.25 -----------AT---VN-------------T-----------I---------------------ST--1664.48.27 -----------AT---VN-------------T-----------I---------------------LT--1664.48.28 -----------AT---VN-------------T-----------I--D------------------LT--1664U01 ---I-------A.---VN-------------T-----------I----------------------T--1664U02 -----------A.Q--VG-------------T-----------I---------------------LT--1664U03 -----------A.Q--VN-L-----------T-----------I---------------------LT--1664U04 -----------A.Q--VS-------------T-----------I--------------R------LT--1664U05 ---I-------A.---VN------------RT-----------I-K--------------------T--1664U06 -----------AT---VN-------------T-----------I---------------------LT-

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202 HXB2 KAR VL AEA MS QVT NS ATI MM QRG NF RNQ RK IVK CFN CG KEG HT ARN CR APR KK GCW KC--R---R---R---R---R--------------------------------------------------------------------------------------------------*-GKE----------------R-----------------R--RK--------------------------------------------------GH------------ET--R---RP------R-R----------------------------QMK-----------------E---PI-P----E--E--E--EP--P--------HI--I--I------------------------------DC--------------------N--------------------------------------TERS--S--S--S--SG----S-----------S--------S--S--S--------------S--S--S--S--S--S--S--S--S--S-QA----------------------------------P------------------------NFL-----------------------------------------------------------------------------------------GKI-----------------------------------------------------------------------------------------WP-----------------------------------------------------------SYK-H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--HQ-H--H--H--H--H--H--H--H--H--H--H--HGR---------------K-K-------------K---------------------------PGN-----K--K--K--K--------------------------------------------------------------------------FL-----------------------------------F----------------------SQSR-N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--NPET-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-TPTA-----------------------------------------------------------------V-----------------------PP-------------EES FR---S---S---S---S---S---S---SG--S-T-S---S---S---S-N-S---S---S-N-T---S-NVS---S---S---S---S---S-N-S---S---S-N-S---S-N-S---S SGVF-EF-EF-EF-EF-EF-DF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-EF-E ET-----------------------------------N-----------------------TTP-.--.--.--.--.--.--.--.--.--.--.--.--.--.--.--.--.-N.--.--.--.--.-N.--.--.--.--.--.--.--.PQS-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-SKQE--D--D--D--D--D--D--D--G--D--D--D--D--D--D--D--DN-D--D--D--D--D--D--D--D--D--D--D--D--D--D PIKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKKK DKE-.--.--.-Y.--.--.--.--.--.--.--.--.--.--.--.--.--.K-.--.--.--.--.--.--.--.--.--.--.--.--.LY-----------------------------------------------------------PLT--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A--A SL-----------------------------------------------------------RSLK--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K-FG-----------------------------------------------------------NDP-G--G--G--G--G--G--G--G--G--G--G--G--G--G--G--G--G--G--G--G--G--G--G--G--G--G--G--G--G--GSS-----------------------------------------------------------Q VFIS 11 Pretherapy 1446U01 ...I----I--------V---K--------------------I-----------1446U02 ...I----I--------V---K--------------------I-----------1446U03 ...I----I--------V---K--------------------I-----------1446U04 ...--------------V---K--------------------I-----------1446U05 ...I-------------V---K--------------------I-----------Posttherapy 1656U01 -----------M---T-V---K--------------------I-------------D1656U10 ---------------TAV------------------------I-------T------1656U11 ---------------TAV------------------------I-------T------1656U12 ---------------TAV---K--------------------I-K-----P------1656U02 -----------M---T-V---K--------------------I--------------1656U03 -----------M---T-V---K--------------------I-------P------1656U04 -----------M---T-V---K--------------------I--------------1656U05 -----------M---T-V---K--------------------I--------------1656U06 -----------M---T-V---K--------------------I-------T-----G1656U07 ---------------TAV------------------------I-------T------1656U08 ---------------TAV----------------------R-I--------------1656U09 ..........-----TAV---K------------------R-I--------------1656C08 -S-----------I-FFL-----H-T----------------I--------Q-----1656.48.01 ....-----------TAV------------------------I--------------1656.48.02 ....-----------TAV------------------------I--------------1656.48.03 ....-----------TAV------------------------I-K------------1656.48.04 ....-----------TAV------------------------I-*------------1656.48.05 ....-----------TAV------------T-----------I--------------1656.48.06 ....-----------TAV---K--------------------I-*------------1656.48.07 ....-----------TAV-------------------S----I-K------------1656.48.08 ....-------M---T-V---K--------------------I-*------------1656.48.09 ....-----------TAV---K--------------------I-K------------1656.48.10 ....S------M---T-V---K--------------------I-K------------1656.48.11 ....-----------TAV---K--------------------I-K------------1656.48.12 ....-----------TAV------------------------I-K-----------

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203 HXB2 KAR VL AEA MS QVT NS ATI MM QRG NF RNQ RK IVK CFN CG KEG HT ARN CR APR KK GCW KC GKE GH QMK DC TER QA NFL---------------------------------------------------------------------------------------------------------------V---------V-----------------------LGKIW--R----R----R----R----R----R----R----R----R----R----R----R----R----R-GKI---------------------------------------------------------------------------------------PSY--H--H--H--H--H--H--H--H--H--H--H--H--H--H WP------------C-------C-------------------------------------KG---------------------------SYK-H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--H-RPG-----------------------------------------GR----------------------------------------------------------NFK-E-K-K-K------------------PGN---------------------------------------------------------------------------------------LQS-----------------------------------------FL-------------F--------------------------------------------RP-------------------S-------QSR---------------------------------------------------------------------------------------EPT---------D-------------------------------PE-----------G--S-------------------------------------------AP--------------G-----G------PTA---------------------------------------------------------------------------------------PEE---------------------Q------------------KPP----------------------------------------------------------SF---------------------------...EES....................................................................................RSG-F--F--F--F--F--F--L--L--L--L--L--L--F--L..FR........................................................VEE-E-E-E-E-E-E-E-E-E-E-E-E-E...FSQ....................................................................................TTT--.--.-A.-A.-A.--.--.--.--.--.--.--.-A.--. ..SR........................................................PP-S-S-S-S-S-S-S-S-S-S-S-S-S-S ...PEP.EP.EP.EP.EP....EP.EP.EP.EP.EP.EP.EP.EP.EP.EP.EP.EP.EP.EP.EP.EP.EP.EP.EP.EP.EP.EP.EPQKQ-----------------------------------------..TASASASASA..SASASASASASASASASASASATASASASASASASASASASASASAEP-Q-QGQ-Q-H-Q-Q-Q-Q-Q-Q-Q-Q-Q ..EPP-PP-PP-PP-PP-..-PP-PP-PP-PP-PP-PP-PP-PP-PP-PP-PP-PP-PP-PP-PP-PP-PP-PP-PP-PP-PP-PP-PP-IDK-------Y-----Y*--------------------------ES----------------------------------------------------------ELN-N-N-N-S-D-N-N-N-N-N-N-N-NFRS--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--FYPLP--P--P--P--P--P--P--P--P--P--P--P--P--P-GV-E-G-G-G-G-E-G-G-G-G-G-G-G-G-G-G-G-E-G-G-G-E-G-G-G-G-G-G-GTSA-A-A-A-A-A-A-A-A-A-A-A-A-A---Q---N-P--A------------ETT-R--------------R----------------------------------G-A---------G-A---------------------LRS-----------------------------------------TP--------A-----------------------------------------A-A-A-A-LF---------------------------PQK---S--S-RS--S-----S-RS-RS-RS-RS-RSRRS-RS-RS-RS-RS-R---S-RS-RS-----S-RS-RS-RSR-S--S--SR-GND--------------------E--------------------QE--------------------------------------------------E-E-E-E-PS---------------------------PID-V--T--T--T-QT--V--T--T--T--T--T--T--T--T--T--T--T--V--T--T-QT--V--T--T--T-QT-QT-QT-QT-SQ -----------E ---KE-------------LYP LT---A---A---A---A---A---A---A---A---A---A---A---A---A---A---A---A---A---A---A---A---A---A---A---A---A---A---A---A---A SLR--------------------------------------------------------------------------------------SL---------------------------------------------------------FGN--------------------------------------------------------------------------------------DPS--------------------------------------------------------------L-------------L--L--L--LSQ------VFIS 12 Pretherapy 1738A02 -----------------V------V-----TI----------L--------R-------R-----------1738A03 --------------P--V------V-S---TI----------L----------------R-----------1738A04 -----------------V------I-----TI----------L----------------R------N----1738A05 ---------------T-V------V-D---TI----------L----------------R-----------1738A06 --------------A--V------V-----PI------R---L----------------R-------------1738O01 -----------------V------V-----PI------R---L--------------------------------1738O03 -----------------V------V------I----------I----------------R---------------1738O04 -----------------V------V-----TI----------L----------------R---------------Posttherapy 1943.48.01 ---------------T-V-----SV-----TI----------L----------------R---------------1943.48.02 ---------------T-V------V-----TI----------L----------------R---------------1943.48.03 ---------------T-V------V-----TI----------L----------------R-E-------D-----1943.48.04 ---------------T-V------V-----TI----------L----------------R---------------1943.48.05 ---------------T-V------V-----TI------R---L----------------R---------------1943.48.06 ---------------T-V------V-----TI----------L----------------R---------D-----1943.48.07 --------V------T-V------V-----TI----------L----------------R---------------1943.48.08 -----------------V------V-----TI----------L----------------R---------------1943.48.10 ---------------T-V------V-----TI----------L----------------R---------------1943.48.11 --------------PT-V------V-----TI----------L----------------R---------D-----1943.48.12 ---------------T-V------V-----TI----------L----------------R---------------1943A01 -----------------V------V-----TI----------L----------------R---------------1943A03 ---------------V-V------V-----PI------R---L--------------------------------1943A05 -----------------V------V-----PI------R---L--------------------------------1943O11 ---------------T-V------V-----TI----------L----------------R---------------1943O12 ---------------T-V------V-----TI----------L----------------R---------D-----1943O13 ---------------T-V------V-----TI----------L----------------R---------------1943O14 ---------------T-V------V-----TI----------L----------------R---------D-----1943O15 ---------------T-V------V-----TI----------L----------------R---------D-----1943O16 ..........-----TPV------V-----TI----------L----------------R---------D-----1943R01 ..........-----TPV------V-----TI----------L----------------R---------D-----HBX2 LAEAMSQVTNSATIMMQRGNFRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQANF VFIS 14 Pretherapy 1329u01 -----------TA----K---------I----------I-K------------------------------1329u02 -----------TA----K---------I----------I-K------------------------------1329u03 -----------TA----K---------I----------I-K------------------------K-----1329u06 -----------TA----E--------TI----------I-K------------------------------1329u07 ----I------TA----K---------I----------I-K-----------------------------Posttherapy 1576.48.02 -----------TA----K--------FI----------I-K------------------------------1576.48.06 -----------TA----K---------I----------I-K-----M------------------------1576.48.07 -----------TA----K---------I----------I-K-----K-----R--------R--------I1576.48.08 -----------TA----K---------I----------I-K------------------------------1576.48.10 -----------TA----K---------I--V-------I-K---T------------------R-------1576.48.11 -----------TA----K-------E-I----------I-K-----M---------V--------------1576.48.12 -----------TA----K---------I----------I-K----R-------------------------1628u01 -----------TA----K------S--I----------I-K-------------------------K----1628u02 -----------TA----K---------I----------I-K------------------------K-----1628u03 -----------TA----K---------I----------I-K-------------------------K------R----H-----------------------F-E--A.-S--1628u04 -----------TA----K------S--I----------I-K--------------------------------R----H-----------------------F-E---.-S----Q---N-P--A------------1628u07 -----------TA----K------S--I----------I-K--------------------------------R----H-----------------------L-E---.-S----Q---N-P--A------------1628u13 -----------TA----K---------I----------I-K----L---------------------------R----HS-GQ-------------------F-E--A.-S----Q---N-P--A------------1628u17 -----------TA----K---------I----------I-K--------------------------------R----H--GQ-------------------F-E--A.-S----Q---N-P--A------------

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204 HXB2 KAR........................----------------------------------------T------VL...............----------------S---------------AEA.....................V-------------------------------------------------MS..............----------V----------------------QVT.........KA-KA--A--A--A--A--A--A--A--A--A--A-EA--A--A--A--A--A--A--A--ANS-A-A.A-----------A-A-A-A-A-A-A-A---A-A---A---A-ATIPILPILPILQIVQIVQIVQIVQIVPILPILPILPILPILPILPILPILQIVPILPILQIVPILQIVPILQIV MM-------------------------------T---------------QRG-------K--K--K--K--K--K--------------------------K-----K--K-----K--K--KNF-----------------------------------------------RNQ-----------------------------------------------------------------------............................................NF.... .....................-...-...-...-...-...-...-...-...-...-...-...-...-...-...-...-RNQ-...-...RKI-----------F--VK CFN-----------------------------------------------------------------------------------------------------------------------CG-----------------------------------------------KEG-------------------V-R---------------------------V--------V-----V-----VHT-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I ARN-K--K--K--K--K--E--K--K--K--K--I--K--K--K--K--K--K--K--K--K--K--K--K--KCR---------APR-----KK GCW-------A---------------------------------------------------------------------------------------------------------------KC GKE GH QMK DC--------------------------------------------------------------------------N--------------------TER---------I--I--P-------------------------------------------------------QAN-P-----------V--------------------------------------------------V------........RQRQRQRQ.................................. ..F..S..-..-AN-AN-AN-AN-..-..-..-..-..-..-..-..-..-..-..-..-..-..-..-..-..LG-----------------------------------------------KIW-------------------V---------------------------------------------------PS-----------------------------------------------YKG-----------------------------------------------------------------------RP-----------------------------------------------GNF-----------------------------------------------------------------------LQ--------------------------I--------------------SRP-K--K--K--------------K--K--K--K--K--K--K--K--K-----K--K--K--K---------EP-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S ...RLERLERLERPERPERPERPERPERLERLERLERLERLERLERLERLERPARLERLERLERLERPARPERPA .TP-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-P-PAPP------------------------D----------------------------------------------EEA-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-ASFR--S-------------------LG-----------------------------------------K-----SGFQF-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-FVE.D-.R-NE-NE-.E-.E-.E-.ET.E-.E-NE-NE-NE-.E-.E-NE-NE-.E-NE-.E-.E-.E-.E-.E-. ....DC.C.............C.C.C.....C.C...C............ ......RDERDE..................MDERDERDE......RDERDE...RDE.............TT... TTNANANA--------..--NANANA----NANA--NA---A-------TPP--S--S--S------------DTS--S--S--S--S--S--S--S--S--S--S--S--S--S--------S QK-------------QEP ID-T---T---T-----------------------T---T---T---T---T--GT---T---T---T---T---T---T---T-----------T-KEL----------------------------------------------------------------G------YP-----------------------------------------------LTS-A--A--A-SA-SA-SA-SA--A--A--A--A--A--A--A--A--A--A--A--A--A-------SA--ALR-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K SLF-----------------------------------------------------------------------GND----------S---------------E--------------------------------------------PS---------SQ --VFIS 15 Pretherapy1868C01 --------1868C02 --------1868C03 ----------1868U01 ----------1868U02 -----------------1868U03 --------------R------1868U04 ---------------------1868U05 ---------------------Postherapy2209.48.02---------------------2209.48.04---------------------2209.48.06---------------------2209.48.07---------------------2209.48.08----G----------------2209.48.09---------------------2209.48.10---------------------2209.48.11---------------------2209.48.12---------------------2209O02 ---------------------2209O03 ---------------------2209O04 ---------------------2209O07 ---------------------2209A03 ---------------------2209A06 ---------------------2209A07 ----------E----------

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205 HXB2 17 erap48.048.048.048.048.048.048.048.148.1hera00.000.000.000.000.000.000.100.100.148.048.048.048.048.048.048.048.048.048.148.148.1 y 1 2 4 5 6 8 9 0 1 py 2 3 5 6 8 9 0 1 2 1 2 3 4 5 6 7 8 9 0 1 2 LAE-----------------------------------------------------------------------------------------AM-----------------------------------------------------------SQV-----------------------------------------------------------------------------------------TN----I---------------------I--------------------------------SAT-------T---------------------------------------------------------------------------------IM------------T----------------------------------------------MQR--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K GN---------------------------------H---------D---------------FRN--------------------------------------------------------------S--------------------------QR-----------------------------------------------------------KIV-------F-----T--F-----F--------------------F-----------F--F--F--F--F--F--F--F--F--F--F--FKC-----------------------------------------------------------FNC----------------------------D------------------------------------------------------------GKE-R--R-----R--R-----------R--R--R--R--R--R-----R--R--R------------------------------------GH-----------------------------------------------------------TARI-KV--I-KI--I--I-KI-KI-KI--I--I--I--I--I--I-KI--I--I--ITKI-KI-KI-QI-KI-KI-KI-KI-KI-KI-KI-K NC----------------------------------------------------S-D---SRAP---------M----------------------------------------------------------------L--------------RK---------M---KGC WK--------------------------------------------------------------------------------------*-----------------------*--------CGK-----------------------------------------------------------------------------------------EG-----------------------------------------------------------HQM----------------R------------------------------------------------------------------------KD-----------------------------------------------------------CTE-----------------------------------------------------------------------------------------RQ--G--------------------------------------------------------ANF------------------T----------------------------------------------------------------------LG------------------------------------------------------------------------------------EKIW-----------PSY--H--H KG RPG---------------N--A-------------------------------------------------------------N---R----------------------------------------------------------------NF LQS RP EPT AP--S----S----S----S---------S----S----S----S-----------------------------S-------------------SC---S----SG---SD---S----S----S----S----S----S----S----S-PEE-------------------------------------------------------------------------K---------------SF-----------------------------------------------------------RSG-F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--..-F--F--F--F--F--F--F--..-F--F--.. VEE-E-E-A-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-ETTT--------------------P-----P--A--A--A--A--A-----A--A--A-----------I-----------------------PP-------------------S-S-S-S-S---S-S-S-----------------A-----QKQ-----------------------------------------------------------------------------------------EP-----------------------------------------------------------IDK-H--H--H--H--H--H--H--H--H--N--N--N--N--N--H--N--NN-N--H--H--H--H--H--H--H--H--H--H--H--HELD-D-D-D-D-D-D-D-D-D-D-D-D-D-D-D-D-D-D-D-D-D-D-D-D-D-D-D-D-DYP.--P--P--P--.--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P--P LT-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A SLR--K--K--K--K--K--K--K--K--K-----------------K-----------K--K--K--K--K--K--K--K--K--K--K--K SL-----------P-----------------------------------------------FGN--S------------------------S-------------------------------------------------------------DP------E------SSQ -------E --E ---------------------VFISPreth1875.-------1875.-------1875.--H-------1875.--H-------1875.-----H--------1875.-------H----------1875.--R----H----------1875.-------H----------1875.-------H---------Postt2434.-------H----------2434.-------H----------2434.-------H----------2434.-------H----------2434.-------H-------E--2434.-------H----------2434.-------H----------2434.-------H-------E--2434.-------H----------2542.-------H----------2542.-------H----------2542.-------H----------2542.-----A-H----------2542.-------H----------2542.-------H----------2542.-------H----------2542.-------H----------2542.-------H----------2542.-------H----------2542.-------H----------2542.-------H---------

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206 HXB2 y y LAE---------------------.--.--.--..-F--...---......--------------------------------LAE------......---...---WL----...-----------------AM------------P-----------.---.-.-----------------------AM----.-..--..------.------------SQV--A--A--------A--A--------A--A----------------------------*---------A-----A------SQV---------...---...--------------------------A--TN------P-P-PHP-----------------------------------------TN------..--..------.------------.SAST-ST-ST-ST-SA-SA-ST-STTST-ST-STTSN-SN-SN-SN-SN-SN-SN-SN-SN-SN-SN-SA-SN-SA-SN-SN-SAT-N--N--NI...-N-..--N--N--N--N--N--N--N--N--N--NTIAMAM-M-M-M-M-MAMAMAMAM-M-M-M-M-M-M-M-M-M-M-M-M-M-M-M-MIM------..T----------------------MMQ---------------------------------------------------------------------------------MQRI-GI-GI-G...I-GI-GI-GI-GI-GI-GI-GI-GI-GI-GI-GI-G RG---------------------------A---A----------------------GN------..-----D-----------------NFR---------------------------------------------------------------------------------FRN---------..-Y--Y--Y--Y--Y--Y--Y--Y--Y--Y--Y--Y-NQG-G-G-G-G-G-G-G-G-G-G-G-G-G-G-G-G-G-G-G-G-G-G-G-G-G-G-QR------L------------------------RKI--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N-XN-RN--N--N--NKIVR--R--R--R-------------------------------------VKI-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-I-KC-------------------------------CFN---------------------------------------------------------------------------------FNC-----------------------------------------------CGK---------------------------------------------------------------------------------GKE-----------------------------------------------EG------------------------------------------------------GH-------------------------------HTA-I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I--I-TARI-KI-KI-KI-KI-KI-KI-KI-KI-KI-KI-KI-KI-KI-KI-KI-K RNK-K-K-K-K-K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--NCRK--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K-CRA--------------PR KKG-------------R----R-------K------------------------------------------------------------------------------------------------------------RKKGC------------R-G----------------------------------------------------------------CW------------------------------------------------------WK-------------------------------KCG---------------------------------------------------------------------------------CGK-----------------------------------------------KE------------K----------------------------------------------------------------EGH-----------------------------------------------GHQ-----------MK-----DCT ER-------------------------------------------------------------P-------------------------------------------------------------------------TERQAN----N----N----N----N---VN---VN---VN---VN---VN---VN----N----N----N----N----N---QAN FL GKI WPS YK---H----H----H----H----H----H----H----H----H----H----H----H----HN---H----H----H----H----H----H----H----H----H----H----H----H----H----H-PSYKG--H----H----H----H----H----H----H----H----H----H----H----H----H----H----H----H-GRP---------------------------------------------------------------------------------RPG-----------------------------------------------GN------------------------------------------------------NF-------------------------------FLQ-P--P-----------P--P-----P--P----------------------L-----------------------------LQS--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N--N SR------------------------------------------------------RP-----------------------T-------PEP---------------------------------------------------------------------L-----------EPT-----------------------------------------------TAS-S-----S-S-S---S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-S-AP------------------------G------PPE-------------------------------------------------------A-------------------------P..-AP-AP-AP-AP-AP-AP-AP-AP-AP-AP-AP-AP-AP-AP-AP-AP ES------------------------------------------------------.EP-P-P-P-P-P-P-P-P-P-P-P-A-P-P-PFRSL-FL-FL-FL-FL-FL-FL-FL-FL-FL-FL-FL-FL-FL-FL-FL-FL-FL-FL-FL-FL-FLKFL-FL-FL-FL-FL-FESF-----------------------------------------------GV-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-ERS-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F ETT-----------------------------------------------------S---------------------------GVE-E--E--E--E--E--E--E--E--E--E--E--E--E--E--E--ETPK-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-K-KAKTK-K-K-K-K-K-K-K-TT-.-.-.-.-.-.-.-.-.-.-.-.-.-.-.-. PQKS--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--TPP-------S----------------------------S----------QE-D-D-D-D-D-D-D-Y-D-D-D-D-D-D-D-D-D-D-D-D-D-D-D-D-D-D-DQK-------------------------------PID------------------Q--------Q--------L--------------------------------------------QEP--T--T--T--T--T--T--T--T--T--T--TR-T--T--TR-T--T KEQGQG--------QG----QG--QGQGQGQGQGQGQGQGQGQGQGQG--QGQGQGID-N-NTN-N-N-N-N-N-N-N-N-N-N-N-N-N LYPM--M--M--M--M--M--T--M--M--T--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--M--KEL------------------Q----------------------------LT-A--A--A--A--S SLR SL-----------------K---K--------------------------------------------------------------------------------------TSLRA--KA--KA--KA--KA--KA--KA--KA--KA--KA--KA--KA--KA--KA--KA--KA--K FGN--------------------------------S------------------------------------------------SLF-----------------------------------------------DP-Q-Q-Q---Q-Q-----Q-Q---Q-Q-Q-Q-Q-Q-Q-Q-Q-Q-Q-Q-Q-Q-Q-QGN-------------------------------SSQ------------------------------------------------------------------------------IRHDPS--*--*--*--*--*--*--*--*--*--*E-*--*E-*--*--*--* SQ-------------------------------VFIS 18 Pretherapy2550U02 -------2550U03 -------2550U04 ----------2550U05 ----------2550U06 -------------2550U07 ---------------S-2550U08 ---------------A-2550U09 ---------------A-2550U10 ---------------A-2550U11 ---------------A-2550U12 ---------------APosttherap2930C01 ---------------A-2930C03 ---------------A-2930C05 ---------------A-2930c06 ---------------A-2930C07 ---------------A-2930.48.01---------------A-2930.48.02---------------A-2930.48.04---------YI----A-2930.48.05---------------A-2930.48.06---------------A-2930.48.07---------------A-2930.48.08---------------A-2930.48.09---------------A-2930.48.10---------------A-2930.48.11---------------A-2930.48.12---------------AHXB2 APQMKDCNFLGKIWYPL VFIS 22 Pretherapy1879C03 -----------------1879C04 -----------------1879C07 -----------------1879C08 ---I------------Posttherap2411c01 -----------------2411c02 -----------------2411c03 -----------------2411c05 -----------------2411c06 -----------------2411c07 -----------------2411.48.02-----------------2411.48.04----T------------2411.48.05-----------------2411.48.06-----------------2411.48.07-----------------2411.48.08-----------------2411.48.09 -----------N---I-G--Y-----------------I-KK----------------------N-------------H---------N--------APP-----F-E--.-------T-N------A--K-------*-2411.48.10 -----------N---I-G--Y-----------------I-KK----------------------N-------------H---------N--------APP-----F-E--.-------T-N------A--K-------*-2411.48.11 -----------N---I-G--Y-----------------I-KK----------------------N-------------H---------N-----G-TDPP-----L-E--.S-T----TMN------A--K-----E-*-2411.48.12 -----------N---I-G--Y-----------------I-KK----------------------N---------T---H---------N--------DPP-----F-E--.S------T-N------A--K-------*-

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207 HXB2 LAE AM SQV TN SAT----------------------T----T----T----T----T----T----T----T----T----T----T----T----T----T----T----T----T---------------------------TNSAT-------------------------------------------------------------------------------IMV-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-V-IMV-V-V-V-V-V-V-V-V-V-V-V-V-V-V-VMQR------------------------------------------------------------------------------MQR-----------------------------------------------GN----------------------------------------------------GN-------------------------------FRN-K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K-FRNL-SL-SL-SL-SL-SL-SL-SL-SL-SL-SL-SL-SL-SL-SL-SL-S QR----------------------------------------------------QR-------------------------------KIV--------------------------------A---------------------------------------------KIVRTIRTIRTIRTI-TIRTIRTI-TI-TI-TI-TI-TI-TIRTIRTI-TI KC----------------------------------------------------KC-------------------------------FNC---------------------------------------------------------Y--------------------FNC-----------------------------------------------GKE------------------------------------------------------------------------------GKE-------------------------------------------Q---GH--I--I--I--I TAR NC--------------------------------------------------------------------------------------------------------ARNC---------------------------------------------------------------RAP------------------------------------------------------------------------------RAP-----------------------------------------------RK------------------------------------K---------------RK-------------------------------KGCR--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--R--KGC------------------------------------------------3058.00.09 -------------V-T----L-S---TI----------L-------------------------------------------------N-------------F-E---A---WK-------------------------------------------------R--WK-------------------------------CGK------------------------------------------------------------------------------CGK------------------------------------------*----EG----------------------------------------------------EG-------------------------------HQM------------------------------------------------------------------------------HQM-----------------------------------------------KD-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-EKD-------------------------------CTE------------------------------------------------------------------------------CTE-----------------------------------------------RQ---------ANF-----LG KIW-----------------------------------------------------------------------------------------------------------------------------*----LGKIW-------------------------------------------------------------------------------PSY KG RPG NF LQS------------------------------------------------------N---------------------------------------------------------------------------NFLQS----N----N----N----N----N----N----N----N----N----N----N----N----N----N----N----N RP----------------------------------------------------RP-------------------------------EP.--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--TEPT-----------------------------------------------..APAPAPAPAPAPAPAPAPGPAPAPAPAPAPVAVPAPAPAPAPAPAPAPAPAPAP------V------------------------TAPP--P--P--P--P--P--P--P--P--PG-P--P--P--P--P---V-PV-P--P--P----QP--P--P--P--P--PEE-----------------------------------------------PE----------S-------------------A---------------------SF-----------V-------------------ESF-----------------------------------------------------------------L------------RSG-F--F--F--F--F-SF--F--F--F--F--F--F--F--F--F--FRS-F-F-F-F-F-F-F-F-F-F-F-F-F-F-F-Y-Y-F-F-F-F-F-F-F-F-FVEE-E-E-E-E-E-E-E-E-E-E-E-E-E-E-EGVE-E--E--E--E--E--E--E--E--E--E--E--E--E--E--E--E--E--E--E--E--E--E--E--E--E--E-TTTA-AA-AA-AA-A--AA-AA-D--A--A--A--A--A--A--A--A--A TT----------------------------------------------------PP-----------------------L-------TPP--S--S--S--S--SS-S--S--S--S--S--S--S--S--S--SS-SS-S--S--S--SS-S--S--S--S--S--SQKQ-----------------------------------------------QK----------------------------------------------------EP-Q-Q-Q-Q-Q-Q-Q-Q-Q-Q-Q-Q-Q-Q-Q-Q QEP--------------------------------------------T--A-----------T--T---------------IDK--Q--Q--Q--Q--Q--Q--Q-EQ--Q--Q--Q--Q--Q--Q--Q--Q IDT-T-T-T---T-T-T-----T-T-T-T-T-T---T-T-T-T-T-T-T-T-T-EL-K-K-K-K-KD*-K-K-K-K-K-K-K-K-K-K KEL-T--------K----QK-QK-QK-QK-QK----QK-QK----------QK----QK--------------------K-YPL-----------------------------------------------YP----------------------------------------------------TS----------------------------------Q--Q-K----------------LTSMD--A--A--D--N--D--D--D--D--D--A--D--D--A--A--A--DT-A--D--A--A--A--A--A--A--D-LRS-----------------------------------------------LR----------------------------------------------------LF-------------------------------SLF------------------------------------------------------------------------------GND-----------------------------------------------GN---S-S---S-S-----S-S-S---S-S-S-S-S-S---S-S-S-S-S----PS-------------------------------DPS---------------------------E--E-----------------E--E--------------------------SQ ----------------SQ------------------------------VFIS 24 Pretherapy 2098U01 ----------H-----2098U03 ----------H-----2098U04 ------------H-----2098U05 ------------H----Posttherapy 2375.48.02 ----------I-------H-----2375.48.03 ----------I-------H-----2375.48.04 ----------I-------H-----2375.48.05 ----------I-------H-----2375.48.06 ----------I-------H-----2375.48.07 ----------I-------H-----2375.48.08 ----------I-------H-----2375.48.09 ----------I-------H-----2375.48.10 ----------I-------H-----2375.48.11 ----------I-------H-----2375.48.12 ----------I-------H-----2375.48.15 -----R----I-------H-----2375.48.16 ----------I-----A-H----R2375.48.17 ----------I-------H-----2375.48.18 ----------I-------H-----2375.48.20 ----------I-------H-----2375.48.24 ------E---I-------H-----2375U01 ---------RI-------H-----2375U02 ----------I-------H-----2375U03 ----------I-------H-----2375U04 ----------I-------H-----2375U05 --------R-I-------H----HXB2 LAEAMSQVGHTRQANFPSYKGRPG VFIS 26 Pretherapy 2612.48.01 ----------L-------------2612.48.02 ----------L-------------2612.48.04 ----------L-------------2612.48.05 ----------L-------------2612.48.06 ----------L-------------2612.48.07 ----------L-------------2612.48.09 ----------L-------------2612.48.10 ----------L-------------2612.48.11 ----------L-------------2612.48.12 ----------L------------Postherapy 3058.00.01 ----------L-------------3058.00.02 ----------L-------------3058.00.03 ----------L-------------3058.00.04 ----------L-------------3058.00.05 ----------L-------------3058.00.07 ----------L-------------3058.00.10 -------------V------L-S--RTI----------L-------------------------------------------------N-------------F-E---A------Q--Q-K----------------3058.00.11 -------------V------L-G---TI----------L-------------------------------------------------N---AKG-------F-E---G------Q--Q-K------------E---3058.00.12 -------------V------L-S---TI----------L-------------------------------------------------N-------------F-E---A------Q--Q-K----------------

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208 HXB2 LAE AM SQV TN SAT IM MQR GN FRN QR KIV KC FNC GKE GH TAR NC RAP RK KGC WK CGK EG HQM KD CTE RQ ANF LG KIW PSY----H----H--------------H-------------------H----H---------H---------H------------------------H---------H----H----H----H----H----H----H----H----H----H KG-----------------------------------------------------------RPG-----------------------------------------------------------------------------------------NF-----------------------------------------------------I-----LQS-----------------------------------------------------------------------------------------RP-----------------------------------------------------------EPT-----S--K--------------------------------------------------------------------------------AP----D-------D---------------------------------------G------PEE----A--A--A-----------------------A-----A--------------A---------------------------------SF-----Y-----------------------------------------------------RSG-F--F--L--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F-----F--F--FVEE-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-ETTT----A-AA-AA--P-----P--P--A--A--A--A--A--A--------A--A--A--A--P--P--P--P--P--P--PN-P--P--PPP-----------------------S---S-S-S-----S-------------L-------QKQ-----------------------------------------------------------------------------------------EP-Q---Q-Q-Q-Q-Q-Q-Q-----Q---Q---------Q---Q-Q-Q-Q-Q-Q-Q-Q-Q-* IDKT--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T--T-EL-----------------------------------------------------------YPL-----------------------------------------------------------------------------------------TSA-A-AYA-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-ALRS-------------K---------------------------------------------------------------------------LF--------------GND PS---L-------------E-------------------E-------------------------------------------------------------------E---E---------SQ -P -----------------------------VFIS 29 Pretherapy 3122.48.01 --------STP-N-------------------------I-K---------------------------------3122.48.02 --------STP-N-------------------------I-K---------------------------------3122.48.03 --------STP-N-------------------------I-H-----K---------------N-----------3122.48.04 -V------STP-N-------------------------I-K-----K---------------N-----------3122.48.06 --------STP-N-------------------------I-K---------------------------------3122.48.07 ---------SP-N-------------------------I-K------------------------------------3122.48.08 ---------SP-N-------------------------I-K------------------------------------3122.48.09 --------STP-N-------------------------I-K------------------------------------3122.48.12 --------STP-N------------E------------I-K-----------------------------------Posttherapy 3501.00.01 --------SSP-N-------------------------I-K------------------------------------3501.00.02 ----------P--------------------------RITKS-----------------------------------3501.00.03 ----------P---------------------------I-KS-----------------------------------3501.00.04 ----------P------------K--------------I-KS-----------------------------------3501.00.05 --------SSP-N-------------------------I-K------------------------------------3501.00.07 ----------P---------------------------I-KS-----------------------------------3501.00.08 ----------P---------------------------I-KS-----------------------------------3501.00.09 ----------P---------------------------I-KS-----------------------------------3501.00.10 ----------P------------H--------------ITKS-TG-T------------------------------3501.00.11 --------SSP-N-------L-----------------I-K------------------------------------3501.00.12 --------SSP-N---------S---------------I-KS-----------------------------------3501.48.02 --------SSP-N-------------------------I-K------------------------------------3501.48.03 --------SSP-N-------------------------I-K------------------------------------3501.48.04 --------SSP-N-------------------------I-K------------------------------------3501.48.06 --------SSP-N-------------------------I-K------------------------------------3501.48.07 --------SSP-N--------------------A----I-K--------A---------------------------3501.48.08 --------SSP-N-------------------------I-K------------------------------------3501.48.09 --------SSP-N-------------------------I-K------------------------------------3501.48.10 --------SSP-N-------------------------I-K------------------------------------3501.48.11 --------SSP-N-------------------------I-K--------------------------R---------3501.48.12 --------SSP-N-------------------------I-K-----------------------------------

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209 HXB2 LAE AM SQV TN SAT IM MQR GN FRN QR KIV KC FNC GKE GH TAR NC RAP RK KGC WK CGK EG HQM KD CTE RQ ANF LG KIW PSY KG RPG------------------------------------------------------------------------------------------------------------------R------------------------NF-------------------------------------------------------LQS-----------------G-----------------------------------------------------------------RP-------------------------------------------------------EPT--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S--S AP-----Q--------------D---------------G-G-----G-----D----PEE-----------------------------------------------------------------------------------SF---------------------L-----L-----L---------------------RSG-F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--F--FVEE-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-E-ETTT--I-----I--------------S-----------------------------------N--------N--------------PP-------------------------------------------------------QKQ-----------------------------------------------------------------------------------EP-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L IDK-----------------------------------------------------------------------------T-----EL-R-M-M-R-M-M-M-R-M-M-M-M-M-M-M-M-M-M-M-M-M-M-M-M-M-M-M-M YPL--------------------------------------------------S--------------------------------TSA-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A-A---A-A-A-A-A-A-A-A-ALRS-K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--K--*--K--K--K--K--K--K--K--KLF------------------------------------------------------------------------------GND--E-----------PS SQ LL-H LL-LL-LL-H LL-LL-LL-LL-H LL-LL-LL-LL-LL-LL-LL-LL-LL-LL-LL-LL-LL-LL-LL-LL-LL-LL-LL-LL-VFIS 30 Pretherapy 2649.48.01 -----------------K--I-----------------I-K-------R------------------------------2649.48.02 ------*----------K--I-----------------I-K--------------------------------------2649.48.03 -----------------K--I-----------------I-K-------R----------------------I-------2649.48.05 -----------------K--I-----------------I-K-------R----------------G----------A--2649.48.06 -----------------K--I-----------------I-K--------------------------------------2649.48.08 -----------------K--I-----------------I-K-------R--------------------------------2649.48.09 -----------------K--I-----------------I-K-------R-------------------------------E2649.48.12 -----------------K--I-----------------I-K-------R------------T------------------Postherapy 3037.00.02 -----------------K--I-----------------I-K----------------------------------------3037.00.03 ------------A----K--I-----------------I-K----------------------------------------3037.00.04 -----------------K--I-----------------I-Q---------------------------------------E3037.00.05 ------------A----K--I-----------------I-K-------------------------S--------------3037.00.06 -----------------K--I-----------------I-K-------R--------------------------------3037.00.07 -----------------K--I-----------------I-Q-----------------------------------T--KE3037.00.08 -----------------K--I--------R--------I-K-----------------------------L----------3037.00.09 -----------------K--I-----------------I-K----------------------------------------3037.00.11 -----------------K--I-----------------I-K----------------------------------------3037.00.12 ------L----------K--I-----------------I-K---------------------------------V------3037.48.01 -----------------K--I-------Q---------I-K----------------------------------------3037.48.02 -----------------K--I-----------------I-K--------------------------------------KE3037.48.03 -----------------K--I-----------------I-K----------------------------------------3037.48.04 -----------------K--I-----------------I-K-----M----------------------------------3037.48.05 -----------------K--IT----------------I-K----------------------------------------3037.48.06 -----------------K--I-----------------I-K----------------------------------------3037.48.07 -----------------K--I-----------------I-K----------------------------------------3037.48.09 -----------------K--I-----------------I-Q----------------------------------------3037.48.10 -----------------K--I-----------------I-K----------------------------------------3037.48.11 -----------------K--I-----------------I-K---------------------------------------

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210 HXB2 LAE AM SQV TN SAT IM MQR GN FRN QR KIV KC FNC GKE GH TAR NC RAP RK KGC1877u01 ---------.NSN--------------A----------V----------------*--------N-------------H-------I---T------A----F-E-----S--1877u03 ---------.NSN-------------T-----------I----------------Q----------------------H-------I---T------A----F-E--------1877u04 ---------.NSN-------------T-----------I----------------Q--Y-------------------H-------I---T------A----F-E--------1877u05 ---------.NSN--------------A----------I----------------Q----------------------H-------I---T------A----F-E-----S--2190.48.01 ---------.NSN-------------------------I----------------Q----------------------H-------I---T------A----F-E--------2190.48.02 ---------.NSN-------------T-----------I----------------Q----------------------H-------I---T------A----F-E-----S--2190.48.03 ---------.NSN-------------T-----------I----------------Q------------------T---H-------I---T------A----F-E-----S--2190.48.04 ---------.NSN-------------S-----------I----------------Q-----------------R----H-------I---T------A----F-ERK------2190.48.05 ---------.NSN-------------T-----------I------------R---Q----------------------H-------I---T------A----F-E-----S--2190.48.07 ---------.NSN-------------S-----------I---------R------Q------------------V---H-------I---T------A----F-E-----S--2190.48.08 ---------.NSN-------------S-----------I----------------Q-----------------R----H-------I---T------A----F-E-----S--2190.48.10 ---------.NSN--I----------S-----------I----------------Q-----------------R----H-------I---T------A-IIEF-EKK------2190.48.11 ---------.NSN-------------S-----------I----------------Q-----------------R----H-------I---T------A----F-E-----S--2190.48.12 ---------.NSN-------------T-----------I----------------Q-----------------R----H-------I---T------A-IIEF-EKK------HBX2 LAEAMSQVTNSATIMMQRGNFRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSYKGRPGNFLQSRPEPTAPPEESFRSGVETTTPPQK1944.48.01 -------A---NV--------K----------------I---------R----------------K------------H--------------S---A-DL-F-E--------1944.48.02 -------A--AT---------K----------------I---------R-----------------------------H--------------S---A-DL-F-EG-------1944.48.03 -------A---NV--------K----------------I---------R-----------T----K------------H--------------S---A-DL-F-EG-------1944.48.04 -------A--AT---------K----------------I---------R-----------------------------H--------------S---A-DL-F-EG-------1944.48.05 -------A--AT---------K----------------I-------K-------------------------------HN-------------SG--A-GLKF-E-N------1944.48.06 -------A---T---------K----------------I----K----R-----------------------------H--------------S---A-DL-F-EG-------1944.48.07 -------A--AT---------K----------------I---------R-----------------------------H-------C------S---A-GI-F-E--------1944.48.08 -------A--AT---------K----------------I---------R-----------------------------H-----------S--S---A-DL-F-EG-------1944.48.10 -------A--AT--V------K----------------I---------R-----------------------------H--------------S---A-GL-F-E--------1944.48.11 -------A---NV--------K----------------I---------R-----------------------------H--------------S---A-GL-F-E--------1944.48.12 -------A----A--------K----------------I---------R-----------------------------H--------------S---A-GL-F-E--------2292u01 --KP---A---T---------K----------------I----K----R-----------------------------H--------------S---A-GL-F-E--------2292u02 -------A---T---------K----------------I----K----R-----------------------------H--------------S---A-GL-F-E--------2292u03 -------A--AT------S--K----------------I---------R-----------------------------H--------------S---A-DL-F-EG-------2292u04 F------A--AT---------K----------------I---------R-----------------------------H--------------S---A-GL-F-E--------2292u05 -------A---T---------K----------------I----K----R-----------------------------H--------------S---A-GL-F-E--------2292.48.01 -------A----A--------K----------------I---------R-----------------------------H--------------S---A-DL-L-EG-------2292.48.03 -------A--AT---------K----------------I---------R-----------------------------H--------------S---A-DL-F-EG-------2292.48.04 -------A--AT---------K----------------I---------R-----------------------------H--------------S---A-DL-F-EG-------2292.48.05 -------A--AT---------K----------------I---------R-------------------------L---H--------------S---A-DL-F-EG-------2292.48.06 -------A--AT---------K----------------I----K----R-----------------------------H--------------SG--A-DL-Y-EG--S----2292.48.07 -------A--AT---------K----------------I----K----R-----------------------------H---Q----------SG--ARGL-F-E--------2292.48.08 -------A--AT---------K----------------I---------R-----------------------------H--------------S---A-DL-F-EG-------2292.48.09 -------A--AT---------K----------------I---------R-----------------------------H--------------S---A-GL-F-E--------2292.48.10 -------A--AT---------K----------------I---------R-----------------------------H--------------S---A-DL-F-EG-------2292.48.12 -------A--AT---------K----------------I---------R-----------------------------H--------------S---A-DL-F-EG------WK CGK EG HQM KD CTE RQ ANF LG KIW PSY KG RPG NF LQS RP EPT AP PEE SF RSG VE TTT PP QKQ EP IDK-LT---LT---LT---LK---LT---LK---LK---LT---LE---LK---LK---LT---LK---LT--EPIDK-QT---QT---QT---QT---QT---QT---QT---QT---QT---QT---QT---QT---QT---QT---QT---QT---QT---QT---QT---QT---QT---QT---QT---QT---QT-EL-M-M-M-M-M-M-M-M-M-M-M-M-M-MEL-------------------------------------------M-----YPL------------------------------------------YPL--------------------------------------------------------------------------TSA-A-A-A-A-A-A-A-A-A-A-A-V-A-TS--------N-----------------------------------A-------QT--------------------LRS-K-----K--K--K--K--K--K--K--K--K--K--K--K-LRS-------------------------------------------------------------------K------LF------------------P---------LF-----------------------L-------------------------GND-----------------------------E------------GND--------------E--E-S------------------------------E-----------E-KE--------PSH-H*H*H-H*H*H*H-H*H*H*H*H*H*PSLL-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L-L SQ ---------------SQ -------------------------VFIF 05 Pretherapy ---Posttherapy ---------Q VFIF 06 Pretherapy ----------Posttherapy -------------

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211 HXB2 K AR VLA EA MSQ VT NSA TI MMQ RG NFR NQ RKI VK CFN CGK EG HTA RN CRA PR KKG CW--------------------------------------------PRKK---------------------------------------------------------------------------KCG---------------------------------GCW--------------------------------------------------------KER-R-R-R-R-R-R-R-R-R-R-KC------------------------------R------GHQ---------------------------------GKE--------------------------------------------------------MK----------------------GH-------------------------------------DCT---------------------------------QMK--------------------------------------------------------ER----------------------DC-------------------------------------QAN---------------------------------TER--------------------------------------------------------FL----------------------QA----------*--------------------------GKI---------------------------------NFL--------------------------------------------------------WPS---------------------------------GKI--------------------------------------------------------YKH-H-H-H-H-HEH-H-H-H-H-WP-------------------------------------GRP---------------------------------SYK-H--H-----H--H--H--H--H--H--H--H--H--H--H--H--H--H--H--HGN----------------------GR-------------------------------------FLQ---------------------------------PGN--------------------------------------------------------SRN--N--N--N--N-PEP TA--------------------------------------------------QSRPE------------K---N---------------------------------------------------------------------------G-PP.--A--A--A--A--A--A--A--A--A--A--APTA-----------------G-----------G--------G-----------------..PPPPPPPPPPPPPPLPPPPPPPPP-------------------------------------EES---------------------------------EES--------------------------------------------------------FR------F--F SGVF-EF-E ET TTP-K----K----K----K----K----K----K----K----K----K----K--ETTTP----------------------------------------------------------------------------------------------PQ KQE PI DKE LY----------------------------------------KELYR---R-----K-SN--R---SN--SN--SN--SN--SN--SN--SN--SN--SN--SN--SN--SN--SN--SN-PLT--A--A--A--A--A--A--A--A--A--APLT--A--A--A--A-----A--A--A--A--A--A--A--A--A--A--A--A--A--A SL--------------------SL--------F----------------------------RSL------------------------------RSLK--K-----K--K--K--K-----K--K--K--K--K--K--K--K--K--K--K-FG--------------------FG-------------------------------------NDP-------------------------------A----T--------A------------NDP----------------E--------------E-----E------------------SS--------------Q VFIF 07 Pretherapy 1557U01 ---------------TA----K-H------T-----------I-K-------A----T--1557U02 ---------------TA----K-H------T-----------I-K-------A----T--1557U03 ---------------TA----K-H------T-----------I-K--------EA----T--1557U04 ---------------TA----K-H------T-----------I-K--------EA----T--1557U05 ---------------TA----K-H------T-----------I-K---------F-EA----T-Posttherapy 1678U02 ---------------TA----K-H-K----T-----------I-------------F-ES----T-----1678U03 ---------------TA----K-H------P-----------I-K-----------F-EA----T-----1678U04 ---------------TA----K-HY-----P-----------I-K-----------F-EA----T-----1678U05 ---------------TA----K-H------P-----------I-K-----------F-EA----T-----1678U06 ---------------TA----K-H-K----T-----------I-K-----------F-ES----T-----1678U07 ---------------TA----K-H------T-----------I-K-------N---F-E HXB2 KARVLAEAMSQVTNSATIMMQRGNFRNQRKIVKCFNCGKEGHTARNCRAFLFRSGVPQKQEPIDSSQ VFIF 08 Pretherapy 1934U01 -----------A.-A-A--V----------------------I----------F-ES-----K----1934U03 -----------A.-A-A--V----------------------I----------F-ES-----K----1934U04 -----------A.--PA-------------T-----------V-K--------F-ES----M-----1934U06 -----------A.-ATA--V--------*-------------I----------F-ES-----K----1934U08 -----------A.-A-A--V----------------------I----------F-ES-----K---Posttherapy 2234.48.02 ....-------A.-A-A--V----------------------I----------F-ES-----K----2234.48.03 ....-------A.-A-A--V----------------------I----------F-ES-----K--FT2234.48.05 ....-------A.-A-A--V----------------------I----------F-ES-----K----2234.48.06 ....-------A.-A-A--V----------------------I----------F-ES-----K----2234.48.07 ....------RA.-A-A--V----------------------I----------F-ES-----K----2234.48.08 ....-------A.-A-A--V----------------------I----------F-ES-----K----2234.48.09 ....-------A.-A-A--V----------------------I----------F-ES-----K----2234.48.10 ....-------A.-A-A--V----------------------I----------F-ES-----K----2234.48.11 ....-------A.-A-A--V----------------------I----------F-ES-----K----2234.48.12 ....-------A.-A-A--V----------------------I----------F-ES-----K----2234U02 -----------A.-A-A--V----------------------I----------F-ES-----K----2234U03 -----------A.-ATA--V--------K-------------I----------F-E------K----2234U04 -----------A.-A-A--V----------------------I----------F-ES-----K----2234U06 -----------A.-A-A--V----------------------I----------F-ES-----K---

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212 XB2 LAEAMSQVTNSATIMMQRGNFRNQRKIVKCFNCGKEGHTARNCRAPRKKGCWK VFIF 09 Pretherapy 1955c01 ----------PT-----------P--T-----------I-K----1955c03 -----------T--------------TA----------I-K---------------1955c05 -----------TN----K----S---T-----------I-K---------------1955c08 ----------PT--------------T-----------I-K---------------2208.48.01 ----------PT--------------T-----------I-K---------------2208.48.02 ----------PT--------------T-----------I-K---------------2208.48.03 -----------TN----K----S---T-----------I-K---------------2208.48.04 ----------PT--------------T-----------I-K---------------2208.48.05 ----------PT--------------T-----------I-K---------------2208.48.06 ----------PT----------S-S-T-----------I-K---------------2208.48.07 ----------PT--------------T-----------I-K---------------2208.48.08 ----------PT--------------T-----------I-K---------------2208.48.11 -----------TN----K----S---T-----------I-K--------------R2208.48.12 -----------TN----K--------T-----------I-K------E-------Posttherapy 2686.48.02 -----------TN-------------T-----------I-K---------------2686.48.03 ----------PT--------------T-----------I-K---------------2686.48.04 ----------PT--------------T-----------I-K---------------2686.48.05 -----------TN----K----S---T-----------I-K---------------2686.48.06 ----------PT--------------T-----------I-K-R-------------2686.48.07 ----V-----PT--------------T-----------I-N---------------2686.48.08 -----------TN-------------T-----------V-K---------------2686.48.09 ----------PT-----K----S---T-----------I-K---------------2686.48.11 ----------PT--------------T-----------I-K---------------2686.48.12 ---V-------T--------------T-----------I-K---------------2686c03 -----------TN----K--------T-----------I-K---------------2686c04 -----------TN----K--------T-----------I-K---------------2686c05 ----------PT--------------T-R---------I-K---------------2686c06 -----------TN----K--------T-----------I-K---------------2686c07 -----------TN----K--------T------V----I-K--K------------2686c08 -----------TN----K--------T-----------I-K---------------2686u02 .........--T--------------T-----------I-K---------------2686u021 .........--T--------------T-----------I-K---------------2686u031 .........--T--------------T-----------I-K---------------2686u04 .........--T--------------T-----------I-K--------------CGKEG HQM---------------A------------------------------------------------------------------------------------------------------------------------------------P--------------------KD-E-------------------------------E-------E-------------------------CTE----S--S--S--S-----S-----S--S--S--S--S--S--S--S-----S--S--S-----S--S--S--S--S--S--S--S--S--S--S--S--SRQ-------------------------------------------------------------------ANF------------------------------------------------V-----V--V-----V--------V--V--V--V--V--V-------------LG-----------------------------E-------------------------------------KIW-----------------------------------------------------------------------------------------------------PSY---------------------------------------------------------------L-------------------------------------KG------------------------------------------------Q------------------RPG-----------------------------------------------------------------------------------------------------NF---------------------------L---------------------------------------LQS-----------------------------N--N-----------------------------R--------------------------------------RP-------------------------------------------------------------------EP.--T--T--.--.--.--.--.--.--.--.--.--.--.--T--T--.--.--.--.--.--.--T--.--.--T--T--.--T--T--T--.--.--.--. ..VPVP......................APAP............AP....APAP..APAPAP........ ..........................................PQSPQS..................PQS......PRSPQS...PQSPQSPQS............ ............................RPRP............RP....RPRP..RPRPRP........ ..T..-..-..-..-..-..-..-..-..-..-..-..-..-EP-EP-..-..-..-..-..-..-EP-..-..-EP-EP-..-EP-EP-EP-..-..-..-..AP-------------------------------------------------------------------PEE---------------------------------------------------T-------------------------------------------------SF-------------------------------------------------------------------RSGKF--F--F-KF--F-F-F-F-F-F-F-F-F-F-F-F-F-V-F-F-F-L-F-F-F-F-F-FMF-F-F-F-F-F VEE--E--E--E-TTT PP-A---A---A---A-QKQ-----------E.-.-.-.-. ............... .......... ............... PI---------E---A------.........---------A--------E-L-K -E---A------.........---------A-------------E---A------.........---------A-------------E---A------.........---------A-------------E---A------.........---------A-------------E-S-A------.........-----------------------E-S-A------.........---------A-------------E---A------.........---------A-------------E-P-A------.........-V-------A-------------E---A-S----.........---------A-------------E---A------.........---------A-------------E---A------LTAPPQKQE---------A-------------E---A------.........---------A-------------E---A------PTGPPQKQE---------A-------------E---A------PAAPPQKQE---------A-------------E---A------PTGPPQKQE---------A-------------E---A------SAAPPQKQE---------A-------------E---A------.........---------A-------------E---A------.........---------A-------------E---A------PTAPPQKQE---------A-------------E---A------.........---------A-------------E---A------.........---------A-------------E---A------.........---------A-------------E---A------.........---------A-------------E---A------.........---------A-------------E---A------.........---------A-------------E---A------PTAPPQKQE---------A-------------E---A------PTAPPQKQE---------A-------------E---A------PTAPPQKQE---------A-------------E---A------PTAPPQKQE---------A------------DKE-----------LY-------PLT--A--A--A--A SL-------RSL-----------FG-------NDPD----------SSQ--------------------H

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APPENDIX D ALIGNMENT OF P6 POL SEQUENCES

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214 HXB2 FREDLAFLQGKAREFSSEQTRA...............NSPTRRELQVWGRDNNSPSEAGADRQGTVSFNF VSIS 01 Pretherapy 2272C01 -------P--E-----------...............-----G--------.--L-K--EN------YSC 2272C04 -------P--E-----A-----NSP............-----G--------.----K--ES------LS2272C03 -------P--E-----------NSPTRGELQGWQNRA--------------.--L----ES------LS2272C05 -------P--E-----------NSPTRGELQVWQTRA--------------.--L----ES----I-LS2272C06 -------P--E-----------NSPTRGELQGWQNRA--------------.-------ES----I-LS2272C07 -------P--E-----------NSPTRGELQGWQNRA--------------.--L----ES------YSC 2272C08 -------P--E-----------NSPTRGELQGWQNRA--------------.-------EN------YSC Postherapy 2774A01 -------P--E-----------NSPTRGELQGWQSRA--------------.--L----ES------LS2774A02 -------P--E-----------NSPTRGELQGWQSRA--------------.--L----ES------LS2774A03 -------P--E-----------NSPTRGELQGWQSRA--------------.--L----ES------LS2774A04 -------P--E-----------NSPTRGELQGWQSRA--------------.--L----ES------LS2774A05 -------P--E-----------NSPTRGELQGWQSRA--------------.--L----ES------LS2774O01 -------P--E-----------NSPTRGELQGWQNRA-----G--------.--L----EN------LS2774O02 -------P--E-----------NSPTRGELQGWQNRA-----G--------.--L----EN------LS2774O03 -------P--E-----------NSPTRGELQGWQNRA-----G--------.--L----EN------LS2774O04 -------P--E-----------NSPTRGELQGWQTRA--S-SG--------.-------EN---A--YSC 2774O05 -------P--E------G----NSPTRGELQGWQTRA--S-SG--------.-------EN------LSHXB2 FREDLAFLQGKAREFSSEQTRA...............NSPTRRELQVWGRDNNSPSEAGADRQGTVSFNF VSIS 03 Pretherapy 1794U04 ----------E----------TEQTRA..........---AS-------------------------LS1794U07 ----------E----------TEQTRA..........---AS-------------------------LS1794U08 ---N------E----------TEQTRA..........I--AS-----------------D-------LS1794U03 ---N------E-----------EQTRA..........---AS-----------------D-------LS1794U06 ---N------E-----------EQTRANSP.......---AS-----*-----------D-------LS1794U09 ---N------E-----------EQTRA..........I--AS---------S---------------LS1794U11 ---N------E-----------EQTRA..........I--AS---------I---------------LS1794U05 ----------E-----------EQTRA..........I--AS---------S---------------LSPosttherapy 2590A03 ---N------E-----------EQTRA..........---AS-------------------------LS2590O01 ----------E-----------NSP............---AS-------------------------LS2590O02 ----------E-----------...............---AS-------------------------LS2590O05 ----------E-----------...............---AS-------------------------LS2590O06 ----------E-----------EQTRT..........---AS---------S---------------LS2590O07 ----------E-----------NSP............---AS-----------------D-------LV2590O04 ----------E----F------NSP............---AS-------------------------LS2590O03 ----------E----F------NSP............---AS--I--------------D-------LS2590A02 ----------E----F------...............---AS--I----------------------LS2590A05 ----------E----F------...............---AS--I----------------------LSHXB2 FREDLAFLQGKAREFSSEQTRA...............NSPTRRELQVWGRDNNSPSEAGADRQGTVSFNF VSIS 04 Pretherapy 1669u02 -------P--E----P------...............-------------------------------SL 1669u03 -------P--E----P------...............-------------------------------SL 1669u01 -------P--E----P------...............---------------------------A---SL 1669u04 -------P--E----P------...............------------G-S----------P-A--LSL 1669u05 -------P--E---I-------...............------K-------S----------P-A--LSL Posttherapy 1779u01 -------P--E---IPP-----...............------------G------K---E------LSL 1779u02 -------P--E----PP-----...............------------G------K---E------LSL 1779u03 -------P--E----P------...............--------------S--------E------LS1779u05 -------P--E----P------...............--------------S----------------SL

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215 2 R ........ R QGTVSFNF ..... --I---SHXB VSIS 25 Pretherapy 2473.48.01 ---N---PS-E---LY---D--...............-----------------L---------N--LS2473.48.02 ---N---PS-E---LY---D--...............-----------------L---------N--LS2473.48.03 ---N-T-PS-E---LY---D--...............-----------------L---------N--LS2473.48.06 ---N---PS-E---LY---D--...............-----------------L---------N--LS2473.48.11 ---N---PS-E--KLY---D--...............-R-N-------------L------*--N--LS2473.48.12 ---N----S-E---LY---D--...............-----------------L---------N--LSHXB2 FREDLAFLQGKAREFSSEQTRA...............NSPTRRELQVWGRDNNSPSEAGADRQGTVSFNF VSIS 27 Pretherapy 5761.48.02 ---N---P--E---L------TNSP............T-----------G---------VE-------G5761.48.12 ---N---P--E---L------TNSP............T-----------G---------VE-------G5761.48.07 ---N---P--E---L------TNSP............T-S---------G---------VE-------G5761.48.04 ---N---P--E---L------TNSP............T---------------------VE-------G5761.48.03 ---N---P--E---L------TNSP............T-----------G---------DE-------G5761.48.06 ---N---P--E---L------TNSP............T-----------G---------DE-------G5761.48.08 ---N---P--E---L------TNSP............T-------*---G---------GE-------G5761.48.11 ---N---P--E---L------TNSP............T---------------------DE-------S5761.48.10 -------P--E---L------TNSP............T-----------G---------DE-------SPosttherapy 6063.00.04 ---N---P--E---L------TNSP............T-----------G---------VE-------G6063.00.05 ---N---P--E---L------TNSP............T-----------G---------VE-------G6063.00.06 ---N---P--E---L------TNRP............T---------------------VE-------S6063.00.10 ---N---P--E---L------TNSP............T---------------------VE-------S6063.00.03 ---N---P--E----------TNSP............T---------------------VE-K-----G6063.00.07 ---N---P--E----------TNRP............T---------------------VE-K-----G6063.00.09 ---N---P--E----------TNSP............T---------------------VE-K-----S6063.00.11 -------P--E----------TNSP............T--------------K------VE-------S6063.00.02 -------P--E----------TNSP............T--------------K------VE-------S6063.00.12 -------P--E----------TNSP............T--------------K------VE-------SHXB2 FREDLAFLQGKAREFSSEQTRA...............NSPTRRELQVWGRDNNSPSEAGADRQGTVSFNF VSIS 28 Pretherapy 5332.48.01 ---N---P-------P------...............I----G---I--G----L---------I---S5332.48.10 ---N---P-------P------...............I----G---I--G----L---------I---S5332.48.02 ---N---P-------P------...............I----G---I-------L---------I---S5332.48.03 ---N---P-------P------...............I----G---I-------L---------I---S5332.48.05 ---N---P-------P------...............I----G---I-------L---------I---S5332.48.06 ---N---P-------P------...............I----G---I-------L---------I---S5332.48.11 ---N---P-------P------...............I----G---I-------L---------I---S5332.48.08 ---N---P-------P------...............I----G---I-------L-----N---I---S5332.48.09 ---N---P-------P------...............I----G---I-------L-----N---I---S5332.48.12 ---N---P-------P------...............I----G---I-------L-----N---I---SPosttherapy 5715.00.01 ---N---P-------P------...............I----G---I-------L---------I---S5715.00.02 ---N---P-------P------...............I----G---I-------L---------I---S5715.00.03 ---N---P-------P------...............I----G---I-------L---------I---S5715.00.04 ---N---P-------P------...............I----G---I-------L---------I---S5715.00.05 ---N---P-------P------...............I----G---I-------L---------I---S5715.00.06 ---N---P-------P------...............I----G---I-------L---------I---S5715.00.07 ---N---P-------P------...............I----G---I-------L---------I---S5715.00.08 ---N---P-------P------...............I----G---I-------L---------I---S5715.00.09 ---N---P-------P------...............I----G---I-------L---------I---S5715.00.10 ---N---P-------P------...............I----G---I-------L---------I---S5715.00.11 ---N---P-------P------...............I----G---I-------L---------I---S571 . 1 2 F RE -D N L AF -L P QG -KA -EF SS PE QT -RA -... ... .. .. . . .N .I S P TR -R G EL -Q VWG I-DN -N SP -L S E AG -ADR --5. 00 -.. . -

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216 2 D SSEQTRA....NSP GADRQGTVS.FNF HXB VFIS 02 Pretherapy 1850.48.01 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 1850.48.02 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 1850.48.03 ---N---P--------------.......-----G------G.....----F----T-G--P--.LSL 1850.48.04 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 1850.48.05 ---N---P--------------.......-----G------G....E---------T-G--P--.LSL 1850.48.07 --KN---P--------------.......-----G------G.....----L----T-G--P--.LSL 1850.48.08 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 1850.48.10 ---N-G-P---G----------.......-----G------G.....----L----T-G--P--.LSL 1850.48.09 ---N---P--------------.......D----G------G.....----L----T-G--P--.LSL 1850.48.11 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 1850.48.12 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSA Posttherapy 2088002 ---N---P--------------.......-----GK-----G.....----L----T-G--P--.LSL 2088004 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 2088006 ---N---P--------------.......-----G------G.....----L----T-G--P--.PSL 2088007 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 2088a01 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 2088a03 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 2088a04 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 2088a02 ---N---P--------------.......-----G------G.....----L----T-G--L--.LSL 2088001 -------P--------------.......-----G------G.....----L----T-G--L--.LSL 2088o05 -------P--------------.......-----G------G.....----L----T-G--L--.LSL 2088.48.01 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 2088.48.02 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 2088.48.03 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 2088.48.04 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 2088.48.05 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 2088.48.06 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 2088.48.07 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 2088.48.08 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 2088.48.09 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 2088.48.10 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 2088.48.11 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL 2088.48.12 ---N---P--------------.......-----G------G.....----L----T-G--P--.LSL F RE LA FL Q G KA REF ... TR R EL Q V WG R .. . .. D N NS P SEA

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217 HXB2 FREDLAFLQGKAREFSSEQTRA.......NSPTRRELQVWGR.....DNNSPSEAGADRQGTVS.FNF VFIS 10 Pretherapy 1470C01 ---N---P------L-------...QTRA-------------.....-S--LAT--D----P--.-S1470C02 ---N---P------L-------...QTRA-------------.....-S--LAT--D----P--.-S1470C04 ---N---P------L-------...QTRA-------------.....-S--LAT--D----P--.-S1470C05 ---N---P------L-------...QTRA-------------.....-S--LAT--D----P--.-S1470C07 ---N---P------L-------...QTRA-------------.....-S--LAT--D----P--.-S1470C08 ---N---P------L-------...QTRA-------------.....-S--LAT--D----P--.-S1470A03 ---N---P------L-------...QTRA-------------.....-S--LAA--D----P--.-S1470A04 ---N---P------L-------...QTRA-------------.....-S--LAA--D----P--.-S1470C03 ---N---P------L-------...QTRA-------------.....-S--LAA--D----P--.-S1470C06 ---N---P------L-------...QTRA-------------.....-S--LAA--D----P--.-S1470A02 ---N---P--------------...QTRA-------------.....-S--LAA--D----P--.-S1470O06 ---N---P--------------...QTRA-------------.....-S--LAA--D----P--.-S1470A01 ---N---P-R------------...QTRA-------------.....-S--LAA--D----P--.-S1470O01 ---N---P-R------------...QTRA-------------.....-S--LAA--D----P--.-S1470O05 ---N---P-R------------...QTRA-------------.....-S--LAA--D----P--.-S1470U01 ---N---P-------P------...QTRA-------------.....-S--LAA--D----P--.-S1470U02 ---N---P-------P------...QTRA-------------.....-S--LAA--D----P--.-S1470U03 ---N---P-------P------...QTRA-------------.....-S--LAA--D----P--.-S1470U04 ---N---P-------P------...QTRA-------------.....-S--LAA--D----P--.-S1470U05 ---N---P-------P------...QTRA-------------.....-S--LAA--D----P--.-S1470O02 ---N---Q--------------...QTRA-------------.....-S--LAA--D----P--.-SPosttherapy 1664U01 ---N---P--------------...QTRA-------------.....-S--LAA--D----P--.-S1664U02 ---N---P--------------...QTRA-------------.....-S--LAA--D----P--.-S1664U03 ---N---Q--------------...QTRA-------------.....-S--LAA--D----P--.-S1664U04 ---N---Q--------------...QTRA-------------.....-S--LAA--D----P--.-S1664U05 ---N---Q--------------...QTRA-------------.....-S--LAA--D----P--.-S1664U06 ---N---Q--------------...QTRA-------------.....-S--LAA--D----P--.-S1664.48.17 ---N---Q--R-----------...QTRA-------------.....-S--LAA--D----P--.-S1664.48.26 ---N---Q------L-------...QTRA-------------.....-S--LAA--D----P--.-S1664.48.18 ---N---Q--------------...QTRA-------------.....-S--LAA--D----P--.-S1664.48.19 ---N---Q--------------...QTRA-------------.....-S--LAA--D----P--.-S1664.48.22 ---N---Q--------------...QTRA-------------.....-S--LAA--D----P--.-S1664.48.24 ---N---Q--------------...QTRA-------------.....-S--LAA--D----P--.-S1664.48.25 ---N---Q--------------...QTRA-------------.....-S--LAA--D----P--.-S1664.48.27 ---N---Q--------------...QTRA-------------.....-S--LAA--D----P--.-S1664.48.21 ---N---P--------------...QTRA-------------.....-S--LAA--D----P--.-S1664.48.23 --K----P---PG---------...QTRA-------------.....-S--LAA--D----P--.-S1664.48.28 -------P----G---------...QTRA-------------.....-S--LAA--D----P--.-S

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218 2 F PSEAGADRQGTVS.FNF HXB VFIS 11 Pretherapy 1446U01 --KN---P--E--------N--.......-----G---L--G.....--P.L----QE-.R---.-S1446U02 --KN---P--E---I----N--.......-----G---L--G.....--P.L----QE-.R---.-S1446U03 --KN---P--E---I----N--.......-----G---L--G.....--P.L----QE-.R---.-S1446U04 --KN---P--E---I----N--.......-----G---L--G.....--P.L----QE-.R---.-S1446U05 --KN---P--E---I----N--.......-----G---L--G.....--P.L----QE-.R---.-SPosttherapy 1656C05 ---N---P--E----F---N-T.......---A--KR-L---.....--P.L----QE-.R---.-S1656C08 ---N---P--E----F---N-T.......---A--KR-L---.....--P.L----QE-.R---.-S1656U10 ---N---P--E--------N-T.......H--A-G---L--G.....--P.L----QE-.RA--.-S1656U11 ---N---P--E--------N-T.......H--------L--G.....--P.L----QE-.RA--.-S1656U12 ---N---P--E--------N-T.......H--------L--G.....--P.L----QE-.RA--.-S1656U08 ---N---P--E--------N-T.......H---G-K--L--G.....--P.L----QE-.RA--.-S1656U06 ---N---P--E--------N--.......H---GG---L--G.....--P.L----QE-.RA--.-S1656U07 ---N---P--E--------N--.......H---GG---L--G.....--P.L----QE-.RA--.-S1656U09 ---N---P--E--------N--.......H---G----L---.....--P.L----QE-.RA--.-S1656U01 ---N---P--E--K-----N--.......H---G----L---.....--P.L----QE-.RA--.-S1656U02 ---N---P--E--K-----N--.......H---GG---L--G.....--P.L----QE-.RA--.-S1656U03 ---N---P--E--K-----N--.......H---GG---L--G.....--P.L----QE-.RA--.-S1656U05 ---N---P--E--K-----N--.......H---GGK--L---.....--P.L----QE-.RA--.-S1656U04 ---N---P--E--K-----N--.......----G----L--G.....--P.L----QE-.RA--.-S1656.48.02 ---N---P--E--------N--.......-----G---L--G.....--P.L----QE-.R---.-S1656.48.04 ---N---P--E--------N--.......-----G---L--G.....--P.L----QE-.RA--.-S1656.48.05 ---N---P--E--------N--.......-----G---L--G.....--P.L----QE-.RA--.-S1656.48.08 ---N---P--E--------N--.......-----G---L--G.....--P.L----QE-.RA--.-S1656.48.10 ---N---P--E--------N--.......-----G---L--G.....--P.L----QE-.RA--.-S1656.48.12 ---N---P--E--------N--.......----GG---L--G.....--P.L----QE-.RA--.-S1656.48.03 ---N---P--E--------N--.......----GG---L--G.....--P.L----QE-.RA--.-S1656.48.01 ---N---P--E--------N--.......----GG---L--G.....--P.L----QE-.RA--.-S1656.48.07 ---N---P--E--------N--.......----GG---L--G.....--P.L----QE-.RA--.-S1656.48.06 ---N---P--E--------N--.......----GG---LR-G.....--P.L----QE-.RA--.-S1656.48.09 ---N---P--E--------N--.......----GG---LR-G.....--P.L----QE-.RA--.-S1656.48.11 ---N---P--E--------N--.......----GG---LR-G.....--P.L----QE-.RA--.-SF RE D L AF L QG K ARE SS E QT R A .. . .. . . NS P TR R EL Q V WG R .. . .. D NNS

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219 2 A R.....DNNSPSEAGADRQGTVS.FNF HXB VFIS 12 Pretherapy 1738O01 ---N---P--E-----------.......-------------.....----------SG-----.LSL 1738A03 ---N---P--E-----------.NSPTRAI----G-------.....----L------------.LSL 1738O04 ---N---P--E-----------.NSPTRAI----G-------.....----L------------.LSL 1738O03 ---N---P--E-----------.NSPTRAI----G-------.....----L------------.LSL 1738A04 ---N---P--E---L-------.NSPTRTI----G-------.....----L------------.LSL 1738A05 ---N---P------L-------.NSPTRAI----G-------.....----L------------.LSL 1738A06 ---N---P------L-------.NSPTRAI----G-------.....--S-F------------.LSL Posttherapy 1943A01 ---N---P--E-----------.NSPTRAI----G-------.....----L------------.LSL 1943A03 ---N---P--E-----------.NSPTRAI----G-------.....----L------------.LSL 1943O11 ---N---P--E-----------.NSPTRAI----G-------.....----L------------.LSL 1943O12 ---N---P--E-----------.NSPTRAI----G-------.....----L------------.LSL 1943O13 ---N---P--E-----------.NSPTRAI----G-------.....----L------------.LSL 1943A05 ---N---P--E-----------.NSPTRAI----G-------.....-S--------GG-----.LSL 1943O14 ---N---P--E---L-------.NSPTRAI----G-------.....--S-F--G---------.LSL 1943O15 ---N---P--E---L-------.NSPTRAI----G-------.....--S-F--G---------.LSL 1943O16 ---N---P--E---L-------.NSPTRAI----G-------.....--S-F--G---------.LSL 1943R01 ---N---P--E---L-------.NSPTRAI----G-------.....--S-F--G---------.LSL 1943U01 -------P--E-----------.......------------G.....----L-----EG---I-.-S1943U04 -------P--E-----------.......------------G.....----L-----EG---I-.-S1943U06 -------P--E-----------.......------------G.....----L-----EG---I-.-S1943U08 -------P--E-----------.......------------G.....----L-----EG---I-.--1943.48.01 ---N---P--E-----------.NSPTRAI----G-------.....----L------------.LSL 1943.48.02 ---N---P--E-----------.NSPTRAI----G-------.....----L------------.LSL 1943.48.03 ---N---P--E-----------.NSPTRAI----G-------.....----L------------.LSL 1943.48.04 ---N---P--E-----------.NSPTRAI----G-------.....----L------------.LSL 1943.48.05 ---N---P--E-----------.NSPTRAI----G-------.....----L------------.LSL 1943.48.07 ---N---P--E-----------.NSPTRAI----G-------.....----L------------.LSL 1943.48.08 ---N---P--E-----------.NSPTRAI----G-------.....----L------------.LSL 1943.48.10 ---N---P--E-----------.NSPTRAI----G-------.....----L------------.LSL 1943.48.12 ---N---P--E-----------.NSPTRAI----G-------.....----L------------.LSL 1943.48.06 ---N---P--E---L-------.NSPTRAI----G-------.....----L----------I-.LSL 1943.48.11 ---N---P--E---L-------.NSPTRAI----G-------.....-S--------GG-----.LSL HXB2 FREDLAFLQGKAREFSSEQTRA.......NSPTRRELQVWGR.....DNNSPSEAGADRQGTVS.FNF VFIS 14 Pretherapy 1329u01 ---N---P--E---I-P-----.......-----G------G......D--L--------EPA-.-S1329u02 ---N---P------I-P-----.......-----G------G......D--L--------EPA-.-S1329u06 ---N---P------I-P-----.......-----G------G......DS-L--------EPA-.-S1329u03 ---N---P------I-------.......-----G------G......DS-L--------EPA-.-S1329u07 ---N---P------I-------.......-----G------G......DS-L--------EPA-.-SPosttherapy 1576.48.02 ---N---P--E-----------.......-----G------G......D--L--------EPA-.-S1576.48.06 ---N---P--E-----------.......-----G---A--G......D--L--------EPA-.-S1576.48.08 ---N---P--E-----------.......-----G---A--G......D--L--------EPA-.-S1576.48.10 ---N---P--E-----------.......-----G---A--G......D--L--------EPA-.-S1576.48.12 ---N---P--E-----------.......-----G---A--G......D--L--------EPA-.-S1576.48.07 ---N---P--E-----------.......-R---G---A--G......D--L--------EPA-.-S1576.48.11 ---N---P-RE-----------.......-R---G---A--G......D--L--------EPA-.-S1628u02 ---N---P--E-----------.......-----G---A--G......D--L--------EPA-.-S1628u07 ---N---P--E-----------.......-----G---A--G......D--L--------EPA-.-S1628u04 ---N---P--E-----------.......-----G------G......D--L--------EPA-.-S1628u01 ---N---P--------P-----.......-----G------G......DS-F--------EPA-.-S1628u03 ---N---P--------P-----.......-----G------G......DS-F--------EPA-.-S1628u13 ---N---P-RRPG*--------.......-----G------G......DS-L--------EPA-.-S1628u17 ---N---P--RPG*--------.......-----G------G......DS-L--------EPA-.-SF RE DL FL QG K AR E F SS E QT R A .. . .. . . NS P TR R EL Q VWG

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220 HXB2 FREDLAFLQGKAREFSSEQTRA.......NSPTRRELQVWGR.....DNNSPSEAGADRQGTVS.FNF VFIS 15 Pretherapy 68U01 -S8U0 S18 186 1868U03 ---------------------E...QTRA----S-------G.....-----------------.LS1868U04 ---T------E----------E...QTRA----S-------G.....-----------------.LS1868C01 ----------E----------E...QTRA----S----LP--.....EC--L----D-------.LS1868C03 ----------E----------E...QTRA----S--------KLQGREC--L----D-------.LS1868C04 ----------E----------E...QTRA----S--------KLQGREC--L----D-------.LSPosttherapy 2209A03 ---N------E----------E...QTSA----S-------G.....-----------------.LS2209A07 ---N------E----------E...QTSA----S------RG.....----L----D-------.LS2209O03 ----------E----------E...QTRA----S------RG.....----L----D-------.LS2209O04 ----------E----------E...QTRA----S------RG.....----L----D-------.LS2209O07 ----------E----------E...QTRA----S--------.....----L----D-------.LS2209A06 ----------E----------E...QTRA----S--------...DNN----------------.LS2209O02 ----------E----------E...QTRA----S--------KLQGREC--L----D-------.LS2209.48.12 ---N------E----------E...QTSA----S------RG.....----L----D-------.LS2209.48.02 ----------E----------E...QTRA----S------RG.....----L----D-------.LS2209.48.08 ----------E----------E...QTRA----S------RG.....----L----D-------.LS2209.48.09 ----------E----------E...QTRA----S------RG.....----L----D-------.LS2209.48.04 ----------E----------E...QTRA----S--------KLQGREC--L----D-------.LS2209.48.06 ----------E----------E...QTRA----S--------KLQGREC--L----D-------.LS2209.48.07 ----------E----------E...QTRA----S--------KLQGREC--L----D-------.LS2209.48.10 ----------E----------E...QTRA----S--------KLQGREC--L----D-------.LS2209.48.11 ----------E----------E...QTRA----S--------KLQGREC--L----D-------.LSHXB2 FREDLAFLQGKAREFSSEQTRA.......NSPTRRELQVWGR.....DNNSPSEAGADRQGTVS.FNF VFIS 17 Pretherapy 1875.48.01 ---N---P------L-------.......I------------.....-----------T--S--SLS1875.48.02 ---N---P------L-------.......I------------.....-----------T--S--SLS1875.48.04 ---N---P------L-------.......I------------.....-----------T--S--SLS1875.48.08 ---N---P------L-------.......I------------.....-----------T--S--SLS1875.48.10 ---N---P------L-------.......I------------.....-----------T--S--SLS1875.48.06 ---N---P------L-T-----.......-------------.....-----------T--S--SLS1875.48.09 ---N---P------L-------.......I------------.....--T--------T--S--S-S1875.48.11 ---N---P------L-------.......I------------.....--T--------T--S--S-S1875.48.05 ---N---P-W-G--L-------.......I------------.....-----------T--S--.-SPosttherapy 2434.00.09 ---N---P------L-------.......I------------.....-----------T--S--SLS2434.00.02 ---N---P------L-------.......-------------.....--S-L-----NK--S--S-S2434.00.03 ---N---P------L-------.......-------------.....--S-L-----NK--S--S-S2434.00.05 ---N---P------L-------.......-------------.....--S-L-----NK--S--S-S2434.00.06 ---N---P------L-------.......-------------.....--S-L-----NK--S--S-S2434.00.08 ---N---P------L-------.......-------------.....--S-L-----NK--S--S-S2434.00.10 ---N---P------L-------.......-------------.....--S-L-----NK--S--S-S2434.00.12 ---N---P------L-------.......-------------.....--S-L-----NK--S--S-S2434.00.11 ---N---P------L-------.......-------------.....--S-L-----NK--S--S-S2542.48.03 ---N---P--*---L-------.......I------------.....-----------T--S--SLS2542.48.05 ---N---P------L-------.......I------------.....-----------T--S--SLS2542.48.07 ---N---P------L-------.......I------------.....-----------T--S--SLS2542.48.10 ---N---P--Q---L-------.......I------------.....-----------T--S--SLS2542.48.02 ---N---P--E---L-------.......I------------.....-----------T--S--SLS2542.48.06 ---N---P--E---L-------.......I------------.....-----------T--S--SLS2542.48.04 ---N---P--E---L-------.......I------------.....-----------T--S--SLS2542.48.08 ---N---P--E---L-------.......I------------.....-----------T--S--SLS2542.48.11 ---N---P--E---L-------.......I------------.....-----------T--S--SLS2542.48.01 ---N---P--E---L-------.......I--------..--.....-----------T--S--SLS2542.48.09 ---N---P--E---L-------.......I--------..--.....-----------T--S--SLS2542.48.12 ---N---P--E---L-------.......I--------..--.....-----------T--S--SLS2 --N N ----------E E .. .. . . QT QT R R AA--S -S ----G G .. .. . . .. .. ---------.L -.L --

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221 2 A QVWGR.....DNNSPSEAGADRQGTVS.FNF HXB VFIS 18 Pretherapy 2550U04 ---N---P-R------------.......-------P---RG.....--QTL----S----D--.-S2550U09 ---N---P-R------------.......-------P---RG.....--QTL----S----D--.-S2550U05 ---N---P-R------------.......-------P---RG.....--QTL----S----D--.-S2550U02 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-S2550U03 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-S2550U08 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-S2550U11 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-S2550U06 ---N---P-R------------.......I------P---RG.....--QTL----S----D--.-I2550U07 ---N---P-R------------.......I------P---RG.....--QTL----S----D--.-I2550U10 ---N---P-R------------.......I------P---RG.....--QTL----S----D--.-S2550U12 ---N---P-R------------.......I------P---RG.....--QTL----S----D--.-SPosttherapy 2930C01 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-S2930C02 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-S2930C03 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-S2930C04 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-S2930C05 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-S2930c06 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-S2930C07 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-S2930.48.09 ---N---P-R------------.......I------P---RG.....--QTL----S----D--.-S2930.48.01 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-S2930.48.02 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-S2930.48.04 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-S2930.48.05 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-S2930.48.06 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-S2930.48.07 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-S2930.48.08 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-S2930.48.10 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-S2930.48.11 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-S2930.48.12 ---N---P-R------------.......I------P---RG.....--QTL----S--P-D--.-SHXB2 FREDLAFLQGKAREFSSEQTRA.......NSPTRRELQVWGR.....DNNSPSEAGADRQGTVS.FNF VFIS 22 Pretherapy 1879C03 -------P--------------....NSPT------------......D-------D-K-----.-SL 1879C04 -------P--------------....NSPT------------......D-------D-K-----.-SL 1879C07 ---N---P--------------....NSPT------------......D-F-----D-K-----.-SL 1879C08 -------P--------------....NSPT------------......D-------D-K-----.-SL Posttherapy 2411c01 -------P--------------....NSPT------------......D-------D-K-----.-SL 2411c02 -------P--------------....NSPT------------......D-------D-K-----.-SL 2411c03 -------P--------------....NSPT------------......D-------D-KP----.-SL 2411c04 -------P--RXK--------S....NSPT------------......D-------D-K-----.-SL 2411c05 -------P--------------....NSPT------------......D-------D-K-----.-SL 2411c06 -------P--------------....NSPT------------......D-------D-K-----.-SL 2411c07 -------P--------------....NSPT------------......D-------D-K-----.-SL 2411c08 -------P--------------....NSPT------------......DTP-----D-K-----.-SL 2411.48.02 -------P--------------....NSPT------------......D-------D-*-----.-SL 2411.48.04 -------P-----------N--....NSPT------------......D-------D-K-----.-SL 2411.48.05 -------P--------------....NRPTX-S---------......DK------D-K-----.-SL 2411.48.06 -------P--------------....NSPT------------......D-------D-K-----.-SL 2411.48.07 -------P--------------....NSPT------------......D-------D-K-----.-SL 2411.48.08 -------P--------------....NSPT------------......D-------D-K-----.-SL 2411.48.09 -------P--------------....NSPT------------......D-------D-K-----.-SL 2411.48.10 -------P--------------....NSPT------------......D-------D-K-----.-SL 2411.48.11 -------P--------------....NRP-R--------R--......DK-H----D-E-----.-SL 2411.48.12 -------P--------------....NSPTR-----------......DE------D-K-----.-SL F RE DL FL QG K AR E F SS E QT R A .. . .. . . NS P TR REL

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222 HXB2 FREDLAFLQGKAREFSSEQTRA.......NSPTRRELQVWGR.....DNNSPSEAGADRQGTVS.FNF VFIS 24 Pretherapy 2098U04 ---N---P-------P------....NSST-----------G.....----L-----N---A--.-G2098U03 ---N---P-------P------....NSST-----------G.....----L-----N---A--.-G2098U01 ---N---P-------P------....NSST-----------G.....----L-----N--E-I-.-G2098U05 ---N---P-------P------....NSST-----------G.....----L-----N--E-I-.-GPosttherapy 2375U01 ---N---P-------P------....NSST-----------G.....----L-----N---A--.-G2375U02 ---N---P-------P------....NSST-----------G.....----L-----N---A--.-G2375U03 ---N---P-------P------....NSST-----------G.....----L-----N---A--.-G2375U04 ---N---P-------P------....NSST-----------G.....----L-----N---A--.-G2375U05 ---N---P-------P------....NSST-----------G.....----L-----N--E---.-G2375.48.02 ---N---P-------P------....NSST-----------G.....----L-----N---A--.-G2375.48.08 ---N---P-------P------....NSST-----------G.....----L-----N---A--.-G2375.48.11 ---N---P-------P------....NSST-----------G.....----L-----N---A--.-G2375.48.12 ---N---P-------P------....NSST-----------G.....----L-----N---A--.-G2375.48.17 ---N---P-------P------....NSST-----------G.....----L-----N---A--.-G2375.48.09 ---N---P-------P------....NSST-----------G.....----L-----N-PE-I-.-G2375.48.07 ---N---P-------P------....NSST-----------G.....----L-----N-PE-I-.-G2375.48.04 ---N---P-------P------....NSST-----------G.....----L-----N-PE-I-.-G2375.48.05 ---N---P-------P------....NSST-----------G.....----L-----N-PE-I-.-G2375.48.06 ---N---P-------P------....NSST-----------G.....----L-----N-PE-I-.-G2375.48.10 ---N---P-------P------....NSST-----------G.....----L-----N-PE-I-.-G2375.48.18 ---N---P-------P------....NSST-----------G.....----L-----N-PE-I-.-G2375.48.20 ---N---P-------P------....NSST-----------G.....----L----DN---A--.-G2375.48.24 ---N---P-----e-P------....NSS------------G.....--K-L----DN---A--.-G2375.48.15 ---N---P-------P------....NSS------------G.....--K-L-----N---A--.-G2375.48.16 ---N---P-------P------....NSST-----------G.....--K-L-----N-PE-I-.-G2375.48.03 ---N---P-------P------....NSST-----------G.....--K-L-----N-PE-I-.-GHXB2 FREDLAFLQGKAREFSSEQTRA.......NSPTRRELQVWGR.....DNNSPSEAGADRQGTVS.FNF VFIS 26 Pretherapy 2612.48.01 ----------------------.......-----G-------.....G-S---------PRK--.--2612.48.02 ----------------------.......-----G-------.....G-S---------PRK--.--2612.48.04 ----------------------.......-----G-------.....G-S---------PRK--.--2612.48.05 ----------------------.......-----G-------.....G-S---------PRK--.--2612.48.07 ----------------------.......-----G-------.....G-S---------PRK--.--2612.48.09 ----------------------.......-----G-------.....G-R---------PRK--.--2612.48.06 ----------------------.......-----G-------.....--S---------PRK--.--2612.48.10 ----------------------.......-----G-------.....--S---------PRK--.--2612.48.11 ----------------------.......-----G-------.....--S---------PRK--.--2612.48.12 ----------------------.......-----G-------.....--S---------PRK--.--Posttherapy 3058.00.04 ----------------------.......-----G-------.....--S---------PRK--.--3058.00.05 ----------------------.......-----G-------.....--S---------PRK--.--3058.00.10 ----------------------.......-----G-------.....--S---------PRK--.--3058.00.11 ----------------------.......-----G-------.....--R---------PRK--.--3058.00.01 --------------L-------.......-----G-------.....--S---------PRK--.--3058.00.02 --------------L-------.......-----G-------.....--S---------PRK--.--3058.00.03 --------------L-------.......-----G-------.....--S---------PRK--.--3058.00.07 --------------L-------.......-----G-------.....--S---------PRK--.--3058.00.09 --------------L-------.......-----G-------.....--S---------PRK--.--3058.00.12 --------------L-------.......-----G-------.....--S---------PRK--.--

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223 HXB2 FREDLAFLQGKAREFSSEQTRA.......NSPTRRELQVWGR.....DNNSPSEAGADRQGTVS.FNF VFIS 29 Pretherapy 3122.48.03 --K-----------L----A--.......----G-------G.....GS---------------.-S2.4 -G-------G.....GS---------------.-S312 3122.48.07 --------------L----A--.......----G-------G.....-----------------.-S3122.48.08 --------------L-------.......----G-------G.....-T---------------.-S3122.48.09 --------------L-------.......----G-------G.....-T---------------.-S3122.48.06 -------P------L----A--.......----G-------G.....-T---------------.-S3122.48.01 ---N---P-----------A--.......----G-------G.....-----------------.-S3122.48.02 ---N---P--------------.......----G-------G.....-S---------------.-S3122.48.12 ---N---P-----------A--.......----G-------G.....-S---------------.-SPosttherapy 3501.00.07 -------------------A--.......----G--------.....----L--------R---.-S3501.00.08 -------------------A--.......----G--------.....----L--------R---.-S3501.00.12 -------------------A--.......----G--------.....-S---------------.-S3501.00.02 -------------------A--.......----G--------.....-S---------------.-S3501.00.04 -------------------A--.......----G--------.....-S---------------.-S3501.00.09 -------------------A--.......----G--------.....-S---------------.-S3501.00.10 -------------------A--.......----G--------.....-S---------------.-S3501.00.01 ---N---P-----------A--.......----G--------.....-S---------------.-S3501.00.03 ---N---P-----------A--.......----G-------G.....-S--L------------.-S3501.00.05 ---N---P-----------A--.......----G-------G.....-S--L------------.-S3501.00.11 ---N---P-----------A--.......----G-------G.....-S--L------------.-S3501.48.02 ---N---P-----------A--.......----G-------G.....-T---------------.-S3501.48.03 ---N---P-----------A--.......----G-------G.....-T---------------.-S3501.48.06 ---N---P-----------A--.......----G-------G.....-T---------------.-S3501.48.07 ---N---P-----------A--.......----G-------G.....-T---------------.-S3501.48.08 ---N---P-----------A--.......----G-------G.....-T---------------.-S3501.48.09 ---N---P-----------A--.......----G-------G.....-T---------------.-S3501.48.04 ---N---P-----K-----A--.......----G-------G.....-T---------------.-S3501.48.10 ---N---P-----K-----A--.......----G-------G.....-T---------------.-S3501.48.11 ---N---P-----K-----A--.......----G-------G.....-T---------------.-S3501.48.12 ---N---P-----K-----A--.......----G-------G.....-T---------------.-S8. 0 4 -K ----L --A .. . .. . . ---

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224 2 L VWGR.....DPSE HXB VFIS 30 Pretherapy 2649.48.12 ---N------E----------T.......I----G------G.....--K-----------E--.-S2649.48.01 ---N------E----------T.......I----G------G.....--------------E--.-S2649.48.05 ---N------E----------T.......I----G------G.....--------------E--.-S2649.48.02 ---N------E----------T.......I----G------G.....--------------D--.-S2649.48.03 ---N------E-----------.......I----G------G.....--------------D--.-S2649.48.06 ---N------E-----------.......I----G------G.....--------------D--.-S2649.48.08 ---N------E-----------.......I----G------G.....--------------D--.-S2649.48.09 ---N------E-----------.......I----G------G.....--------------D--.-SPosttherapy 3037.00.02 ---N------E----------T.......I----G------G.....--------------D--.-S3037.00.05 ---N------E----------T.......I----G------G.....--------------D--.-S3037.00.03 ---N------E----------T.......I----G------G.....--------------D--.-S3037.00.08 ---N------E----------T.......I----G------G.....--------------D--.-S3037.00.09 ---N------E----------T.......I----G------G.....--------------D--.-S3037.00.12 ---S------E----------T.......I----G------G.....--------------D--.-S3037.00.04 ---N------E-----------.......IR---G--K---G.....--------------D--.-S3037.00.07 ---N------E-----------.......I----G--K---G.....--------------D--.-S3037.00.11 ---N------E-----------.......I----G--K---G.....--------------D--.-S3037.00.06 ---N------E-----------.......I----G------G.....--------------D--.-S3037.48.03 ---N------E----------T.......I----G------G.....--------------D--.-S3037.48.04 ---N------E----------T.......I----G------G.....--------------D--.-S3037.48.06 ---N------E----------T.......I----G------G.....--------------D--.-S3037.48.07 ---N------E----------T.......I----G------G.....--------------D--.-S3037.48.10 ---N------E----------T.......I----G------G.....--------------D--.-S3037.48.11 ---N------E----------T.......I----G------G.....--------------D--.-S3037.48.01 ---N------E----------T.......IR---G------G.....--------------D--.-S3037.48.09 ---N------E----------T.......IR---G------G.....--------------D--.-S3037.48.02 ---N------E----------T.......IR---G------G.....--K-----------D--.-S3037.48.05 ---N------E----------T.......IR---G------G.....--K-----------D--.-SF RE D LAF QG K AR E F SS E QT R A .. . .. . . NS P TR R ELQ NNS AG A DR Q GT V S .F N F

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225 HXB2 FREDLAFLQGKAREFSSEQTRA..........NSPTRRELQVWGRDNNSPSEAG.........ADRQGTVSFNF VFIF 05 Pretherapy 1877u04 ---N---P--E----Y---N--..........----S-----------------.........-----D---S1877u01 ---N---P--E----Y---D--..........----S------------L----.........-----D---S1877u05 ---N---P--E----Y---D--..........----S------------L----.........-E---D---S1877u03 ---N---P--E----H---D--..........----S-----------------.........-----D---SPosttherapy 2190.48.01 ---N---P--E----Y---D--..........----S-----------------.........-----D---S2190.48.02 ---N---P--E----H---N--..........----S------------L----.........-E---D---S2190.48.03 ---N---P--E----H---N--..........----S------------L----.........-E---D---S2190.48.05 ---N---P--E----H---N--..........----S------------L----.........-E---D---S2190.48.07 ---N---P--E----H---N--..........----S------------L----.........-E---D---S2190.48.08 ---N---P--E----H---N--..........----S------------L----.........-E---D---S2190.48.11 ---N---P--E----H---N--..........----S------------L----.........-E---D---S2190.48.04 ---N---P--E----H---N--..........----S-------KE--------.........----RD---S2190.48.10 ---N---P--E----H---D--..........----S-DHR---KE--------.........----RD---S2190.48.12 ---N---P--E----H---D--..........----S-DHR---KE--------.........----RD---SHXB2 FREDLAFLQGKAREFSSEQTRA..........NSPTRRELQVWGRDNNSPSEAG.........ADRQGTVSFNF VFIF 06 Pretherapy 1944.48.01 ---N---P------L-P-----..........I---S-GP----G---------.........----------1944.48.02 ---N---P------L-P-----..........I---S-GP----G---------.........----------1944.48.03 ---N---P------L-P-----..........I---S-GP----G---------.........----------1944.48.08 ---N---P------L-P-----..........I---S-GP----G---------.........----------1944.48.04 ---N---P------L-P-----..........I---S-GP----G---------.........----------1944.48.06 ---N---P------L-P-----..........I---S-GP----G---------.........--------LD1944.48.10 ---N---P------L-P-----..........I---S-GP----G---------.........--------LD1944.48.11 ---N---P------L-P-----..........I---S-GP----G---------.........--------LD1944.48.12 ---N---P------L-P-----..........I---S-GP----G---------.........--------LD1944.48.07 ---N---P------LPP-----..........I---S-GP----G---------.........----R---LD1944.48.05 ---N---P------LPP-----..........IR--S-GP----G---------.........----R---LDPosttherapy 2292u04 ---N---P------L-P-----..........I---S-GP----G---------.........----------2292u01 ---N---P------L-P-----..........I---S-GP----G---------.........--------L-2292u02 ---N---P------L-P-----..........I---S-GP----G---------.........--------L-2292u05 ---N---P------L-P-----..........I---S-GP----G---------.........--------L-2292u03 ---N---P------L-P-----..........I---S-GP----G---------.........--------LD2292.48.06 ---N---P------L-P-----..........IR--S-GP----G---------.........----------2292.48.05 ---N---P------LPP-----..........I---S-GP----G---------.........----------2292.48.03 ---N---P------L-P-----..........I---S-GP----G---------.........----------2292.48.10 ---N---P------L-P-----..........I---S-GP----G---------.........----R---L-2292.48.04 ---N---P------L-P-----..........I---S-GP----G---------.........--------LD2292.48.08 ---N---P------L-P-----..........I---S-GP----G---------.........--------LD2292.48.12 ---N---P------L-P-----..........I---S-GP----G---------.........--------LD2292.48.01 ---N---P------L-P-----..........I---S-GP----G---------.........--------LD2292.48.07 ---N---P------LPP-----..........IR--S-GP----G---------.........--------LD2292.48.09 ---N---P------LPP-----..........I---S-GP----G---------.........--------LD

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226 HXB2 FREDLAFLQGKAREFSSEQTRA..........NSPTRRELQVWGRDNNSPSEAG.........ADRQGTVSFNF VFIF 07 Pretherapy 1557U01 -------P--E-----------NSP.......T---G-------GE---R----.........D--------S1557U02 -------P--E-----------NSP.......T---G-------GE---R----.........D--------S1557U03 -------P--E-----------NSP.......T---G-------GE---R----.........D--------S1557U04 -------P--E-----------NSP.......T---G-------GE---R----.........D--------S1557U05 -------P--E-----------NSP.......T---G-------GE---R----.........D--------SPosttherapy 1678U03 -------P--E-----------NSP.......T---G-------GE---R----.........D--------S1678U04 ---N---P--E------K----NSP.......T---G-------GE---R----.........D--------S1678U05 -------P--E-----------NSP.......T---G-------GE---R----.........D--------S1678U07 -------P--E-----------NSP.......T---G-------GE---R----.........D--------S1678U02 -------PR-E-----------NSP.......T---G-------GE---L----.........D--------S1678U06 -------P--E-----------NSP.......T---G-------GE---L----.........D--------SHXB2 FREDLAFLQGKAREFSSEQTRA..........NSPTRRELQVWGRDNNSPSEAG.........ADRQGTVSFNF VFIF 08 Pretherapy 1934U01 -------P--E-----------..........------------G----L----.........-EG---I--S1934U03 -------P--E-----------..........------------G----L----.........-EG---I--S1934U08 -------P--E-----------..........------------G----L----.........-EG---I--S1934U06 -------P--E-----------..........------------G----L----.........-EG-QSI--SPosttherapy 2234U04 -------P--E-----------..........---------I--G----L----.........-EG-QSI--S2234U06 -------P--E-----------..........------------G----L----.........-EG-QSI--S2234U02 ---N---P--E-----------..........------------G----L----.........-EG-QSI--S2234U03 ---N---P--E-----------..........------------G---------.........-EG-QSI--S2234U08 ---N---P--E-----------..........------------G---------.........-EG-QSI--S2234.48.03 -------P--E-----------..........---------I--G----L----.........-EG-QSI--S2234.48.05 ---N---P--E-----------..........------------G----L----.........-EG-QSI--S2234.48.06 ---N---P--E-----------..........------------G----L----.........-EG-QSI--S2234.48.08 ---N---P--E-----------..........----*-------G----L----.........-EG-QSI--S2234.48.09 ---N---P--E-----------..........------------G----L----.........-EG-QSI--S2234.48.11 ---N---P--E-----------..........------------G----L----.........-EG-QSI--S2234.48.12 ---N---P--E-----------..........------------G----L----.........-EG-QSI--S2234.48.07 ---N---P--E-----------..........-R----------G----L----.........-EG-QSI--S2234.48.02 ---N---P--E-----------..........-R----------G----L----.........-EG-QSI--S2234.48.10 ---N---P--E-----------..........-R----------G----L----.........-EG-QSI--S

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227 HXB2 FREDLAFLQGKAREFSSEQTRA..........NSPTRRELQVWGRDNNSPSEAG.........ADRQGTVSFNF VFIF 09 Pretherapy 1955c01 ----------E-----------NSP.......------------G--S------.........--------LS1955c03 ----------E-----------NSP.......------------G--S------.........--------LS1955c05 ----------E-----------..........------------G--S------.........--------LS1955c08 ----------E-----------..........------------G--S------.........--------LS1955c06 ----------E-----------..........------------G--S------ADSSPSEAG--------LS2208.48.12 ----------E-----------NSPTSEQTRA------------G--S------.........--------LS2208.48.01 ----------E-----------..........----------R-G--S------.........--------LS2208.48.02 ----------E-----------..........----------R-G--S------.........--------LS2208.48.03 ----------E-----------..........------------G--S------.........--------LS2208.48.04 ----------E-----------..........------------G--S------.........--------LS2208.48.05 ----------E-----------..........------------G--S------.........--------LS2208.48.08 ----------E-----------..........------------G--S------.........--------LS2208.48.11 ----------E-----------..........------------GADS------.........--------LS2208.48.06 ----------------------..........------------GVDS------.........--------LS2208.48.07 ----------------------..........------------GVDS------.........--------LSPosttherapy 2686c01 ----------E-----------NSPTSEQTRA------------G-DS------.........--------LS2686c02 ----------E-----------NSPTSEQTRA------------G-DS------.........--------LS2686c03 ----------E-----------NSPTSEQTRA------------G-DS------.........--------LS2686c04 ----------E-----------NSPTSEQTRA------------G-DS------.........--------LS2686c06 ----------E-----------NSPTSEQTRA------------G-DS------.........--------LS2686c07 ----------E-----------NSPTSEQTRA------------G-DS------.........--------LS2686c08 ----------E-----------NSPTSEQTRA------------G-DS------.........--------LS2686c05 ----------E-----------..........------------G--S------.........--------LS2686u02 ----------E-----------..........------------G--S------ADSSPSEAR--------LS2686u021 ----------E-----------..........------------G--S------ADSSPSEAR--------LS2686u031 ----------E-----------..........------------G--S------ADSSPSEAR--------LS2686u04 ----------E-----------..........------------G--S------ADSSPSEAR--------LS2686.48.02 ----------E-----------NSPTSEQTRA------------G--S------.........--------LS2686.48.09 ----------E-----------NSPTSEQTRA----------R-G--S------.........--------LS2686.48.04 ----------E-----------..........------------G--S------.........--------LS2686.48.11 ----------E-----------..........------------G--S------.........--------LS2686.48.03 ----------E-----------..........------------G--S------ADSSPSEAG--------LS2686.48.12 ----------E-----------..........------------G--S------ADSSPSEAR--------LS2686.48.07 ----------E-----------..........------------G--S------ADRSPSEAG--------LS2686.48.05 ----------E-----------..........------------G--S------TDRSPSEAG--------LS2686.48.06 ----------E-----------..........------------G--S------AGSSPSEAG--------LS2686.48.08 ----------E-----------..........------------G--S------VGSSPSEAG--------LS

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APPENDIX E ALIGNMENT OF ENVELOPE SEQUENCES

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229HXB2 CTDLK NDT NTNSSSGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGK IGNMRQAHCNISRAKWNNTLKQIASKLREQFGN VSIS 01 Pretherapy 2272 00 01 ---CTSNC-TS-T--KENI----K-----T-NR-NRIK----L-NE--V-K--HS-SN-I-RR-Q--SND-------MTKA-V------------S-HA-----FAT-DI--DI------L---E--D----V-G-------2272 00 02 ---CTSNC-TS-T--KENI----K-----T-NR-NRIK----L-NE--V-K---S-SN-I-RR-Q--SN--------MTKA-V------------S-HA-----FAT-DI--DI------L---E--D----V-G-------2722 00 03 ---CTSNC-TS-T--KENI----K-----T-NR-NRIK----L-NE--V-K---S-SN-I-RR-Q--SN--------MTKA-V------------S-HA-----FAT-DI--DI------L---E--D----V-G-------2722 00 05 ---CTSNC-TS-T--KENI----K-----T-NR-NRIK----L-NE--V-K---S-SN-I-RR-Q--SN--------MTKA-V------------S-HA-----FAT-DI--DI------L---E--D----V-G-------2722 00 06 ---CTSNC-TS-T--KENI----K-----T-NR-NRIK----L-NE--V-K---S-SN-I-RR-Q--SN--------MTKA-V------------S-HA-----FAT-DI--DI------L---E--D----V-G-------2722 00 07 ---CTS -TSNCNDTNWE-STTANNSSGKN-----K-F---T-NR-D-MK------N---V-K---S-SN-V-RG-Q--SN--------MTKV-V-----------RS-N------YAT-DI--DI------L---E--D----V-G-------2722 00 08 ---CTSNC-TS-T--KENI----K-----T-NR-NRIK----L-NE--V-K---S-SN-I-RR-Q--SN--------MTKA-V------------S-HA-----FAT-DI--DI------L---E--D----V-G-------2272 00 10 ---CTSNC-TS-T--KENI---EK-----T-NR-NRIK----L-NE--V-K---S-SN-I-RR-Q--SN--------MTKA-V------------S-HA-----FAT-DI--DI------L---E--D----V-G-------2272 00 11 ---CTSNC-TS-T--KENI----K-----T-NR-NRIK----L-NE--V-K---S-SN-I-RR-Q--SN--------MTKA-V------------S-HA-----FAT-DI--DI------L---E--D----V-G-------2272 00 12 ---CTS -TSICNWGSTTS-T--KENI----K-----T--R-N-IK----L-N---V-K-G-S-SD-I-R--Q--SN--------MTKA-V------------S-NA-----FAT-DI--DI------L---E--D----V-G-------Posttherapy 2774 00 01 ---CTSNC--S-SNKN -----K-----T-NR-NRIR----L-NE--V-K---S-SN-I-RR-Q--SN--------MTKA-VV----A------S-H--A--ICAT-DI--DI------L---E--D----V-G-------2774 00 02 ---CTSNC--S-SNKN -----K-----T-NR-NRIR----L-NE--V-K---S-SN-I-RR-Q--SN--------MTKA-I------------S-H--A--IYAT-DI--DI------L---E--D----V-G-------2774 00 03 ---CTSNC--S-SNKN -----K-----T-NR-NRIR----L-NE--V-K--DS-SN-I-RR-Q--SN--------MTKA-V------------S-H--A--IYAT-DI--DI------L---E--D--T-V-G-------2774 00 04 ---CTSNC--S-SNKN -----K-----T-NR-NRIR----L-NE--V-K---S-GN-I-RR-Q--SN---A----MTKA-V------------S-H--A--IYAT-DI--DI------L---E--D----V-G-------2774 00 05 ---CTSNC--S-SNKN -----K-----T-NR-NRIR----L-NE--V-K---S-SN-I-RR-Q--SN--------MTKA-V-----T------S-H--A--IYAT-DI--DI------L---E--D----V-G-------2774 00 06 ---CTSNC--S-SNKN -----K-----T-NR-NRIR----LSNE--V-K---S-SN-I-RR-Q--SN--E-----MTKA-V------------S-H--A--IYAT-DI--DI------L---E--D----V-G-------2774 00 07 ---CTSNC--S-SNKN -----K-----T--R-NRIR----L-NE--V-K---S-SN-I-RR-Q--SN--------MTKA-V-----T------S-H--S--IYAT-DI--DI------L---E--D----V-G-------2774 00 08 ---CTSNC--S-SNKN -----K-----T-NR-NRIR----L-NE--V-K---S-SN-I-RR-Q--SN--------MTKA-V------------S-H--A--IYAT-DI--DI------L---E--D----V-G-------2774 00 09 ---CTSNC--S-SNKN ----TK--VD-T-NR-NRIR----L-NE--V-T---S-SN-I-RR-Q--SN--------MTKA-V------------S-H--A--IYAT-DI--DI------L---E--D--T-V-G-------2774 00 10 ---CTSNC--S-SNKN -----K-----T-NR-NRIR----L-TE--V-K---S-SN-I-RR-Q--SN--------MTKA-V------------S-H--A--IYAT-DI--DI------L---E--D----V-G-------2774 00 11 ---CTSNC--S-SNEN -----K-----T-NR-NRIR----L-NE--V-K---S-SN-I-RR-Q--SN--------MTKA-V------------S-H--A--IYAT-DI--DI------L---E--D----V-G-------2774 00 12 ---CTSNC--P S-SNKN -E---K-----A-NR-NRIR----L-NE--V-K---S-SN-I-RR-Q--SN--------MTKA-V------------S-H--V--IYAT-DI--DI------L---E--D----V-G-------HXB2 CTDLK NDT NTNSS SGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPID NDT TSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKI GNMRQAHCNISRAKWNNTLKQIAS VSIS 03 Pretherapy 1914 00 06 -----NT-L-KTTTSSPTASSS--GR-K-G--M-------T----D----N--L--T--LVK--KK --Y-T-I--D--S--T-------KEP-Q------------S-H------YAT-E-I-DI--------S---KD--QK-VT 1914 00 08 -----NT-L-KTTTSSPTISSS--KE-K-G--M-------T----D----N--L--T--LVK--KK --Y-T-I--D-----T-------KEP-Q------------S-N------YAT-Q-I-DI------V------D--H--VK 1914 00 05 -----TT-LNATPTPTSSS--KE-KGG--M-------T--L-D----N--L--T--LVK--KK --Y-T-I--D--S--T-------KDP-Q------------S-H------FAT-E-I-DI--------SK--KS--Q---K 1914 00 11 -----TT-LNATPTPTSSS--KE-K-G--M-------T----D----N--L--T--LVK--QK --Y-T-I--D--S--T-------KDP-Q------------S-H------FAT-E-I-DI------V--EQ-SI--Q--VE 1914 00 04 -----TT-LNATPTP-S-GR-K-G--M-------T----D-M-----L-----LVQ-GDNK--Y-T-I--D--S-ST-------KDP-Q------------S-H------FAT-E-I-DI------V------D--H--VK 1914 00 07 -----NT-LKTTTTTPS-GE-K-G----------T----D-M-----L-----LVQ-GD -KS NPY-T-I--D--S--T-------KDP-Q------------S-H------FAT-E-I-DI--------SKN--S--Q---T 1914 00 02 -----NT-LKTTTTTPS-GE-K-G----------T----D-M-----L-----LVQ-GD -KS NPY-T-I--D--S--T-------KDP-Q------------S-H------FAT-E-IRDI--------SKN--S--Q---T 1914 00 12 ----INVINATTNT---KE-K-G--M-------T----D-M-----L-----LVQ-GD -KS NPY-T-I--D--S--T-------KEP-Q------------S-H---S--YAT-E-I-DI--------S---KD--Q--VT 1914 00 03 -N--TDLMNANNTN---EMK-G--M-------T----D-I-----L-D---LV---NG -RS -T-I--D-L---T-------KEP-Q------------S-N------Y-T-Q-I--I------V--V--D---Q--VK 1914 00 09 -N--TDLMNANNTN--GEMK-G--M-------T----D-I-----L-----LV---NG -RS -T-I--D-----T-------KDP-Q------------S-N------YAT-Q-I-DI------V------D--H--VK Posttherapy 2357 00 06 -----NT-L-KTTTSSPIVSR-T--G-K-E--M-------T----DR---N--L--T--LVQ--NK --Y-T-I--D--S--T-------KDP-Q------------S-H---S--YAT-E-I-DI--------S---KD--Q--VT 2357 00 11 -----NT-L-KTTTSSPTSST-T--G-K-E--M-------T----D----N--L--T--LVL--NK --Y-T-I--D--S--T-------KDP-Q------------S-H---S--YAT-E-I-DI--------S---KD--Q--VT 2357 00 05 -----TT-SDAATNR---GE-K-G--MR------T----N-M-----L-----LV---KG--T-I--D--S--T-------KEP-Q------------S-H------YAT-Q-I-DI------V-G----D-VH-VVI 2357 00 09 -----TT-SDAATNR---GE-K-G--MR------T----D-M--N--L--T--LVK--EG--T-I--D-----T-------KDP-Q-D----------S-H------YAT-Q-I-DI------V-----KD--Q--VK 2357 00 02 ---V-ATNT-D-GGR--G--M-------T----D-M--N--L--T--LVQ--EK --S ---T-I--D-----T-------KEP-Q-H----------S-H-LV---YAT-Q-I-DIK-----V-G----D--H-VVI 2357 00 10 -----NDLKNV--TTSNSGGMK-G--M-------T----D-M-----L-----LV---ES -R-I--D-L---T-------KEP-Q-----------RS-N------YAT-Q-I-DI------V-KV------Q--VK 2357 00 04 ----S -TDPKNTAA-TTR-R-G-K-G--M-------T----D-M--N--L--T--LVK--EG--T-I--D-----T-------KDP-Q------G-----S-H------YATEQ-I-DI-----SV-K----S--Q---K 2357 00 01 ----TNLVNVTNTN--GEM-----M-------T----D-M-----L-----LV---ND -KSYNDTR--R-I--D-----T-------KDP-Q-D----------S-H------YAT-Q-I-DI------V-G----D--H-VVI 2357 00 03 ----TNLVNVTNTN--GEM-----M-------T----D-M-----L-----LV---ND -KSYNDTR--R-I--D-L---T-------KDP-Q-D----------S-H------YAT-Q-I-DI------V-G----D--H-VVI 2357 00 12 ----TNLVNVTNTN--GEM-----M-------T----D-M-R---L-----LV---ND -KSYNDTR--R-I--D-L---T-------KDP-Q------G----RS-N------YAT-Q-I-DI------V--E------Q---K

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230HXB2 CTDLKNDT NTNSS SGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPID NDTTSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGK IGNMRQAHCNISRAKWNNTL VSIS 04 Pretherapy 2141 00 04 -S--V-CTND-SNSSCT NDS NSTKNR-SEEETGRK-M------VT-D--D--KE-N-L--N---VQ-GND-TS -R-I--E---N----------S--I------------S-P------YAT-EI--DI------L-GEQ--D-2141 00 05 -S--V-CTN--SNSSCT NDS NSTKNR-SEEETGRK-M------VT-D--D--KE-N-L--N---VQ-GND-TS -R-I--E---N----------S--I------------S-P------YAT-EI--DI------L-GEQ--D-2141 00 06 -S--V-CTN--SNSSCT NDS NSTKNR-SEEETGRK-M------VT-D--D--KE-N-L--N---VQ-GND-TS -R-I--E---N----------S--I------------S-P------YAT-EI--DI------L-GEQ--D-2141 00 07 -S--V-CTN--SNSSCT NDS NSTKNR-SEEETGRK-M------VT-D--D--KE-N-L--N---VQ-GND-TS -R-I--E---N----------S--I------------S-P------YAT-EI--DI------L-GEQ--D-2141 00 08 -S--V-CTN--SNSSCT NDS NSTKNR-SEEETGRK-M------VT-D--D--KE-N-L--N---VQ-GND-TS -R-I--E---N----------S--I--------S---S-P------YAT-EI--DI------L-GEQ--D-2141 00 11 -S--V-CTN--SNSSCT NDS NSTKNR-SEEETGRK-M------VT-D--D--KE-N-L--N---VQ-GND-TS -R-I--E-L-N----------S--I------------S-P------YAT-EI--DI------L-GEQ--D-2141 00 12 -S--V-CTN--SNSSCT NDS NSTKNR-SEEETGRK-M------VT-D--D--KE-N-L--N---VQ-GND-TS -R-I--E---N----------S--I------------S-P------YAT-EI--DI------L-GEQ--D-2141 00 13 -S--V-CTN--SNSSCT NDS NSTKNR-SEEETGRK-M------VT-D--D--KE-N-LS-N---VQ-GND-TS -R-I--E---N----------S--I------------S-P------YAT-EI--DI------L-GEQ--D-2141 00 01 --N-V-CTK--TTSSSSCTNNTTTKASNSSINSWK-M------VT-N--D-----N-L--N---VR--ND TS -R-I--E---N----------S--V------------S-P------YAT-DI--DI---------EQ----2141 00 02 ----V-CTK--TTSSSSCTNNNTTKASNSSINSWK-M------VT-D--D--KE-N-L--N---VQ-GND-TS -R-I--E---N----------S--I------------S-P------YAT-EI--DI------L-GEQ--D-2141 00 03 ----V-CTK--TTSSSSCTNNNTTKASNSSINSWK-M------VT-N--D--KE-N-L--N---VQ-GND-TS -R-I--E---N----------S--V------------S-P------YAT-DI--DI------L-GEQ----2141 00 09 -S--V-CTN--TTSSSSCTNNNTTKASNSSINSWK-M------VT-N--D-----N-L--N---VR--ND TS -R-I--E---N----------S--I------------S-P------YAT-EI--DI------L-GEQ--D-2141 00 10 ----V-CTK--TTSSSSCTNNNTTKASNSSINSWK-M------VT-N--D-----N-L--N---VR--ND TS -R-I--E---N----------S--I--R---------S-P------YAT-EI--DI------L-GEQ--D-Posttherapy 1836 00 01 ----V-CP Y -GSTSNSNCT NNS NSTKNS-SWEE-GRE-M------VT-N--D---N-N-L--N---VQ-GND-TS -R-I--Q--------------S--V------------S-P------YAT-DI--DI------L-GEQ----1836 00 03 -S--V-CT ---SITSNSNCT NYS NSSNKNSWEK-GRE-M--W---VT-N--D---N-N-L--N---VQ-GYDYTS -R-I--Q--------------S--V------------S-P------YAT-DI--DI------L-EEQ----1836 00 06 ----V-CT -N-GSTSNSNCT NNS NSTKNS-SWEE-GGE-M------VT-N--D---N-N-L--N---VQ-GND-TS -R-I--E---N----------S--V--------Y---S-HA-----YAT-DI--DI------L-GEQ--Y-1836 00 10 -S--V-CT ---SITSNSSCT NNS NSSNKNSWEK-GRE-MR-----VT-N--D---N-N-P--N---VQ-GND-TS -R-I--Q--------------S--V------------S-P------YAT-DI--DI------L-EEQ----1836 00 11 S---V-CT -H-TTTSYSNCT NDS NSTKNSTSWEE-GRE-M------VT-NL-D---N-N-L--N---VQ-GNDDTS -R-I--E-V-N--Q-------S--V-----SYHY---S-P------YAT-DI--DI------L-GEQ--D— HXB2 CTDLK NDT NTNSS SGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPID NDTTSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGK IGNMRQAHCNISRAKWNNTLKQIASK VSIS 25 Pretherapy 2473 00 01 --L-D-M-----L---P-VV---GD-TS -R-I-FE-IS-S---------Q--V-----------RD-HM ------YAT-DI---I---Y------D--R--R--VE2473 00 02 --N--K-CELE-CTNPNNKNQTSNNS TVG-TE---------T--L-D-M-----L---P-VV---GD-TS -R-I--D-I-N----------E--A------------S-P------YAT-EI--DI----------E-----E---K2473 00 03 --N--K-CELE-CT DSKTLVNNSNSGR-G-TE-----P---T--L-D-M-----L---P-VV---GD-TS -R-I--D-I-N----------E--A------------S-P------YAT-DI---I-------T--D--K--G---K2473 00 04 --N--K-CELE-CT DSKTLVNNSNSGR-G-TE-----P---T--L-D-M-----L---P-VV--GGD-TS -R-I--E-I--DT--------Q--V-----------RG-HM ------YAT-DI---I----------D--R--R--VE2473 00 05 -----K-CGLM-TT QANSS NGEVR-TE-M-------T--L-D-M-----L---P-VV---GD-TS -R-I--D-L------------K--A---I--------S-Q------YAT-DI---I-------T--D--K--G---K2473 00 07 -----K-CGLM-TT QANSS NGEVR-TE-M-------T--L-D-M-----P--RP--V---GD-TS -R-I--E-I--DT--------Q--V-----------RG-HM ------YAT-DI---I----------D--R--R--VE2473 00 09 -----K-CGLM-TT QANSS NGEVR-TE-M-------T--L-D-M-----L--RP--V---GD-TS -R-I--D--------------E--A-K-I--------S-Q------YAT-DI---I---Y------D--R--R--VE2473 00 10 -----K-CGLM-AT QANSS NGEVR-TV---------T--L-D-M-----L---P-VV---GD-TS -R-I--E-I--DT--------Q--V----G------RD-HM ------YAT-DI---I---Y------D--R--R--VE2473 00 11 -----K-CGLM-TT QANSS NGGVR-TE-M-------T--L-D-M-----L--RP--V---GD-TS -R-I--D-I-N----------E--A------------S-P------YAT-DI---I-------T--D--K--G---K2473 00 12 -----K-CGLM-TT QANSS NGEVR-TE-M-------T--L-D-M-----L-CRP--V---GD-TS -R-I--D-L------------K--A---I--------S-Q------YAT-EI--DI----------E-----E---KPosttherapy 2887 00 02 --N-KSCTELE---CT ---------T--L-D-M-----L--RT--V--EGD-TS -R-I--E-I---T--------Q--V-----------RG-H------YAT-DIV-DI----------N--R--R--VE2887 00 04 --N-KSCTELE---CT ---------T--L-D-M-----L--RT--V--EGD-TS -R-I--E-I---T--------Q--V-----------RG-H------YAT-DIV-DI----------N--R--R--VE2887 00 08 -----TKANSG SGEVR-TE-M-------T--L-D-M-----L--RP--V---GD-TS -R-I--D--------------K--A-K-I--------S-Q------YAT-EI--DI-------T--D-----G--VK2887 00 09 --N-KSCTELE---CT ------S--T--L-D-M-----L--RT--V--EGD-TS -R-I--E-I---T--------Q--V---A-------RG-H------YAT-DIV-DI----------N--R--R--VE2887 00 10 --N-KSCTELE---CT ---------T--L-D-M-----L--RT--V--EGD-TS -R-I--D--------------K--A-K-I--------S-Q------YAT-EI--DI-------T--D-----G--VK2887 00 11 --N-KSCTELE---CT ---------T--L-D-M-----L--RT--V--EGD-TS -R-I--E-I---T--------Q--V-----------RG-H------YAT-DIV-DI----------N--R--R--VE2887 00 12 --N-KSCTELE---CT ---------T--L-D-M-----L--RT--V--EGD-TS -R-I--E-I---T--------Q--V-K-I--------S-Q------YAT-EI--DI-------T--D-----G--VK

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231HXB2 CTDLK NDT NTNSSSGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPID NDT TSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGK IGNMRQAHCNISRAKWNNTLKQIASKLR VSIS 27 Pretherapy 5523 00 02 --N--V -N-NST---TSGKELME-M-------T-A--D-------LL-Q---V---NKKD -KS NTNY-N-R-I--E-I-N---------KDPI-------------S-HM ---K--YAT-EV--DI--------G----K--GE--N--5523 00 05 -N---V -N-SST---TSGEGLMK-M-------T-A--D-------L------V---DDKNT --S -K-R-I--E-I-N---------KDP-------LS-----S-PM ---K--YAT-EV---I-------------E--GK-VN--5523 00 06 -N---V -N-SST---TSGEGLMK-M-------T-A--D-------L------V---DDKNT --S -K-R-I--E-I-N---------KDP-------LS-----S-PM ---K--YAT-EV---I-------------E--GK-VN--5523 00 07 --N--V -S---TSGKELME-M-------T-A--D-------L------V---DDKNT --S -K-R-I--E-I-N---------KDP-------LS-----S-PM ---K--YAT-EV---I-------------E--GK-VN--5523 00 08 --N--V -S---TSGKEFME-M-------T-A--D-------L------V---NN --SKSNTNY-N-R-I--E-I-N---------KDPI-------------S-HM ---K--YAT-EV--DI--------GVQ--K--E---N--5523 00 11 --N--V -N-NST---TSGKELME-M-------T-A--D-------L------V---NKKD -KS NTNY-N-R-I--E-I-N---------KDPI-------------S-HM ---K--YAT-EV--DI--------G----K--GE--N--5523 00 12 --N--V -N-NST---TSGKELME-M-------T-A--D-------L------V---NKKD -KS NTNY-N-R-I--E-I-N---------KDPI-------------S-HM ---K--YAT-EV--DI--------G----K--GE--N--Posttherapy 6063 00 01 ----N-SST---NSEEGLRQ-M-------T-A--D-------L------VS--NDEDTS-NNNS-N-R-I--E-I-N---------KDP--------S-----S-SM ------YAT-EV--DI-------------D--GK--N--6063 00 07 --N--VN-AS ST---TSGEELMQ-M-------T-A--D-------L------V---NDKD -NS NTNY-N-R-I--E-I-N---------KDP--------S-----S-SM ------YAT-EV--DI----------Q--D--GK--N--6063 00 08 ----N-SST---NSEEGLRQ-M-------T-A--D-------L------VS--NDEDTS-NNNS-K-R-I--E-I-N---------KDP--------S-----S-SM ------YAT-EV--DI-------------D--GK--N--6063 00 10 ----N-SST---NSEEGLRQ-M-------T-A--D-------L------VS--NDEDTS-NNNS-K-R-I--E-I-N---------KDP--------S-----S-SM ------YAT-EV--DI-------------D--GK--N--6063 00 12 ----N-SST---NSEEGLRQ-M-------T-A--D-------L------VS--NDEDTS-NNNS-K-R-I--E-I-N---------KDP--------S-----S-SM ------YAT-EV--DI-------------D--GK--N--6063 00 11 ----N-SST---NSEEGLRQ-M-------T-A--D-------L------VS--NDEDTS-NNNS-K-R-I--E-I-N---------KDP--------S-----S-SM ------YAT-EV---I--------GV---E--RK--N--HXB2 CTDLK NDTNTNSS SGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPID NDT TSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKI GNMRQAHCNISRAKWNNTLKQIASKLREQF VSIS 28 Posttherapy 5601 00 01 ---DLN-A-K---TKTNS-SWEE------------VTATR-D-M-Q---L-----VV---DDK -K-NTSYGLNR-I--S--S-----------E--V------------S-HA-----YAT-E-I-DI-K-------VV-D----R--I-----5601 00 02 ---DLN-A-----TSTNSTSWEK------------VTATR-D-M-Q---L-----VV---DDRT -TS YKLNR-I--S--S-----------E--A-----------RS-H------YAT-E-I-DI-K--------A-----GK-VI-----5601 00 04 ---DLN-A-----TNTNSTS*EK------------VTATR-D-I-Q---L-----VV---DDK -K-HTSYKLNR-I--S--S-----------E--V------------S-HV A-----YAT-E-I-DI-K--------A-----EK-VI-----5601 00 05 ---DLN-A-----TNTNSTSWEK------------VTATR-D-M-Q---L-----VV--EDDKDKT-TS YKLNR-I--S--S-----------E--A------------S-H------YAT-E-I-DI-K--------A------K-VI-----5601 00 07 ---DLN-A-K---T NNS-SWEE------------VTATR-D-M-Q---L-----VV---DDK -K-NTSYGLNR-I--S--S-----------E--V------------S-HA-----YAT-E-I-DI-K-------VA-D----R--I-----5601 00 08 ---DLN-A-----TNTNSTSWEK------------VTATR-D-M-Q---L-----VV--EDDKDKT-TS YKLNR-I--S--S-----------E--A------------S-H------YAT-E-I-DI-K--------A------K-VI-----5601 00 09 ---ELN-A-----T NNS-SWEKT-----------VTATR-D-M-Q---L-----VV---DDK -K-NTSYGLNR-I--S--S-----------E--V------------S-H------YAT-E-I-DI-K--------A-----GK-VI-----5601 00 10 ---DLN-A-K---TKTNS-SWEE------------VTATR-D-M-Q---L-----VV---DDK -KADTSYKLNR-I--S--S-----------E--V------------S-H------YAT-E-I-DI-K--------A------R-VI--K--5601 00 11 ---DLN-A-----TDTNSTSWEK------------VTATR-D-M-Q---L-----VV--EDDKDKT-TS YKLNR-I--S--S-----------E--A------------S-H------YAT-E-I-DI-K--------A------R--I--K--5601 00 12 ---DLN-A-----TNTNSTS*EK------------VTATR-D-I-Q---L-----VV---DDK -K-HTSYKLNR-I--S--S-----------E--V------------S-HA-----YAT-E-I-DI-K--------A-----EK-VI-----5601 00 13 ---DLN-A-K---TKTNS-SWEE------------VTATR-D-M-Q---L-----VV---DDK -K-NTSYGLNR-I--S--S-----------E--V------------S-HA-----YAT-E-I-DI-K-------VA-D----R--I-----5601 00 14 ---DLN-A-K---TKTNS-SWEE------------VTATR-D-M-Q---L------V---NDN -K--R-I--S--S----I------E--V------------S-HA-----YAT-E-I-DI-K--------A------K-VI-----5601 00 15 ---DLN-A-----TNTNSTSWEK------------VTATR-D-M-Q---L-----VV--EDDKDKT-TS YKLNR-I--S--S----I------E--V------------S-HA-----YAT-E-I-DI-K--------A------R--M-----5601 00 16 ---DLN-A-----TNTNSTSWEK------------VTATR-D-M-R---L-----VV--EDDKDKT-TS YKLNR-I--S--S-----------E--V------------S-HA-----YAT-E-I-DI-K--------A-----EK-VI-----5601 00 17 ---VLK-A-----TNTNSRS--E--M------VTATR-D---Q---L------V---NDN -K--R-I--S--S----I------E--V------------S-HA-----YAT-E-I-DI-K--------V------R--M-----

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232HXB2 CTDLK NDT NTNSS SGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPID NDTTSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGK IGNMRQAHCNISRAKWNNT VFIS 02 Pretherapy 1850C01 ----EKN -TR AIELTN--T TATTPATTTTSTNTN-SHVKEIRE-MT---V-TT-G-GN-L-TNH-L-----LMS--NEYY NFI-IH-E-L-N--TP-----KDP-------H----I-G-QL -----VIATK-V--DI-----TL------S1850C02 ----EKN -TS AYSTTN--T TTSTPATTTTSTNTN-SHLKEIRE-MT---V-TT-G-GN-L-TNH-L-----LMS--NEYY KFI-NH-E-L-N---------KDP------------I-G-QL -----VIATK-I--DI-----TL-1850C03 ----EKN -TS ANSTTN--T TTT PATTTTSTNTN-SHLKE RR-MT-----TT-G-GN-L-NNH-L-----LMS--NEYY NFI-IH-E-L-N---------KDP------G-------G-QL -----VIATK-I--DI-----TL------S1850C04 ----EKN -TS ANSTTN--T TTT PATTTTSTNTN-SHLKEIRE-MT-----TT-G-GN-L-TNH-L-----LMS--K-TNFI-IH-E-L-N---------KDP------------I-G-QL -----VIATK-I--DI-----TL------S1850C05 ----EKN -TS PNSTTN--T TTT PATATTSTNTN-SHLKEIRE-MT----KTT-G-GN-L-TNH-L-----LMS--K-TNFI-IH-E-L-N---------KDP------------I-G-QL -----VIATK-I--DI-----TL------SPosttherapy 2192C01 --G-EKD -TS ANSTTN--T TTT PATTTTS IRE-MT-----TT-G-GN-L-TNH-L-----LMS--QSTNFI-IH-E-L-N----T----KDP------------I-G-QL -----VIATK-IT-DT-----TL-----Y 2192C02 --G-EKN -TS ANSTTN--T TTT PATTTTS IRE-MT-----TT-G-GD-L-TNH-L-----LMS--QSTNFI-VH-E-L-N---------KDP------------I-G-QL -----VIATK-IT-DT-----TL-----2192C03 --G-EKN -TS ANSTTNH-T TTT PATTTTS IREKMT-----TT-G-GN-L-TNH-L-----LMS--QSTNFI-IH-E-L-N---------KDP------------I-G-QL -----VIATK-IT-DT-----TL-------2192 00 01 ----EKN -TS ANSTTN-NT TTSTPATTTTSTNTN-SHWKEIRE-MT-----TT-G-GN-L-TN--L-----LMS--K-TNFI-IH-E-L-N---------KDP------------I-G-QL -----VIATK-IT-DI---Y--VTAEQ--SA 2192 00 02 ----EKN -TS ANSTTN-NT TTSTPATTTTSTNTN-SHWKEIRE-MT-----TT-G-GN-L-TN--L-----LMS--K-TNFI-IH-E-L-N---------KDP------------I-G-QL -----VIATK-IT-DI---Y--VTAEQ--SA 2192 00 03 ----EKN -TS ANSTTN-NT TTSTPATTTTSTNTN-SHWKEIRE-MT-----TT-G-GN-L-TN--L-----LMS--K-TNFI-IH-E-L-----------KDP------------I-G-QL -----VIATK-IT-DI---Y--VTAEQ--SA 2192 00 04 ----EKN -TS ANSTTN-NT TTSTPATTTTSTNTN-SHWKEIRE-MT-----TT-G-GN-L-TN--L-----LMS--K-TNFI-IH-E-L-N---------KDP------------I-G-QL -----VIATK-IT-DI---Y--VTAEQ--SA 2192 00 05 ----EKN -TS ANSTTN-NT TTSTPATTTTSTNTN-SHWKEIRE-MT-----TT-G-GN-L-TN--L-----LMS--K-TNFI-IH-E-L-N---------KDP------------I-G-QL -----VIATK-IT-DI---Y--VTAEQ--SA 2192 00 07 ----EKN -TS ANSTTN-NT TTSTPATTTTSTNTN-SHWKEIRE-MT-----TT-G-GN-L-TN--L-----LMS--K-TNFI-IH-E-L-NS--------KDP------------I-G-QL -----VIATK-IT-DI---Y--VTAEQ--SA 2192 00 08 ----EKN -TS ANSTTN-NT TTSTPATTTTSTNTN-SHWKEIRE-MT-----TT-G-GN-L-TN--L-----LMS--K-TNFI-IH-E-L-N---------KDP------------I-G-QL -----VIATK-IT-DI---Y--VTAEQ--SA 2192 00 09 ----EKN -TS ANSTTN-NT TTSTPATTTTSTNTN-SHWKEIRE-MT-----TT-G-GN-L-TN--L-----LMS--K-TNFI-IH-E-L-N---------KDP------------I-G-QL -----VIATK-IT-DI---Y--VTAEQ--SA

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233HBX2 CTDLKNDT NTNSSSGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPID NDT TSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKI GNMRQAHCNISRAKWNNTLKQIASKLRE VFIS 10 Pretherapy 1470 00 01 --A-S NEGNATT-YS-SL----------T--LQD-MK----L--TF--V-LESGT-GK-R-V--E--------------E--V------------S-PM -----LYAT-A-I-DI---------E---E---R--I---1470 00 03 --A-S NEGNTTT-YS-SL--M-------T--LQD-MK----L--TF--V-LESGT--K-R-V--E--------------E--V------------S-P-----LYAT-A-I-DIG--------E---E--ER--I---1470 00 04 --A-SINEGNATT --GL--M-------T--LQD-RK-D--L--TF--V-LENDPSENGTNGNK-R-V--E---N----------E--V------------S-PM -----LYAT-A-I-DI---------E---E---R--I---1470 00 05 --V-RINEGNTTT-YS-SL--M-------T--LQD-MKR---L--TF--V-LESGT-GK-R-V--E--------------E--V------------S-SM ---K-LYAT-A-I-DI---------E---E---R--I---1470 00 06 --A-S NEGNATT --GL--M-------T--LQD-RK-D--L--TF--V-LESGT-GK-R-A--E--------------E--V------------S-PM -----LYAT-A-I-DI---------E---E---R--I---1470 00 07 --A-SINEGNATT-Y--GL--M-------T--LQD-RK-D--L--TF--V-LESGT-GK-R-V--E--------------E--V------------S-PM ---K-LYAT-A-I-DI---------E---E--QR--I---1470 00 08 --V-S NERNATA-N--GL--M-------T--LQDERKED--L--TF--V-LESGT-GK-R-V--E--------------E--V------------S-PM -----LYAT-A-I-DI---------E---E---R--I---1470 00 09 --A-S SNATT-YS-SL--M-------T--LQDERKED--L--TF--V-LESGT-GN K-R-V--E---N----------E--V------------S-PM -----LYAT-A-I-DI---------E---E---R--I---1470 00 10 --A-STNKGNATT S-L--M-------T--LQD-RK-D--L--TF--V-LESGT-GK-R-V--E--------------E--V------------S-PM ---S-LYAT-A-I-DI---------E---E--ER--I---1470 00 12 -INERNATT-Y--GL--M-------T--LQDERKQD--L--TF--V-LESGT-GK-R-V--E--------------E--V------------S-P---S-LYAT-A-I-DI---------E---E--ER--I---1470 00 13 --A-SINEGNATT-Y SL--M-------T--LQD-RKED--L--TS--V-LESGT-GK-R-V--E--------------E--V------------S-SM ---G-LYAT-A-I-DI---------E---E--QR--I---G 1470 00 15 --A-S NEGNATT SNGL--M-------T--LQDEMK-D--L--TF--V-LESGT-GK-R-V--E--------------E--V------------S-SM ---G-LYAT-A-I-DI---------E---E---R--I---1470 00 16 -SINERNATT-Y--GL--M-------T--LQDERKED--L--TF--V-LESGT-GK-R-V--E---------V----E--V------------S-PM ---K-LYAT-A-I-DI---------E---E---R--I---1470 00 17 --A-S NEGNTTT-YS-SL--M-------T--LQD-RK-D--L--TS--V-LESGT-GK-R-V--E--------------E--V------------S-HM -----LYAT-A-I-DI---------E---E---R--I---Posttherapy 1664u01 --AINKENTTT-Y--GL--M-------T--LQDEMK-D--L--TF--V-LESGT-GK-R-V--E--------------E--V------------S-PM ---GVLYAT-A-T-DI---------E---E---R--I---G 1664u02 --A-SINEGKATT-Y--GL--M-------T--LQD-RK-D--L--TF--V-LESGT-GK-R-VG-E---N----------E--V------------S-HM -----LYAT-A-I-DI---------E---E---R--I---1664 13A -A -A-SI N-Y--GL--M-------T--LQD-RK-D--L--TF--V-LESGT-GK-R-V--E--------------E--VN-----------S-PM -----LYAT-A-I-DI---------E---E---R--I---1664 14A --A-SI T-Y--GL--M--------P-LQD-RK-D--L--TF--V-FESGT-GK-R-VG-E---N----------E--V----G-------S-HM -----LYAT-A-I-DI---------E---E---R--I---1664 15S --VNEGNATA-N--GL--K-------T--LQDERKQD--L--TF--V-LESGT-ST-R-VGP -E--------------E--V----G-------S-PM -----LYAT-A-I-DI---------E---E--TR--I---1714 48 06 --ASNEGNTT -NYSSSL--M-------T--LQD*RKQD--L--TF--V-LESGT-GK-R-V--E---N----------E--V------S-----S-SM ---G-LYAT-A-I-DIK----S---E---E---R--I---1714 48 22 --ASNEGNTT -NYSSSL--M-------T--LQDERKQD--L--TF--V-LESGT-GK-R-V--E---N----------E--V------S-----S-S---G-LYAT-A-I-DIK--------E---E---R--I---1714 48 18 --ASNEGNTT -NYNSGL--M--S----T--LQDERKQD--L--TF--V-LESGT-GK-R-V--E--------------E--V------------S-SM ---K-LFAT-A-I-DI---------E---E--ER--I---1714 48 14 --ASNEGNTT -NYNSGL--M-------T--LQDERKQD--L--TF--V-LESGT-GK-R-V--E---N----------E--V------S-----S-S---G-LYAT-A-I-DIK--------E---E---R--I---1714 48 02 --VNEGNATA-NYSSSL--M-------T--LQDERKQD--L--TF--V-LESGT-GK-R-V--E--------------E--V------------S-PM ---K-LFAT-A-I-DI---------E---E---RV-I---1714 48 09 --VNEGNATATNYSSSL--M-------T--LQDERKQD--L--TF--V-LESGT-GK-R-I--E--------------E--V------------S-S-----LYAT-A-I-DI---------E---E--TR--I---1714 48 05 --VNEGNATASN--GL--M-------T--LQDERKQD--L--TF--V-LESGT-GK-R-V--E---N-------R--E--V------S-----S-S---G-LYAT-A-I-DIK--------E---E--RR--I---1714 48 16 --VNEGNATA-N--GL--M-------T--LQDERKQD--L--TF--V-LESGT-GK-R-V--E--------------E--V------------S-TM -----LYAT-A-I-DI---------E---E--TR--I---1714 48 01 --TSNERNATATNYSSSL--M-------T--LQDERKQD--L--TF--V-LESGT-GK-R-V--E---N----------E--V------S-----S-SM ---G-LYAA-A-I-DI-----S---E---E---R--I---1714 48 07 --TSNERNATATNYSSSL--M-------T--LQDERKQD--L--TF--V-LESGT-GK-R-V--E------R-------E--V------------S-S-----LYAT-A-I-DIK--------E---E--TR--I---1714 48 10 --TSNERNATATNYSSSL--M-------T--LQDERKQD--L--TF--V-LESGT-VK-R*V--E---N----------E--V------S-----S-SM ---G-LYAT-A-I-DI------V--E---E---RV-I---1714 48 12 --TSNERNATATNYSSSL--M-------T--LQDERKQD--L--TF--V-LESGT-GK-R-V--E--------------E--V------------S-SM ---S-LFAT-A-I-DI---------E---E---RV-I---1714 48 17 --TSNERNATATNYSSSL--M-------T--LQDERKQD--L--TF--V-LESGT-GK-R-V--E---G----------E--V------------S-HM ---GTLYAT-A-I-DIK--------E---E--ER--I---1714 48 21 --TSNERNATATNYSSSW--M-------T--LQDERKQD--L--TF--V-LESGT-GK-R-V--E---N----------E--V------S-----S-SM -----VYAT-A-I-DI---------E---E---RV-I---1714 48 13 -NINERNVTG-NIGL--M-------T--LQDERKQD-TL--TF--V-LESGT-GK-R-V--E--------------E--V------------S-S---S-LYAT-A-I-DI---------E---E--ER--I---1714 48 11 -NINERNATV-NIGL--M-------T--LQDERKQD--L--TF--V-LESGT-GK-R-V--E--------------E--V------------S-S-----LYAT-A-I-DI---------E---E--TR--I---1714 48 20 -NINERNATA-NIGL--M-------T--LQDERKQD--L--TF--V-LESGT-GK-R-V--E--------------E--V------------S-SM ---G-LYAT-A-I-DI---------E---E---R--I---1714 48 03 -NINERNATA-NIGL--M-------T--LQDERKQD--L--TF--V-LESGT-GK-R-V--E---N----------E--V------S-----S-SM ---G-LFAT-A-I-DI---------E---E---R--I---1714 48 23 -NINEGNATA-N--GL--M-------T--LQDERKQD--L--TF--V-LESGT-GK-R-V--E--------------E--V------------S-SM ---G-LYAT-A-I-DI---------E---E---RV-I---

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234HXB2 CTDLK NDT NTNSS SGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPID NDT TSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFV TIGKI GNMRQAHCNISRAKWNNTLKQIASKL VFIS 11 Pretherapy 1181 00 01 -SNA -IQSK-NNRATG-NETR--E--M-------T--L-D-IK----L-----VV---NE-NSRNN DSGK-R-I--E--SN----------E--V------------G-------SFY-AD--I-DI------L--TQ--------VT-1181 00 02 -SNANK-AEVDGTK--A---------VT--L-D--K----L-----VV---NE-NSRNN DSGK-RWI--E--SN----------E--V----------S-G-------SFY-TE--I-DI------L-G-Q--------VT-1181 00 04 -SNANK-AEVDGTK--A---------VT--L-D--K----L-----VV---NE-NSRNN DSGK-R-I--E--SN----------E--V------------S-Q----FH-T-D-I-DI------L---Q------HVVT-1181 00 07 -SNA -IQSG-KTNATG-NETR--E--M-------T--L-D-IK----L------V---NE-KSNSNNNGSGN-R-I--E--SN----------E--V------------G-------SFY-TD--I-DI------L-G-Q--------VT-1181 00 09 -SNA -I-QSGN---TTGDTGGKWKK--E----------T-N--D-IK----L-----VV--EDNDNKTNN GSGG-R-I--E--SN----------E--V------------S-Q----FH-T-D-I-DI------L---Q-------VVT-1181 00 10 -SNA -IQSK-NNRATG-NETR--E--M-------T--L-D-IK----L-----VV---NE-NSRNN DSGK-R-I--E--SN----------E--V------------G-------SFY-TD--I-DI------L--TQ--------VT-S 1181 00 11 -SNA -IQSG-KTNATG-NETR--E--M-------T--L-D-IK----L------V---NE-N-NN GSGN-R-I--E--SN----------E--V------------G-------SFY-TD--I-DI------L-G-Q--------VT-1181 00 12 -SNA -I-QSGN---TTGDTGGKWKK--E----------T-N--D-IK----L-----VV--EDNDNKTNN GSGG-R-I--E--SN----------E--V------------S-Q----FH-T-D-I-DI------L---Q-------VVT-Posttherapy 1604 11S -SNA -IQSG-KTNAAG-DGTK--A--M------VT--L-D-IK----L-----VV---HK-NSNP GAGN-R-I--E--SN---------KEP-V------------G-------SFY-TD--V-DI------L---Q--------VT-1604 13A -STA -IQSG-NKNATG-SETR-KE--M-------T--L-D-IK--F-L------V---NE-NSNSNNNSSGK-R-I--E--SN----------E--V------S-----S-Q----FH-T-D-I-DI------F---Q-------VVT-1604 14A -SNA DIQSG-NKNATGNSETR-KE--M-------T--L-D--K--F-L------V---NE-NSNSNNNSSGK-R-I--E--SN----------E--V------------S-Q----FH-T-D-I-DI------L---Q-------VVT-1604 00 04 -SNA -IQSG-NKNATG-SETR-KE--M-------T--L-D-IK----L-----VV---NK-NS NNSSGK-R-I--E--SN---------KEP-V------S-----S-Q----FH-T-D-I-DI------L---Q-------VVT-1604 00 05 -SNA -IQSG-NKNATG-SETR-KE--M-------T--L-D--K----L------V---NE-NSNSNNNSSGK-R-I--E--SN----------E--V------S-----S-Q----FH-T-D-I-DI------L---Q-------VVT-1604 00 06 -SNA -IQSG-KINAAG-NETR--E--M------VT--L-D-IK----L-----VV---NK-NS NNGSGN-R-I--E--SN---------KEP-V------S-----S-Q----FH-T-D-I-DI------L---Q-------VVT-1604 00 08 -SNA -IQSG-KTNAAG-DGTK--A--M------VT--L-D-IK-D--L-----VV---NK-NS NNGSGN-R-I--E--SN---------KEP-V------S-----S-Q----FH-T-D-I-DI------L---Q-----NTVVT-1604 00 09 -SNA -IQSG-NKNATG-SEAR-KE--M-------T--L-D--K----L------V---NE-NSNSNNNSSGK-R-I--E--SN---------KEP-V------------G-------SFY-TD--V-DI------L-G-Q--------VT-1604 00 11 -SNV NKNATG-SETR-KE--M-------T--L-D--K----L-----VV---NK-NS NNGSGN-R-I--E--SN---------KEP-V------------G-------SFY-TD--V-DI------L---Q--------VT-1604 00 12 -SNA -IQSG-NKNATG-SETR-KE--M-------T--L-D-IK----L-----VV---NK-NS NNSSGK-R-I--E--SN----------E--V------S-----S-Q----FH-T-D-I-DI------L---Q-------VVT-1604 00 13 -SNA -IQSG-KTNAAG-DGTK--A--M------VT--L-D-IK----L-----VV---NK-NS NNGSGN-R-I--E--SN-------*-KGP-V------S-----S-Q----FH-T-D-I-DI------L---Q--------VT-1656 48 02 -SNA -IQSG-KTNAAG-NETK--E--M------VT--L-D-IK----L-----VV---NK-NS NNGSGN-R-I--E--SN----------E--V------S-----S-Q----FH-T-D-I-DI------L---Q----F--VVT-1656 48 04 -SNA -IQSG-KTNAAG-DGTN--A--M------VT--L-D-IK----L-----VV---NK-NS NNGSGN-R-I--E--SN----------E--V------------G-------SFY-TD--T-DI------L-G-Q--------VT-1656 48 05 -SNA -IQSG-KTNTAGGNETK--A--M------VT--L-D-IK----LL----VV---NK-TS NNGSGNNR-I--E--SN--Y-------E--V------SY----S-QDT--FH-T-D-I-DI------L---Q-------VVT-1656 48 06 -SNA -IQSG-NKNATG-NETR--E--M------VT--L-D-IK----L-----VV---NK-NS NNGSGN-R-M--E--SN---------KEP-V------------G-------SFY-TD--V-DI------L---Q--------VT-1656 48 07 -SNA -IQSG-KTNAAG-NETK--E--M------VT--L-D-IK----L-----VV---NK-NS NNGSGN-R-I--E--SN---------KEP-V------------G-------SFY-TD--V-DI------L---Q--------VT-1656 48 10 -SNA -IQSG-KTNAAG-NETR--E--M------VT--L-D-IK----L-----VV---N -NS NNGSGK-R-I--E--SN----------E--V------S-----S-Q----FH-T-D-I-DI------L---Q--------VT-1656 48 12 -SNA -IQSG-KINAAE-NETR--E--M------VT--L-D-IK----P-----VV---NK-NS NNGSGK-R-I--E--SN---------REP-V------S-----S-Q----FH-T-D-I-DI------L---Q--------VT-1656 48 15 -SNA -IQSG-KINAAG-NETR--E--M------VT--L-D-IK----L-----VV---NK-NS NNGSGN-R-I--E-SSN---------KEP-V------------G-------SFY-TD--VEDI------L---Q--------VT-1656 48 18 -SNA -IQSG-QTNAAG-NETR--E--M------VT--L-D-IK----L-----VV---NK-NS NNGSGN-R-I--E--SN---------KEP-V------------G-------SFY-TERVI-DI------L-GTQ--------VT-1656 48 22 -SNA -IQSG-KTNAAG-NETR--E--M------VT--L-D-IK----L-----VV---NK-NS NNGSGN-R-I--E--SN---------KEP-V------S-----S-Q----FH-T-D-I-DIK-----L---Q-------VVT*1656C05 -SNA -IQSG-KTNAAG-NEQR--A--M------VT--L-D-IK----L-----VV---NK-NS NNGSGN-R-I--E--SN----------E--V------------G-------SFY-TD--I-DI------L-G-Q--------VT-1656C06 -SNG -TQSG-KTNAAG-NETR--AE-M------VT--L-D-IK----L-----VV---NK-NS NNGSGN-R-I--E--SN----------E--V------------G-------SFY-TD--I-DI--E---L-G-Q--------VT-

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235HXB2 CTDLKNDTNTNSS SGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDTTSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGK IGNMRQAHCNISRAKW VFIS 12 Pretherapy 1738 48 1 --V-I-TAAPRT TATSTTTTSTTTT-INLGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-IN-E-IS----------KEP---T-V--G-----SVHM ---S-LFAT-AIT-DI-R---TLNGT-1738 48 2 --V-I-TAAP STAA STTTTSTTTT-INLGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E-IS----------KEP---T-V--G-----SVHM ---S-LFAT-AIT-DI-R---TLNGT-1738 48 3 --V-I-TAAP STAATSTTTTSTTTT-ISLGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E-IS----------KEP---T-V--G-----SVHM ---S-LFAT-AIT-DI-R---TLNGT-1738 48 4 --V-IA -TAAP STTATSTTITSTTTT-INVGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E-IS----------KEP---T-V-LG-----SVHM ---S-LFAT-AIT-DI-R---TLNGT-1738 48 5 --V-I-TAAPRT TATSTTTTSTTTT-INLGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-IN-E-IS----------KEP---T-V--G-----SVHM ---S-LFAT-AIT-DI-R---TLNGT-1738 48 6, --RV -V-M---T VVSSANATVGNMTE-MR-----VT-G-GSE-RR---L--N--LKR-K NN-M-IH-E-IS----------KEP---E-V--G----RS-----S-LLAT-TIT-DI-K-----T--N1738 48 7 --V-I-TAAPST TAPSNTTISTTTT-INVGNMTE-MR------T-G-GN-L-----------VM--K--SQ--M-VN-E--SN---------KEP---T-V--G-----SVHM ---S-PFAT-AI--DT-R---TLNGT-1738 48 8 --V-I-TAATA TSTATTSTTTT-INVGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E-IS----------KEP---T-V--G-----SVHM ---S-LFAT-AI--DP-R---TLNGT-1738 48 9 --V-I-TAATA TSTATTSTTTTGINVGNMTE-MR------T-G-GN-L-----L-----VM--KT-SQ--M-VN-E-IS----------KEP---T-V--G-----SVHM ---S-LFAT-AIS-DI-R---TLNGT-1738 48 12 --V-I-TAAPST TAASTTTTSTTTT-INVGNMTE-MR------T-G-GN-L-R---L-----VM--K--SQ--M-VN-E--SN---------KEP---T-V--G-----SVHM ---S-LFAT-AI--DT-R---TPNGT-1738 48 13 --V-I-TAAPST T TTSTTTT-INLGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E-IS----------KEP---T-V--G-----SVHM ---S-LFAT-AIT-DI-R---TLNGT-1738 48 14 --V-I-TAATA TSTATTSTTTTGINVGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E-IS----------KEP---T-V--G-----SVHM ---S-LFAT-AI--DP-R---TLNGT-1738 48 15 --V-I-TAAPST TA STTTTSTTTT-INVGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E--SN---------KEP---T-V--G-----SV-M ---S-LFAT-AI--DT-R---TLNGT-1738 48 16 --V-I-TAATA TSTATTSTTTTGINVGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E-IS----------KEP---T-V--G-----SVHM ---S-LFAT-AI--DP-R---TLNGT-1738 48 17 --V-I-TAAPST IATSTATTSTTTT-INVGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E--SN---------KEP---T-V--G-----SVHM ---S-LFAT-AI--DT-R---TLNGS-1738 48 18 --V-IA -TAAPST TATSTSTTSTTTT-INVGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E--SN---------KEP---T-V--G-----SVHM ---S-LFAT-AI--DT-R---TLNGT-1738 48 20 --V-I-TAATATSTA TTSTTTT-INVGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E-IS----------KEP---T-V--G-----SVHM ---S-LFAT-AI--DP-R---TLNGT-1738 48 21 --V-IA -TAAPST TATSTSTTSTTTT-INVGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E-IS----------KEP---T-V--G-----SVHM ---S-LFAT-AIT-DI-R---TLNGT-1738A01 --KV -V-I---T VVSNENSTVGNMTE-MR-----VT-G-GNE-RR---L--N--LKH-KEN-M-IH-E-IS----------KEP---E-V--G----RS-----S-LLAT-TIT-DI-R-Y---T--N1738A03 -STTTTSTTTT-INVGNMTE-MR-----VT-G-GNE-RR---L--N--LKH-KEN-M-IH-E-IS----------KEP---E-V--G----RS-----S-LYAT-AIT-DI-K-Y---T--N1738A04 --KV -V-I---T VVSSVNATVGNMTE-MR-----VT-G-GNE-RR---L--N--LKH-KEN-M-IH-E-IS----------KEP---E-V--G----RS-----S-LLAT-AI--DI-K-Y-I-NET-1738A05 --KV -V-IA--T VVSSANATVGNMTE-MR-----VT-G-GNE-RR---L--D--LKH-NE NN-M-IH-E-IS----------KEP---E-I--G----RS-----S-LYAT-AIT-DI-K-Y---T--N1738A06 --KV -V-I---T VVSSANATVGNMTE-MR-----VT-G-GNE-RR---L--N--LKH-KEN-M-IH-E-IS----------KEP---E-I--G----RS-----S-LYAT-AIT-DI-K-Y---T--N1738O04 --V-I-TAAPST TATSPTTTSTTTT-INVGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E-IP----------KEP---E-V--G----RS-----S-LLAT-TIT-DI-K-Y-I-NGT-1738O05 --VATST TATSATTTSTTTT-INVGNMTE-MR------T---GS-L-R---L-----VM--K--SQ--M-VN-E-IS----------KEP---T-V--G-----SVHM ---S-LFAT-AIT-DI-R---TLNGT-1738O06 --V-IA -TAAPST TAPSNTTTSTTTT-INVGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E--SN---------KEP---T-V--G-----SVHM ---S-LFAT-AI--DT-R---TLNGT-Posttherapy 1943 48 1 --V-I-TAATATSTA TTSTTTT-INVGNMTEDMR-----VP-G-GNE-RR---L*DN--LKH-QE HN-M-IH-E-IS----------KEP---E-V--G----RS-----S-LLAT-AIV-DI-K-Y-I-NET-1943 48 2 --V-I-TAATATSTA TTSTTTT-INVGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E--SN---------KEP---T-V--G-----SVHM ---S-LFAT-AI--DP-R---TLNGT-1943 48 4 --VAI-TAATATSTA TTSTTTT-INVGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E--SN---------KEP---T-V--G-----SVHM ---S-LFAT-AI--DP-R---TLNGT-1943 48 7 --V-I-TAATATSTA TTSTTTT-INVGNMTE-MR------T-G-GN-L-----L-----VM--T--SQ--M-VN-E--SN----T----KEP---T-V--G-----SVHM ---S-LFAT-AI--DP-R---TLNGT-1943 48 8 --V-I-TTATNT TATSAATTSTTTT-INVGNMTEDMR-----VP-G-GNE-RR---L--N--LKH-TE HN-M-IH-ESIS----------KEP---E-V--G----RS-----S-LLAT-AI--DP-R---TLNGT-1943 48 9 --KV -VSI---T VFSSENSTVGNMTE-MR-----GH-G-GNE-RR---L--R--LKR-TE HN-M-IH-E-IS----------KEP---E-V--G----RS-----S-LLAT-TIT-DI-K-Y---T--N1943 48 10 --V-I-TAATATSTA TTSTTTT-INVGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E--SN---------KEP---T-V--G-----SVHM ---S-LFAT-AI--DP-R---TLNGT-1943 48 12 --V-I-TAATATSTA TTSTTTT-INVGNMTE-MR------T-G-GN-L---C-L-----VM--K--SQ--M-VN-E--SN---------KEP---T-V--G-----SVHM ---S-LFAT-AI--DP-R---TLNGT-1943 48 13 --V-I-TTATNT TATSAATTSTTTT-INVGNMTEDMR-----VP-G-GNE-RR---L--N--LKH-TE HN-M-IH-E-IS----------KEP---E-V--G----RS-----S-LLAT-AI--DP-R---TLNGT-1943 48 14 --V-I-TAATATSTA TTSTTTT-INVGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E--SN---------KEP---T-V--G-----SVHM ---S-LFAT-AI--DP-R---TLNGT-1943 48 15 --V-I-TAATATSTA TTSTTNT-INVGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E--SN---------KEP---T-V--G-----SVHM ---S-LFAT-AI--DP-R---TLNGT-1943 48 16 --V-I-TAATATSTA TTSTTTT-INVGNMTEDMR-----VP-G-GNE-RR---L--N--LKH-TE HN-M-IH-E-IS---R------KEP---T-V--G-----SVHM ---S-LFAT-AI--DP-R---TLNGT-1943 48 17 --V-I-TAATATSTA TTSTTTT-INVGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E--PN---------KEP---T-V--G-----SVHM ---S-LFAT-AI--DP-RS--TLNGT-1943 48 18 --V-I-TAATATSTA TTGTTTT-INVGNMTE--R------T-G-GN-L-----L-----VM--K--SQ--M-VN-E--SN---------KEP---T-V--G-----SVHM ---S-LFAT-AI--DP-R---TLNGT-1943 48 20 --V-I-TTATNT TATSAATTSTTTT-INVGNMTEDMR-----VP-G-GNE-RR---L--N--LKH-TE HN-M-IH-E-IS----------KEP---E-V--G----RS-----S-LLAT-AI--DP-R---TLNGT-1943 48 21 --V-I-TAATATSTA TTSTTTT-INVGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E--SN---------KEP---T-V--G-----SVHM ---S-LFAT-AI--DP-R---TLNGT-1943 48 22 --V-I-TAATATSTA TTSTTTT-INVGNMTE-MR------T-G-GN-L-----L-----VM--K--SQ--M-VN-E--SN---------KEP---T-V--G-----SVHM ---S-LFAT-AI--DP-R---TLNGT-1943A01 --TV -V-I---T VVSSENSTVGNMTE-MR-----VT-G-GN-L ---L------RQTN-NS -M-VH-E-IS----------KEP-G-E-V--G----RS-----S-LLAT-AIV-DI-K-Y-I-NET-1943A03 --V-I-TAAP STTASGTTTTSTTTT-INVGNMTE-MR------T---GS-L-R---L-H---V---K--SQ--M-VN-E--SN---------KEP---E-V--G----RS-----S-LLAT-AIV-DI-K-Y-I-NET-1943A04 --EV -V-I---T VVSSNINVGNMTE-MR------T-G-GN-L-R---L------MQTN-NS -M-VN-E-IS----------KEP---T-V--G-----SVHM ---S-LFAT-AIL-D--R---TLNGT-1943A05 --KV -V-I---T VVSSANATVGNMTE-MR-----VT-G-GNE-RR---L--N--LKH-KEHN -M-IH-E-IS----------KEP---E-VG-G----RS-----S-LLAT-TIT-DI-K-Y---T--N1943A06 --KV -V-I---T VVSSANATVGNMTE-MR-----VT-G-GNE-RR---L------MQTN--S -M-VN-E-IS----------KEP---E-V--G----RS-----S-LYAT-AIT-DI-K-Y---T--N1943O02 --V-I-TAATATSTA TTSNTTT-INVGNMTE-MR-----VT-G-GNE-RR---L--N--LKH-NEHN -M-IH-E-IS----------KEP---E-V--G----RS-----S-LLAT-AIV-DI-K-Y-I-NET-

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236HXB2 CTDLK NDT NTNSS SGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPID NDT TSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKIGNMRQAHCNISRAKWNNTLKQIAS VFIS 14 Pretherapy 1329 48 01 ---VI -GS-GT SSRDRGGLIKT--------K----V-D--K---SL--R--VV-LEDNPD-SSYRN-R-I--E--SN----------Q---------S-----GVHM ---G-LY-TNI--DI------L------D--R--VI 1329 48 02 ---VI -GS-AT SGIDRGGLIKT--------K-A--V-D--K---SL--R--VV-LEDNPD-SSYRN-R-I--E--SN----------Q---------S-----GVHM ---G-LY-TNI--DI------L-T----D--R--VI 1329 48 04 ---AI -GS-GT SRIDRGGLIKT--------K-T--V-D--K---SL-----VV-LEDNPD-SSYRN-R-I--E--SN----------Q---------S-----GVHM ---G-LY-TNI--DI------L-T----D--R--VI 1329 48 06 ---VI -GS-GT SRIDRGGLIKT--------K-T--V-D--K---SL--R--VV-L-DNHD-S-YR--R-I--E--SN----------Q---------S-----GVHM ---G-LY-TNI-EDI------L-T----D--R--VI 1329 48 07 ---VI -GS-AT SSIDRGGLIKT--------K-T--V-D--K---SLS-R--VV-LEDKHD-SSYS--R-I--E--SN----------Q---------S-----GVHM ---G-LY-TNI--DI------L-T----D--R--VI 1329 48 08 ---VI -GS-GT SSVDRGGLIKT--------K-T--V-D--K---SL--R--VV-LEDNPD-SSYRN-R-I--E--SN----V-----Q---------S-----GVHM ---G-LY-TNI--DI------L-*V---D--R--VI 1329 48 09 ---VI -GSR-DT -IGGLIKT----------T--V-D------SL------V--KDN --SNS--R----E---N----------Q--A------S-----GVP---G--Y-QNI--DI------L---D-----R---I 1329 48 10 ---VI -GS-AT SSIDRGGLIKT--------K-T--V-D--K---SL--R--VV-L-DNHD-SSYR--R-I--E--SN----------Q---------S-----GVHM ---G-LY-TNI--DI------L-T----D--R--VT Posttherapy 1576C01 ---VI -GS-GT SRIDRGGLIKT--------K----V-D--K---SL--R--VV-LEDNND-SSYRN-R-I--E--SN--R-------Q---------S-----GVHM ---G-LY-TNI--DI------L-T----D--R--VI 1576C02 --N -VGTS-GT NINGGLI------------T--V-D--K---SLL----VV-LEDNHD-RSYSN-R-I--E--SN----------Q---------S-----GVHM ---G-LY-TNI--DI------L-T----D--R--VI 1576C03 --N -VGTS-GT NINGGLI------------T--V-D--K---SL-----VV-LEDNHD-RSYSN-R-I--E--SN----------Q---------S-----GVHM ---G-LY-TNI--DI------L------DA-R--VI 1576 00 04 --N -VGTS-GT NINGGLI------------T--V-D--K---SL-----VV-LEDNHD-RSYSN-R-I--E--SN----------Q---------S-----GVHM ---G-LY-TNI--DI------L-T----D--R--VI 1576 00 09 ---VI -GS-GT SSRDRGGLIKT--------K----V-D--K---SL--R--VV-LEDNPD-SSYRN-R-I--E--SN----------Q---------S-----GVHM ---G-LY-TNI--DI------L------D--R--VI 1576 00 10 ---VS -GS-AT RSIDRGGLIKT--------K-A--V-D--K---SL--R--VV-LEDNPD-SSYRN-R-I--E--SN----------Q---------S-----GVHM ---G-LF-TNI--DI------L------D--R--VI 1576 48 02 --N -VGTS-GT NINGGLI------------T--V-D--K---SL-----VV-LEDNHD-RSYSN-R-I--E--SN----------Q---------S-----GVHM ---G-LY-TNI--DI------L------D--R--VI 1576 48 03 --N -VGTS-GT NINGGLI------------T--V-D--K---SL-----VV-LEDNHD-RSYSN-R-I--E--SN----------Q---------S-----GVHM ---G-LY-TNI--DI------L-T----D--R--VI 1576 48 04 --N -VGTS-GT KINGGLI------------T--V-D--K---SL-----VV-LEDNHD-RSYSN-R-I--E--SN----------Q---------S-----GVHM ---G-LY-TNI--DI------L-T----D--R--VI 1780 00 01 ---EV -GRS-GT SNST NSISG-LGLI--------T---T--V-D--K---SL-----VV-MNND-SSYR--R-I--E--SN----------Q---------S-----GVHM ---G-LYATNI--DI------L--T---D--R--VI 1780 00 02 ---EV -GRS-GT SNST NSISG-LGLI------------T--V-D--K---SL-----VV-MNND-SSYR--R-I--E--SN----------Q---------S-----GVHM ---G-LYATNI--DI----Y-L--T---D--R--VI 1780 00 03 ---EV -GRS-GT SNST NSISG-LGLI------------T--V-D--K---SL-----VV-MNND-SSYR--R-I--E--SN----------Q---------S-----GVHM ---G-LYATNI--DI------L--T---D--R--VI 1780 00 04 ---EV -GRS-GT SNST NSISG-LGLI------------T--V-D--K---SL-----VV-MNNDHSSYR--R-I--E--SN----------Q---------S-----GVHM E--GPLYATNI--DI----G-L--T---D--R--VI 1780 00 06 ---EV -GRS-GT SNST NSISG-LGLI------------T--V-D--K---SL-----VV-MNND-SSYR--R-I--E--SN----------Q---------S-----GVHM ---G-LYATNI--DI------L--T---D--R--VI 1780 00 07 ---EV -GRS-GT SNST NSISG-LGLI------------T--V-D--K---SL-H---VV-MNND-SSYR--R-I--E--SN----------Q---------S-----GVHM ---G-LYATNI--DI------L--T---D--R--VI 1780 00 08 ---EV -GRS-GT SNST NSISG-LGLI------------T--V-D--K---SL-C---VV-MNND-SSYR--R-I--E--SN----------Q---------S-----GVHM ---G-LYATNI--DI------L--T---D--R--VI 1780 00 09 ---EV -GRS-GT SNST NSISG-LGLI------------T--V-D--K---SL-----VV-MNND-SSYR--R-I--E--SN----------Q---------S-----GVHM ---G-LYATNI--DI------L--T---D--R--VI 1780 00 10 ---EV -GRS-GT SNST NSISG-LGLI------------T--V-D--K---SL-----VV-MNND-SSYR--R-I--E--SN----------Q-----G---S-----GVHM ---G-LYATNI--DI------L--T---D--R--VI 1780 00 11 ---EV -GRS-GT SNST NSISG-LGLI------------T-NV-D--K---SL-----VV-MNND-SSYR--R-I--E--SN----------Q---------S-----GVHM ---G-LYATNI--DI--T---L--T---D--R--VI 1780 00 12 ---EV -GRS-GT SNST NSISG-LGLI------------T--V-D--K---SL-----VV-MNND-SSYR--R-I--E--SN----------Q---------S-----GVHM ---G-LYATNI--DI------L--T---D--R--VI

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237HXB2 CTDLK NDT NTNSS SGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPID NDT TSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGK IGNMRQAHCNISRAKWNNTL VFIS 15 Pretherapy 1868c01 -----K-PP ---N-TTNSTTNTTNSS KGLMEQM-----D-T----DRR-----L------VK-EEEK -KS IGGNYS--R-I--D-------------TEP-Q-D-I-------RS-N------YAT-DI--DI---Y-TVKKT-*-DS1868c02 -I---KDPP ---N-TTNSTTNTTNSS KGLMEQM-----D-T----DRR-----L------VK-EEEK -KS IGGNYS--R-I--D--S----------TEP-Q-D-I-------RS-N------YAT-DI--DI---*-TVKKT---D-1868 48 1 -----K-T-N-N-TTNSTTNNNNSS KGLMEQM-----D-T----DRR-----LY-----VK-EEE I-KS IDSNYS--R-I--D-------------TEP-Q-D-I-------RS-N------YAT-DI--DI---Y-TVNKT---D-1868 48 2 -----K-T-N-N-TTNSTTNNNNSS KGLMEQM-----D-T--V-DRR-----L------VN-EEVK HKS IGGNYS--R-I--D-------------TEP-Q-D-I-------RS-N------YAT-DI--DI---Y-TVNKT---D-1868 48 3 ----NN-T-N-NTTTNSTTNNNNSN KGLMEQM-----D-T----YRR-----LYH----VE-EEEK -ES IGGDYS--R-I--D--S----------TEP-Q-D-I-------RS-N------YAT-DI--DI---Y-TVNKT---D-1868 48 4 --E-M -VYA-DSGE-T-EQKM-------T----DRM-----L------VQ--EEKI-SS SNNS--R-I--D-------------KDP-P---I-------RS-N------YAT-DI---I-----KVNKT---D-1868 48 6 -----K-T-N-N-TTNSTTNNNNSS KGLMEQM-----D-T----DRR-----L------VK-EEVL -KS IGGNYS--R-I--D-------------KEP-Q-D-I-------RS-N------YAT-DI--DI---Y-TVNKT---D-1868 48 8 -----K-T-N-N-TTNNTTNNNNSS KGLMEQM-----D-T----DRR-----L------VK-EEEK -KS IGGNYS--R-I--D-------------TEP-Q-D-I-------RS-N------YAT-DI--DI---Y-TVNKT---D-1868 48 9 -----K-T-N-N-TTNSTTNNNNSS KGLM-QM-----D-T----DRR-----L------VK-EEEK HKS TGGKYS--R-I--D-------------TEP-Q-D-I-------RS-N------YAT-DI--DI---Y-TVNKT---D-1868 48 11 -----K-T-N-N-TTNSTTNNNNSS KGLMEQM-----D-T----DRR-----L------VK-EEEK -KS IGGNYS--R-I--D-------------TEP-Q-D-I-------RS-N------YAT-DI--DI---Y-TVNKT-----1868 48 12 ----SR-T-ELG-NTSLENTTNVNNSSTANISSEGIR--M-------T----D-I-----L------V--KEEQD-SS -R-I--A-------------KEY-H---S--G----RS-H------YAT-QI--DT-R----V---E--SAPosttherapy 2248 48 1 -----K-T-N-N-TTNTT SSS EGLME-M-------T----DRI-----L------VQ-EEEK -SSKNSSKDNYS--R-I--D-------------KEP-Q-----------RS-N------YAT-DI---I---Y--VNKT---D-2248 48 2 ----SR-T-ELG-NTSLENTTDSKNTTTANISSEGIR--M-------T----D-I-----L-----LVQ-KEEQD-SS -R-I--A-------------KN--S---S--G----RS-H-----IYAT-QI--DT-R----V-K-E--SAV 2248 48 5 -----K-I-N-NGTTNNS NSS KELMEQM-----D-T----D-I-----L------VK-EEEK -KS IDGNYS--R-I--D--S----------TEP-Q-D-I-------RS-N------YAT-DI--DI-K-Y-TVNKT---D-2248 48 6 -----K-I-N-NGTTNNS NSS KELMEQM-----D-T----D-I-----L------VK-EEEK -KS IDGNYS--R-I--D--S----------TEP-Q-D-I-------RS-N------YAT-DI--DI-K-Y-TVNKT---D-2248 48 7 ----SR-T-ELG-NTSLENTTDSKNTTTANISSEGIR--M-------T----DMI-----L-----LVQ-KEEQD-SS -R-I--A-------------KN--S---S--G----RS-H-----IYAT-QI--DT-R----V-K-E--SAV 2248 48 8 -----K-T-N-N-TTNSTTNNNNSS KGLMEQM-----D-T----DRR-----L------VK-EEEK -KS TDGNYS--R-I--D-------------TEP-Q-D-I-------RS-N------YAT-DI--DI---Y-TVNKT---D-2248 48 10 -----K-I-N-NGTTNNS NSS KELMEQM-----D-T----D-I-----L------VK-EEEK -KS IDGNYS--R-I--D--S----------TEP-Q-D-I-------RS-N------YAT-DI--DI-K-Y-TVNKT---D-2248 48 11 -----K-T-N-N-TTNTT SSS EGLME-M-------T----DRI-----L------VQ-EEEK -SSKNSNKDNYS--R-I--D-------------KEP-Q-----------RS-N------YAT-DI---I---Y-TVNKT---D-2248 48 12 -----K-T-N-N-TTNTT SSS EGLME-M-------TA---DRI-----L------VQ-EEEK -SSKNSSKDNYS--R-I--D-------------KEP-Q-----------RS-N------YAT-DI--DI-K-Y-TVNKT---D-2248 48 15 -----K-T-N-N-TTNTT SSS EGLME-M-------T----DRI-----L------VQ-EEEK -SSKNSRKDNYS--R-I--D-------------KEP-Q-------Y---RS-N------YAT-DI---I---Y--VNKT---A-HXB2 CTDLK NDT NTNSS SGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPIDNDT TSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKI GNMRQAHCNISRAKWNNTLKQIAFGN VFIS 17 Pretherapy 1766 00 02 ---PVW -TGI-AT SYYANNS SR----DST---T-PL-D--E----LV--N-VV--N-E-NS--FI-IH-E-V-----------KEP-Q-------YIKTG--H------T-TKT-E-QI---Y----WT---T--Y-VV-NI 1766 00 03 ---PVW -TG--AT SYYANNS SR------T---T-PL-D--E----LL--N-VV--N-E-NS--FI-IH-E-L-----------KEP-Q-------YIQTR--H------T-TKT-K-QI---Y--V-WTN--T--Y-VV-NK 1766 00 09 ---PVW -TGI-AT SYYANNS SR------T---T-PL-D--E----LL--N-VV--N-E-NS-NFI-IH-E--S----------KEP-P---A--------S-H------Y-T-S-I-QI--------K----D--L-V--N1766 00 11 ---PVW -TG--AT SYYANNS SR------T---T-PL-D--E----LL--N-VV--N-E-NS--FI-IH-E-------------KEP-Q--------IKTG--H------Y-TKT-E-QI---Y----WT---T--H-VV-KK 1766 00 13 ---PVW -I-TG-A-NT TATSYYANNS SR------T---T-PL-D--E----LL--N-VV--N-E-NR--FI-IH-E-L-----------KEP-Q-------YIQTR--H------H-TKT-E-QI---Y--V-WTN--T--Y-VV-NK 1766 00 14 ---PMW -T-SG-V--TSTNTTGYYANNS SR------T---T-VL-D--E----LL--N-VV--N-E-NS--FI-IH-E-------------KEP-Q------------S-H------YAT-A-V-QI---Y----K----Y--L-V--NK 1766 00 12 ---YEGSNN-ITNANNS -----V---T---T-PV-D-------IL--S-VVSMN-E-NS---R-I--E-L-----------KEP-Q-------YIHTR--H------T-TKT-E-QI---Y----WT---D--L-V--NK 1766 00 05 ---YEGSNN-ITNANSS -----V---T-S-T-PV-D-------IM-QS-VVS-N-E-NS---R-I--E----Y--------KKP-P--WA--------S-H------Y-T-S-I-QI--------KT---A--LKV--HK 1766 00 10 --NYEGSNN-ITNANSS -----V---T---T-P--D-----F-IL--S-VVSMN-E-NS---R-I--E-------------KDP-K--------IKTR--H------Y-TKT-E-KI--------K----D--H-VV-KK Posttherapy 2434 00 06 ---PVW -TSG-VT TGYYANNS SR------T---TKTL-D--E----LL--N-VV--N-E-NS-NFI-IH-E-L-N---I-----KEP-Q-------YIQTR--H------T-TKT-E-QI---Y--V-WTN--T--Y-VV-NK 2434 00 09 ---PVW -TSG-VT TGYYANNS SR------T---T-PL-D--E----LL--N-VV--N-E-NS-NFI-IH-E-L-N---------KDP-K-------YIKTG--H------T-TKN-E-KI---Y--V-WT---T--Y-VV-NK 2434 00 10 ---PVW -TSG-VT TGYYANNS SR------T---T-PL-D--E----LL-RN-VV--N-E-NS-NFI-IH-E-L-N---------KGP-K-------YIRTG--Y------T-TKN-E-KI---Y--V-WT---T--Y-VV-NK 2434 00 01 ---YEGSNN-IIKANSS -----V---T---T-PV-D-------IL--S-VVSMN-E-NS---R-I--E-------------KEP-Q------------S-H------Y-T-S-I-QI--------KT---A--NKV-LI2434 00 04 ---YEGSNN-IIKANSS -----V---T---T-P--D-------IL--S-VVSMN-E-NS---R-I--E-------------KEP-Q------------S-H------Y-T-S-I-QI--------KT---A--NKV--NK 2434 00 07 ---YEGSNN-ITKANSS ---A-V---T---T-PL-D--E----LL-RN-VV--N-E-NS-NFI-IH-E-L-N---------KDP-K-------YIKTG--Q------T-TKN-E-KI---Y--V-WT---T--Y-VV-NK 2434 00 08 ---YEGSNN-ITKANSS -----V---T---T-P--D-------IL--S-VVS-N-E-NS---R-I--E-L-N---I-----KEP-Q-------YIQTR--H------T-TKN-E-KI---Y--V-WT---T--Y-VV-NK 2434 48 01 ---PVW -TSG-VT TGYYANNS SR------T---T-PL-D--A----LL--N-VV--N-E-NS-NFI-IH-E-L-N---I-----KEP-Q-------YIQTR--H------T-TKT-E-QI-L-Y--V-WTN--T--Y-VV-NK 2434 48 02 ---PVW -TSG-VT TGYYANNS SR------T---T-PL-D--E----LL--N-VV--N-E-NS-NFI-IH-E-L-N---I-----KEP-Q-------YIQTR--H------T-TKT-E-QI---Y--V-WTN--T--Y-VV-NK 2434 48 03 ---PVW -TSG-VT TGYYANNS SR------T---T-PL-D--E----LL--N-VV--N-E-NS-NFI-IH-E-L-N---I-----KEP-Q-------YIQTR--H------T-TKT-E-QI---Y--V-WTN--T--Y-VV-NK 2434 48 04 ---PVW -TSG-VT TGYYANNS SR------T---T-PL-D--E----LL--N-VV--N-E-NS-NFI-IH-E-L-N---I-----KEP-Q--R----YIQTR--H------T-TKT-E-QI---Y--V-WTN--T--Y-VV-NK 2434 48 05 ---PVW -TSG-VT TGYYANNS SR------T---T-PL-D--E----LL--N-VV--N-E-NS-NFI-IH-EKL-N---I-----KEP-Q-------YIQTR--H------T-TKT-E-QI---Y--V-WTN--T--Y-VV-NK 2434 48 06 ---PVW -TSG-VT TGYYANNS SR------T---T-PL-D--E----LL--N-VV--N-E-NS-NFI-IH-E-P-N---I-----KEP-Q-------YIQTR--H------T-TKT-E-QI---Y--V-WTN--T--Y-VV-NK

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238HXB2 CTDLKNDTNTNSSSGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPID NDTTSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGK IGNMRQAHCNISRAKWNNTLKQIASKLREQ VFIS 18 Pretherapy 2304 00 01 ---MG-A--A-NSLGP-------------T--L-D-M-R---T-----LV--SEN-NSA--R-I--A-----T-I------E--Q------------G-TL ----VYY-T-QI--DI-K-------V------Q--VR----2304 00 05 ---MG-A--A-NSLGP-------------T--L-D-M-R---T-----LV--SEN--SA--R-I--A-----T-I------E--Q------------G-TL ----VYY-T-QI--DI-K-------V------Q--VR----2304 00 06 ---VG-A--AT SLGP-------------T--L-D-M-R---T-----LV--SEN--SA--R-I--A-----T-I------E--Q------------G-TL ----VYY-T-QI--DI-K-------V------Q--VRR---2304 00 07 ---VG-A--AT-SLGP-------------T--L-D-M-R---T-----LV--SEN--SA--R-I--A-----T-I------E--Q------------G-TL ----VYY-T-QI--DI-K--R----V------Q--VR----2304 00 08 ---MG-A----NSLGP-------------T--L-D-M-R---T-----LV--SEN--SA--R-I--A-----T-I------E-I-------------G-TL ----VYY-T-QI---I-K-------T-*----Q--VR----2304 00 09 ---MG-A--A-NSLGP-------------T--L-D-M-R---T-----LV--SEN--SA--R-I--A-----T-I------E---------------G-TL -----YY-T-QI--DI-K-------V------Q--VR----2304 00 10 ---MG-A--AT SLGP-------------T--L-D-M-R---T-----LV--SEN--SA--R-I--A-----T-I------E---------------G-TL ----VYY-T-QI--DI-K-------V------Q--VR----2304 00 11 ---MG-A----NSLGP-------------T--L-D-M-R---T-----LV--SEN--SA--R-I--A-----T-I------E--Q------------G-TL ----VYY-T-QI--DI-K-------V------Q--VR----2304 00 12 ---VG-A--A-NSLGP-------------T--L-D-M-R---T-----LV--SEN--SA--R-I--A-----T-I------E---------------G-TL ----VYY-T-QI--DI-K-------T------Q--VR----Posttherapy 2930 48 01 ---MG-A----NSLGP-------------T--L-D-M-R---T-H---LV--SEN-NNA--R-I--A-----T-I------E--Q------------G-TL ----VYY-T-QI--DI-K-------V------Q--VR----2930 48 02 ---MG-A----NSLGP-------------T--L-D-M-R---T-----LV--SEN-NSA--R-I--A-----T-I------E--Q------------G-TL ----VYY-T-QI--DI-K--------------Q--VR--K-2930 48 03 ---MG-A----NSLGP-------------T--L-D-M-R---T-----LV--SEN-NSA--R-I--A-----T-I------E---------------G-TL ----VYY-T-QM--DI-K-------T------Q--VR----2930 48 04 ---MG-A----NSLGP-------------T--L-D-M-R---T-----LV--SEN-NSA--R-I--A-----T-I------E-----------S---G-TL ----VYY-T-QI--DI-K-------T------Q--VR----2930 48 05 ---MG-A----NSLGP-------------T--L-D-M-R---T-----LV--SEN-NSA--R-I--A-----T-I------E---------------G-TL ----VYY-T-QI--DI-K-------V------Q--VR----2930 48 06 ---MG-A----NSLGP-------------T--L-D-M-R---T-----LV--SEN-NSA--R-I--A-----T-I------E--Q------------G-TL ----VYY-T-QI--DI-K-------V------Q--VR----2930 48 07 ---MG-A----NSLGP-------------T--L-D-M-R---T-----LV--SEN-NSA--R-I--A-----T-I------E---------------G-TL ----VYY-T-QI--DI-K-------V------Q--VR----2930 48 08 ---MG-A----NSLGP-------------T--L-D-M-R---T-----LV--SEN-NSA--R-I--A-----T-I------E---------------G-TL ----VYY-T-QI--DI-K-------T------Q--VR----2930 48 09 ---MG-A----NSLGP-------------T--L-D-M-R---T-----LV--SEN-NSA--R-I--A-----T-I------E---------------G-TL ----VYY-T-QI--DI-K-------T------Q--VR----2930 48 10 ---MG-A----NSLGP-------------T--L-D-M-R---T-----LV--SEN-NSA--R-I--A----YT-I------E--Q------------G-TL ----VYY-T-QI--DI-K----V---------Q--VR----2930 48 11 ---MG-A----NSLGP-------------T--L-D-M-R---T-----LV--SEN-NSA--R-I--A-----T-I------E--Q------------G-TL ----VYY-T-QI--DI-K-------V------Q--VR----2930 48 12 ---MG-A----NSLGP-----------K-T--L-D-M-R---T-----LV--SEN-NSA--R-I--A-----T-I------E--Q------------G-TL ----VYY-T-QI--DI-K--------------Q--VR--K-2930 48 13 ---MG-A----NSLGP-------------T--L-D-M-R---T-----LV--SEN-NSA--R-I--A-----T-I------E---------------G-TL ----VYY-T-QI--DI-K-------T------Q--VR----2930 48 14 ---MG-A--AT-SLGP-------------T--L-D-M-R---T-----LV--SEN-NSA--R-I--G-----T-I------E--Q------------G-TL ----VYY-T-QI--DI-K-------T------Q--VR----2930 48 15 ---MG-A----NSLGP-------------T--L-D-M-R---T-----LV--SEN-NSA--R-I--A-----T-I------E--Q------------G-TL ----VYY-T-QI--DI-E--------R-----Q--VR----2930 48 16 ---MG-A----NSLGP-------------T--L-D-M-R---T-----LV--SEN-NSA--R-I--A-----T-I------E--Q------------G-TL ----VYY-T-QI--DI-K--------------Q--VR----

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239HXB2 CTDLK NDTNTNSS SGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPID NDT TSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKI GNMRQAHCNISRAKWNNTLKQIASKLREQ VFIS 22 Pretherapy 2041 00 01 --AYW ---YG-DTKANNSKNWGR-DR---QS----VT----D--R--H-LL-RT-LV---ND-SS SSTRNIS-R-I-I--S--SN-------H--Q-----------------H----SW--TKS-T-DI-K-Y-----VA--K--Q--VE---K2041 00 03 --AYG ---YG-DTSANNNRSWER-DR---Q-----VT----D--R--H-LL-RT-LV---ND-SS SSTRNIS-R-I-I--S--SN------A---Q------------I----H----SW--T-D-I-D -K-Y-----VA--K--Q--VE---K2041 00 04 --AYG -N-YG-DTSANNNKNWGR-DR---Q-----VT----D--R--H-LL-RT-LV---ND-SS SSTRNISKR-I-I--S--SN-------H--Q------I----------H----SW--TKS-T-DI-K-Y-----VA--K--Q--VE---K2041 00 05 --AYW -N-YG-DTKANNNTSWGR-D----Q-----VT----D--R----LL-RI-LV---SR-R-I--S--SN----------Q---------------S-H-----WFAT-D-I-DI-K-Y-----TA-EK--Q--VE---K2041 00 06 --AYW ---YG-D KANNNT-WGR-DR-G-Q-----VT-G--D--R--H-LL-RT-LV---NN-SSSSSTRNISKR-I-I--S--SN--------P-Q---------------S-H-----WFAT-D-I-DI-K-Y-----TA-EK--Q--VE---K2041 00 07 --AYWK--KANNNKNWGR-DR---Q-----VT----D--R--H-LL-RT-LV---ND-SS SNTRNISKR-I-I--S--SN-------H--Q-----------------H----SW--TKS-T-DI-K-Y-----VA--K--Q--VE---K2041 00 09 --AYW ---YG-DTKANNNKNWGR-DR---Q-----VT-G--D--R--H-LL-RT-LV---ND-SS SSTRNIS-R-I-I--S--SN-------H--Q-----------------H----SW--TKS-T-DI-K-Y-----VA--K--Q--VE---K2041 00 10 --AYW ---YG-DTKANNNTSWGR-D----Q-----AT----D--R----LL-RI-LV---SR-R-I--S--SN----------Q---------------S-H-----WFAT-D-I-DI-K-Y-----TA-EK--Q--VE---K2041 00 11 --AYWK--KANNNKNWGR-DR---Q-----VT----D--R--H-LL-RT-LV---ND-SS SSTRNISKR-I-I--S--SN----------Q------------I----H----SW--T-S-K-DIGK-------VA--K--Q--VE---KPosttherapy 2411 00 01 --AYG ---YG-DTKANNNRSWER-DR---Q-----VT----D--R--H-LL-RT-LV--NND-SS SSTRNISKR-I-I--S--SN-------H--Q---------S-------H----SW--TKS-I-DI-K-Y-----TA-EK--Q--VE---K2411 00 02 --AYG ---YG-DTKANNNRSWER-DR---Q-----VT----D--R--H-LL-RT-LV--NND-SS SSTRNISKR-I-I--S--SN-------H--Q---------S-------H----SW--TKS-I-DI-K-Y--V--TA-EK--Q--VE---K2411 00 03 --AYG ---YG-DTKANNNRSWER-DR---Q-----VT----D--R--H-LL-RT-LV--NND-SS SSTRNISKR-I-I--S--SN-------H--Q---------S-------H----SW--TKS-I-DI-K-Y-----TA-EK--Q--VE---K2411 00 04 --AYG ---YR-DTKANNNRSWER-DR---Q-----VT----D--R--H-LL-RT-LV--NND-SS SSTRNISKR-I-I--S--SN-------H--Q---------S-------H----SW--TKS-I-DI-K-Y-----TA-EK--Q--VE---K2411 00 05 --AYG ---YG-DTSANNNRSGER-DR---Q-----VT----D--R--H-LL-RT-LV---ND-SS SSTRNIS-R-I-I--S--SN-------H--Q---------S-------H----SW--TKS-I-DI-K-Y-----TA-EK--Q--VE---K2411 00 06 --AYG ---YG-DTKANNNRSWER-DR---Q-----VT----D--R--H-LL-RT-LV--NND-SS SSTRNISKR-I-I--S--SN-------H--Q---------S-------H----SW--TKS-I-DI-K-Y-----TA-EK--Q-MVE---K2411 00 07 --AYG ---YG-DTSANNNRSGER-DR---Q-----VT----D--R--H-LL-RT-LV---ND-SS SSTRNIS-R-I-I--S--SN-------H--Q----------H-I----H----SW--T-D-I-D -K-Y-----VA--E--Q--VE---K2411 00 08 --AYG ---YG-DTKANNNRSWER-DR---Q-----VT----D--R--H-LL-RT-LV--NND-SS SSTRNISKR-I-I--S--SN-------H--Q---------S-------H----SW--TKS-I-DI-K-Y-----TA-EK--Q--VE---K2411 00 09 --AYG ---YG-DTKANNNRSWER-DR---Q-----VT----D--R--H-LL-RT-LV--NND-SS SSTRNISKR-I-I--S--SN-------H--Q---------S-------H----SW--TKS-I-DI-K-Y-----TA-EK--Q--VE---K2411 00 10 --AYW ---YG-DTKANNNTIWGR-D----Q-----VT----D--R--H-LL-RT-LV---ND-SS SSTRNIS-R-I-I--S--SN-------H--Q---------S-------H----SW--TKS-I-DI-K-Y-----TA-EK--Q--VE---K2411 00 11 --AYG ---YG-DAKANNNRSWER-DR---Q-----VT----D--R--H-LL-RT-LV--NND-SS SSTRNISKR-I-I--S--SN-------H--Q---------S-------H----SW--TKS-I-DI-K-Y-----TA-EK--Q--VE---K2411 00 12 --AYG ---YG-DTKANNNRSWER-DR---Q-----VT----D--R--H-LL-RT-LV--NND-SS SSTRNISKR-I-I--S--SN-------H--Q---------S-------H----SW--TKS-I-DI-K-Y-----TA-EK--Q--VE---K2411 00 14 --AYG ---YG-DTKANNNRSWER-DR---Q-----VT----D--R--H-LL-RT-LV--NND-SS GSTRNISKR-I-I--S--S--------H--Q---------SS------H----SW--TKS-I-DI-K-Y-----TA-EK--Q--VE---K2411 00 15 --AYG ---YG-DTKANNNRSWER-DR---Q-----VT----D--R--H-LL-RT-LV--NND-SS SSTRNISKR-I-I--S--SN-------H--Q---------S-------H----SW--TKS-I-DI-K-Y-----TA-EK--Q---E---K2411 00 17 --AYG ---YG-DTKANNNRSWER-DR---Q-----VT----D--R--H-LL-RT-LV--NND-SS SSTRNISKR-I-I--S--SN-------H--Q---------S-------H----SW--TKS-I-DI-K-Y-----TA-EK--Q--VE---K2411 48 02 --AYG ---YG-DTKTNNNTIWRR-D----Q-----VT-G--D--R----LL-RI-LV---SR-R-I--S--SN-------H--Q---------------S-H-----WFAT-D-I-DI-K-Y-----TA-EK--Q--VE---K2411 48 05 --AYG -N-HG-DTSANNNTSWER-D----Q-----VT-G--D-MR----LL-RI-LV---SR-R-I--S--SN----------Q---------------S-H-----WFAT-D-I-DI-K-Y----GTA-EK--Q--VE---K2411 48 06 --AYG -N-HG-GTKTNNNTIWGR-D----Q-----VT----D--R--H-LL-RT-LV--SSR-R-I--S--SN-------H--Q---------S-------H----SCA-AQR-I-DISK-Y-----TA-EK--Q--VE---K2411 48 12 --AYG -N-HG-DTKTNNNTIWGR-D----Q-----VT----D--R--H-LL-RT-LV---SR-R-I--S--SN-------H--Q---------S-------H----SW--TKS-I-DI-K-Y-----TA-VK--Q--VE---K2411 48 14 --AYG -N-HG-DTKTNNNTIWGR-D----Q-----VT----D--R--H-LL-RT-LV---SR-R-I--S--SN----------Q---------------S-H-----WFAT-D-L-DI-K-Y-----TA-EK--Q--VE---KHXB2 CTDLK NDTNTNSS SGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPID NDTTSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGK IGNMRQAHCNISRAKWNNTLKQIASKLREQ VFIS 24 Posttherapy 2375 00 02 -SNW-NT-A-TTTPTSTTSTPASNSS SEGK--E------T--VT-N--D-MRE-H-L-----VV---NS-TS -R-I--A--SN----------E---------------S-T------HAT-GI--DI---Y----STQ------KVVE----2375 00 04 -SNW-NT-A-TTTPTSTTSTPASNSS SEGK--E------T--VT-N--D-MRE-H-L-----VV---SS-TS -R-I--A--SN--E-----F-E---------Y-----S-T------HAT-GI--DI---Y----STQ------KVVE----2375 00 07 -SNW-NT-A-TTTPTSTTSTPASNSS SEGK--E------T--VT-N--D-MRE-H-LL----VV---SS-TS -R-I--A--SN--------F-E---------------S-T------HAT-GI--DI---Y----STQ------KVVE----2375 00 12 -SNW-NT-A-TTTPTSNTSTPASNSS SEGK--E------T--VT-N--D-MRE-H-LI----VV---SS-TS -R-I--A--SN--------F-E---------------S-T------HAT-GI--DI---Y----STQ------KVVE----2375 00 06 -SNW-NTSA-TTTPTSTTSTPASNSS SEGK--E------T--VT-N--D-MRE-H-L-----VV---NS-TS -R-I--A-LSN--------F-E---------------S-T------HAT-GI--DI---Y----STQ------KVVE----2375 00 11 -SNW-NTSA-TTTPTSTTSTPASNSS SEGK--E------T--VT-N--D-MRE-H-L-----VV---NS-TS -R-I--A-LSN--------F-E---------------S-T------HAT-GI--DI---Y----STQ------KVVE----2375 48 07 -SNW-NT-A-TTASPSTTSTTASTSS SEGK--E------T--VT-N--D-M-----L-----VV---RS-TS -R-I--A--SN----------E--------------RS-PM ---K--Y-T EI--DI--------SGN-T----K-VE----2375 48 08 -SNW-NT-A-TTASPSTTSTTTSTSS SEGK--E------T--VT-N--D-M-----L-----VV---RS-TS -R-V--A--SN----------E--------------RS-PM ---K--Y-T EI--DI--------SGN-T----K-VE----2375 48 10 --NW-NT-A-TT STPSPTNSS EGK--E------T--VT-N--D-I-----L-----VV---DS-TS -R-I--A--SN----------E--------------RS-PM ---K--Y-T EI--DI--------SGN-T----K-VE----2375 48 03 -S-T-A -TITPTPTSNSS SEGK---------T--VT-N--DRMRE---L-----VV---GS-TS -R-I--A--SN--------F-E---------------S-TM ---KV-YAT EI---I--------SGN-T----KVVE----2375 48 06 -S-T-A -TITPTPTSNSS SEGK---------T--VT-N--D--RE---L-----VV---KS-TS -R-I--A--SN----------E---------------S-T------HAT DI--DI--------STQ------KVVE----2375 48 01 -SNW -N-KA-TT STPTTTSTSS SEGR--E------T--VT-N--D--RE---L-----VV---KS-TS -R-I--A--SN----------E---------------S-T------HAT DI--DI--------STQ------KVVE----2375 48 04 -SNW -N-KA-TT STPTTTSTSS SEGK--E------T--VT-N--D--RE---L-----VV---KS-TS -R-I--A--SN----------E----------D----S-T------HAT DI--DI--------STQ------KVVE----2375 48 12 -SNW -N-KA-TT STPTTTSTSS SEGK--E------T--VT-N--D--RE---L-----VV---KS-TS -R-I--A--SN----------E---------------S-T------HAT DI--DI--------STQ------KVVE----2375 48 14 -SNW -N-KA-TT STPTTTSTSS SEGK--E------T--VT-N-----RE---L-----VV---KS-TS -R-I--A--SN----------E---------------S-T------HAT DI--DI--------S-Q------KVVE----

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240HXB2 CTDLK NDT NTNSS SGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPID NDTTSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKI GNMRQAHCNISRAKWNNTLKQIA VFIS 26 Pretherapy 2491 00 02 ---NL -S-RTDL-NT ANTT VANNGTNWER-------------T-N--D-I-----L-----VV--GSD --S -R-I--E-----V--------E---------------S-H------YAT-E-V--I---------T--DK--k--2491 00 03 ---ELN -NS T-L--T TNTT NANNGTIWGR-D----T------T-N--D-M-----L-----VV--GSD -AS -R-I--E-----V--------E---------------S-H------YAT---V-DI---------T------GR-2491 00 06 ---ELN -NS T-L--T TNTT NANNGTIWGR-D-----------T-N--D-M-----L-----VV--GSD -AS -R-I--ED----VQ-------E---NK----------S-H------YAT---V-DIK--------TR-----GR-2491 00 07 ---ELN -NS T-L--T TNTT NANNGTIWGR-D-----------T-N--D-M-----L-----VV--GSD -AS -R-I--E-----V--------E---------------S-H------YAT---V-DI---------T------GR-2491 00 10 ---ELN -NS T-L--T TNTT NANNGTIWGR-D-----------T-N--D-M-----L-----VV--GSD -AS -R-I--E-----V--------E---------------S-H------YAT---V-DI---------T------GR-2491 00 11 ---ELN -NS T-L--T TNTT NANNGTIWGR-D-----------T-N--D-M-----L-----VV--GSD -AS -R-I--E-----V--------E---------------S-H------YAT---V-DI---------T------GR-2491 00 12 ---ELN -NS T-L--T TNTT NANNGTIWGR-D-----------T-N--D-M-----L-----VV--GSD -AS -R-I--E-----V--------E---------------S-H-L----YAT---V-DI---------T------GR-Posttherapy 3058 00 01 ---NL -N-THVN-VTANNNTANTTVANNGTIWGK-D-----------T-N--D-I-----L-----VV--GSD --S -R-I--E-----V--------E---------------S-H------YAT-E-V--I---------T------GR-3058 00 02 ----N -IDVN-ET SWGK-------------T-N--D-M-----L--------D -TS -R-I--E---N-V--------E--------------RS-N------YATDQ-I--I-------------------V 3058 00 11 ----N -IDVN-ET SWGK-------------T-N--D-M-----L--------D -TS -R-I--E---N-V---T----E----------Y---RS-N------YAT---I-DI---------T---------V 3058 00 03 ----N -T-NTDL-NT TNTTTVNNVTVT SWEK-------------T-N--D-M-----L------V-DN----R-I--E---N-V--------E---V-----------S-N------YAT-Q-V-DI--------G-----A--H-V 3058 00 04 ---KL N-NS T-L--T TNTTNAENVT SWEK--T----------T-N--D-I-----L-----VV--GSD --S -R-I--E-----V--------E---------------S-H------YAT-E-V--I---------T--D------3058 00 06 ---KLT -TAVN-NT TNTTGANNGT IWGK-------------T-N--D-I-----L-----VV--GSD -AS -R-I--E-----V--------E---------------S-H------YAT-E-V--I--------G-----A--H-V 3058 00 12 ---KLT -T-AV-N-TT NTTGANNGT IWGK-------------T-N--D-M-----L--------D -TS -R-I--E---N-V--------E---------------S-N------YATDQ-I--I-------------------V 3058 48 01 ---ELNT-S-NL-N--TNLNSTTNTTNANNGTIWGK-D-----------T-N--D-I-----L-----VV--GSN -AS -R-I--E-----VI-------E---------------S-H------YAT-Q-V-DI---------T--D---ER-3058 48 02 ---ELNT-S-NL-N--TNLNSTTNTTNANNGTIWGK-D-----------T-N--D-I-N---L-----VV--GSN -AS -R-I--E-----V--------E---------------S-H------YAT-Q-V-DI---------T--D---ER-3058 48 03 ---ELNT-S-NL-N--TNLNSTTNTTNANNGTIWGK-D-----------T-N--D-I-----L-----VV--GSN -AS -R-I--E-----V---T----E-----W---------S-H------YAT-Q-V-DI---------T--D---ER-3058 48 07 ---ELNT-S-NL-N--TNLNSTTNTTNANNGTIWGK-D-----------T-N--D-I-----L-----VV--GSN -AS -R-I--E-----V--------E---------------S-H------YAT-Q-V-DI---------T--D---ER-3058 48 08 ---ELNT-S-NL-N--TNLNSTTNTTNANNGTIWGK-D-----------T-N--D-I-----L-----VV--GSN -AS -R-I--E-----V--------E---------------S-H------YAT-Q-V-DI---------T--D---ER-3058 48 09 ---ELNT-S-NL-N--TNLNSTTNTTNANNGTIWGK-D-----------T-N--D-I-----L-----VV--GSN -AS -R-I--E-----V--------E---------------S-H------YAT-Q-V-DIG--------T--D---ER-3058 48 10 ---ELNT-S-NL-N--TNLNSTTNTTNANNGTIWEK-D-----------T-N--D-I-----L-----VV--GSN -AS -R-I--E-----V--M-----E---------------S-H------YAT-Q-V-DI-----D---T--DTP*NR-3058 48 11 ---ELNT-S-NL-N--TNLNGTTNTTNANNGTIWGK-D-----------T-N--D-I-----L-----VV--GSN SAS -R-I--E-----V--------E---------------S-H------YAT-Q-V-DI---------T--D---ER-3058 48 12 ---ELNT-S-NL-N--TNLNSTTNTTNANNGTIWGK-D-----------T-N--D-I-----LY----VV--GSN -AS -R-I--E-----V--------E---------------S-H------YAT-Q-V-DI---------T--D---ER-3058 48 13 ---ELNT-S-NL-N--TNLNSTTNTTNANNGTIWGK-D-----------T-N--D-I-----L-C---VA--GSN -AS -R-I--E-----V--------E---------------S-H------YAT-Q-V-DI---------T--D---ER-HXB2 CTDLK NDT NTNSSSGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPID NDTTSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKI GNMRQAHCNISRAKWNNTLKQIASKLREQ VFIS 29 Pretherapy 3122 00 02 ---WG -N---NNWET------------ST----D-M-----V---S--V---NV--S---R-I-FE-L-N----------E--T------Y-----S-H--VG-YYLT-D-I-DI-K------G-S--KS-Q---I----3122 00 03 ---WG -N---NNWET------------ST----D-M-----V---S--V---NV--S---R-I-FE-V-N----------E--T------------S-H------Y-T-D-I-DI-K------G-S--K--Q---I----3122 00 04 ---WG -N---NNWET------------ST----D-M-----V---S--V---NV--S---R-I-FE-L-N----------E--T------------S-H--V--YY-T-D-I-DI-K------G-S--K--Q---M----3122 00 05 ---WG -N---NNWET------------ST----D-M-----V---S--V---NV--S---R-I--E--S--T-N------V--P------------S-H------Y--RRYI-DI-K------G-S--K--Q---I----3122 00 07 ---WG -N---NNLET------------ST----D-M-----V---S--V---NV--S---R-I-FE-L-N----------E--T------------S-H------Y-T-D-I-DI-K------G-S--KA-QP--I----3122 00 08 ---WG -N---NNWET------------ST----D-M-----V---S--V---NV--S---R-I-FE-L-N----------E--T------------S-H------Y-T-D-I-DI-K------G-S--K--Q---I----3122 00 11 ---WG -N---NNLET------------ST----D-M-----V---S--V---NV--S---R-I-FE-L-N--R-------E--T------------S-H--VMNNINT-D-I-DI-K------G-S--K--Q--GI---V3122 00 12 ---WG -N---NNWET------------ST----D-M-----G---S--V---NV--S---R-I-FE-L-N---------KE--T------YY----S-H------Y-T-D-I-DI-K------G-S--K--Q---I----Posttherapy 3501 00 01 ----G -----NG NWG-------------SA----D-M-----V---S--V---NV--S---R-I-FE-L-N----------E--T------------S-H------Y-T-E-V-DI-K------G-A--K--Q---I---A3501 00 02 ----G -----NNWG-------------ST----D-M-----V---S--V---NV--S---R-I-FE-L-N----------E--T------------S-H------Y-T-E-V-DI-K------G-A--K--Q---I----3501 00 03 ----G -----NNWG-------------ST----DQM-----V--TS--V---NV--S---R-I-FE-L-N----------E--T------------S-H------Y-T-E-V-DI-K------G-A--K--Q---I--S-3501 00 05 ----G -----NNWG-------------ST----D-M-----V--RS--V---NVD-N---R-I-FE-V-N-------H--E--T------------S-H------Y-T-E-V-DI-K------G-A--K--Q---I----3501 00 06 ----G -----NNWG-------------ST----D-M-----V---S--V---NV--S---R-I-FE-L-N----------E--T-------H----S-H------Y-T-E-V-DI-K------G-A--K--Q---I---A3501 00 07 ----G -----NNWG-------------ST----D-M-----V---S--V---NV--S---R-I-FE-L-N----------E--T------------S-Y------Y-T-E-V-DI-K------G-A--K--Q---I----3501 00 08 ----G -----NNWG-------------NT----D-M-R---V---S--V---NV--S---R-I-FE-L-N----------E--T------------S-H------Y-T-E-V-DI-K------G-A--K--Q---I--SAP 3501 00 09 ----G -----NNWG-------------ST----D-M-----V---S--V---NV--S---R-I-FE-L-N----------E--T------------S-H------Y-T-E-V-DI-K------G-A--K--Q---I----3501 00 10 ----G -----NNWG-------------ST----D-M-----V---F--V---NV--S---R-I-FE-L-N----------E--T------------S-H------Y-T-E-V-DI-K------G-A--K--Q---I---AP 3501 00 11 ----G -----NNWG-------------ST----D-M-----V---S--V---NV--S---R-I-FE-L-N----------E--T------------S-H------Y-T-E-V-DI-K------G-A--K--Q---I---AP 3501 00 12 ----G -----NNWG-------------ST----D-M-----V---S--V---NV--S---R-I-FE-L-N----------E--T------------S-H------Y-T-E-V-DI-K------G-A--K--Q---I----3501 00 13 ----G -----NNWG-------------ST----DRM-----V---S--V---NV--S---R-I-FE-L-N----------E--T------------S-H------Y-T-E-V-DI-K------G-A--K--Q---I----3501 00 14 ----G -----NNWG-------------ST----D-M-----V---S--V---NV--S---R-I-FE-L-N----------E--T------------S-H------Y-T-E-V-DI-K------G-A--K--Q---I----3501 00 15 ----G -----NNWG-------------ST----D-M-----V---S--V---NV--S---R-I-FE-L-N----------E--T------------S-H------Y-T-E-V-DI-K------G-A--K--Q---I----3501 00 16 ----G -----NNWG-------------ST----D-M-----V---S--V---NV--S---R-I-FE-L-N-T--------E--T------------S-H------Y-T-E-V-DI-K------G-A--K--Q---I----

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241HXB2 CTDLK NDT NTNSS SGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPID NDT TSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKI GNMRQAHCNISRAKWNN VFIS 30 Pretherapy 2649 00 06 --SSRTA--NTTNNS SREMI----------T-NM-DR------L--N---V---ER -NS TNY-N-R-I--E-I---T-N------K----H-I--S----RSVHM ------Y-T-D-I-DI----------N-T2649 00 01 --SSRTA--NTTNNS SREMI----------T-NM-DR------L--N---V---EKNNNA-YS ANY-N-R-L--E-I---T-N------K----H-V--S----RS-N------Y-T-E-I-DI--------KTN*T2649 00 02 --SSRTA--NTTNNS SREMI----------T-NM-DR------L--N---V---EKNNNA-YS ANY-N-R-L--E-I---T-N------K----H-V--S----RS-N------Y-T-E-I-DI---Q----KTN*T2649 00 07 --SSRTA--NTTNNS SREMI----------T-NM-DR------L--N---V---EKNNNA-YS ANY-N-R-L--E-I---T-N------K----H-V--S----RS-N------Y-T-E-I-DI--------KTN*T2649 00 09 ---NL -T-NNLR-TT NNS SGEPI-R----------T--M-N-------L---P--V---ER -NS TNY-N-R-M--E-I---T-N------K----H-I-------RSVHM ------Y-T-D-I-DI----------N-T2649 00 08 ---KL -I-NDLR-ATNNTATTNNTTNNSTNEMV----------T-N--DR------LL-N---V---EKNN -VS YSTNYNN-R-L--E-I---T-N---H--E--K------S----RS-N------Y-T-E-I-DI---------KD--2649 00 10 ---NL -T-NNLR-TTNTT TNNSSREMI----------T-NM-DR------P------V---EKTTNA-YS DNY-N-R----E-I---T-N------K----H-V--S----RS-N------Y-T-E-I-DI--------KTN*T2649 00 04 ---DLR-T-NNS S-ETI------------T--M-H-------L--N---V---EK -TS -K-R-I--E-I---T-N------K----H-V--S----RS-N------Y-T-E-I-DI--------STN-T2649 00 05 ---DLR-T-NNS S-ETI------------T--M-H-------L--N---V---EK -TS -K-R-I--E-I---T-N------K----H-I-------RSVHM D-----Y-T-D-I-DI----------N-T2649 00 12 ---DLRDT-NNS S-ETI------------T--M-H-------L--N---V---EK -TS -K-R-I--E-I---T-N------K----H-I-------RSVHM ---K--Y-T-D-I-DI----------N--Posttherapy 3037 00 04 ---NL -I-NNSK-ITNNASITNNTANNSSREMI----------T--L-DR------L------V---KNNANY-N-R-L--E-I---T-N------E--V-Q-V--S-----S-N------Y-T-E-I-DI---------KD--3037 00 06 ---KL -I-NITN-TVTTNNTTNNSTN EMV-------S--T--L-DG------L------V---NKSANY-N-R-L--E-I---T-N------E--V-Q-V--S-----S-N------Y-T-E-I-DI---------KD--3037 00 08 ---NL -I-NNLR-ITNNTITTNITTNNSSKEMI----------T--L-DR------LY-----V-V-EKKK -SS TNYSANY-N-R-M--E-I---T-N---H--E--K------S----RS-N------Y-T-E-I-DI---------ED--K 3037 00 12 ---NL -I-NNL--NTTTTNNTTNNS TNEMV----------T--L-DR-K----L------V---EKNNSAEGEKNNNNTSNSAKY-N-R-M--E-I---T-N------K----H-I-------RSVHM ---K--Y-T-D-I-DI---------VN-T3037 00 02 ---NL -I-NNL--NTTTTNNTTNNS TNEMV----------T--L-DR-K----L------V---EKNNSAEGEKNNNNTSNSAKY-N-R-M--E-I---T-N------E--V-Q-M--S-----S-N------Y-T-D-I-DI--------STN-T3037 00 09 ---DLR-T-TN-S GKPI------------T--M-H-------L--N---V---EKNNT NY-N-R-I--E-I---T-N------K----H-I-------RSVHM ---K--Y-T-D-I-DI---------VN-T3037 00 10 ---DLR-T-TN-S GKPI------------T--M-H-------L--N---V---EKNNT NY-N-R-I--E-I---T-N------K----H-I-------RSVHM ---K--Y-T-D-I-DI-*-------VN-T3037 00 03 ---NL -I-NNL--NTTTTNNTTNNS TNEMV----------T--L-DR-K----L------V---EKNNSAEGEKNNNNTSNSAKY-N-R-M--E-I---T-D------K----H-I-------RSVHM ---K--Y-T-D-I-DI---------VN-T3037 48 01 --SSRTP--ITTDNNST EMM----------T--L-DR-K----L------V---EKNNNV-NS ANY-N-R-M--E-I---T-N---H--E--K------S----RS-N------Y-T-E-I-DI---------ED--3037 48 02 --SSRTP--ITTNNNST EMM----------T--L-DR-K----L------V---EKNNNV-NS ANY-N-R-M--E-I---T-N---H--E--K------S----RS-N------Y-T-E-I-DI---------ED--3037 48 03 --SSRTP--ITTDNNST EMM----------T--L-DR-K----L------V---EKNNNV-NS ANY-N-R-M--E-I---T-N---H--E--K------S----RS-N------Y-T-E-I-DI---------ED--3037 48 04 --SSRTP--ITTNNNST EMM----------T--L-DR-K----L------V---EKNNNV-NS ANY-N-R-M--E-I---T-N---H--E--K------S----RS-N------Y-T-E-I-DI---------ED--3037 48 07 --SSRTP--ITTNNNST EMM----------T--L-DR-K----L------V---EKNNNV-NS ANY-N-R-M--E-I---T-N---H--E--K------S----RS-N------Y-T-E-I-DI---------ED--3037 48 08 --SSRTP--ITTNNNST EMM----------T--L-DR-K----L------V---EKNNNV-NS ANY-N-R-M--E-I---T-N---H--E--K------S----RS-N------Y-T-E-I-DI---------ED--3037 48 09 --SSRTP--ITTNNNST EMM----------T--L-DR-K----L------V---EKNNNV-NS ANY-N-R-M--E-I---T-N---H--E--K------S----RS-N------Y-T-E-I-DI---------ED--3037 48 11 --SSRTP--ITTNNNST EMM----------T--L-DR-K----L------V---EKNNNV-NS ANY-N-R-M--E-I---T-N---H--E--K------S----RS-N------Y-T-E-I-DI---------EDR-3037 48 12 --SSRTP-HITTNNNST AMM----------T--L-DR-K----L--Q---V---EKNHNV-NS GNYNN---K--E-I---T-N---H--E--K------S----RS-N------Y-T-E-I-DI---------ED--3037 48 13 --SSRTP--ITTNNNST EMM----------T--L-DG-K----L------V---EKNNNV-NS ANY-N-R-M--E-I---T-N---H--E--K------S----RS-N------Y-T-E-I-DI---------ED--3037 48 14 --SSRTP--ITTNNNST EMM----------T--L-DR-K----L------V---EKNNNV-NS ANY-N-R-M--E-I---T-N---H--E--K------S----RS-N------Y-T-E-I-DI---------ED--

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242HXB2 CTDLKNDT NTNSS SGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPI DND TTSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQR GPGRAFVTIGK IGNMRQAHCNISRAKWNNTLKQIASKLRE VFIF 05 Pretherapy 1469M01 ---V--CII-GTITCTDLD TKNSSGGMRE-MR------T-G--D-L-----L--RT--V--N---NTDN-----I--A--------------D--T------G----RGHI------F-T-DI--DI-K-----NLT------EK-VR--G1469M02 ---V--CII-GTITCTDLD TKNSSGGMRE-MR------T-G--D-P-----L--RT--V--N---NTDN-----I--A--------------D--T------G----RGHI------F-T-DI--DI-K-----NLT------EK-VR--G1469M05 ---V--CII-GTITCTDLD TKNSSGGMRE-MR------T-G--D-L-----L--RT--V--N---NTDN-N---I--A--------------D--T------G----RGHI------F-T-DI--DI-K-----NLT------EK-VR--G1469M07 ---V--CII-GTITCTDLD TKNSSGGMRE-MR------T-G--D-L-----L--RT--V--N---NTDN-----I--A--------------D--T------G----RGHI------F-T-DI--DI-K-----NLT------EKLDR--GPosttherapy 2190 00 04 ---V--CY--GTINCTDVDHDKTKSSSGGMRE-MR------T-G--D-L-----L--RT--V--N-K-IIDN---R-I--A--------------D--T------G----RGHI------F-T-DI--DI-K-----NFT------EK-V----2190 00 05 ---V--CY--GTINCTDVDHDKTKSSSGGMRE-MR------T-G--D-L-----L--RT--V--N-K-IIDN---R-I--A--------------D--T------G----R----GHI----T-Y-T-D --DI-K-S---NLT------EK-VR---2190 00 07 ---V--CII-GTITCTDLD TKNSSGGMRE-MR------T-G--D-L-----L--RT--V--N-K-IIDN---R-I--A---------V----D--T------G----RG---GHI----T-F-T-DI--DIIK-----NST------EK-VR---2190 00 11 ---V--SY--GTINCTDVDPDTTKSSSGGMRE-MR------T-G--D-L-----L--RT--V--N-K-IIDN---R-I--A--------------ET--------G----RGHI------F-T-DI--DIKK-----NLT------EK-VR---HXB2 CTDLK NDT NTNSSSGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPID NDTTSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGK IGNMRQAHCNISRAKWNNTLKQIASKLREQ VFIF 06 Pretherapy 1944 00 01 -S-T-IR-G-TP-TWEID-KE----------T---GD-MK--H-L-NR--VV--YNNGEHD-V-R-I--D-------I---H--E--R------------S-TM ------Y-T-DI---I---Y-----EE-SK--EKVVK----K 1944 00 02 -S-T-IR-G-TP-TWEIN-KE----------T---GE-MK--H-L-NR--VV--YNNGEHD-I-R-I--D-------I---H--E--R------------S-TM ------Y-T-DI---I---Y-----EE-SK--EKVVK----K 1944 00 03 -S-T-IR-G-TP-TWEIDTKE----------T---GD-MK--H-L-NR--VV--YNNGEHD-V-R-I--D-------I------K--R------------S-TM ------Y-T-DI---I---Y-----EE-SK--EKVVK----K 1944 00 04 -S-T-IR-G-TP-TWEID-KE----------T---GD-MK--H-L-NR--VV--YNNGEHD-I-R-I--D-----------H--E--R------------S-TM ------Y-T-DI---I---Y-----EE-SK--EKVVK----K 1944 00 05 -S-T-IR-G-TP-TWEID-KE----------T---GD-MK--H-L-NR--VV--YNNGEHD-V-R-I--D-------I---H--E--R------------S-TM ------Y-T-DI---I---Y-----KE-SK--EKVVK----K 1944 00 06 -S-T-IR-G-TP-TWEID-KE----------T---GD-MK--H-L-NR--VV--YNNGEHD-I-R-I--D-----------H--E--R------------S-TM ------Y-T-DI---I---Y-----EE-SK--EKVVK----K 1944 00 07 -S-T-IR-G-TP-TWEID-KE----------T---GD-MK--H-L-NR--VV--YNNGEHD-I-R-I--D-------I---H--E--R------------S-TM ------Y-T-DI---I---Y-----EE-SK--EKVVK----K 1944 00 08 -S-T-IR-G-TP-TWEINLKE----------T---GD-MK--H-L-NR--VV--YNNGEHD-I-R-I--D-------I------K--R------I-----S-TM ------Y-T-DI---I---Y-----EE-SK--EKVVK----K 1944 00 09 -S-T-IR-G-TP-TWEID-KE----------T---GD-MK--H-L-NR--VV--YNNGEHD-V-R-I--D-------I---H--E--R------------S-TM ------Y-T-DI---I---Y-----EE-SK--EKVVK----K 1944 00 10 -S-T-IR-G-TP-TWEID-KE----------T---GE-IK--H-L-NR--VV--YNNGEHD-I-R-I--D-------I---H--E--R------------S-TM ------Y-T-DI---I---Y-----KE--E---KAVK----K 1944 00 12 -S-T-IR-G-TP-TWEID-KE----------T---GE-IK--H-L-NR--VV--YNNGEHD-I-R-I--D-------I---H--E--R------------S-TM ------Y-T-DI---I---Y-----EE-SK--EKVVK----K Posttherapy 2230 00 01 -S-TTIR-G-TP-TWEID-KD-DL-------T---GE-MK--H-L-NR--VV--YNNGEHD-I-R-I--D--------------K-IR------------S-TM ------Y-T-DI---I---Y-----E--SK--EKVVK----K 2230 00 02 -S-T-IR-G-TP-TWEIDTKE----------T---GE-MK--H-L-NR--VV--YNNGEHD-I-R-I--D-----------H--E--R---------K-R--TM ----VYY-T-EIV-DI-K-Y-----EE--K--EKVVK----K 2230 00 03 -S-T-IR-G-TP-TWEID-KE----------T---GE-MT--H-L-NR--VV--YNNGEHD-I-R-I--D-----------H--E--R---------K-R--TM ----VYY-T-EIV-DI-K-Y-----EE--K--EKVVK--PGK 2230 00 04 -S-T-IR-G-TP-TWEID-KE----------T---GE-MK--H-L-NR--VV--YNNGEHD-I-R-I--D--------------K-IR------------S-TM ------Y-T-DI---I---Y-----E--SK--EKVVK----K 2230 00 05 -S-T-IR-G-TP-TWEID-KE----------T---GE-MK--H-LLNR--VV--YNNGEHD-I-R-I--D-----------H--E--R---------K-R--TM ----VYY-T-EIV-DI-K-Y-----EE--K--EKVVK----K 2230 00 06 -N-A -I--T -TWEIQ-------------T----DG-K--H-L-NR--VV-EEDG-T-R-I--D--------------K-IR------------S-TM ------Y-T-DI---I---Y-----E--SK--EKVVK----K 2230 00 07 -S-T-IR-G-TP-TWEID-KE----------T---GE-MK--H-L-NR--VV--YNNGEHD-I-R-I--D--------------K-IR------------S-TM ------Y-T-DI---I---Y-----E--SK--EKVVK----K 2230 00 08 -S-T-IR-G-TP-TWEID-KE----------T---GE-MK--H-L-NR--VV--YNNGEHD-I-R-I--D--------------K-IR------------S-TM ------Y-T-DI---I---Y-----E--SK--EKVVK----K 2230 00 09 -S-T-IR-G-TP-TWEID-KE----------T---GE-MK--H-L-NR--VV--YNNGEHD-I-R-I--D--------------K-IR------------S-TM ------Y-T-DI---I---Y-----E--SK--EKVVK----K 2230 00 10 -N-A -I--T -TREIQ-------------T----DG-K--H-L-NR--VV-EEDG-T-R-I--D--------------K-IR------------S-TM ------Y-T-DI---I---Y-----E--SK--EKVVK----K 2230 00 11 -S-T-IR-G-TP-TWEID-KE----------T---G--MK--H-L-NR--VV--YNNGEHD-I-R-I--D--------------K-IR------------S-TM ------Y-T-DI---I---Y-----E--SK--EKVVK----K 2230 00 12 -S-T-IR-G-TP-TWEID-KE----------T---GE-MK--H-L-NR--VV--YNNGEHD-I-R-I--D--------------K-IR------------S-TM ------Y-T-DI---I---Y-----E--SK--EKVVK----K

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243HXB2 CTDLKNDT......NTNSS..SGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPID...NDT...TS.YKLTSC...SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKI.GNMRQAHCNISRAKWNNTLKQIASKLREQFGN VFIF 07 Pretherapy 1535.1A --...-A-....SI-KGNATTNYS.SSSL--M-------T--LQD-RK-D--L--TF--V-LESGT-G-.....K-R-V-R...-E---N----------E--V------------S-PM..-----LYAT-A-I-GI---------E---E---R--I------K1535.2A --...---....SI-KGNATTNYS.SSSL--M-------T--LQD-RK-D--L--TF--V-LESGT-G-.....K-R-V-R...-E---N----------E--V------------S-PM..-----LYAT-A-I-GI---------E---E---R--I------K1535.3A --...-A-....SI-KGNATTNYS.SSSL--M-------T--LQD-RK-D--L--TF--V-LESGT-G-.....K-R-V--...-E---N----------E--V------------S-PM..-----LYAT-A-I-GI---------E---E---R--I------K1535.4A --...-A-....SI-KGNANTNYS.SSSL--M-------T--LQD-RK-D--L--TF--V-LESGT-G-.....K-R-V--...-E---N----------E--V------------S-PM..-----LYAT-A-I-GI---------E---E---R--I------K1535.5A --...-A-....SI-EGNATTKYK.SSGL--M-------T--LQD-RK-D--L--TF--V-LESGT-G-.....K-R-V--...-E--------------E--V------------S-PM..---S-LYAT-A-I-DI---------E---E--ER--I------KA 1535.6A --...-A-....SI-EGNATTNYN.SSGL--M-------T--LQD-RK-D--L--TF--V-LESGT-G-.....K-R-V--...-E--------------E--V------------S-PM..---S-LYAT-A-I-DI---------E---E--ER--I------K1535.7A --...-A-....SI-KGNATTNYS.SSSL--M-------T--LQD-RK-D--L--TF--V-LESGT-G-.....K-R-V-R...-E---N----------E--V------------S-PM..-----LYAT-A-I-GI---------E---E---R--I------KX 1535.8A --...-A-....SI-KGNATTNYK.SSGL--M-------T--LQD-RK-D--L--TF--V-LESGT-G-...............-E---N----------E--V------------S-PM..-----LYAT-A-I-DI---------E---E---R--I------KD 1535.9A --...-V-....SI-KGNATTNYS.SSSL--M-------T--LQD-RK-D--L--TF--V-LESGT-G-.....K-R-V--...-E---N----------E--V------------S-PM..-----LYAT-A-I-GI---------E---E---R--I------K1535.12A --...-A-....SI-KGNATTNYS.SSSL--M-------T--LQD-RK-D--L--TF--V-LESGT-G-.....K-R-V--...-E---N----------E--V------------S-PM..-----LYAT-A-I-GI---------E---E---R--I------K1535.13A R-...-A-....SIKKENATTNYS.SSSL--MQ------T--LQD-RK-D--L--TF--V-LESGT-G-.....D-RVV-R...-E---N----------E--V------------S-PM..-----LYAT-A-I-GI---------E---E-W-R--I----L-XA 1535.14A --...-A-....SI-KGNATTNYS.SSSL--M-------T--LQD-RK-D--L--TF--V-LESGT-G-.....K-R-V--...-E---N----------E--V------------S-PM..-----LYAT-A-I-GI---------E---E---R--I------KPosttherapy 1678,160 ---W.-NNDTTANTTKGNTTN-STENL-T--------DVT-D--D-TRT-N-L------V--TDITDKPSDV--.-R-I--...-E---N---I------E---------------G---..-----IFQAT--I-DI-R-Y-T-RT----KA-N--VE------R1678.4SK ---W.-N-DTTNAT-RTNNTSNKIEN-------------T-N--DE--T-N-L--N---V--LDISKKNSSV-N.-R-I--...-E---N---I------E-----------T-K-G---..-----IFQAT-RI-DI-R-Y-T-N-T---E--S--VK-------1678.5SK ---W.-NNDTTANTTKGNTTT-SKENL-T--------DVT-D--D-TRT-N-L------V--TDIKDKPSDV--.-R-I-R...-E---N--RS------E-------------SNG-H-..------FAT-RVT-DI-R-Y-K--KT---K--S--VG------KT 1678.6SK ---W.-N-DTTNAT-RTNNTSNKIEN-------------T-N--DE--T-N-L--N---V--LDISKKNSSV-N.-R-I-R...-Q---N---I------E-----------T-K-G---..-----IFQAT-RI-DI-R-Y-T--KT---K--SR-VE-------HXB2 CTDLKNDT NTNSS SGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPID NDTTSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKI GNMRQAHCNISRAKWNNTLKQIASKLREQFGN VFIF 08 Pretherapy 1934 48 04 -I--R-KS-GTNTN-SIWANM----------VA-RT-D-------L-XR--LEQ--TNRSNT-S--R-I--A--S--G-------KEP--------G----RS-HL ------HAT-D-I-DI--------IT------R--VK-----YN1934 48 06 -I--R-KS-GTNTN-SIWANM----------VA-RT-D-------L--R--LEQ--TNRSNT-S--R-I--A--S--G-------KEP--------G----RS-HL ----S-HAT-D-I-DI--------IT------R--VK-----YN1934 48 08 -I--R-KS-GTNTN-SIWANM----------VA-RT-D-------L--R--LEQ--TNRSNT-S--R-I--A--S--G-------KEP---D----G----RS-HL ----S-HAT-D-I-DI-*------IT------R--VK-----YN1934 48 11 -I--R-KS-GTNTN-SIWANM----------VA-RT-D-------L--R--LEQ--TNRSNT-S--R-I--A--S--G-------KEP-----A--G----RS-HL ----S-HAT-D-I-DI--------IT------R--VK-----YN1934 48 13 -I--R-KS-GTNTN-SIWANM----------VA-RT-D-------L--R--LEQ--TNRSNT-S--R-I--A--S--G-------KEP--------G----RS-HL ----S-HAT-D-I-DI--------IT------R--VK-----YN1934 48 14 -I--R-KS-GTNTN-SIWANM----------VA-RT-D-------L--R--LEQ--TNRSNT-S--R-I--A--S--G-------KEP--------G----RS-HL ----S-HAT-D-I-DI--------IT------R--VK-----YN1934 48 16 -I--R-KS-GTNTN-SIWANM----------VA-RT-D-------L--G--LEQ--TNRSNT-S--R-I--A--S--G-------KEP--------G----RS-HL ----S-HAT-D-I-DI--------IT------R--VK-----YN1934 48 10 -I--R-KS-GTNTN-SIWANM---V------VA-RT-D-A-----L--R--LEQ--ADRGNT-SN-R-I--A--S--G-------KEP--------G----RS-HL ----S-HAT-D-I-DI--------IT------R--VK-----YN1934 48 05 -I------IRGN-GTSIN-SIWGNM-----------T--K-------N-L------V--EANKTVN-NK-R-IK-E-----G-------KEP--------G----RG-H----VIY-T-R-I-DI--------I----D-----VK-----Y-1934 48 07 -I------IRGN-GTSIN-SIWGNM-----------T--K-------N-L------V--EANKTVN-NK-R-IK-E-----G-------KEP--------G----RG-H-----IY-T-R-I-DI--------IT------R--VK-----YN1934 48 12 -I------IRGN-GTSIN-SIWGNM-----------T--K-------N-L------V--EANKTVN-NK-R-IK-E-----G-------KEP--------G----RG-H----VIY-T-R-I-DI--------I----D-----VK-----Y-1934 48 03 -I----V-IKGN--TNIN-SIWGNM-----------T--K-------N-L------V--EANKTVN-NK-R-IK-E-----G-------KEP--------G----RG-H-----IY-T-R-I-DI--------I----D-----VK-----Y-Posttherapy 2234 00 03 -I----KS-GTNTN-SIWANM----------VT-RT-D-------LL-R--LEQ--ADRGNT-SN-R-I-FA--S--S-------KEP--------G----RS-HL ----S-HAT-D-I-DI--------IT------R--VK-----YN2234 00 04 -I----KS-GTNTN-SIWANM----------VT-RT-D-------LL-R--LEQ--ADRGNT-SN-R-I--A--S--S-------KEP--------G----RS-HL ----S-HAT-D-I-DI--------IT------R--VK-----YN2234 00 07 -I--R-KS-GTNTN-SIWANM----------VT-RT-D-------L-----LEQ--ADRGDT-NN-R-I--A--S--G-------KEP--------G----RS-HL ----S-HAT-D-I-DI--------IT------R--VK-----YN2234 00 08 -I----KS-GTNTN-SIWANM----------VT-RT-D-------LL-R--LEQ--ADRGNT-SN-R-I--A--S--S-------KEP--------G----RS-HL ----S-HAT-D-I-DI--------IT------R--VK-----YN2234 00 10 -I--R-KS-GTNTN-SIWANM----------VT-RT-D-------L--R--LEQ--TDRGNT-S--R-I--A--S--S-------KEP--------G----RS-HL ----S-HAT-D-I-DI--------IT------R--VK-----YN2234 00 11 -I----KS-GTNTN-SIWANM----------VT-RT-D-------LL-R--LEQ--ADRGNT-SN-R-I--A--S--S-------KEP--------G----RS-HL ----S-HAT-D-I-DI--------IT------R--VKR----YN2234 00 12 -I----KS-GTNTN-SIWANM----------VT-RT-D-------LL-R--LEQ--ADRGNT-SN-R-I--A--S--S-------KEP--------G----RS-HL ----S-HAT-N-IEDIK---G---IT------R--VK-----YN2234 00 06 -I----N-IKGN--TNTN-SIRGNM-----------T-RT-D-----N-L------V--EA NKTG-NK-R-IK-E-----G-------KEP--------G----RG--L ----KIY-TRR-I-DI-R------IT---D-----VK-----Y-

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244HXB2 CTDLK NDT NTNSS SGRMIMEKGEIKNCSFNISTSIRGKVQKEYAFFYKLDIIPID NDTTSYKLTSC SVNFTDNAKTIIVQLNTSVEINCTRPNNNTRKRIRIQRGPGRAFVTIGKI GNMRQAHCNISRAKWNNTLKQIASKLRE VFIF 09 Pretherapy 1955 00 01 ---YN -SS GKWEQ----G-Q------T-G--D-M---H-L---V-V----DNK-KS-N-M-R--E--SN------I---K----D-V-------RG-H---S--Y-T-Q-I--I-H-Y----KGN-SD-----VT--G1955 00 02 ---YN -SS GKWEQ----G-Q------T-G--D-M---H-L---V-V----DNK-KS-N-M-R--E--SN------I---K----D-V-------RG-H---S--Y-T-Q-I--I-H-Y----KGN-SD-----VT--G1955 00 03 ---YN -SS GKWEQ----G-Q------T-G--D-M---H-L---V-V----DNK-KS-N-M-R--E--SN------I---K----D-V-------RG-H---S--Y-T-Q-I--I-H-Y----KGN-SD-----VT--G1955 00 04 ---YN -SS GKWEQ----G-Q------T-G--D-M---H-L---V-V----DNK-KS-N-M-R--E--SN------I---K----D-V-------RG-H---S--Y-T-Q-I--I-H-Y----KGN-SD-----VT--G1955 00 07 ---YN -SS GKWEQ----G-Q----D-T-G--D-M---H-L---V-V----DNK-KS-N-M-R--E--SN------I---K----D-V-------RG-H---S--Y-T-Q-I--I-H-Y----KGN-SD-----VT--G1955 00 08 ---YN -SS GKWEQ----G-Q------T-G--D-M---H-L---V-V----DNK-KS-N-M-R--E--SN------I---K----D-V-------RG-H---S--Y-T-Q-I--I-H-Y----KGN-SD-----VT--G1955 00 09 ---YN -SS GKWEQ----G-Q------T-G--D-M---H-L---V-V----DNK-KS-N-M-R--E--SN------I---K----D-V-------RG-H--VS--Y-T-Q-I--I-H-Y----KGN-SD-----VT--G1955 00 10 ---YN -SS GKWEQ----G-Q------T-G--D-M---H-L---V-V----DNK-KS-N-M-R--E--SN------I---K----D-V-------RG-H---S--Y-T-Q-I--I-H-Y---NKGN-SD-----VT--G1955 00 12 ---YN -SS GKWEQ----G-Q------T-G--D-M---H-L---V-V----DNK-KS-N-M-R--E--SN------I---K----D-V-------RG-H---S--Y-T-Q-I--I-H-Y----KGN-SD-----VT--G1955 00 14 ---YN -SS GKWEQ----G-Q------T-G--D-M---H-L---V-V----DNK-KS-N-M-R--E--SN------I---K----D-V-------RG-H---S--Y-T-Q-I--I-H-Y----KGN-SD-----VT--G1955 00 15 ---YN -SS GKWEQ----G-Q------T-G--D-M---H-L---V-V----DNK-KS-N-M-R--E--SN------I---K----D-V-------RG-H---S--Y-T-Q-I--I-H-Y----KGN-SD-----VT--GPosttherapy 2686 00 01 --NFEK-T-NNNDTSN-----TKTTSS S-EQVD-------T---T----D-MRS-HVL--NT-VV-MS ----I-R--E--S-----------E--I----------K-G-F-----VY-T-N-I-DI-------T-KD--------VA--G2686 00 02 --NFEK-T-NNNDTSN-----TKTTSS S-EQVD-------T---T----D-MRS-H-L--NT-VV-MS ----I-R--E--S-----------E--I----------K-G-F-----VY-T-N-I-D--------T-KD--------VA--G2686 00 03 --NFEK-T-NNNDTSN-----TKTTSS S-EQVD-------T---T----D-MRS-H-L--NT-VV-MS ----I-R--E--S-----------E--I----------K-G-F-----VY-T-N-I-DI-------T-KD--------VA--G2686 00 04 --NFEK-T-NNNDTSN-----TKTTSS S-EQVD-------T---T----D-MRS-H-L--NT-VV-MS ----I-R--E--S-----------E--I----------K-G-F-----VY-T-N-I-DI-------T-KD--------VA--G2686 00 05 --NFEK-T-NNNDTSN-----TKTTSS S-EQVD-------T---T----D-MRS-H-L--NT-VV-MS ----I-R--E--S-----------E--I----------K-G-F-----VY-T-N-I-DI-------T-KD--------VA--G2686 00 06 --NFEK-T-NNNDTSN-----TKTTSS S-EQVD-------T---T----D-MRS-H-L--NT-VV-MS ----I-R--E--S-----------E--I----------K-G-F-----VY-T-N-I-DI-------T-KD--------VA--G2686 00 07 --NFEK-T-NNNDTSN-----TKTTSS S-EQVD-------T---T----D-MRS-H-L--NT-VV-MS ----I-R--E--S-----------E--I----------K-G-F-----VH-T-N-I-DI-------T-KD--------VA--G2686 00 08 --NFEK-T-NNNDTSN-----TKTTSS S-EQVD-------T---T----D-MRS-H-L--NT-VV-MS ----I-R--E--S-----------E--I----------K-G-F-----VY-T-N-I-DI-------T-KD--------VA--G2686 00 09 --NFEK-T-NNNDTSN-----TKTTSS S-EQVD-------T---T----D-MRS-H-L--NT-VV-MS ----I-R--E--S-----------E--I----------K-G-F-----VY-T-N-I-DI-------T-KD--------VA--G2686 00 10 --NFEK-T-NNNDTSN-----TKTTSS S-EQVD-------T---T----D-MRS-H-L--NT-VV-MS ----I-R--E--S-----------E--I----------K-G-F-----VY-T-N-I-DI-------T-KD--------VA--G2686 00 11 --NFEK-T-NNNDTSN-----TKTTSS S-EQVD-------T---T----D-MRS-H-L--NT-VV-MS ----I-R--E--S-----------E--I----------K-G-F-----VY-T-N-I-DI-------T-KD--------VA--G2686 00 12 --NFEK-T-NNNDTSN-----TKTTSS S-EQVD-------T---T----D-MRS-H-L--NT-VV-MS ----I-R--E--S-----------E--I----------K-G-F-----VC-T-N-I-DI-------T-KD--------VA--G

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272 BIOGRAPHICAL SKETCH Sarah Jane Koch was born in 1979 to Su san and Leslie Koch, and grew up in Hopkinton, Massachusetts, with her sister s Lori and Tracy. She graduated from Worcester Academy in 1997, and received he r Bachelor of Science degree in biology from Emory University in Atlanta, Geor gia, in May of 2001. Sarah entered the Interdisciplinary Program in Biomedical Scie nces at the University of Florida in the summer of 2001, and was awarded an Alumni Fellowship. Sarah plans to pursue a postdoctoral position in North Caro lina after she earns her PhD.