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Oligomerization of the L Subunit of the Measles Virus RNA-Dependent RNA Polymerase


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OLIGOMERIZATION OF THE L SUBU NIT OF THE MEASLES VIRUS RNA-DEPENDENT RNA POLYMERASE By EMMANUEL GEORGE VROTSOS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS FOR THE DEGREE MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2003

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ACKNOWLEDGMENTS I would like to begin by thanking my parents Vickie and George Vrotsos. Their support and encouragement throughout the years has allowed me to accomplish many important things. I would also like to express thanks to the Moyer lab for all their help and support. I have a great appreciation for Dr. Sue Moyer, in whose lab I did this work. She provided me with the academic advice and encouragement to complete my degree. I would also like to thank the members of my supervisory committee, Dr. Henry Baker and Dr. David Bloom, who have overseen my advancement toward this degree. ii

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS..................................................................................................ii LIST OF TABLES...............................................................................................................v LIST OF FIGURES...........................................................................................................vi ABSTRACT......................................................................................................................vii CHAPTER 1 INTRODUCTION........................................................................................................1 Viral Genes...................................................................................................................1 Infection........................................................................................................................4 Viral Replication Proteins.............................................................................................5 Experimental Design..................................................................................................11 2 MATERIALS AND METHODS...............................................................................13 Cells and Viruses........................................................................................................13 Antibodies...................................................................................................................14 Plasmids......................................................................................................................14 Primers........................................................................................................................14 Cloning pTM1 FLAG MV L (EcoRI) and pTM1 HA MV L (EcoRI).......................15 Subcloning Remaining MV L Gene into HA and FLAG pTMI Plasmids.................16 Growing and Purifying pTM1 HA MV L and pTM1 FLAG MV L Clones..............16 Constructing C-Terminal Truncations pTM1 FLAG MV L SacI and pTM1 FLAG MV L SmaI............................................................................................................18 In Vitro Transcription/Translation (TNT)..................................................................18 Measles CAT Minigenome Assay..............................................................................18 Infection and Transfection..........................................................................................21 Protein Analysis..........................................................................................................22 Immunoprecipitation...................................................................................................23 3 RESULTS...................................................................................................................24 Creation of Full Length HA and FLAG MV L and Truncated FLAG MV L Mutants..................................................................................................................24 iii

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In Vitro Protein Synthesis...........................................................................................25 CAT Assay to Test the Full Length FLAG and HA MV L Mutants for Functional Activity..................................................................................................................25 Polymerase Complex Formation of the FLAG MV L Proteins with MV P...............29 FLAG MV L SacI Mutant Protein Interaction with HA MV L Protein.....................29 FLAG MV L SmaI Mutant Protein Interaction with HA MV L Protein....................30 HA MV L 408 Mutant Protein Interaction with FLAG MV L Protein......................33 4 DISCUSSION.............................................................................................................38 LIST OF REFERENCES...................................................................................................43 BIOGRAPHICAL SKETCH.............................................................................................47 iv

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LIST OF TABLES Table page 1-1 MV L 408 mutant interactions with MV P (David Holmes)....................................12 3-1 MV L 408 mutant interactions with MV L..............................................................37 v

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LIST OF FIGURES Figure page 1-1 Genetic map of measles virus genome RNA ............................................................2 1-2 Paramyxovirus............................................................................................................3 1-3 Sendai virus L protein schematic. Conserved domains I-VI.....................................8 1-4 Primary amino acid sequence of measles virus L 408 mutant protein.....................10 2-1 Epitope creation using PCR amplification...............................................................17 2-2 Illustration of pTMI FLAG MVL plasmid...............................................................19 2-3 Comparison of full length FLAG MV L with C-terminal truncated FLAG MV Ls20 3-1 In vitro expression of all the epitope tagged proteins using the TNT coupled transcription translation ...........................................................................................26 3-2 Ability of wild type and tagged measles virus polymerase complexes to perform in vitro transcription and replication in a mini-genome system (CAT assay).........28 3-3 FLAG MV L mutants binding to MV P....................................................................31 3-4 FLAG MVL SacI HA MV L binding.....................................................................32 3-5 FLAG MV L SmaI HA MV L complex formation................................................34 3-6 FLAG MVL HA MV L 408 mutant binding..........................................................36 vi

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science OLIGOMERIZATION OF THE L SUBUNIT OF THE MEASLES VIRUS RNA-DEPENDENT RNA POLYMERASE By Emmanuel George Vrotsos December 2003 Chair: Sue A. Moyer Major Department: Molecular Genetics and Microbiology The measles virus RNA-dependent RNA polymerase is composed of the L and P proteins. By using HA and FLAG tagged measles full-length and truncated L proteins, we showed that the P binding site on L resides within amino acids 1-1197 and 1-874 of L protein. These results are consistent with previous data that showed that the N-terminal 408 amino acids of the L protein contain the P binding domain (Horikami et al. 1994). Previous studies have shown that the Sendai virus L protein gives intragenic complementation and forms an oligomer where the L-L interaction site was mapped to the N-terminal half of the protein. We showed that measles L also forms an oligomer, and that the L-L interaction site resides in amino acids 1-1197, 1-874, and 1-408. Site-directed mutations in the N-terminal 408 amino acids of measles L, which abolish P binding, do not affect L-L complex formation. Therefore the L and P binding sites on L are overlapping but are mediated by different amino acids. vii

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CHAPTER 1 INTRODUCTION Measles virus is a member of the Paramyxovirus family in the Order Mononegavirales. The measles virus is an enveloped virus containing a single-stranded, nonsegmented negative-stranded RNA genome. The Order Mononegavirales consists of three families: Paramyxoviridae, which includes both measles and Sendai viruses; Rhabdoviridae, which includes vesicular stomatitis virus (VSV) and rabies virus; and Filoviridae, which includes Ebola and Marburg viruses. There are two Subfamilies in the Paramyxoviridae family: Paramyxovirinae and Pneumovirinae. There are three Genera in the Paramyxovirinae subfamily: Paramyxovirus, Rubulavirus, and Morbillivirus (which includes the measles virus). Viral Genes The measles virus genome size is 15894 nucleotides and encodes six mRNAs that follow the linear order of 3 leader-N-(P/C/V)-M-F-H-L 5 (Lamb and Kolakofsky 2001) (Figure 1-1). A schematic of the virion is shown in Figure 1-2. The nucleocapsid protein (N) has an important role in replication and transcription. The nucleocapsid encapsidates the genome RNA into an RNase-resistant nucleocapsid, which is used as the template for RNA synthesis. The RNA-dependent RNA polymerase is made up of two subunits: the P protein, or phosphoprotein; and the L protein, or large protein (2183 amino acids). The P gene encodes two regulatory proteins (C and V). The RNA-dependent RNA polymerase recognizes the encapsidated genome as a template for viral transcription and replication. The matrix (M) protein plays an important role in virus assembly. 1

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2 5 3 M 1473 P/C / 1657 N 1688 H 1949 L 6639 F 2377 Figure 1-1. Genetic map of the measles virus genome RNA. Lamb, R.A., and D. Kolakofsky. Paramyxoviridae pgs 689-724. Fundamental Virology 4th edition. Lippincott, Williams, and Wilkins. Philadelphia, PA 2001. The gene size is drawn to scale. Gene boundaries are shown by vertical lines. The size of each gene is given as number of nucleotides.

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3 F (Fusion protein)M (Membrane protein)Transcriptase Complex L (Large Polymerase)N (Nucleocapsidprotein)P (Phosphoprotein)VirionRNALipid bilayerHemagglutinin Figure 1-2. Schematic diagram of a paramyxovirus Lamb, R.A., and D. Kolakofsky. Paramyxoviridae pgs 689-724. Fundamental Virology 4 th edition. Lippincott, Williams, and Wilkins. Philadelphia, PA 2001.. The lipid bilayer is shown as the gray concentric circle. The viral matrix protein is shown as a black concentric circle. Inserted through the viral membrane are the hemagglutinin (H) attachment glycoprotein and the fusion (F) glycoprotein. Inside the virus is the negative strand virion RNA, which is encapsidated with the N protein. Associated with the nucleocapsid are the L and P proteins, and together this complex has RNA-dependent RNA transcriptase activity.

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4 The matrix protein functions as the central organizer of viral morphogenesis by interacting with the cytoplasmic tails of the integral membrane proteins, the lipid bilayer, and the nucleocapsids. The hemagglutinin (H) protein interacts with the CD 46 protein. This brings the virus to the cell, which allows the fusion (F) protein to interact with the cell membrane (Lamb and Kolakofsky 2001). Infection The measles virus enters the host through the upper respiratory passages; and the primary site of infection is the respiratory epithelium. The integral membrane proteins H and F are involved in cell attachment and fusion of the viral envelope with the plasma membrane of the host cell. Once the membranes fuse, the nucleocapsid can then enter the cytoplasm of the host cell and begin viral transcription. The entire life cycle of the measles virus takes place in the cytoplasm. Upon infection of the host cell, the viral RNA polymerase initiates transcription starting at the 3 end of the genome. Transcription of the nucleocapsid is sequential and begins by transcribing (+) leader sequence followed by the remaining genes in order from the 3 end. The RNA polymerase fails to reinitiate roughly 30% of the time, thereby leading to a gradient of transcription with the leader RNA being the most abundant and the L mRNA being the least abundant. All the mRNAs are capped methylated, and polyadenylated (Galinski and Wechsler 1991). In replication, the polymerase copies the entire genome, ignoring the gene junctions for mRNA synthesis, creating a full length (+) sense antigenomic RNA which is encapsidated with N simultaneously with its synthesis, which serves as a template for the replication and encapsidation of (-) sense genome RNA (Myers and Moyer 1997). The nucleocapsid protein of the new nucleocapsids interacts with the M protein to help form the viral core. At the same time, H and F are processed through the

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5 Golgi apparatus and are accumulating on the plasma membrane of the host cell. Once the core (containing a nucleocapsid and a few copies of the RNA polymerase) is complete, it will bud at the site of H and F concentration on the host membrane, creating the next generation of virus (Lamb and Kolakofsky 2001). The switch from transcription to replication appears to be regulated by accumulation of the N protein (Arnheiter et al. 1985). Once these proteins accumulate, the RNA polymerase ignores cis acting signals of the gene junctions during replication (Vidal and Kolakofsky 1990). Viral Replication Proteins The measles virus N protein (562 amino acids) is encoded by the first gene in the linear order of the genome. After the leader RNA, the N mRNA is the most abundant viral transcript present in an infected cell. The N protein must interact with the viral genome to form the nucleocapsids and it also must interact with other viral proteins to complete the viral life cycle. The formation of the nucleocapsid is essential in that it allows the polymerase complex to recognize the genome and renders the genome RNA ribonuclease resistant (Buchholz et al. 1994). In order to tightly encapsidate the genome the N protein must interact with neighboring N proteins forming a multimer of N proteins which associate with the RNA genome (Buchholz et al. 1993). The N protein of the nucleocapsid also interacts with the M protein. This interaction brings the encapsidated genome to the plasma membrane of the infected cell so that the virus may bud (Stricker et al. 1994). Calain and Roux (1993) proposed the rule of six (which states that there is one nucleocapsid protein for every six nucleotides in the nucleocapsid, so genome length must be a multiple of six). The measles P protein (507 amino acids) was named for its highly phosphorylated state. The P protein binds with L to form the RNA polymerase. The P protein functions

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6 in stabilization and folding of L, which is unstable in the absence of P (Horikami et al. 1992, Horikami and Moyer 1994). The P protein of Sendai virus was shown to be at tetramer by biophysical and crystallographic studies (Tarbouriech et al. 2000). The measles P protein is an oligomer (Harty and Palese 1995) but its exact composition is not known. The P gene has the ability to produce a number of polypeptide products (including the P, V, and C proteins) by using an overlapping reading frame (for C) and by a process of transcription known as RNA editing (for V), which causes a reading frame shift on translation (Lamb and Kolakofsky 2001). The V mRNA uses the same transcription start site as the P gene, but a nontemplated G residue is incorporated at nucleotide 751 of its mRNA. This causes a frameshift that enables the V protein to be identical to the P protein for the first 231 amino-terminal amino acids. The last 68 amino acids are translated from the V reading frame (Tober et al. 1998). The V protein is not associated with intracellular viral particles and has been shown to be dispensable for measles virus infection in cell culture (Schneider et al. 1997). The start site for the measles C gene is 22 nucleotides downstream of the P and V protein start site on the P mRNA. The protein is in the +1 frame relative to the P protein. The translation of the C protein is due to ribosomal scanning because of the stronger Kozak sequence compared to the P and V start site. The C protein, like the V protein, is not needed for the MV infection cycle in cell culture (Radecke and Billeter 1996). The C protein downregulates viral RNA synthesis (Lamb and Kolakofsky 2001, Reutter et al. 2001).

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7 The measles virus large (L) protein (2183 amino acids) is the least abundant of the structural proteins, since its mRNA is at the 5 end of the genome and therefore the last, and least abundant transcription product. As stated before, the L and P proteins form a complex, and both of these components are required for polymerase activity with the N:RNA template. The measles virus L amino acid sequence has been compared to the L proteins of other () strand RNA viruses and six conserved regions shown in Figure 1-3 have been found (Blumberg et al. 1988; Poch et al. 1990; Sidhu et al. 1993). The specific functions of these domains have not been identified. Studies have shown that polyadenylation, methyltransferase, and kinase activities are associated with the L protein in vesicular stomatitis virus (Hammond and Lesnaw 1987; Hunt and Hutchinson 1993), and a kinase activity is associated with the Sendai L protein (Einberger et al. 1990). Domains II, III, and VI contain motifs found in other polymerases, where domains II and III have been proposed to make up the polymerase module of the L protein (Jablonski et al. 1991; Muller et al. 1994). Domains I, IV and V have no recognizable motifs (Horikami and Moyer 1995). Site directed mutations were constructed by substitution of multiple amino acids at conserved regions in the Sendai L protein. These different substitutions at conserved amino acids in domains I-VI in the Sendai L protein gave varying phenotypes. The majority of the mutations completely inactivated the L protein for all aspects of RNA synthesis. In some cases phenotypes from inactivation to partial activities were observed. The differences in activity depend on the nature of the amino acid that was substituted. The inactive L mutants, however, were still able to bind P protein and the complex was able to bind nucleocapsids (Chandrika et al. 1995; Corteses et al. 2000; Feller et al.

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8 Figure1-3. Sendai virus L protein schematic. Conserved domains I-VI are shown as shaded boxes with their amino acid boundaries indicated above. (Poch et al. 1990)

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9 2000; Smallwood et al. 1999). It was initially thought that each domain had its own catalytic activity, but with further studies it was concluded that the activities of the polymerase are not simply compartmentalized and that each domain contributes to multiple steps in viral RNA synthesis. These domains can, however, function in trans. It has been demonstrated that intragenic complementation between pairs of coexpressed inactive L mutants can restore RNA synthesis (Smallwood et al. 2002). This shows that the L protein forms an oligomer with multiple independent domains each of which exhibits several functions Previous experiments in the measles virus have shown that the N-terminal 408 amino acids of the L protein contain the P binding domain (Horikami et al. 1994). The site on the measles virus P protein that the L protein binds resides in the C-terminal half of the P protein (Harty and Palese). The L binding site was mapped to a region of P protein encompassing the C-terminal amino acids 412-478 (Smallwood et al. 1994). Measles L protein truncations were made in the past to test L-P binding. A C-terminal deletion of 1775 a.a. was made in MV L protein, leaving 408 N-terminal amino acids that still bind P (Horikami and Moyer 1994). Twelve different site directed mutations were constructed in MV L 408 (Joyce Feller and David Holmes, unpublished). The mutants were constructed by changing clustered charged or hydrophobic amino acids to alanine (Figure 1-4). An HA epitope tag was cloned on the N-terminus of each mutation (Bayram Cevik et al. 2003). The twelve mutant truncations were named the following: MV L 491, 492, 494, 495, 496, 497, 498, 499, 500, 501, 508, 502. The amino acid sequence of the wild type 408 is shown in Figure 1-4. The WT 408 gave 100% binding to P. MV L 494, 501,

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10 MDSLSVNQIL YPEVHLDSPI VTNK IVAIL E YARVPHAYSL EDPTLCQNIK 491 HRLKNGFSNQ MIINNVEVGN VI KSKLR SYP AHSHIPYPNC NQDLFNIEDK 492 ESTRKIRELL KKGNSLYSKV SDKVFQCLRD TNSRLGLGS E LRED IKEKVI 494 NLGVYMHSSQ WFEP FLFWF T VKTEMRSVIK SQTHTCHRRR HTPVFFTGSS 495 VELLI SRDLV AIISKESQHV YYLTFE VLLI YCDVIEGRLM TETAMTIDAR 496 497 YTE LLGRV RY MWKLIDGFFP ALGNPTYQ IV AML EPLSLAY LQLRDITVEL 498 499 RGAFLNHCFT EIHDVLDQNG FSDEGTYHEL IEALDY IFI T DDIHLTGE IR 500 501 SFF RSFGHP S LEAVTAA ENV RKYMNQPKVI VYETLMKGHA IFCGIIINGY 508 502 Figure 1-4. Primary amino acid sequence of measles virus L 408 mutant protein. The underlined bold amino acids represent areas where charged or hydrophobic amino acids were changed to alanine (Joyce Feller and David Holmes) the mutant number is in bold below the underlined bold amino acid.

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11 508, 502 all bound P at roughly 30%. MV L 491 gave 10% binding while the rest of the mutants showed no binding to P (Table 1-1). In Sendai virus it has been shown that the L protein forms an oligomer (Smallwood et al. 2002) and the L-L interaction site resides in the amino acids 1-174. This L oligomerization does not depend on P protein. Site directed mutations in the N-terminal 347 amino acids of L which abolish P binding, do not affect the L-L complex formation (Cevik et al. 2003). This shows that while the L and P binding sites are overlapping they are mediated by different amino acids. Protein subunits bind one another through noncovalent interactions. Multiple noncovalent interactions can form between complementary molecules, causing them to bind tightly. The types of noncovalent interactions that can occur are hydrophobic, van der Waal, ionic, and hydrogen bonding. The mode in which the subunits of the measles virus RNA polymerase bind is unknown. Experimental Design The purpose of this project is to determine if measles L forms an oligomer and to identify the binding site for the L-L complex. C-terminal truncations were made in the L protein with an epitope tag in order to determine where on the protein the binding region resides. Site directed mutations were created in MV L 408 by Dr. Joyce Feller (University of Florida) by changing clustered charged or hydrophobic amino acids to alanine. It is thought that these charged and hydrophobic amino acids might be responsible for protein-protein interactions. The amino acids are replaced by alanine because the small methyl side group is unlikely to disturb the tertiary structure of the protein (Cunningham and Wells, 1989; Bass et al. 1991). This approach allowed for the identification of the amino acids that are required for L-L oligomerization.

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12 Table 1-1. MV L 408 mutant interactions with MV P. The table lists the12 MV L 408 mutants plus wild type 408 and their interaction with the MV P. The type of mutation for each mutant is also listed. MV L 408 Mutant L-P Interaction Type of Mutation MV L 408 wt +++ NONE MV L 491 +/H to A MV L 492 C to A MV L 494 + C to A MV L 495 H to A MV L 496 H to A MV L 497 H to A MV L 498 H to A MV L 499 H to A MV L 500 H to A MV L 501 + H to A MV L 502 + C to A MV L 508 + MV to SV +++ refers to strong interaction. + refers to slight interaction, approx. 30% of wild type. +/refers to little or no interaction, approx. 10% of wild type. refers to no interaction. H to A = hydrophobic to alanine C to A = charged to alanine MV to SV = measles virus to Sendai virus (Holmes and Moyer 2002)

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CHAPTER 2 MATERIALS AND METHODS Cells and Viruses Human lung carcinoma cells (A549, American Type Culture Collection; Manassas, VA) were grown at 37 C with 5% CO 2 in Eagles Minimum Essential Medium (F11, Gibco BRL; Langley, OK) supplemented with 8% fetal bovine serum (FBS, Gibco BRL; Langley, OK), 1% nonessential amino acids (100X NEAA, Mediatech; Herndon, VA), 1% penicillin and streptocmycin solution (pen/strep, penicillin 5000 I.U./mL and streptomycin 5000 g/mL, Mediatech; Herndon, VA), 2 mM L-glutamine (200 mM, Mediatech; Herndon, VA), and 1mM sodium pyruvate (100mM, Mediatech; Herndon, VA). The cells were cut 1:6 or 1:12 every three or four days respectively, using 1X trypsin-EDTA (ICN Biochemicals, Inc; Costa Mesa, CA). Recombinant vaccinia virus expressing phage T7 RNA polymerase (VVT7) (Fuerst et al. 1986) was provided by Dr. Edward Niles (Suny Buffalo, NY). Ten 15 cm dishes with a confluent monolayer of Vero cells were infected with VVT7 at a multiplicity of infection (m.o.i.) of 0.05 pfu/cell. The cells were scraped four days post infection and pelleted at 7,000 rpm for 30 min at 4C in a J10 rotor (Beckman). The pellets were resuspended in PBS (phosphate buffered saline; 4.3 mM sodium phosphate, 1.5 mM potassium phosphate monobasic, 137 mM NaCl, 2.7 mM KCl pH7.2). The PBS was supplemented with 1% pen/strep. The cells were subjected to freeze/thaw cycles and then sonicated to produce a consistent solution of virus. The VVT7 titer was determined 13

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14 by plaque assays on A549 cells. The virus was then aliquoted and stored at -80 C. The MVA virus utilized in the CAT assay was provided by Dr. Bernard Moss (NIH). Antibodies The following antibodies were utilized for immunoprecipitation (IP) assays: FLAG mouse and HA monoclonal antibody (Sigma; St. Louis, MO). Rabbit mouse IgG (Southern Biotechnology Associates, Inc; Birmingham, AL) was used as secondary antibody to bind FLAG. Protein A Agarose beads (Sigma; St. Louis, MO) were used to bring down immuno-complexes bound to FLAG and Protein G agarose beads (Sigma; St. Louis, MO) were used to bring down immuno-complexes bound to HA. Plasmids The following plasmids encoding the measles virus phosphoprotein (P), C, nucleocapsid (NP), and the L proteins: pBS MV-P/C; pBS MV-N; and pBS MV-L were provided by Dr. W. Bellini (CDC; Atlanta, GA) and Dr. M. Billeter (Zurich, Switzerland). MV p107CAT minigenome plasmid encoding the chloramphenicol acetyl transferase (CAT) gene cloned between measles virus conserved termini was provided by Dr. S. Udem (New Jersey Medical School, Newark, NJ) (Sidhu et al. 1995). CMPI (measles P without measles C open reading frame) contains a stop codon in the C protein open reading frame downstream of the Y2 start codon such that only the P protein is expressed (Reutter et al. 2001). pTMI HA MV L 408 mutants were created by Dr. Joyce Feller, David Holmes, and subcloned into the tagged pTMI plasmid by Dr. Bayram Cevik. Primers Plasmids containing the measles L gene linked to a tagged epitope were made using polymerase chain reaction (PCR). In order to create the measles L gene with an epitope,

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15 two upstream primers containing the appropriate epitope and part of the measles L gene were created. A NcoI site was made upstream of the epitope to allow subcloning. The FLAG MV L primer (5ATG GCTATCGATT ACAAGGATGA CGATGACAAG CTTGCAATGG ACAGCTTAAG CGTTAAC 3) and the HA MV L primer (5ATG GCTTACCCAT ACGATGTTCC AGATTACGCT AGCTTGGGTG GTCCAATGGA CAGCTTAAGC GTTAAC 3) were used in conjunction with the downstream primer, SM 482 (5 CAGGGACTTC GCTAGCTGTG GAGACGGC 3), downstream of the EcoRI site in MV L, to create an HA MV L and a FLAG MV L. Cloning pTM1 FLAG MV L (EcoRI) and pTM1 HA MV L (EcoRI) The PCR reaction was carried out using the Vent DNA polymerase with the following temperatures profiles: 0:30 min at 94C for denaturation, 0:30 min at 42C for annealing temperature, and 0:30 min at 72C for primer extension. There were 28 cycles in all, followed by 4:00 min at 72C for a final primer extension. The primers were used to create a PCR product of 420 base pairs using the wild type pAel MV L plasmid as the template. The PCR products were purified using phenol chloroform extraction and ethanol precipitation. The two purified PCR products, with an HA and FLAG tag, were digested with NcoI and EcoRI overnight at 37C. A pTM1 plasmid was digested with NcoI and EcoRI and then shrimp alkaline phosphatase (SAP) treated, SAP (1 l) to the digest, incubated at 37 C for 30 minutes and heat inactivate at 65 C for 15 minutes, to prevent self ligation. The digested products along with the digested pTM1 plasmid were separated on a 1.2 % agarose gel. The desired bands (HA MV L and FLAG MV L = 420 bp, pTM1= 5300 bp) were eluted into 15% PEG (polyethylene glycol) in 1X TAE (40 mM Tris, 0.11% glacial acetic acid, and 1mM EDTA) and precipitated with NaOAC and ethanol. Each epitope tagged MV L product was ligated into the digested pTM1 plasmid

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16 using 20 units of T4 DNA Ligase (20,000 units/ml) incubated at 16 C overnight. The ligated products were transformed into DH5 competent cells and plated on LB (Luria broth)/ampicillin (100 g/ml) plates. Multiple colonies were scrapped from LB/AMP plate and PCR screened for the insert using the same PCR primers used to create them. A positive sample for each clone was inoculated in 25 ml of LB/AMP and purified using a Qiagen midiprep kit. The samples were then sequenced by the DNA Core at the University of Florida to assure that the clones contained their epitopes and had the correct sequence. Subcloning Remaining MV L Gene into HA and FLAG pTMI Plasmids The pTM1 HA MV L (EcoRI) and pTM1 FLAG MV L (EcoRI) clones were digested with EcoRI restriction enzyme and SAP treated to prevent self ligation. PAel MV L was digested with EcoRI restriction enzyme. The MV L fragment (6300bp) was ligated with the pTM1 HA and pTM1 FLAG (5700bp). The clones were transformed into DH5 competent cells and plated on LB/ampicillin plates. The colonies were PCR screened using original primers to check for inserts. To assure the clones contained an insert in the correct orientation, the clones were digested with StuI restriction enzyme that cut at one site inside the insert and analyzed on a 1.2% agarose gel to check the size. Figure 2-1 gives an overview of the total cloning process. Growing and Purifying pTM1 HA MV L and pTM1 FLAG MV L Clones To grow log phase cultures positive colonies were inoculated in 2 ml of LB with ampicillin (100 g/ml) and incubate for 5-6 hour (or until turbid). To grow large preparation cultures 200 l of log phase culture was inoculated into 50 ml of LB with ampicillin (100 g/ml). The plasmids were purified with the Qiagen midiprep kit according to the manufacturers protocol.

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17 pAelMVL MVL T7 Promoter HA Epitope PCR SM 482 (downstream primer) pTM1 HAMVL MVL T7 Promoter FLAGEpitope pTM1 FLAGMVL MVL Promoter PCR products 420 bp EcoRI EcoRI NcoI EcoRI NcoI NcoI EcoRI NcoI EcoRI EcoRI NcoIEcoRIT7 EcoRI EcoRI Figure 2-1. Epitope creation using PCR amplification. The procedure was done using upstream primers that contained the appropriate epitope (HA or FLAG) and a downstream primer that contained part of the measles virus L gene. pAeL MV L was used as the template for the PCR reaction. After the PCR product is formed, it was cloned into pTMI. Then the remaining EcoRIEcoRI portion of measles L gene was cloned into the epitope tagged vectors to yield the full length tagged L genes.

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18 Constructing C-Terminal Truncations pTM1 FLAG MV L SacI and pTM1 FLAG MV L SmaI The pTMI FLAG MV L SacI was made by digesting pTM1 FLAG MV L with restriction enzyme SacI (Figure 2-2). The digested plasmid was separated on a 1.2% agarose gel and the pTM1 FLAG MV L SacI product giving a protein of 1197 amino acids was eluted, leaving the remaining fragment. The same procedure was done to the pTMI FLAG MV L SmaI plasmid, except the truncation was up to the SmaI restriction site giving a protein of 874 amino acids (Figure 2-2). The truncated plasmids were religated using T4 DNA ligase, and transformed into DH5 competent cells. Plasmid preparations were prepared as above. Figure 2-3 shows the final product of the truncations compared to the wild type MV L. In Vitro Transcription/Translation (TNT) An in vitro coupled transcription translation reaction was done using a TNT kit (Promega; Madison, WI). This kit allows for transcription and translation to occur simultaneously in the same reaction. The plasmids were incubated with T7 RNA polymerase, rabbit reticulocyte lysate, and 3 H leucine for 2 hours at 37C and the products separated on a 7.5% SDS-PAGE gel. Measles CAT Minigenome Assay A549 cells at 80 % confluence in 35 mm dishes were infected with MVA at a multiplicity of infection of 2.5 in F11 infection media at 37C for 1 hour. Following infection, the inoculum was removed and the dishes were washed with Opti-MEM supplemented with 1% pen/strep. Opti-MEM with 1% pen/strep was then added to each dish, 0.8 for 35 mm or 2.4 for 60 mm. The following plasmids were transfected in Lipofectin (Invitrogen) and incubated for 48 hours: 1.7 g of CMPI (measles P without

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19 SacI (4881) FLAG Epitope Sma I (8133) IRES MV-L SmaI (4204) Sac I (8141) T7 promote r pTMI FLAG MVL 12052 bp Figure 2-2. Illustration of pTMI FLAG MVL plasmid. The plasmid contains a T7 promoter followed by the IRES and FLAG epitope fused to the 5 end of the MV L gene. The figure shows the restriction sites that were used to create the C-terminal truncations.

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20 FLAGepitope T7 promote r IRES p TMI FLAG MV L 2183 amino acids FLAGepitope IRES T7 promote r FLAGepitope T7 promote r IRES p TMI FLAG MV L-SacI 1197 amino acids p TMI FLAG MV L-SmaI 874 amino acids Figure 2-3. Comparison of full length FLAG MV L with C-terminal truncated FLAG MV Ls. The figure shows the truncated FLAG MV L genes with respect to the full length FLAG MV L gene. The size of the proteins produced are given in amino acids.

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21 measles C open reading frame), 0.83 g of MV p107CAT minigenome, 0.83 g of pBS MV-N, 0.16 g of pAel MV L, 0.10 g of pTM1 HA MV L and 0.10 g of pTM1 FLAG MV L. The cells were scraped into 200 l of 0.25M Tris-HCL, pH 7.8/0.5% Triton X-100. The procedure was carried out using a CAT ELISA kit (Roche; Nutley, NJ) according to the manufacturers protocol. The data was analyzed using a microtiter plate reader (Tecan). Infection and Transfection A549 cells at 80 % confluence in 35 or 60 mm dishes were infected with VVT7 or MVA at a multiplicity of infection of 2.5 in F11 infection media (F11 supplemented with 14 mM HEPES pH 7.4, 1 % pen/strep, and 2 mM L-glutamine) at 37C for 1 hour. The volumes of the infection media used were 0.3 ml for 35 mm dishes and 0.5 ml for 60 mm dishes. Following infection, the media was removed and the dishes were washed with Opti-MEM supplemented with 1% pen/strep. Opti-MEM with 1% pen/strep was then added to each dish, 0.8 for 35 mm or 2.4 for 60 mm. Unsupplemented Opti-MEM, combined with lipofectin (Bethesda Research Laboratories; Bethesda, MD) and the appropriate plasmid DNA in lipofectin/Opti-MEM (3l of lipofectin/1g of DNA) were transfected for 18 hours or 48 hours for the CAT assay. For binding assays, the transfection media was aspirated 18 hours post transfection. The cells were then washed with PBS and a pulse labeling media, consisting of Dulbeccos Modified Eagles Medium without methionine, cysteine or L-glutamine (Sigma Cell Culture; St. Louis, MO), 1% pen/strep, 2 mM L-glutamine, 14 mM HEPES, pH 7.4 and unsupplemented F11, was added to each dish for 15 minutes. Immediately after the 15 minutes, Express35 S (100 Ci/ml) (New England Biolabs;

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22 Beverly, MA) was added, 75 and 150 Ci for 35 and 60 mm dishes, respectively. The dishes were then incubated at 37C for 2 hours. Protein Analysis To test for protein expression, 80 % confluent A549 cells in 60 mm dishes were infected with vaccinia virus T7 (VVT7) at a multiplicity of infection of 2.5 in F11 infection media at 37C for 1 hour. For the L-P interaction, cells were transfected with pGEM MV P (5.0 g) and pTM1 FLAG MV L (1 g) or 0.50 g of either of the pTM1 FLAG MV L truncations for 18 hours. For the L-L interactions, cells were transfected with pGEM MV P (5.0 g) and pTM1 HA MV L (0.25 g alone and 1.0 g when expressed with either truncated MV L). pTM1 FLAG MV L SacI (0.5 g) was used when expressed alone and 1.0 g with pTM1 HA MV L. pTM1 FLAG MV L SmaI (0.5 g ) was used when expressed alone and 1.0 g with pTM1 HA MV L. To test for L-L interaction using pTMI HA MV L 408s (MV L 408 wt, MV L 491, MV L 494, MV L 495, and MV L 496), cells were transfected with pGEM MV P (5.0 g), pTM1 FLAG MV L (1 g), and 0.25 g of the 408 mutants. The cells were then labeled with Express35 S (100 Ci/ml) in medium with no cysteine and methionine for 2 hours. The cytoplasmic extracts were prepared by scraping into 165 l of RM Salts [0.1M Hepes pH 8.0, 0.05 M NH 4 Cl, 7 mM KCl, 4.5 mM MgAc] + 0.25% NP-40 lysis buffer [0.15 M NaCL, 50 mM Tris-HCl (pH 8.0), 1% Nonidet P-40 (NP-40), and 1g/ml aprotinin]. The cell lysate was cleared by centrifugation for 30 min at 15,000 rpm at 4 C. 10 l of the sample was used to analyze total protein and 155 l was used for immunoprecipitation. Both the totals and immunoprecipitations were separated and analyzed by 7.5% SDSPAGE gel.

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23 Immunoprecipitation 1% NP-40 lysis buffer (100l) was added to the 155 l sample of RM Salts + 0.25% lysis buffer containing the cell extracts. The extracts were incubated 30 minutes at 4 C with 40 l of Protein A or G Agarose beads (Sigma; St. Louis, MO). The extracts were centrifuged for 3 minutes at 10,000 rpm and the supernatant was transferred, while disposing of the pellet for preabsorption. FLAG or HA (2 g) monoclonal antibody was then added and incubated while rocking at 4 C for 1 hour. The secondary antibody, rabbit mouse IgG, is then added and incubated while shaking at 4 C for 1 hour. To bring down the immunocomplexes, Protein A or G agarose beads are added again and incubated at 4 C for 30 minutes. The beads are pelleted and then washed 2X with 800 l of 1% NP-40 Lysis buffer and finally resuspended in 35 l of 2X lysis buffer. The immunoprecipitations were separated on a 7.5% SDS-PAGE gel.

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CHAPTER 3 RESULTS Creation of Full Length HA and FLAG MV L and Truncated FLAG MV L Mutants In order to show protein interactions through coimmunoprecipitation experiments, two different epitopes, HA and FLAG, were created on the N-terminal side of the MV L protein through modification of the L gene as described in Materials and Methods. This process was done through the use of PCR amplification using an upstream primer (containing either the HA or FLAG epitope) and a downstream primer (containing a small section of the MV L gene). The PCR product, containing the epitope and part of the MV L gene was subcloned into the pTMI plasmid (Figure 2-1). The samples were then sequenced by the DNA Core at the University of Florida to assure that the clones contained their epitopes and had the correct sequence. The remaining part of the MV L gene was then subcloned into the vector, as described in Materials and Methods. pTMI HA MV L and pTMI FLAG MV L are the products that were created. In order to determine where the binding site resides on the MV L protein, C-terminal truncations were made. If the truncated MV L protein is still able to bind to the full length MV L protein then the deleted amino acids are not essential for protein-protein interactions. pTMI FLAG MV L was truncated at two different sites. pTMI FLAG MV L SacI was made by digesting pTM1 FLAG MV L with restriction enzyme SacI, as described in Materials and Methods, giving a protein of 1197 amino acids (Figure 2-3). The pTMI FLAG MV L SmaI plasmid, was truncated up to the SmaI restriction site, as described in Materials and Methods, giving a protein of 874 amino acids (Figure 2-3). 24

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25 In Vitro Protein Synthesis The proper expression of each of the epitope tagged, full length and truncated, measles L proteins was first determined in vitro by using the TNT coupled transcription translation system (Promega). The plasmids were incubated with T7 RNA polymerase, rabbit reticulocyte lysate, and 3 H leucine for 2 hours at 37C and the products separated on a 7.5% SDS-PAGE gel, as described in Materials and Methods. Figure 3-1 shows the products of all 4 proteins as well as the Sendai virus L protein, which is used as wild type L marker protein. Sendai L protein is used to compare with the measles L protein because of their similar size and expression. In the absence of plasmid no background bands are present (Figure 3-1, lane 1). The Sendai L protein shows the correct size and expression of the protein (Figure 3-1, lane 2). The two epitope tagged full length L proteins, HA MV L and FLAG MV L, are both expressed and the size is smaller than the Sendai L protein as expected (Figure 3-1, lanes 3 and 4). HA MV L is expressed less well because the DNA concentration was less. In all future experiments the DNA concentrations of the tagged proteins were the same. The truncated measles L proteins, FLAG MV L SacI and FLAG MV L SmaI, are shown as truncated proteins of the expected sizes with respect to the size of the wild type L protein (Figure 3-1, lanes 5 and 6). CAT Assay to Test the Full Length FLAG and HA MV L Mutants for Functional Activity The functional activity of the full length tagged MV L proteins was tested in a minigenome reporter system. The measles virus CAT assay is a minigenome system that tests a viral polymerase complex for its ability to perform transcription and replication in a mammalian expression system. A549 cells are infected with MVA-T7 and transfected

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26 Sendai Virus LSendai Virus LFlag MV LFlag MV LHA MV LHA MV LFLAG MV L FLAG MV L SacISacIFLAG MV L FLAG MV L SmaISmaIMV LMV L--SacISacI MV LMV L--SacISacI MV LMV L--SmaISmaI MV LMV L--SmaISmaI Full length LFull length L MockMock Figure 3-1. In vitro expression of all the epitope tagged proteins using the TNT coupled transcription translation system. The various indicated plasmids were incubated with T7 RNA polymerase, rabbit reticulocyte lysate, and 3 H leucine for 2 hours at 37C and the products separated on a 7.5% SDS-PAGE. Lanes 1 and 2 are the mock and the Sendai virus L protein (shown by an asterisk). Lanes 3-6 show all the epitope tagged mutants. The positions of the full length and truncated L proteins are indicated.

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27 in triplicate with the following plasmids: pBS MV-N, CMPI (measles P without measles C open reading frame), MV p107 CAT minigenome, and wt or tagged full length L proteins, as described in Materials and Methods. The plasmids are all driven by the T7 promoter. The CAT mini genome expresses a negative sense RNA, which contains the CAT gene located between conserved measles virus termini. The genome RNA is encapsidated by the MV N protein and then used as a template for transcription and replication by the viral polymerase. If the functional polymerase complex forms there is transcription and replication, which leads to the production of the CAT mRNA and the CAT protein. The cell extracts were prepared and the remaining reactions were carried out using a CAT ELISA kit (Roche). The data was analyzed using a microtiter plate reader (Tecan). Figure 3-2A shows the results of the CAT assays. This test showed that the The negative control (Figure 3-2A, lane 1), which contains sample buffer but no plasmid showed no activity, as expected. The positive control (Figure 3-2A, lane 2), which contains 200 pg of CAT enzyme, showed a good functional activity. The mock sample was transfected with the minigenome DNA alone and showed no activity (Figure 3-2A, lane 3), showing the template alone gave no activity. The wild type MV L (MV L pAeL) showed functional activity, comparable to positive control (Figure 3-2A, lane 4). The FLAG and HA MV L proteins (Figure 3-2A, lanes 5 and 6 respectively) gave almost identical results. Both tagged L proteins had an activity level approximately 40% of the wild type showing the tag did have some effect on protein function. Nonetheless there was still activity. Figure 3-2B shows the results for the positive control test. The graph shows that the reaction is linear.

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28 A 00.10.20.30.40.50.60.70.80.91NegativecontrolPostitivecontrolMockMV LPAeLFlag MVLHa MV L O.D. B 00.20.40.60.811.21.4Negative100 pg200 pg300 pg400 pg500 pgCAT enzyme (picograms)O.D. Figure 3-2. CAT assay testing the ability of wild type and tagged measles virus polymerase complexes to perform in vitro transcription and replication in a mini-genome system. A549 cells were infected with MVA-T7 at an m.o.i. of 2.5 and then transfected with the appropriate plasmids for 48 hours. Cell extracts were prepared and the CAT activity was measured and described in the Materials and Methods. Figure 3-2A is a graphical representation of CAT activity, each bar represents the average value of an experiment done in triplicate. The error bars show the standard deviation. 3-2B shows the positive control test, which shows that the reaction is linear.

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29 Polymerase Complex Formation of the FLAG MV L Proteins with MV P After it was shown that the tagged MV L proteins all synthesized proteins with the correct size and expression, their ability to bind to the MV P protein to form a polymerase complex was tested. The full length and truncated FLAG MV L proteins were co-expressed with the MV P protein and were tested for binding in an immunoprecipitation assay. A549 mammalian cells were infected with VVT7 and transfected with the following plasmids: pGEM MV P and pTM1 FLAG MV L or either of the pTM1 FLAG MV L truncations for 18 hours. The transfected cells were labeled with Express35 S for 2 hours. Analysis of total cytoplasmic cell extracts showed that all the proteins were synthesized (Figure 3-3A). The immunoprecipitation assay was then carried out on the cell extracts, and separated on a SDS-PAGE gel, as described in Materials and Methods. When the samples expressing mock or MV P alone were immunoprecipitated with FLAG antibody, MV P was not detected (Figure 3-3B, lanes 1 and 2). These data show that the FLAG antibody is specific for the appropriate epitope tagged protein. Immunoprecipitation with FLAG antibody of an extract expressing both MV P and the FLAG MV L proteins showed that MV P coimmunoprecipitated with the FLAG MV L, FLAG MV L SacI, and FLAG MV L SmaI indicative of an L-P interaction (Figure 3-3B, lanes 3-5, respectively). FLAG MV L SacI Mutant Protein Interaction with HA MV L Protein After it was shown that the FLAG MV L mutants were still able to bind P and form the polymerase complex, the truncated FLAG MV L mutants were individually tested for their ability to bind to L. FLAG MV L SacI (1197 amino acids) was tested for its ability to bind to full length HA MV L and form an oligomer. A549 mammalian cells

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30 were infected with VVT7 and transfected with the following plasmids: pGEM MV P alone or together with pTM1 HA MV L or pTM1 FLAG MV L SacI. Analysis of total cytoplasmic cell extracts showed that all the proteins were synthesized (Figure 3-4A). The immunoprecipitation assay was then carried out on the cell extracts, and separated on a SDS-PAGE gel as described in Materials and Methods. When the samples expressing the proteins not tagged with FLAG were immunoprecipitated with FLAG antibody, there was only a very slight background band for HA MV L (Figure 3-4B, lane 3). In this experiment there are significant background bands for the MV P protein (Figure 3-4B, lanes 2 and 3), but our interest is with the L-L interaction. These data shows that the FLAG antibody is specific for the appropriate epitope tagged protein and does not bring down the HA MV L when expressed alone (Figure 3-4B, lane 3 and 4). Immunoprecipitation with FLAG antibody of an extract expressing both FLAG MV L SacI and HA MV L showed that increased HA MV L coimmunoprecipitated with the FLAG MV L SacI indicative of an L-L interaction (Figure 3-4B, lane 5). FLAG MV L SmaI Mutant Protein Interaction with HA MV L Protein It was shown above that FLAG MV L SacI (1197 amino acids) was able to bind full length HA MV L. Therefore the smaller MV L truncation, FLAG MV L SmaI (874 amino acids) was also tested for its ability to bind to full length HA MV L and form an oligomer. A549 mammalian cells were infected with VVT7 and transfected with the following plasmids: pGEM MV P alone or with pTM1 HA MV L or pTMI FLAG MV L SmaI and pTM1 FLAG MV L SmaI and pTM1 HA MV L. Analysis of total cytoplasmic cell extracts showed that all the proteins were synthesized (Figure 3-5A).

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31 MV PMV P FLAG MV L FLAG MV L Sma I Sma I FLAG MV LFLAG MV L FLAG MV L FLAG MV L SacISacI MV PMV P FLAG MV L FLAG MV L SmaISmaI FLAG MV LFLAG MV L FLAG MV L FLAG MV L SacISacI MOCKMOCKFLAG MV L + MV PFLAG MV L + MV PFLAG MV L FLAG MV L SmaISmaI+ MV P+ MV PFLAG MV L FLAG MV L SacISacI+ MV P+ MV PMV P aloneMV P aloneMOCKMOCKFLAG MV L + MV PFLAG MV L + MV PFLAG MV L FLAG MV L SmaISmaI+ MV P+ MV PFLAG MV L FLAG MV L SacISacI+ MV P+ MV PMV P aloneMV P aloneIP w/ IP w/ FLAGFLAGTOTALSTOTALSAB Figure 3-3. FLAG MV L mutants binding to MV P. A) Total cytoplasmic extract. B) Immunoprecipitation with FLAG antibody. VVT7 infected cells were either not transfected (Mock) or transfected with the indicated FLAG MV L, FLAG MV L SacI, or FLAG MV L SmaI plasmids in the presence of MV P plasmid. The cells were transfected for 18 hours and then labeled with Express 35 S for 2 hours. The positions of the L and P proteins are indicated.

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32 MOCKMOCKHA MV L + MV PHA MV L + MV PFLAG MV L FLAG MV L SacISacI+ HA MV L + MV + HA MV L + MV FLAG MV L FLAG MV L SacISacI+ MV P+ MV PMV P aloneMV P alone MV PMV P HA MV LHA MV L FLAG MV L FLAG MV L SacISacI MV PMV P HA MV LHA MV L FLAG MV L FLAG MV L SacISacI MOCKMOCKHA MV L + MV PHA MV L + MV PFLAG MV L FLAG MV L SacISacI+ HA MV L +MV P+ HA MV L +MV PFLAG MV L FLAG MV L SacISacI+MV P+MV PMV P aloneMV P aloneIP w/ IP w/ FLAGFLAGTOTALSTOTALSAB Figure 3-4. FLAG MVL SacI HA MV L binding. A) Total cytoplasmic extract. B) Immunoprecipitation with FLAG antibody. VVT7 infected cells were either not transfected (Mock) or transfected with the indicated FLAG MV L SacI and/or HA MV L in the presence of MV P. The cells were transfected for 18 hours and then labeled with Express 35 S for 2 hours. The positions of the L and P proteins are indicated.

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33 The immunoprecipitation assay was then carried out on the cell extracts, and separated on a SDS-PAGE gel as described in Materials and Methods. When the samples expressing the non FLAG tagged proteins were immunoprecipitated with FLAG antibody, there was only a very slight background band for HA MV L (Figure 3-5B, lane 3), but again there was backgound bands for the MV P protein (Figure 3-5B, lanes 2 and 4). These data shows that the FLAG antibody does not bring down the HA MV L when expressed with MV P. Immunoprecipitation with FLAG antibody of an extract expressing both FLAG MV L SmaI and HA MV L showed that significant HA MV L coimmunoprecipitated with FLAG MV L SmaI indicative of an L-L interaction (Figure 3-5B, lane 5). HA MV L 408 Mutant Protein Interaction with FLAG MV L Protein After it was shown that the full-length MV L protein was still able to bind to the truncated proteins, FLAG MV L SacI and FLAG MV L SmaI, the HA MV L 408 mutants were tested for their ability to bind full length FLAG MV L. To test for L-L interaction using HA MV L 408s (MV L 408 wt, MV L 491, MV L 494, MV L 495, and MV L 496), cells were transfected with pGEM MV P, pTM1 FLAG MV 408 mutants, and MV L 408 wt when expressed alone with MV P. Analysis of total cytoplasmic cell extracts showed that all the FLAG MV L proteins were synthesized (Figure 3-6A). The HA MV L 408 mutants are not visible due to the large amount of background. The immunoprecipitation assay was then carried out on the cell extracts, and separated on a SDS-PAGE gel, as described in Materials and Methods. When the samples expressing the individual proteins were immunoprecipitated with FLAG antibody, only the FLAG tagged proteins were detected, with little background for HA

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34 IP w/ IP w/ FLAGFLAGMOCKMOCKHA MV L + MV PHA MV L + MV PHA MV L + FLAG MV L HA MV L + FLAG MV L SmaISmaI+ MV P + MV P FLAG MV L FLAG MV L SmaISmaI+ MV P+ MV PFLAG MV L FLAG MV L SmaISmaIMV PMV PHA MV LHA MV L TOTALSTOTALSFLAG MV L FLAG MV L SmaISmaIMV PMV PHA MV LHA MV L MV P aloneMV P aloneMOCKMOCKHA MV L + MV PHA MV L + MV PHA MV L + FLAG MV L HA MV L + FLAG MV L SmaISmaI+ MV P+ MV PFLAG MV L FLAG MV L SmaISmaI+ MV P+ MV PMV P aloneMV P alone AB Figure 3-5. FLAG MV L SmaI HA MV L complex formation. A) Total cytoplasmic extract. B) Immunoprecipitation with FLAG antibody. VVT7 infected cells were either not transfected (Mock) or transfected with the indicated FLAG MV L SmaI and/or HA MV L in the presence of MV P. The cells were transfected for 18 hours and then labeled with Express 35 S for 2 hours. The positions of the L and P proteins are indicated.

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35 MV L 408 wt (Figure 3-6B. lanes 1 and 2). These data shows that the FLAG antibody is specific for the FLAG tagged proteins. Immunoprecipitation with FLAG antibody of an extract expressing both FLAG MV L and each HA MV L 408 mutant showed that the HA MV L 408s coimmunoprecipitated with FLAG MV L indicative of an L-L interaction (Figure 3-6B, lanes 3-7). Table 3-1 compares the L and P interactions with the HA MV L 408 mutants.

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36 HA MV L 408 mutantsHA MV L 408 mutantsFLAG MV LFLAG MV LMV L 408 wtMV L 408 wtFLAG MV LFLAG MV LFLAG MV L + HA MV L 408 wtFLAG MV L + HA MV L 408 wtFLAG MV L + HA MV L 495FLAG MV L + HA MV L 495FLAG MV L + HA MV L 491FLAG MV L + HA MV L 491FLAG MV L + HA MV L 494FLAG MV L + HA MV L 494FLAG MV L + HA MV L 496FLAG MV L + HA MV L 496 FLAG MV LFLAG MV L MV L 408 wtMV L 408 wtFLAG MV LFLAG MV LFLAG MV L + HA MV L 408 wtFLAG MV L + HA MV L 408 wtFLAG MV L + HA MV L 495FLAG MV L + HA MV L 495FLAG MV L + HA MV L 491FLAG MV L + HA MV L 491FLAG MV L + HA MV L 494FLAG MV L + HA MV L 494FLAG MV L + HA MV L 496FLAG MV L + HA MV L 496TOTALSTOTALSIP w/ IP w/ FLAGFLAGAB Figure 3-6. FLAG MVL HA MV L 408 mutant binding. A) Total cytoplasmic extract. B) Immunoprecipitation with FLAG antibody. VVT7 infected cells were transfected with the indicated FLAG MV L and/or HA MV L 408s in the presence of MV P. The cells were transfected for 18 hours and then labeled with Express 35 S for 2 hours. The positions of the L and P proteins are indicated.

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37 Table 3-1. MV L 408 mutant interactions with MV L. The table lists the 4 MV L 408 mutants plus wild type 408 and their interaction with the MV L. The type of mutation for each mutant is also listed. MV L 408 Mutant L-P Interaction L-L Interaction Type of Mutation MV L 408 wt +++ +++ NONE MV L 491 +/+++ H to A MV L 494 + +++ C to A MV L 495 +++ H to A MV L 496 +++ H to A +++ strong interaction. + slight interaction (approx. 30% of wild type). +/little or no interaction (approx. 10% of wild type). no interaction. H to A = hydrophobic to alanine C to A = charged to alanine

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CHAPTER 4 DISCUSSION The purpose of this work was to determine if measles L formed an oligomer and to identify the binding site for the L-L complex. Previous work in the Sendai virus showed that the L protein forms an oligomer (Smallwood et al. 2002) and the L-L interaction site resides in the amino acids 1-174. This L oligomerization does not depend on the binding of P protein. Site directed mutations in the N-terminal 347 amino acids of L which abolish P binding (Holmes and Moyer 2002), do not affect the L-L complex formation (Cevik et al. 2003). This shows in Sendai virus that the L and P binding sites are in the same region, but are mediated by different amino acids. In order to test the L-L interactions in measles epitope tags were constructed onto the N-terminus of the MV L protein. This allowed the interactions to be tested with an immunoprecipitation assay. An HA and FLAG tag were used as the epitopes and are immunoprecipitated with their appropriate antibody. Both clones were sequenced at the DNA Core at the University of Florida to assure that the clones contained their epitopes and had the correct sequence. C-terminal deletions were made to measles FLAG L producing two truncated L proteins, FLAG MV L SacI and FLAG MV L SmaI, (1197 a.a. and 874 a.a. respectively). The tagged full length and mutant proteins were synthesized in the in vitro coupled transcription and translation system and both full length and mutant genes were shown to produce proteins of the correct size (Figure 3-1). Through the use of coimmunoprecipitation experiments it was shown that the MV L protein truncated to a.a. 874 was still able to interact with MV P (Figure 3-3), which is 38

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39 consistent with previous work that showed the N-terminal 408 amino acids of MV L are required for P binding (Horikami et al. 1994). It was shown in the Paramyxovirus simian virus 5 (SV5) through the use of coimmunoprecipitation assays that the N-terminal half of the L protein contains sequences important for stable L-P complex formation and that the C-terminal half of L is not directly involved in these interactions with P (Parks, 1994). In a study of the L protein of human parainfluenza virus type 3 (hPIV3) the N-terminus was shown to be necessary for polymerase function. Deletion of nonconserved amino acids 2-15 abolished both P binding and viral transcription (Malur et al. 2002). Thus the N-terminal half of L is required for P binding in a number of paramyxoviruses. In Sendai virus, using sequential deletions in the P protein (568 amino acids), the site on the protein where the L protein binds was mapped to the C-terminal half of the P protein. The L binding site was mapped to a region of P protein encompassing amino acids 412-478. This region lies between the nucleocapsid binding domain (amino acids 345-411 and 479-568), which suggests that the L and NP protein binding domains on P do not overlap (Smallwood et al. 1994). Similar results were found in bovine respiratory syncytial virus (BRSV). The results indicate two independent N binding sites exist on the P protein (amino acids 161-180 and 221-241). The L binding site was mapped to a region of P encompassing amino acids 121-160, which suggests that N and L protein binding domains on the BRSV P protein also do not overlap (Khattar et al. 2001). Previous work was done with the human parainfluenza type 2 virus to determine the region on the P protein that interacts with the L protein. Through the use of truncated P proteins it was shown that the amino acids 278-353 on the P protein are essential for

PAGE 47

40 the binding to the L protein (Nishio et al. 2000). In human parainfluenza virus type 1 (hPIV1), however, it was shown that two regions (residues 387-423 and 511-568) on P protein are required for the functional interaction with hPIV1 L. These regions do overlap with the binding domain for nucleocapsids (Bousse et al. 2001). Thus while the C-terminal portion of P is required for L binding in all paramyxoviruses, the details of the site are specific for each virus. Through the use of coimmunoprecipitation experiments it was shown that the truncated MV L proteins to a.a. 874 were still able to interact with full length L and form an oligomer (Figures 3-4 and 3-5). Therefore the L-L binding site resides in the N-terminal 1-874 a.a. of MV L. This is consistent with the similar L-L binding site on Sendai L (Smallwood et al. 2002). The Sendai virus L protein was compared to that of other negative strand RNA viruses and six conserved region were found (Poch et al. 1990). It was shown that these domains can function in trans because intragenic complementation between pairs of inactive L mutants can restore viral RNA synthesis. Oligomerization of L in the polymerase complex was suggested by these experiments and demonstrated directly by the coimmunoprecipitation of differentially epitope-tagged full length and truncated Sendai virus L proteins (Smallwood et al. 2002). Twelve site directed mutants were constructed in the truncated MV L 408 by Dr. Joyce Feller and Dr. David Holmes to test for the requirement of specific amino acids in the L-P interaction (Table 1-1). The mutagenesis strategy was changing clustered hydrophobic or charged amino acids to alanine. Four of these 408 mutants (MV L 491, 494, 495, 496) as well as wt MV L 408 were used here to test for L-L interactions. A coimmunoprecipitation experiment showed that the wt MV L 408 and all four mutants

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41 were able to bind full length L (Figure 3-6). This is a strong indication that the L binding site on the L protein resides within the amino terminal 408 amino acids for measles virus. Therefore all four mutants were able to bind L compared to only one, MV L 494, which binds P (Holmes and Moyer 2002). So while the L and P binding sites on L are in the same region, they are mediated by different amino acids. Similar experiments were done to test the amino acids required for the L-L interaction in Sendai virus. It was shown that L oligomerization does not depend on P protein and N-terminal fragments of L from amino acids 1-1146 through amino acids 1-174 all bind wild type L. N-terminal deletions of amino acids 1-189 and amino acids 1-734 have lost the ability to form the L-L complex as well as the L-P complex. These data are consistent with the L-L interaction site residing in amino acids 1-174 (Cevik et al. 2003). To further characterize this L-L interaction site-directed mutations in the N-terminal 347 amino acids were created to determine whether those amino acids are essential for L oligomerization. These mutations either abolished or greatly reduced P binding (Holmes and Moyer 2002) but did not affect L-L complex formation. This suggests that the L and P binding sites on L are in the same region but are mediated by different amino acids as is also the case for MV L. Most of the L mutations changed clustered hydrophobic amino acids to alanine, with the exception of one, which changed a charged reside to alanine. This would suggest that the L-L interaction may be mediated by charged residues or hydrophobic amino acids in regions that were not altered (Cevik et al. 2003). We propose that L oligomerizes and then binds the P oligomer which allows the proper folding of L as it is thought to occur in Sendai virus.

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42 Future experiments to further characterize the measles L-L oligomerization include larger C-terminal truncations in order to determine a more precise binding region. After the binding region has been narrowed down, site directed mutations in the binding region to change clustered hydrophobic and charged amino acids to alanine should be created. We showed that some charged and hydrophobic to alanine changes did not affect the L-L interaction in the amino acids 1-408, therefore the creation of different mutations in a smaller region should test if the L-L interaction depends on a charged or hydrophobic interaction between the proteins.

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LIST OF REFERENCES Arnheiter, H., Davis, N.L., Wertz, G., Schubert, M., and R.A. Lazzarini. 1985. Role of the nucleocapsid protein in regulating vesicular stomatitis virus RNA synthesis. Cell. 41, 259-267 Bass, S.H., Mulkerrin, M.G. and J.A. Wells. 1991. A systematic mutational analysis of hormone-binding determinants in the human growth hormone receptor. Proc. Natl. Acad. Sci. U.S.A. 88, 4496-4502. Blumberg, B.M., Crowley, J. C., Silverman, J.I., Menonna, J., Cook, S.D., and P.C. Dowling. 1988. Measles virus L protein evidences elements of ancestral RNA polymerase. Virology 164, 487-497. Bousse, T., Takimoto, T., Matrosovich, T., and A. Portner. 2001. Two regions of the P protein are required to be active with the L protein for human parainfluenza virus type 1 RNA polymerase activity. Virology 283, 306-314. Buchholz, C., Spehner, D., Drillien, R., Neubert, W., and H. Homann. 1993. The conserved N-terminal region of Sendai virus nucleocapsid protein NP is required for nucleocapsid assembly. J. Virol. 67, 5803-5812. Buchholz, C., Retzler, C., Homann, H., and W. J Neubert. 1994. The carboxy-terminal domain of Sendai virus nucleocapsid protein is involved in complex formation between phosphoprotein and nucleocapsid-like particles. Virology 204, 770-776. Calain, P., and L. Roux. 1993. The rule of six, a basic feature for efficient replication of Sendai virus defective interfering particle RNA. J. Virol. 67, 4822-4830. Cevik, B., Smallwood, S., and S.A. Moyer. 2003. The L-L oligomerization domain resides at the very N-terminus of the Sendai virus L RNA polymerase protein. Virology 313, 525-536. Chandrika, R., Horikami, S.M., Smallwood, S., and S.A. Moyer. 1995. Mutations in conserved domain I of the Sendai virus L polymerase protein uncoupletranscription and replication. Virology 213, 325-363. Cortese, C.K., Feller, J.A., and S.A. Moyer. 2000. Mutations in domain V of the Sendai virus L polymerase protein uncouple transcription and replication and Differentially affect replication in vitro and in vivo. Virology 277, 387-396. Cunningham, B.C. and J.A. Wells. 1989. High resolution epitope mapping of hGH-receptor interactions by alanine scanning mutagenesis. Science 244, 1081-1085. 43

PAGE 51

44 Das, T., Schuster, A., Schneider-Schaulies, S., and A.K. Banerjee. 1995. Involvement of cellular casein kinase II in the phosphorylation of measles virusP protein: identification and phosphorylation sites. Virology 211, 218-226. Einberger, H., Mertz, R., Hofschneider, P.H., and W.J. Neubert. 1990. Purification, renaturation, and reconstituted protein kinase activity of the Sendaivirus large (L) protein: L protein phosphorylates the NP and P proteins in vitro. J. Virol. 64, 4274-4280. Escoffier, C., Manie, S., Vincent, S., Muller, C.P., Billeter, M., and D. Gerlier. 1999. Role of the M2-1 transcription antitermination protein of respiratory syncytial virus in sequential transcription. J. Virol. 73, 5852-5864. Feller, J.A., Smallwood, S., Horikami, S.M., and S.A. Moyer. 2000. Mutations in conserved domains IV an VI of the (L) subunit of the Sendai virus RNApolymerase gives a spectrum of defective RNA synthesis phenotypes. Virology 269, 426-439. Fuerst, T.R., Niles, E.G., Studier, F.W., and Moss, B. 1986. Eukaryotic transient expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 83: 8122-8126. Galinski, M.S. and S.L. Wechsler. 1991. The molecular biology of the paramyxoviurs genus. In The Paramyxovirusis, D.W. Kingsbury, ed. (New York: Plenum Press), pp. 410-480. Hammond, D.C., and J.A. Lesnaw. 1987. Functional analysis of hypomethylation variants of the New Jersey serotype of vesicular stomatitis virus. Virology 160, 330-335. Harty, R.N. and P. Palese. 1995. Measles virus phosphoprotein (P) requires the NH2and COOH-terminal domains for interactions with the nucleoprotein (N) but only the COOH terminus for interactions with itself. J. Gen. Virol. 76, 2863-2867. Holmes, D.E., and S.A. Moyer. 2002. The phosphoprotein (P) binding site resides in the N terminus of the L polymerase subunit of sendai virus. J. Virol.. 76, 3078-3083. Horikami, S.M., Curran, J., Kolakofsky, D., and S.A. Moyer. 1992. Complexes of Sendai virus NP-P and P-L proteins are required for defective interfering particle genome replication in vitro. J. Virol.. 66, 4901-4908. Horikami, S.M., and S.A. Moyer. 1994. An amino-proximal domain of the Lprotein binds to the P protein in the measles virus RNA polymerase complex. Virology 205, 540-545. Horikami, S.M., and S.A. Moyer. 1995. Alternative amino acids at a single site in the Sendai virus L protein produce multiple defects in RNA synthesis in vitro. Virology 211, 577-582.

PAGE 52

45 Hunt D.M., and K.L. Hutchinson. 1993. Amino acid changes in the L polymerase protein of vesicular stomatitis virus which confer aberrant polyadenylation and temperature sensitive phenotypes. Virology 193, 786-793. Jablonski, S.A., Luo, M., and C.D. Morrow. 1991. Enzymatic activity of poliovirus RNA polymerase mutants with single amino acid changes in theconserved YGDD amino acid motif. Virology 65, 4565-4572. Khattar, S.K., Yunus, A.S., and S.K. Samal. 2001. Mapping the domains on the phosphoprotein of bovine respiratory syncytial virus required for N-P and P-L interactions using a minigenome system. J. Gen. Virol. 82, 775-779. Lamb, R.A., and D. Kolakofsky. Paramyxoviridae pgs 689-724. Fundamental Virology 4 th edition. Lippincott, Williams, and Wilkins. Philadelphia, PA 2001. Malur, A.G., Choudhary, S.K., De, B.P., and A.K. Banerjee. 2002. Role of a highly conserved NH 2 terminal domain of the human parainfluenza virus type 3 RNA polymerase. J. Virol. 76, 8101-8109. Muller, R., Poch, O., Delarue, M, Bishop, D. H., and M. Bouloy. 1994. Rift Valley fever virus L segment: correction of the sequence and possible functional role of newly identified regions conserved in RNA dependentpolymerases. J. Gen. Virol. 75, 1345-1352. Myers, T.M., and S.A. Moyer. 1997. An amino-terminal domain of the Sendai virus nucleocapsid protein is required for template function in viral RNA synthesis. J. Virol. 71, 918-924. Nishio, M., Tsurudome, M., Ito, M., and Y. Ito. 2000. Mapping of domains on the Human parainfluenza type 2 virus P and NP proteins that are involved in the interaction with the L protein. Virology 273, 241-247. Parks, G.D. 1994. Mapping of a region of the paramyxovirus L protein required for the formation of a stable complex with the viral phosphoprotein P. J. Virol. 68, 4862-4872. Poch, O., Blumberg, B.M., Bougueleret, L., and N. Tordo. 1990. Sequencecomparison of five polymerases (L proteins) of unsegmented negative-strand RNA viruses: theoretical assignment of functional domains. J. Gen. Virol. 71, 1153-1162. Radecke, F., and M.A. Billeter. 1996. The nonstructural C protein is not essentialfor multiplication of Edmonston B strain measles virus in cultured cells. Virology 217, 418-421. Reutter G.L., Corteses-Grogan, C., Wilson, J., and S.A. Moyer. 2001.Mutations in the measles virus C protein that up regulate viral RNA synthesis. Virology. 2001 285(1) 100-109.

PAGE 53

46 Schneider, H., Kaelin, K., and M.A. Billeter. 1997. Recombinant measles viruses defective for RNA editing and V protein synthesis are viable in cultured cells. Virology. 227, 314-322. Sidhu, M.S., Chan, J., Kaelin, K., Spielhofer, P., Radecke, F., Schneider, H., Masurekar, M., Dowling, P.C., Billeter, M. A., and S.A. Udem. 1995. Rescue of synthetic measles virus minireplicons: measles genomic termini direct efficient expression and propagation of a reporter gene. Virology 208, 800-807. Sidhu, M.S., Menonna, J.P., Cook, S.D., Dowling, P.C., and S. A. Udem. 1993. Canine distemper virus L gene: sequence and comparison with related viruses. Virology 193, 50-65. Smallwood S, Cevik B, and S.A. Moyer. 2002. Intragenic complementation and oligomerization of the L subunit of the sendai virus RNA polymerase. Virology 304, 235-245. Smallwood, S., Hovel, T., Neubert, J., and S A. Moyer. 1999. Different substitutions at conserved amino acids in domains II and III in the Sendai L RNA polymerase protein inactivated viral RNA synthesis. Virology 304, 135-145. Smallwood, S., Ryan, K.W., and S.A. Moyer. 1994. Deletion analysis defines a carboxyl-proximal region of Sendai virus P protein that binds to the polymerase L protein. Virology 202, 154-163. Stricker, R., Mottet, G., and L. Roux. 1994. The Sendai virus matrix protein appears to be recruited in the cytoplasm by the viral nucleocapsids to function in viral assembly and budding. J. Gen. Virol. 75, 1031-1042. Tarbouriech, N., Curran, J., Ebal, C., Ruigrok, R. W., and W. P. Burmeister. 2000. On the domain structure and the polymerization state of the Sendai virus P protein. Virology 266, 99-109. Tober, C., Seufert, M., Schneider, H., Billeter, M.A., Johnston, I.C., Niewiesk, S., ter, M., V, and S. Schneider-Schaulies. 1998. Expression of measles virus V protein is associated with pathogenicity and control of viral RNA synthesis. J. Virol. 72, 8124-8132. Vidal, S., and D. Kolakofsky. 1990. Modified model for the switch from Sendai virus transcription to replication. J. Virol. 63, 1951-1958.

PAGE 54

BIOGRAPHICAL SKETCH Emmanuel Vrotsos was born in Miami, Florida on December 26, 1977. At the age of six he moved to Fort Lauderdale, Florida. He graduated from Cooper City High School in 1996 and earned his baccalaureate degree in Zoology from the University of Florida (UF) in 2001. He received his Masters of Science degree from UF in December 2003, and began working toward a Ph.D. at the University of Central Florida, in the Biomolecular Science program. 47


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Material Information

Title: Oligomerization of the L Subunit of the Measles Virus RNA-Dependent RNA Polymerase
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
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OLIGOMERIZATION OF THE L SUBUNIT OF THE MEASLES VIRUS
RNA-DEPENDENT RNA POLYMERASE
















By

EMMANUEL GEORGE VROTSOS


A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF
FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2003













ACKNOWLEDGMENTS

I would like to begin by thanking my parents Vickie and George Vrotsos. Their

support and encouragement throughout the years has allowed me to accomplish many

important things. I would also like to express thanks to the Moyer lab for all their help

and support.

I have a great appreciation for Dr. Sue Moyer, in whose lab I did this work. She

provided me with the academic advice and encouragement to complete my degree. I

would also like to thank the members of my supervisory committee, Dr. Henry Baker and

Dr. David Bloom, who have overseen my advancement toward this degree.















TABLE OF CONTENTS
Page

A C K N O W L E D G M E N T S .................................................................................................. ii

LIST OF TABLES ............................................................................. v

L IST O F FIG U R E S .... .............................. ....................... ........ .. ............... vi

ABSTRACT .............. ..................... .......... .............. vii

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

V ira l G e n e s ............................................................... ..................................... 1
Infection.......................... .............4
V iral R eplication Proteins................................................... .............................. 5
Experim mental D design .................. ...................................... .. ........ .... 11


2 M ATERIALS AND M ETHOD S ........................................ ......................... 13

Cells and Viruses ................................. ............................... ........ 13
A n tib o d ie s ............................................................................................................. 1 4
P la sm id s .........................................................................................14
P rim ers ............................................................................. .... ..................... 14
Cloning pTM1 FLAG MV L (EcoRI) and pTM1 HA MV L (EcoRI) .....................15
Subcloning Remaining MV L Gene into HA and FLAG pTMI Plasmids ..............16
Growing and Purifying pTM1 HA MV L and pTM1 FLAG MV L Clones ...........16
Constructing C-Terminal Truncations pTM1 FLAG MV L SacI and pTM1 FLAG
M V L Sm aI ...................................................................................................... 18
In Vitro Transcription/Translation (TN T) ...................................... ........... ....18
M easles CA T M inigenom e A ssay ................................................................... 18
Infection and Transfection ................................. .......................... ....... 21
Protein A nalysis................................................... 22
Im m u n precipitation ............................................................................... 2 3


3 R E S U L T S ...................................................................2 4

Creation of Full Length HA and FLAG MV L and Truncated FLAG MV L
M u ta n ts ................................................................2 4









In V itro P rotein Synthesis................................................... .................................2 5
CAT Assay to Test the Full Length FLAG and HA MV L Mutants for Functional
A c tiv ity ................. ........ ....... ....................... ............... ......................... 2 5
Polymerase Complex Formation of the FLAG MV L Proteins with MV P...............29
FLAG MV L SacI Mutant Protein Interaction with HA MV L Protein ...................29
FLAG MV L Smal Mutant Protein Interaction with HA MV L Protein ..................30
HA MV L 408 Mutant Protein Interaction with FLAG MV L Protein ....................33


4 D ISCU SSION ................................................................ ...... .......... 38

L IST O F R EFE R E N C E S ............................................................................. ............. 43

BIOGRAPH ICAL SKETCH ...................................................... 47









LIST OF TABLES


Table page

1-1 MV L 408 mutant interactions with MV P (David Holmes)...............................12

3-1 M V L 408 mutant interactions with M V L.. ................................. ............... 37









LIST OF FIGURES


Figure pge

1-1 Genetic m ap of m easles virus genom e RNA ........................................ .................2

1-2 P aram y x ov iru s ............. ............. .. ......... ..... .............. ................ .. 3

1-3 Sendai virus L protein schematic. Conserved domains I-VI..................................8

1-4 Primary amino acid sequence of measles virus L 408 mutant protein............... 10

2-1 Epitope creation using PCR amplification. ....................................................... 17

2-2 Illustration of pTMI FLAG MVL plasmid......................................................19

2-3 Comparison of full length FLAG MV L with C-terminal truncated FLAG MV Ls 20

3-1 In vitro expression of all the epitope tagged proteins using the TNT coupled
transcription translation ......................... .................... .. ........ .. ...... ............26

3-2 Ability of wild type and tagged measles virus polymerase complexes to perform
in vitro transcription and replication in a mini-genome system (CAT assay) .........28

3-3 FLAG M V L mutants binding to M V P ........................................ ............... 31

3-4 FLAG MVL SacI HA MV L binding................................................ ...............32

3-5 FLAG MV L Smal HA MV L complex formation. .............................................34

3-6 FLAG MVL HA MV L 408 mutant binding..................................... ...............36














Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

OLIGOMERIZATION OF THE L SUBUNIT OF THE MEASLES VIRUS
RNA-DEPENDENT RNA POLYMERASE

By

Emmanuel George Vrotsos

December 2003

Chair: Sue A. Moyer
Major Department: Molecular Genetics and Microbiology

The measles virus RNA-dependent RNA polymerase is composed of the L and P

proteins. By using HA and FLAG tagged measles full-length and truncated L proteins,

we showed that the P binding site on L resides within amino acids 1-1197 and 1-874 of L

protein. These results are consistent with previous data that showed that the N-terminal

408 amino acids of the L protein contain the P binding domain (Horikami et al. 1994).

Previous studies have shown that the Sendai virus L protein gives intragenic

complementation and forms an oligomer where the L-L interaction site was mapped to

the N-terminal half of the protein. We showed that measles L also forms an oligomer,

and that the L-L interaction site resides in amino acids 1-1197, 1-874, and 1-408.

Site-directed mutations in the N-terminal 408 amino acids of measles L, which abolish P

binding, do not affect L-L complex formation. Therefore the L and P binding sites on L

are overlapping but are mediated by different amino acids.














CHAPTER 1
INTRODUCTION

Measles virus is a member of the Paramyxovirus family in the Order

Mononegavirales. The measles virus is an enveloped virus containing a single-stranded,

nonsegmented negative-stranded RNA genome. The Order Mononegavirales consists of

three families: Paramyxoviridae, which includes both measles and Sendai viruses;

Rhabdoviridae, which includes vesicular stomatitis virus (VSV) and rabies virus; and

Filoviridae, which includes Ebola and Marburg viruses. There are two Subfamilies in the

Paramyxoviridae family: Paramyxovirinae and Pneumovirinae. There are three Genera

in the Paramyxovirinae subfamily: Paramyxovirus, Rubulavirus, and Morbillivirus

(which includes the measles virus).

Viral Genes

The measles virus genome size is 15894 nucleotides and encodes six mRNAs that

follow the linear order of 3' leader-N-(P/C/V)-M-F-H-L 5' (Lamb and Kolakofsky 2001)

(Figure 1-1). A schematic of the virion is shown in Figure 1-2. The nucleocapsid protein

(N) has an important role in replication and transcription. The nucleocapsid encapsidates

the genome RNA into an RNase-resistant nucleocapsid, which is used as the template for

RNA synthesis. The RNA-dependent RNA polymerase is made up of two subunits: the

P protein, or phosphoprotein; and the L protein, or large protein (2183 amino acids). The

P gene encodes two regulatory proteins (C and V). The RNA-dependent RNA

polymerase recognizes the encapsidated genome as a template for viral transcription and

replication. The matrix (M) protein plays an important role in virus assembly.











H
1949


Genetic map of the measles virus genome RNA. Lamb, R.A., and D.
Kolakofsky. "Paramyxoviridae" pgs 689-724. Fundamental Virology 4th
edition. Lippincott, Williams, and Wilkins. Philadelphia, PA 2001. The
gene size is drawn to scale. Gene boundaries are shown by vertical lines.
The size of each gene is given as number of nucleotides.


N P/C/
1688 1657


Figure 1-1.


II









Hemagglutinin
/


7M (



M (Membr


ic~ rif


ane protein)


Schematic diagram of a paramyxovirus Lamb, R.A., and D. Kolakofsky.
"Paramyxoviridae" pgs 689-724. Fundamental Virology 4th edition.
Lippincott, Williams, and Wilkins. Philadelphia, PA 2001.. The lipid
bilayer is shown as the gray concentric circle. The viral matrix protein is
shown as a black concentric circle. Inserted through the viral membrane
are the hemagglutinin (H) attachment glycoprotein and the fusion (F)
glycoprotein. Inside the virus is the negative strand virion RNA, which is
encapsidated with the N protein. Associated with the nucleocapsid are the
L and P proteins, and together this complex has RNA-dependent RNA
transcriptase activity.


Figure 1-2.


Lipid bilayer




Virion RNA






Transcriptase Complex
L (Large Polymerase)
N (Nucleocapsid protein)
P (Phosphoprotein)


F (Fusion protein)









The matrix protein functions as the central organizer of viral morphogenesis by

interacting with the cytoplasmic tails of the integral membrane proteins, the lipid bilayer,

and the nucleocapsids. The hemagglutinin (H) protein interacts with the CD 46 protein.

This brings the virus to the cell, which allows the fusion (F) protein to interact with the

cell membrane (Lamb and Kolakofsky 2001).

Infection

The measles virus enters the host through the upper respiratory passages; and the

primary site of infection is the respiratory epithelium. The integral membrane proteins H

and F are involved in cell attachment and fusion of the viral envelope with the plasma

membrane of the host cell. Once the membranes fuse, the nucleocapsid can then enter

the cytoplasm of the host cell and begin viral transcription. The entire life cycle of the

measles virus takes place in the cytoplasm. Upon infection of the host cell, the viral

RNA polymerase initiates transcription starting at the 3' end of the genome.

Transcription of the nucleocapsid is sequential and begins by transcribing (+) leader

sequence followed by the remaining genes in order from the 3' end. The RNA

polymerase fails to reinitiate roughly 30% of the time, thereby leading to a gradient of

transcription with the leader RNA being the most abundant and the L mRNA being the

least abundant. All the mRNAs are capped methylated, and polyadenylated (Galinski

and Wechsler 1991). In replication, the polymerase copies the entire genome, ignoring

the gene junctions for mRNA synthesis, creating a full length (+) sense antigenomic

RNA which is encapsidated with N simultaneously with its synthesis, which serves as a

template for the replication and encapsidation of (-) sense genome RNA (Myers and

Moyer 1997). The nucleocapsid protein of the new nucleocapsids interacts with the M

protein to help form the viral core. At the same time, H and F are processed through the









Golgi apparatus and are accumulating on the plasma membrane of the host cell. Once the

core (containing a nucleocapsid and a few copies of the RNA polymerase) is complete, it

will bud at the site of H and F concentration on the host membrane, creating the next

generation of virus (Lamb and Kolakofsky 2001). The switch from transcription to

replication appears to be regulated by accumulation of the N protein (Amheiter et al.

1985). Once these proteins accumulate, the RNA polymerase ignores cis acting signals

of the gene junctions during replication (Vidal and Kolakofsky 1990).

Viral Replication Proteins

The measles virus N protein (562 amino acids) is encoded by the first gene in the

linear order of the genome. After the leader RNA, the N mRNA is the most abundant

viral transcript present in an infected cell. The N protein must interact with the viral

genome to form the nucleocapsids and it also must interact with other viral proteins to

complete the viral life cycle. The formation of the nucleocapsid is essential in that it

allows the polymerase complex to recognize the genome and renders the genome RNA

ribonuclease resistant (Buchholz et al. 1994). In order to tightly encapsidate the genome

the N protein must interact with neighboring N proteins forming a multimer of N proteins

which associate with the RNA genome (Buchholz et al. 1993). The N protein of the

nucleocapsid also interacts with the M protein. This interaction brings the encapsidated

genome to the plasma membrane of the infected cell so that the virus may bud (Stricker et

al. 1994). Calain and Roux (1993) proposed the rule of six (which states that there is one

nucleocapsid protein for every six nucleotides in the nucleocapsid, so genome length

must be a multiple of six).

The measles P protein (507 amino acids) was named for its highly phosphorylated

state. The P protein binds with L to form the RNA polymerase. The P protein functions









in stabilization and folding of L, which is unstable in the absence of P (Horikami et al.

1992, Horikami and Moyer 1994). The P protein of Sendai virus was shown to be at

tetramer by biophysical and crystallographic studies (Tarbouriech et al. 2000). The

measles P protein is an oligomer (Harty and Palese 1995) but its exact composition is not

known. The P gene has the ability to produce a number of polypeptide products

(including the P, V, and C proteins) by using an overlapping reading frame (for C) and by

a process of transcription known as RNA editing (for V), which causes a reading frame

shift on translation (Lamb and Kolakofsky 2001).

The V mRNA uses the same transcription start site as the P gene, but a

nontemplated G residue is incorporated at nucleotide 751 of its mRNA. This causes a

frameshift that enables the V protein to be identical to the P protein for the first 231

amino-terminal amino acids. The last 68 amino acids are translated from the V reading

frame (Tober et al. 1998). The V protein is not associated with intracellular viral

particles and has been shown to be dispensable for measles virus infection in cell culture

(Schneider et al. 1997).

The start site for the measles C gene is 22 nucleotides downstream of the P and V

protein start site on the P mRNA. The protein is in the +1 frame relative to the P protein.

The translation of the C protein is due to ribosomal scanning because of the stronger

Kozak sequence compared to the P and V start site. The C protein, like the V protein, is

not needed for the MV infection cycle in cell culture (Radecke and Billeter 1996). The C

protein downregulates viral RNA synthesis (Lamb and Kolakofsky 2001, Reutter et al.

2001).









The measles virus large (L) protein (2183 amino acids) is the least abundant of the

structural proteins, since its mRNA is at the 5' end of the genome and therefore the last,

and least abundant transcription product. As stated before, the L and P proteins form a

complex, and both of these components are required for polymerase activity with the

N:RNA template. The measles virus L amino acid sequence has been compared to the L

proteins of other (-) strand RNA viruses and six conserved regions shown in Figure 1-3

have been found (Blumberg et al. 1988; Poch et al. 1990; Sidhu et al. 1993). The specific

functions of these domains have not been identified. Studies have shown that

polyadenylation, methyltransferase, and kinase activities are associated with the L protein

in vesicular stomatitis virus (Hammond and Lesnaw 1987; Hunt and Hutchinson 1993),

and a kinase activity is associated with the Sendai L protein (Einberger et al. 1990).

Domains II, III, and VI contain motifs found in other polymerases, where domains II and

III have been proposed to make up the polymerase module of the L protein (Jablonski et

al. 1991; Muller et al. 1994). Domains I, IV and V have no recognizable motifs

(Horikami and Moyer 1995). Site directed mutations were constructed by substitution of

multiple amino acids at conserved regions in the Sendai L protein. These different

substitutions at conserved amino acids in domains I-VI in the Sendai L protein gave

varying phenotypes. The majority of the mutations completely inactivated the L protein

for all aspects of RNA synthesis. In some cases phenotypes from inactivation to partial

activities were observed. The differences in activity depend on the nature of the amino

acid that was substituted.

The inactive L mutants, however, were still able to bind P protein and the complex

was able to bind nucleocapsids (Chandrika et al. 1995; Corteses et al. 2000; Feller et al.
















P/C/V M F HN


575 a'"
^^ "


4 0 4
- z vr


c$l

4


2228 3a


I.


I N


Figurel-3. Sendai virus L protein schematic. Conserved domains I-VI are shown as
shaded boxes with their amino acid boundaries indicated above. (Poch et al.
1990)


3- and of
(- genome
1i* NP


55 524 5
nt ,


I I I I I I


568 o 348 s 565 as


c
c









2000; Smallwood et al. 1999). It was initially thought that each domain had its own

catalytic activity, but with further studies it was concluded that the activities of the

polymerase are not simply compartmentalized and that each domain contributes to

multiple steps in viral RNA synthesis. These domains can, however, function in trans.

It has been demonstrated that intragenic complementation between pairs of coexpressed

inactive L mutants can restore RNA synthesis (Smallwood et al. 2002). This shows that

the L protein forms an oligomer with multiple independent domains each of which

exhibits several functions

Previous experiments in the measles virus have shown that the N-terminal 408

amino acids of the L protein contain the P binding domain (Horikami et al. 1994). The

site on the measles virus P protein that the L protein binds resides in the C-terminal half

of the P protein (Harty and Palese). The L binding site was mapped to a region of P

protein encompassing the C-terminal amino acids 412-478 (Smallwood et al. 1994).

Measles L protein truncations were made in the past to test L-P binding. A C-terminal

deletion of 1775 a.a. was made in MV L protein, leaving 408 N-terminal amino acids that

still bind P (Horikami and Moyer 1994). Twelve different site directed mutations were

constructed in MV L 408 (Joyce Feller and David Holmes, unpublished). The mutants

were constructed by changing clustered charged or hydrophobic amino acids to alanine

(Figure 1-4).

An HA epitope tag was cloned on the N-terminus of each mutation (Bayram Cevik

et al. 2003). The twelve mutant truncations were named the following: MV L 491, 492,

494, 495, 496, 497, 498, 499, 500, 501, 508, 502. The amino acid sequence of the wild

type 408 is shown in Figure 1-4. The WT 408 gave 100% binding to P. MV L 494, 501,









MDSLSVNQIL YPEVHLDSPI VTNKIVAILE YARVPHAYSL EDPTLCQNIK
491
HRLKNGFSNQ MIINNVEVGN VIKSKLRSYP AHSHIPYPNC NQDLFNIEDK
492
ESTRKIRELL KKGNSLYSKV SDKVFQCLRD TNSRLGLGSE LREDIKEKVI
494
NLGVYMHSSQ WFEPFLFWFT VKTEMRSVIK SQTHTCHRRR HTPVFFTGSS
495
VELLISRDLV AIISKESQHV YYLTFEVLLI YCDVIEGRLM TETAMTIDAR
496 497
YTELLGRVRY MWKLIDGFFP ALGNPTYQIV AMLEPLSLAY LQLRDITVEL
498 499
RGAFLNHCFT EIHDVLDQNG FSDEGTYHEL IEALDYIFIT DDIHLTGEIR
500 501
SFFRSFGHPS LEAVTAAENV RKYMNQPKVI VYETLMKGHA IFCGIIINGY
508 502


Figure 1-4. Primary amino acid sequence of measles virus L 408 mutant protein. The
underlined bold amino acids represent areas where charged or
hydrophobic amino acids were changed to alanine (Joyce Feller and David
Holmes) the mutant number is in bold below the underlined bold amino
acid.









508, 502 all bound P at roughly 30%. MV L 491 gave 10% binding while the rest of the

mutants showed no binding to P (Table 1-1).

In Sendai virus it has been shown that the L protein forms an oligomer (Smallwood

et al. 2002) and the L-L interaction site resides in the amino acids 1-174. This L

oligomerization does not depend on P protein. Site directed mutations in the N-terminal

347 amino acids of L which abolish P binding, do not affect the L-L complex formation

(Cevik et al. 2003). This shows that while the L and P binding sites are overlapping they

are mediated by different amino acids.

Protein subunits bind one another through noncovalent interactions. Multiple

noncovalent interactions can form between complementary molecules, causing them to

bind tightly. The types of noncovalent interactions that can occur are hydrophobic, van

der Waal, ionic, and hydrogen bonding. The mode in which the subunits of the measles

virus RNA polymerase bind is unknown.

Experimental Design

The purpose of this project is to determine if measles L forms an oligomer and to

identify the binding site for the L-L complex. C-terminal truncations were made in the L

protein with an epitope tag in order to determine where on the protein the binding region

resides. Site directed mutations were created in MV L 408 by Dr. Joyce Feller

(University of Florida) by changing clustered charged or hydrophobic amino acids to

alanine. It is thought that these charged and hydrophobic amino acids might be

responsible for protein-protein interactions. The amino acids are replaced by alanine

because the small methyl side group is unlikely to disturb the tertiary structure of the

protein (Cunningham and Wells, 1989; Bass et al. 1991). This approach allowed for the

identification of the amino acids that are required for L-L oligomerization.









Table 1-1. MV L 408 mutant interactions with MV P. The table lists thel2 MV L 408
mutants plus wild type 408 and their interaction with the MV P. The type of
mutation for each mutant is also listed.


MV L 408 Mutant


L-P Interaction


Type of Mutation


MV L 408 wt

MV L 491

MV L 492

MV L 494

MV L 495

MV L 496

MV L 497

MV L 498

MV L 499

MV L 500

MV L 501

MV L 502


NONE

Hto A

C to A

C to A

Hto A

Hto A

Hto A

Hto A

Hto A

Hto A

Hto A

C to A


MV L 508


MV to SV


+++ refers to strong interaction.
+ refers to slight interaction, approx. 30% of wild type.
+/- refers to little or no interaction, approx. 10% of wild type.
- refers to no interaction.
H to A = hydrophobic to alanine
C to A = charged to alanine
MV to SV = measles virus to Sendai virus
(Holmes and Moyer 2002)














CHAPTER 2
MATERIALS AND METHODS

Cells and Viruses

Human lung carcinoma cells (A549, American Type Culture Collection; Manassas,

VA) were grown at 370 C with 5% CO2 in Eagle's Minimum Essential Medium (F 11,

Gibco BRL; Langley, OK) supplemented with 8% fetal bovine serum (FBS, Gibco BRL;

Langley, OK), 1% nonessential amino acids (100X NEAA, Mediatech; Herndon, VA),

1% penicillin and streptocmycin solution (pen/strep, penicillin 5000 I.U./mL and

streptomycin 5000 [tg/mL, Mediatech; Herndon, VA), 2 mM L-glutamine (200 mM,

Mediatech; Herndon, VA), and ImM sodium pyruvate (100mM, Mediatech; Herndon,

VA). The cells were cut 1:6 or 1:12 every three or four days respectively, using 1X

trypsin-EDTA (ICN Biochemicals, Inc; Costa Mesa, CA).

Recombinant vaccinia virus expressing phage T7 RNA polymerase (VVT7) (Fuerst

et al. 1986) was provided by Dr. Edward Niles (Suny Buffalo, NY). Ten 15 cm dishes

with a confluent monolayer of Vero cells were infected with VVT7 at a multiplicity of

infection (m.o.i.) of 0.05 pfu/cell. The cells were scraped four days post infection and

pelleted at 7,000 rpm for 30 min at 40C in a J10 rotor (Beckman). The pellets were

resuspended in PBS (phosphate buffered saline; 4.3 mM sodium phosphate, 1.5 mM

potassium phosphate monobasic, 137 mM NaC1, 2.7 mM KC1 pH7.2). The PBS was

supplemented with 1% pen/strep. The cells were subjected to freeze/thaw cycles and

then sonicated to produce a consistent solution of virus. The VVT7 titer was determined









by plaque assays on A549 cells. The virus was then aliquoted and stored at -800 C. The

MVA virus utilized in the CAT assay was provided by Dr. Bernard Moss (NIH).

Antibodies

The following antibodies were utilized for immunoprecipitation (IP) assays:

a FLAG mouse and a HA monoclonal antibody (Sigma; St. Louis, MO). Rabbit a mouse

IgG (Southern Biotechnology Associates, Inc; Birmingham, AL) was used as secondary

antibody to bind a FLAG. Protein A Agarose beads (Sigma; St. Louis, MO) were used to

bring down immuno-complexes bound to a FLAG and Protein G agarose beads (Sigma;

St. Louis, MO) were used to bring down immuno-complexes bound to a HA.

Plasmids

The following plasmids encoding the measles virus phosphoprotein (P), C,

nucleocapsid (NP), and the L proteins: pBS MV-P/C; pBS MV-N; and pBS MV-L were

provided by Dr. W. Bellini (CDC; Atlanta, GA) and Dr. M. Billeter (Zurich,

Switzerland). MV pl07CAT minigenome plasmid encoding the chloramphenicol acetyl

transferase (CAT) gene cloned between measles virus conserved termini was provided by

Dr. S. Udem (New Jersey Medical School, Newark, NJ) (Sidhu et al. 1995). CMPI

(measles P without measles C open reading frame) contains a stop codon in the C protein

open reading frame downstream of the Y2 start codon such that only the P protein is

expressed (Reutter et al. 2001). pTMI HA MV L 408 mutants were created by Dr. Joyce

Feller, David Holmes, and subcloned into the tagged pTMI plasmid by Dr. Bayram

Cevik.

Primers

Plasmids containing the measles L gene linked to a tagged epitope were made using

polymerase chain reaction (PCR). In order to create the measles L gene with an epitope,









two upstream primers containing the appropriate epitope and part of the measles L gene

were created. A Ncol site was made upstream of the epitope to allow subcloning. The

FLAG MV L primer (5'ATG GCTATCGATT ACAAGGATGA CGATGACAAG

CTTGCAATGG ACAGCTTAAG CGTTAAC 3') and the HA MV L primer (5'ATG

GCTTACCCAT ACGATGTTCC AGATTACGCT AGCTTGGGTG GTCCAATGGA

CAGCTTAAGC GTTAAC 3') were used in conjunction with the downstream primer,

SM 482 (5' CAGGGACTTC GCTAGCTGTG GAGACGGC 3'), downstream of the

EcoRI site in MV L, to create an HA MV L and a FLAG MV L.

Cloning pTM1 FLAG MV L (EcoRI) and pTM1 HA MV L (EcoRI)

The PCR reaction was carried out using the Vent DNA polymerase with the

following temperatures profiles: 0:30 min at 940C for denaturation, 0:30 min at 420C for

annealing temperature, and 0:30 min at 720C for primer extension. There were 28 cycles

in all, followed by 4:00 min at 720C for a final primer extension. The primers were used

to create a PCR product of 420 base pairs using the wild type pAel MV L plasmid as the

template. The PCR products were purified using phenol chloroform extraction and

ethanol precipitation. The two purified PCR products, with an HA and FLAG tag, were

digested with Ncol and EcoRI overnight at 370C. A pTM1 plasmid was digested with

Ncol and EcoRI and then shrimp alkaline phosphatase (SAP) treated, SAP (1 [l) to the

digest, incubated at 370 C for 30 minutes and heat inactivate at 650 C for 15 minutes, to

prevent self ligation. The digested products along with the digested pTM1 plasmid were

separated on a 1.2 % agarose gel. The desired bands (HA MV L and FLAG MV L = 420

bp, pTM1= 5300 bp) were eluted into 15% PEG (polyethylene glycol) in IX TAE (40

mM Tris, 0.11% glacial acetic acid, and ImM EDTA) and precipitated with NaOAC and

ethanol. Each epitope tagged MV L product was ligated into the digested pTM1 plasmid









using 20 units of T4 DNA Ligase (20,000 units/ml) incubated at 160 C overnight. The

ligated products were transformed into DH5a competent cells and plated on LB (Luria

broth)/ampicillin (100 [tg/ml) plates. Multiple colonies were scrapped from LB/AMP

plate and PCR screened for the insert using the same PCR primers used to create them. A

positive sample for each clone was inoculated in 25 ml of LB/AMP and purified using a

Qiagen midiprep kit. The samples were then sequenced by the DNA Core at the

University of Florida to assure that the clones contained their epitopes and had the correct

sequence.

Subcloning Remaining MV L Gene into HA and FLAG pTMI Plasmids

The pTM1 HA MV L (EcoRI) and pTM1 FLAG MV L (EcoRI) clones were

digested with EcoRI restriction enzyme and SAP treated to prevent self ligation. PAel

MV L was digested with EcoRI restriction enzyme. The MV L fragment (-6300bp) was

ligated with the pTM1 HA and pTM1 FLAG (z5700bp). The clones were transformed

into DH5a competent cells and plated on LB/ampicillin plates. The colonies were PCR

screened using original primers to check for inserts. To assure the clones contained an

insert in the correct orientation, the clones were digested with StuI restriction enzyme that

cut at one site inside the insert and analyzed on a 1.2% agarose gel to check the size.

Figure 2-1 gives an overview of the total cloning process.

Growing and Purifying pTM1 HA MV L and pTM1 FLAG MV L Clones

To grow log phase cultures positive colonies were inoculated in 2 ml of LB with

ampicillin (100 [tg/ml) and incubate for 5-6 hour (or until turbid). To grow large

preparation cultures 200 [l of log phase culture was inoculated into 50 ml of LB with

ampicillin (100 [tg/ml). The plasmids were purified with the Qiagen midiprep kit

according to the manufacturer's protocol.



















I P- PCR


Nml ERoRI PCR products 420 bp


NEc rom
EcoR \
l T7 Promotero \


Eco


MVL
,EcoRI


pTM1 HA MVL


pTM1 FLAG MVL


Epitope creation using PCR amplification. The procedure was done using
upstream primers that contained the appropriate epitope (HA or FLAG)
and a downstream primer that contained part of the measles virus L gene.
pAeL MV L was used as the template for the PCR reaction. After the
PCR product is formed, it was cloned into pTMI. Then the remaining
EcoRI- EcoRI portion of measles L gene was cloned into the epitope
tagged vectors to yield the full length tagged L genes.


Figure 2-1.


I1. II









Constructing C-Terminal Truncations pTM1 FLAG MV L SacI and pTM1 FLAG
MV L Small

The pTMI FLAG MV L SacI was made by digesting pTM1 FLAG MV L with

restriction enzyme SacI (Figure 2-2). The digested plasmid was separated on a 1.2%

agarose gel and the pTM1 FLAG MV L SacI product giving a protein of 1197 amino

acids was eluted, leaving the remaining fragment. The same procedure was done to the

pTMI FLAG MV L Smal plasmid, except the truncation was up to the Smal restriction

site giving a protein of 874 amino acids (Figure 2-2). The truncated plasmids were

religated using T4 DNA ligase, and transformed into DH5a competent cells. Plasmid

preparations were prepared as above. Figure 2-3 shows the final product of the

truncations compared to the wild type MV L.

In Vitro Transcription/Translation (TNT)

An in vitro coupled transcription translation reaction was done using a TNT kit

(Promega; Madison, WI). This kit allows for transcription and translation to occur

simultaneously in the same reaction. The plasmids were incubated with T7 RNA

polymerase, rabbit reticulocyte lysate, and 3H leucine for 2 hours at 370C and the

products separated on a 7.5% SDS-PAGE gel.

Measles CAT Minigenome Assay

A549 cells at 80 % confluence in 35 mm dishes were infected with MVA at a

multiplicity of infection of 2.5 in F11 infection media at 370C for 1 hour. Following

infection, the inoculum was removed and the dishes were washed with Opti-MEM

supplemented with 1% pen/strep. Opti-MEM with 1% pen/strep was then added to each

dish, 0.8 for 35 mm or 2.4 for 60 mm. The following plasmids were transfected in

Lipofectin (Invitrogen) and incubated for 48 hours: 1.7 [g of CMPI (measles P without










FLAG Epitope
IRES
T7 promoter


Sac/(4881


SmaI(4204


MV-L


Smai (8133)


Sa(8141)


1 I I I


pTMI FLAG MVL
12052 bp


Illustration of pTMI FLAG MVL plasmid. The plasmid contains a T7
promoter followed by the IRES and FLAG epitope fused to the 5' end of
the MV L gene. The figure shows the restriction sites that were used to
create the C-terminal truncations.


Figure 2-2.










T7 promoter





FLAG- epitope
IRES

T7 promoter


pTMI FLAG MV L
2183 amino acids


pTMI FLAG MV L-SacI
1197 amino acids


FLAG- epitope


IRES
T7 promc


pTMI FLAG MV L-SmaI
874 amino acids


Comparison of full length FLAG MV L with C-terminal truncated FLAG
MV Ls. The figure shows the truncated FLAG MV L genes with respect
to the full length FLAG MV L gene. The size of the proteins produced are
given in amino acids.


Figure 2-3.


I I









measles C open reading frame), 0.83 tg of MV pl07CAT minigenome, 0.83 tg of pBS

MV-N, 0.16 tg ofpAel MVL, 0.10 tg ofpTM1 HAMVL and 0.10 tg ofpTM1

FLAG MV L. The cells were scraped into 200 ptl of 0.25M Tris-HCL, pH 7.8/0.5%

Triton X-100. The procedure was carried out using a CAT ELISA kit (Roche; Nutley,

NJ) according to the manufacturers protocol. The data was analyzed using a microtiter

plate reader (Tecan).

Infection and Transfection

A549 cells at 80 % confluence in 35 or 60 mm dishes were infected with VVT7 or

MVA at a multiplicity of infection of 2.5 in F 11 infection media (F 11 supplemented with

14 mM HEPES pH 7.4, 1 % pen/strep, and 2 mM L-glutamine) at 370C for 1 hour. The

volumes of the infection media used were 0.3 ml for 35 mm dishes and 0.5 ml for 60 mm

dishes. Following infection, the media was removed and the dishes were washed with

Opti-MEM supplemented with 1% pen/strep. Opti-MEM with 1% pen/strep was then

added to each dish, 0.8 for 35 mm or 2.4 for 60 mm. Unsupplemented Opti-MEM,

combined with lipofectin (Bethesda Research Laboratories; Bethesda, MD) and the

appropriate plasmid DNA in lipofectin/Opti-MEM (3dl of lipofectin/1 tg of DNA) were

transfected for 18 hours or 48 hours for the CAT assay.

For binding assays, the transfection media was aspirated 18 hours post

transfection. The cells were then washed with PBS and a pulse labeling media,

consisting of Dulbecco's Modified Eagle's Medium without methionine, cysteine or L-

glutamine (Sigma Cell Culture; St. Louis, MO), 1% pen/strep, 2 mM L-glutamine, 14

mM HEPES, pH 7.4 and unsupplemented F11, was added to each dish for 15 minutes.

Immediately after the 15 minutes, Express-35S (100 iCi/ml) (New England Biolabs;









Beverly, MA) was added, 75 and 150 jiCi for 35 and 60 mm dishes, respectively. The

dishes were then incubated at 370C for 2 hours.

Protein Analysis

To test for protein expression, 80 % confluent A549 cells in 60 mm dishes were

infected with vaccinia virus T7 (VVT7) at a multiplicity of infection of 2.5 in Fl 1

infection media at 370C for 1 hour. For the L-P interaction, cells were transfected with

pGEM MV P (5.0 ig) and pTM1 FLAG MV L (1 jg) or 0.50 jg of either of the pTM1

FLAG MV L truncations for 18 hours. For the L-L interactions, cells were transfected

with pGEM MV P (5.0 ig) and pTM1 HA MV L (0.25 jg alone and 1.0 jg when

expressed with either truncated MV L). pTM1 FLAG MV L SacI (0.5 ig) was used

when expressed alone and 1.0 jg with pTM1 HA MV L. pTM1 FLAG MV L Smal (0.5

jg ) was used when expressed alone and 1.0 jg with pTM1 HA MV L. To test for L-L

interaction using pTMI HA MV L 408s (MV L 408 wt, MV L 491, MV L 494, MV L

495, and MV L 496), cells were transfected with pGEM MV P (5.0 ig), pTM1 FLAG

MV L (1 jg), and 0.25 [ig of the 408 mutants.

The cells were then labeled with Express-35S (100 jiCi/ml) in medium with no

cysteine and methionine for 2 hours. The cytoplasmic extracts were prepared by scraping

into 165 jl of RM Salts [0.1M Hepes pH 8.0, 0.05 M NH4C1, 7 mM KC1, 4.5 mM MgAc]

+ 0.25% NP-40 lysis buffer [0.15 M NaCL, 50 mM Tris-HCl (pH 8.0), 1% Nonidet P-40

(NP-40), and 1 jg/ml aprotinin]. The cell lysate was cleared by centrifugation for 30 min

at 15,000 rpm at 4 o C. 10 il of the sample was used to analyze total protein and 155 il

was used for immunoprecipitation. Both the totals and immunoprecipitations were

separated and analyzed by 7.5% SDS-PAGE gel.









Immunoprecipitation

1% NP-40 lysis buffer (100l) was added to the 155 itl sample of RM Salts +

0.25% lysis buffer containing the cell extracts. The extracts were incubated 30 minutes at

4 C with 40 pl of Protein A or G Agarose beads (Sigma; St. Louis, MO). The extracts

were centrifuged for 3 minutes at 10,000 rpm and the supernatant was transferred, while

disposing of the pellet for preabsorption. aFLAG or a HA (2 tg) monoclonal antibody

was then added and incubated while rocking at 40 C for 1 hour. The secondary antibody,

rabbit a mouse IgG, is then added and incubated while shaking at 40 C for 1 hour. To

bring down the immunocomplexes, Protein A or G agarose beads are added again and

incubated at 40 C for 30 minutes. The beads are pelleted and then washed 2X with 800 ptl

of 1% NP-40 Lysis buffer and finally resuspended in 35 ptl of 2X lysis buffer. The

immunoprecipitations were separated on a 7.5% SDS-PAGE gel.














CHAPTER 3
RESULTS

Creation of Full Length HA and FLAG MV L and Truncated FLAG MV L Mutants

In order to show protein interactions through coimmunoprecipitation experiments,

two different epitopes, HA and FLAG, were created on the N-terminal side of the MV L

protein through modification of the L gene as described in Materials and Methods. This

process was done through the use of PCR amplification using an upstream primer

(containing either the HA or FLAG epitope) and a downstream primer (containing a

small section of the MV L gene). The PCR product, containing the epitope and part of

the MV L gene was subcloned into the pTMI plasmid (Figure 2-1). The samples were

then sequenced by the DNA Core at the University of Florida to assure that the clones

contained their epitopes and had the correct sequence. The remaining part of the MV L

gene was then subcloned into the vector, as described in Materials and Methods. pTMI

HA MV L and pTMI FLAG MV L are the products that were created. In order to

determine where the binding site resides on the MV L protein, C-terminal truncations

were made. If the truncated MV L protein is still able to bind to the full length MV L

protein then the deleted amino acids are not essential for protein-protein interactions.

pTMI FLAG MV L was truncated at two different sites. pTMI FLAG MV L SacI was

made by digesting pTM1 FLAG MV L with restriction enzyme SacI, as described in

Materials and Methods, giving a protein of 1197 amino acids (Figure 2-3). The pTMI

FLAG MV L Smal plasmid, was truncated up to the Smal restriction site, as described in

Materials and Methods, giving a protein of 874 amino acids (Figure 2-3).









In Vitro Protein Synthesis

The proper expression of each of the epitope tagged, full length and truncated,

measles L proteins was first determined in vitro by using the TNT coupled transcription

translation system (Promega). The plasmids were incubated with T7 RNA polymerase,

rabbit reticulocyte lysate, and 3H leucine for 2 hours at 370C and the products separated

on a 7.5% SDS-PAGE gel, as described in Materials and Methods. Figure 3-1 shows the

products of all 4 proteins as well as the Sendai virus L protein, which is used as wild type

L marker protein. Sendai L protein is used to compare with the measles L protein

because of their similar size and expression. In the absence of plasmid no background

bands are present (Figure 3-1, lane 1). The Sendai L protein shows the correct size and

expression of the protein (Figure 3-1, lane 2). The two epitope tagged full length L

proteins, HA MV L and FLAG MV L, are both expressed and the size is smaller than the

Sendai L protein as expected (Figure 3-1, lanes 3 and 4). HA MV L is expressed less

well because the DNA concentration was less. In all future experiments the DNA

concentrations of the tagged proteins were the same. The truncated measles L proteins,

FLAG MV L SacI and FLAG MV L Smal, are shown as truncated proteins of the

expected sizes with respect to the size of the wild type L protein (Figure 3-1, lanes 5 and

6).

CAT Assay to Test the Full Length FLAG and HA MV L Mutants for Functional
Activity

The functional activity of the full length tagged MV L proteins was tested in a

minigenome reporter system. The measles virus CAT assay is a minigenome system that

tests a viral polymerase complex for its ability to perform transcription and replication in

a mammalian expression system. A549 cells are infected with MVA-T7 and transfected













* -
m
=(
!*
e)




IE
raj7


A.M..
A-.:


n

rTj


C,

d r3

=d









Ir
~e~,iri(
UI


In vitro expression of all the epitope tagged proteins using the TNT
coupled transcription translation system. The various indicated plasmids
were incubated with T7 RNA polymerase, rabbit reticulocyte lysate, and
3H leucine for 2 hours at 370C and the products separated on a 7.5% SDS-
PAGE. Lanes 1 and 2 are the mock and the Sendai virus L protein (shown
by an asterisk). Lanes 3-6 show all the epitope tagged mutants. The
positions of the full length and truncated L proteins are indicated.


Figure 3-1.


-- Full length L



N- MV L-Sacl

R- MV L-Smal









in triplicate with the following plasmids: pBS MV-N, CMPI (measles P without measles

C open reading frame), MV p107 CAT minigenome, and wt or tagged full length L

proteins, as described in Materials and Methods. The plasmids are all driven by the T7

promoter. The CAT mini genome expresses a negative sense RNA, which contains the

CAT gene located between conserved measles virus termini. The genome RNA is

encapsidated by the MV N protein and then used as a template for transcription and

replication by the viral polymerase. If the functional polymerase complex forms there is

transcription and replication, which leads to the production of the CAT mRNA and the

CAT protein.

The cell extracts were prepared and the remaining reactions were carried out

using a CAT ELISA kit (Roche). The data was analyzed using a microtiter plate reader

(Tecan). Figure 3-2A shows the results of the CAT assays. This test showed that the

The negative control (Figure 3-2A, lane 1), which contains sample buffer but no plasmid

showed no activity, as expected. The positive control (Figure 3-2A, lane 2), which

contains 200 pg of CAT enzyme, showed a good functional activity. The mock sample

was transfected with the minigenome DNA alone and showed no activity (Figure 3-2A,

lane 3), showing the template alone gave no activity. The wild type MV L (MV L

pAeL) showed functional activity, comparable to positive control (Figure 3-2A, lane 4).

The FLAG and HA MV L proteins (Figure 3-2A, lanes 5 and 6 respectively) gave almost

identical results. Both tagged L proteins had an activity level approximately 40% of the

wild type showing the tag did have some effect on protein function. Nonetheless there

was still activity. Figure 3-2B shows the results for the positive control test. The graph

shows that the reaction is linear.














1
0.9

0.8

0.7

0.6

S0.5

0.4

0.3

0.2

0.1

0









1.4-
1.2
1
S0.8
0 0.6

0.4
0.2
0


Negative Postitive Mock MV L Flag MV Ha MV L
control control PAeL L


Negative 100 pg 200 pg 300 pg 400 pg 500 pg
CAT enzyme (picograms)


CAT assay testing the ability of wild type and tagged measles virus
polymerase complexes to perform in vitro transcription and replication in
a mini-genome system. A549 cells were infected with MVA-T7 at an
m.o.i. of 2.5 and then transfected with the appropriate plasmids for 48
hours. Cell extracts were prepared and the CAT activity was measured
and described in the Materials and Methods. Figure 3-2A is a graphical
representation of CAT activity, each bar represents the average value of an
experiment done in triplicate. The error bars show the standard deviation.
3-2B shows the positive control test, which shows that the reaction is
linear.


Figure 3-2.









Polymerase Complex Formation of the FLAG MV L Proteins with MV P

After it was shown that the tagged MV L proteins all synthesized proteins with

the correct size and expression, their ability to bind to the MV P protein to form a

polymerase complex was tested. The full length and truncated FLAG MV L proteins

were co-expressed with the MV P protein and were tested for binding in an

immunoprecipitation assay. A549 mammalian cells were infected with VVT7 and

transfected with the following plasmids: pGEM MV P and pTM1 FLAG MV L or either

of the pTM1 FLAG MV L truncations for 18 hours. The transfected cells were labeled

with Express-35S for 2 hours. Analysis of total cytoplasmic cell extracts showed that all

the proteins were synthesized (Figure 3-3A).

The immunoprecipitation assay was then carried out on the cell extracts, and

separated on a SDS-PAGE gel, as described in Materials and Methods. When the

samples expressing mock or MV P alone were immunoprecipitated with aFLAG

antibody, MV P was not detected (Figure 3-3B, lanes 1 and 2). These data show that the

FLAG antibody is specific for the appropriate epitope tagged protein.

Immunoprecipitation with aFLAG antibody of an extract expressing both MV P and the

FLAG MV L proteins showed that MV P coimmunoprecipitated with the FLAG MV L,

FLAG MV L SacI, and FLAG MV L Smal indicative of an L-P interaction (Figure 3-3B,

lanes 3-5, respectively).

FLAG MV L SacI Mutant Protein Interaction with HA MV L Protein

After it was shown that the FLAG MV L mutants were still able to bind P and

form the polymerase complex, the truncated FLAG MV L mutants were individually

tested for their ability to bind to L. FLAG MV L SacI (1197 amino acids) was tested for

its ability to bind to full length HA MV L and form an oligomer. A549 mammalian cells









were infected with VVT7 and transfected with the following plasmids: pGEM MV P

alone or together with pTM1 HA MV L or pTM1 FLAG MV L Sac. Analysis of total

cytoplasmic cell extracts showed that all the proteins were synthesized (Figure 3-4A).

The immunoprecipitation assay was then carried out on the cell extracts, and

separated on a SDS-PAGE gel as described in Materials and Methods. When the samples

expressing the proteins not tagged with FLAG were immunoprecipitated with aFLAG

antibody, there was only a very slight background band for HA MV L (Figure 3-4B, lane

3). In this experiment there are significant background bands for the MV P protein

(Figure 3-4B, lanes 2 and 3), but our interest is with the L-L interaction. These data

shows that the FLAG antibody is specific for the appropriate epitope tagged protein and

does not bring down the HA MV L when expressed alone (Figure 3-4B, lane 3 and 4).

Immunoprecipitation with aFLAG antibody of an extract expressing both FLAG MV L

SacI and HA MV L showed that increased HA MV L coimmunoprecipitated with the

FLAG MV L SacI indicative of an L-L interaction (Figure 3-4B, lane 5).

FLAG MV L Smal Mutant Protein Interaction with HA MV L Protein

It was shown above that FLAG MV L SacI (1197 amino acids) was able to bind

full length HA MV L. Therefore the smaller MV L truncation, FLAG MV L Smal (874

amino acids) was also tested for its ability to bind to full length HA MV L and form an

oligomer. A549 mammalian cells were infected with VVT7 and transfected with the

following plasmids: pGEM MV P alone or with pTM1 HA MV L or pTMI FLAG MV

L Small and pTM1 FLAG MV L Smal and pTM1 HA MV L. Analysis of total

cytoplasmic cell extracts showed that all the proteins were synthesized (Figure 3-5A).















A +

i
M
U n


i FLAGMV L


em 40 -FLAG MVL Sad
FLAG MVL Smal






TOTALS


B +


S"---FLAGMVL


i--FLAG MVL Sad


MV LSmal


IP w AG
IP w/oFLAG


FLAG MV L mutants binding to MV P. A) Total cytoplasmic extract. B)
Immunoprecipitation with aFLAG antibody. VVT7 infected cells were
either not transfected (Mock) or transfected with the indicated FLAG MV
L, FLAG MV L Sac, or FLAG MV L Smal plasmids in the presence of
MV P plasmid. The cells were transfected for 18 hours and then labeled
with Express 35S for 2 hours. The positions of the L and P proteins are
indicated.


Figure 3-3.




























4-HA MVL

4-FLAG I L Sac

4-MVP


--HA MV L


"- --FLAG MV L Sac


i- a M4NVP


-
do


IP w/ FLAG


Figure 3-4. FLAG MVL SacI HA MV L binding. A) Total cytoplasmic extract. B)
Immunoprecipitation with aFLAG antibody. VVT7 infected cells were
either not transfected (Mock) or transfected with the indicated FLAG MV
L SacI and/or HA MV L in the presence of MV P. The cells were
transfected for 18 hours and then labeled with Express 35S for 2 hours.
The positions of the L and P proteins are indicated.


A

+
>

i I


TOTALS


-oft.'









The immunoprecipitation assay was then carried out on the cell extracts, and

separated on a SDS-PAGE gel as described in Materials and Methods. When the samples

expressing the non FLAG tagged proteins were immunoprecipitated with aFLAG

antibody, there was only a very slight background band for HA MV L (Figure 3-5B, lane

3), but again there was background bands for the MV P protein (Figure 3-5B, lanes 2 and

4). These data shows that the FLAG antibody does not bring down the HA MV L when

expressed with MV P. Immunoprecipitation with aFLAG antibody of an extract

expressing both FLAG MV L Smal and HA MV L showed that significant HA MV L

coimmunoprecipitated with FLAG MV L Smal indicative of an L-L interaction (Figure

3-5B, lane 5).

HA MV L 408 Mutant Protein Interaction with FLAG MV L Protein

After it was shown that the full-length MV L protein was still able to bind to the

truncated proteins, FLAG MV L SacI and FLAG MV L Smal, the HA MV L 408 mutants

were tested for their ability to bind full length FLAG MV L. To test for L-L interaction

using HA MV L 408s (MV L 408 wt, MV L 491, MV L 494, MV L 495, and MV L

496), cells were transfected with pGEM MV P, pTM1 FLAG MV 408 mutants, and MV

L 408 wt when expressed alone with MV P. Analysis of total cytoplasmic cell extracts

showed that all the FLAG MV L proteins were synthesized (Figure 3-6A). The HA MV

L 408 mutants are not visible due to the large amount of background.

The immunoprecipitation assay was then carried out on the cell extracts, and

separated on a SDS-PAGE gel, as described in Materials and Methods. When the

samples expressing the individual proteins were immunoprecipitated with aFLAG

antibody, only the FLAG tagged proteins were detected, with little background for HA


















B
u +
^a
^ "3
u fc


----H MIVL


I --HAMVL


-- .-.


--FLAG N IVL Sma


4--VP


-MVP
Om 4f <
abi


TOTALS


IP w/o FLAG


Figure 3-5. FLAG MV L Smal HA MV L complex formation. A) Total cytoplasmic
extract. B) Immunoprecipitation with aFLAG antibody. VVT7 infected
cells were either not transfected (Mock) or transfected with the indicated
FLAG MV L Smal and/or HA MV L in the presence of MV P. The cells
were transfected for 18 hours and then labeled with Express 35S for 2
hours. The positions of the L and P proteins are indicated.


A
+

u f0 E
o > ^






35


MV L 408 wt (Figure 3-6B. lanes 1 and 2). These data shows that the FLAG antibody is

specific for the FLAG tagged proteins. Immunoprecipitation with aFLAG antibody of an

extract expressing both FLAG MV L and each HA MV L 408 mutant showed that the

HA MV L 408s coimmunoprecipitated with FLAG MV L indicative of an L-L interaction

(Figure 3-6B, lanes 3-7). Table 3-1 compares the L and P interactions with the HA MV

L 408 mutants.


















s
A





2La
+
i QQ
I -


- ,t 1
O\hO


+ + +l


X.a

B

<
s-

+rrz

? ii^3d id


I, In)
IV *W


2; 2



< aN =I



+T +T
Lr Cr


- -- FLAG MV L


FLAG MV L


,- ----HA MV L 408 mutants




IP w/ aFLAG


TOTALS


Figure 3-6. FLAG MVL HA MV L 408 mutant binding. A) Total cytoplasmic
extract. B) Immunoprecipitation with aFLAG antibody. VVT7 infected
cells were transfected with the indicated FLAG MV L and/or HA MV L
408s in the presence of MV P. The cells were transfected for 18 hours
and then labeled with Express 35S for 2 hours. The positions of the L and
P proteins are indicated.









Table 3-1. MV L 408 mutant interactions with MV L. The table lists the 4 MV L 408
mutants plus wild type 408 and their interaction with the MV L. The type of
mutation for each mutant is also listed.

MV L 408 Mutant L-P Interaction L-L Interaction Type of Mutation
MV L 408 wt +++ +++ NONE
MV L 491 +/- +++ H to A
MV L 494 + +++ C to A
MV L 495 +++ H to A
MV L 496 +++ H to A
+++ strong interaction.
+ slight interaction (approx. 30% of wild type).
+/- little or no interaction (approx. 10% of wild type).
- no interaction.
H to A = hydrophobic to alanine
C to A = charged to alanine














CHAPTER 4
DISCUSSION

The purpose of this work was to determine if measles L formed an oligomer and to

identify the binding site for the L-L complex. Previous work in the Sendai virus showed

that the L protein forms an oligomer (Smallwood et al. 2002) and the L-L interaction site

resides in the amino acids 1-174. This L oligomerization does not depend on the binding

of P protein. Site directed mutations in the N-terminal 347 amino acids of L which

abolish P binding (Holmes and Moyer 2002), do not affect the L-L complex formation

(Cevik et al. 2003). This shows in Sendai virus that the L and P binding sites are in the

same region, but are mediated by different amino acids. In order to test the L-L

interactions in measles epitope tags were constructed onto the N-terminus of the MV L

protein. This allowed the interactions to be tested with an immunoprecipitation assay.

An HA and FLAG tag were used as the epitopes and are immunoprecipitated with their

appropriate antibody. Both clones were sequenced at the DNA Core at the University of

Florida to assure that the clones contained their epitopes and had the correct sequence.

C-terminal deletions were made to measles FLAG L producing two truncated L proteins,

FLAG MV L SacI and FLAG MV L Smal, (1197 a.a. and 874 a.a. respectively). The

tagged full length and mutant proteins were synthesized in the in vitro coupled

transcription and translation system and both full length and mutant genes were shown to

produce proteins of the correct size (Figure 3-1).

Through the use of coimmunoprecipitation experiments it was shown that the MV

L protein truncated to a.a. 874 was still able to interact with MV P (Figure 3-3), which is









consistent with previous work that showed the N-terminal 408 amino acids of MV L are

required for P binding (Horikami et al. 1994). It was shown in the Paramyxovirus

simian virus 5 (SV5) through the use of coimmunoprecipitation assays that the N-

terminal half of the L protein contains sequences important for stable L-P complex

formation and that the C-terminal half of L is not directly involved in these interactions

with P (Parks, 1994). In a study of the L protein of human parainfluenza virus type 3

(hPIV3) the N-terminus was shown to be necessary for polymerase function. Deletion of

nonconserved amino acids 2-15 abolished both P binding and viral transcription (Malur et

al. 2002). Thus the N-terminal half of L is required for P binding in a number of

paramyxoviruses.

In Sendai virus, using sequential deletions in the P protein (568 amino acids), the

site on the protein where the L protein binds was mapped to the C-terminal half of the P

protein. The L binding site was mapped to a region of P protein encompassing amino

acids 412-478. This region lies between the nucleocapsid binding domain (amino acids

345-411 and 479-568), which suggests that the L and NP protein binding domains on P

do not overlap (Smallwood et al. 1994). Similar results were found in bovine respiratory

syncytial virus (BRSV). The results indicate two independent N binding sites exist on

the P protein (amino acids 161-180 and 221-241). The L binding site was mapped to a

region of P encompassing amino acids 121-160, which suggests that N and L protein

binding domains on the BRSV P protein also do not overlap (Khattar et al. 2001).

Previous work was done with the human parainfluenza type 2 virus to determine

the region on the P protein that interacts with the L protein. Through the use of truncated

P proteins it was shown that the amino acids 278-353 on the P protein are essential for









the binding to the L protein (Nishio et al. 2000). In human parainfluenza virus type 1

(hPIV1), however, it was shown that two regions (residues 387-423 and 511-568) on P

protein are required for the functional interaction with hPIV1 L. These regions do

overlap with the binding domain for nucleocapsids (Bousse et al. 2001). Thus while the

C-terminal portion of P is required for L binding in all paramyxoviruses, the details of the

site are specific for each virus.

Through the use of coimmunoprecipitation experiments it was shown that the

truncated MV L proteins to a.a. 874 were still able to interact with full length L and form

an oligomer (Figures 3-4 and 3-5). Therefore the L-L binding site resides in the N-

terminal 1-874 a.a. ofMV L. This is consistent with the similar L-L binding site on

Sendai L (Smallwood et al. 2002). The Sendai virus L protein was compared to that of

other negative strand RNA viruses and six conserved region were found (Poch et al.

1990). It was shown that these domains can function in trans because intragenic

complementation between pairs of inactive L mutants can restore viral RNA synthesis.

Oligomerization of L in the polymerase complex was suggested by these experiments and

demonstrated directly by the coimmunoprecipitation of differentially epitope-tagged full

length and truncated Sendai virus L proteins (Smallwood et al. 2002).

Twelve site directed mutants were constructed in the truncated MV L 408 by Dr.

Joyce Feller and Dr. David Holmes to test for the requirement of specific amino acids in

the L-P interaction (Table 1-1). The mutagenesis strategy was changing clustered

hydrophobic or charged amino acids to alanine. Four of these 408 mutants (MV L 491,

494, 495, 496) as well as wt MV L 408 were used here to test for L-L interactions. A

coimmunoprecipitation experiment showed that the wt MV L 408 and all four mutants









were able to bind full length L (Figure 3-6). This is a strong indication that the L binding

site on the L protein resides within the amino terminal 408 amino acids for measles virus.

Therefore all four mutants were able to bind L compared to only one, MV L 494, which

binds P (Holmes and Moyer 2002). So while the L and P binding sites on L are in the

same region, they are mediated by different amino acids.

Similar experiments were done to test the amino acids required for the L-L

interaction in Sendai virus. It was shown that L oligomerization does not depend on P

protein and N-terminal fragments of L from amino acids 1-1146 through amino acids 1-

174 all bind wild type L. N-terminal deletions of amino acids 1-189 and amino acids 1-

734 have lost the ability to form the L-L complex as well as the L-P complex. These data

are consistent with the L-L interaction site residing in amino acids 1-174 (Cevik et al.

2003). To further characterize this L-L interaction site-directed mutations in the N-

terminal 347 amino acids were created to determine whether those amino acids are

essential for L oligomerization. These mutations either abolished or greatly reduced P

binding (Holmes and Moyer 2002) but did not affect L-L complex formation. This

suggests that the L and P binding sites on L are in the same region but are mediated by

different amino acids as is also the case for MV L. Most of the L mutations changed

clustered hydrophobic amino acids to alanine, with the exception of one, which changed

a charged reside to alanine. This would suggest that the L-L interaction may be mediated

by charged residues or hydrophobic amino acids in regions that were not altered (Cevik et

al. 2003). We propose that L oligomerizes and then binds the P oligomer which allows

the proper folding of L as it is thought to occur in Sendai virus.









Future experiments to further characterize the measles L-L oligomerization include

larger C-terminal truncations in order to determine a more precise binding region. After

the binding region has been narrowed down, site directed mutations in the binding region

to change clustered hydrophobic and charged amino acids to alanine should be created.

We showed that some charged and hydrophobic to alanine changes did not affect the L-L

interaction in the amino acids 1-408, therefore the creation of different mutations in a

smaller region should test if the L-L interaction depends on a charged or hydrophobic

interaction between the proteins.














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BIOGRAPHICAL SKETCH

Emmanuel Vrotsos was born in Miami, Florida on December 26, 1977. At the age

of six he moved to Fort Lauderdale, Florida. He graduated from Cooper City High

School in 1996 and earned his baccalaureate degree in Zoology from the University of

Florida (UF) in 2001. He received his Masters of Science degree from UF in December

2003, and began working toward a Ph.D. at the University of Central Florida, in the

Biomolecular Science program.