Hepatitis C Patients Treated with Interferon-Alpha/Ribavirin Therapy Develop Autoantibodies to Distinct Cytoplasmic Rod/...

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Hepatitis C Patients Treated with Interferon-Alpha/Ribavirin Therapy Develop Autoantibodies to Distinct Cytoplasmic Rod/Ring Structures Composed of CTP/GTP Nucleotide Biosynthetic Enzymes
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Carcamo, Wendy C
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Doctorate ( Ph.D.)
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University of Florida
Degree Disciplines:
Medical Sciences, Molecular Cell Biology (IDP)
Committee Chair:
Chan, Edward K
Committee Members:
Satoh, Minoru
Kasahara, Hideko
Terada, Naohiro
Burne, Robert A

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Subjects / Keywords:
autoantibodies -- ctps -- glutamine -- hcv -- impdh -- rr
Molecular Cell Biology (IDP) -- Dissertations, Academic -- UF
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Medical Sciences thesis, Ph.D.
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Abstract:
Hepatitis C (HCV) is a pandemic disease affecting an estimated 180 million individuals worldwide and infecting another 3-4 million people each year, making HCV a global public health issue. HCV is the main cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Despite significant improvements in antiviral intervention, only approximately 50% of treated patients with HCV clear the virus after treatment. Human autoantibodies serve as useful disease-specific markers for systemic rheumatic and other autoimmune disorders. Cytoplasmic filamentous structures referred to as rods and rings (RR) were identified using human autoantibodies. Our data show that HCV patients who have been treated with pegylated-interferon-alpha and ribavirin (RBV) develop these antibodies. The distinct cytoplasmic rods (~3-10 µm in length) and rings (~2-5 µm in diameter) were visualized in HEp-2 cells by immunofluorescence using HCV patient sera. Co-localization studies revealed that although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and were not associated with known cytoplasmic structures. Two proteins, cytidine triphosphate synthetase (CTPS) and inosine monophosphate dehydrogenase (IMPDH) were identified as components of RR. CTPS and IMPDH are important enzymes in the cytidine triphosphate (CTP) and guanosine triphosphate (GTP) biosynthesis pathways, respectively. Two CTPS inhibitors, 6-diazo-5-oxo-L-norleucine (DON) and acivicin were used to test the effects of CTPS inhibition. RR were formed in the presence of these inhibitors in a concentration-dependent and time-dependent manner. Similar results were observed when cells were treated with RBV or mycophenolic acid (MPA), inhibitors of IMPDH. Additionally glutamine deprivation affects CTP/GTP levels which induced RR at a slower rate compared to CTP/GTP inhibitors. Thus, RR formation might be affected by reduction of intracellular CTP/GTP levels. Interestingly, RR were readily detected in the majority of untreated mouse embryonic stem cells (>95%). Upon retinoic acid-induced differentiation, RR disassembled in these cells, but reformed again when treated with acivicin. RR formation appears to represent a response to a decrease in intracellular levels of CTP or GTP in cancer cell lines, mouse primary cells, and mouse embryonic stem cells. RR autoantibodies may develop in HCV due to direct interaction of HCV and RR induced by pegylated-interferon-alpha and ribavirin therapy.
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Includes vita.
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by Wendy C Carcamo.
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Thesis (Ph.D.)--University of Florida, 2012.
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Adviser: Chan, Edward K.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-08-31

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1 HEPATITIS C PATIENTS TREATED WITH INTERFERON ALPHA / RIBAVIRIN THERAPY DEVELOP A UTOANTIBODIES TO DISTINCT CYTOPLASMIC ROD / RING STRUCTURE S COMPOSED OF CTP/GTP NUCLEOTIDE BIOSYNTHETIC ENZYMES By WENDY CARCAMO A DISSERTATION PR ESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 Wendy Carcamo

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3 To my parents for all their encouragement, love and ne ver ending support ; t o my mentor for all his hard work an d dedication to help me succeed

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4 ACKNOWLEDGMENTS It is with great appreciation and sincere thanks that I acknowledge the following individuals for their professional or personal role in my aca demic career First and foremost, I would like to thank my family for always being there for me. I thank them for being extremely patience with all of my academic career choices. I thank my mentor, Dr. Edward Chan, for supporting me in my PhD endeavor. He has guided me throughout my research and with my future career decisions. His support has allowed for our success. I would also like to thank the members of my committee, Dr. Minoru Satoh, Dr. Naohiro Terada, Dr. Hideko Kasahara, and Dr. Robert Burne for t heir support and advice throughout my research. I would like to thank our collaborators, Dr. Nicola Bizzaro, Dr. Chen Liu, Dr. David Ostrov, Dr. Giovanni Covini, Dr. Kevin Wang, Dr. Lung Ji Chang, Dr. Luis Andrade, Dr. Maria Zajac Kaye, Dr. Katherine Han ko wski and Dr. David Purich who have help ed with our study by providing reagents, samples and scientific discussions. I thank all the current and past Chan lab members for their friendship, assistance and collaboration. I e specially would like to thank Dr. Shangli Lian and Dr. Kaleb Pauley for their support and guidance for perusing my PhD. I would like to thank Dr. Andrew Jakymiw, Dr. Songqing Li, and Dr. Keigo Ikedo for their help when I was just starting out in the lab. A special thanks to Bing Yao, Hyun Min Jung, Dr. Angela Ceribelli, Lan La, Grant Abadal, Brad Pauley, John Calise, Paul Dominguez Gutierrez, Stephanie Tamayo, Claire Krueger, and Dr. Abu Nahid for their help with my experiments.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 13 Autoantibodies in Rheumatic Diseases ................................ ................................ ... 13 Hepatitis C Virus and Disease ................................ ................................ ................ 14 HCV and Its Life Cycle ................................ ................................ ..................... 15 HCV and Autoantibodies ................................ ................................ .................. 16 2 INDUCTION OF CYTOPLASMIC RODS AND RINGS STRUCTURES BY INHIBITION OF THE CTP AND GTP SYNTHETIC PATHWAY IN MAMMALIAN CELLS ................................ ................................ ................................ .................... 19 Backg round ................................ ................................ ................................ ............. 19 Materials and Methods ................................ ................................ ............................ 20 Cell Culture ................................ ................................ ................................ ....... 20 Embryonic Stem Cell Culture and Differentiation ................................ ............. 21 Mouse Cardiomyocyte Collection and Preparation ................................ ........... 21 Autoantibody Reagents ................................ ................................ .................... 21 Immunoprecipitation ................................ ................................ ......................... 21 Indirect Immunofluorescence ................................ ................................ ........... 22 Induction of RR Formation Using CTPS or IMPDH Inhibitors ........................... 22 siRNA Knockdown ................................ ................................ ............................ 23 Transfection of GFP Plasmid ................................ ................................ ............ 23 Western Blotting ................................ ................................ ............................... 23 Statistical Analysis ................................ ................................ ............................ 24 Results ................................ ................................ ................................ .................... 24 Rods and Rings in the Cytoplasm ................................ ................................ .... 24 Identification of CTPS1 and IMPDH2 as RR Components ............................... 26 Expression of Rods and Rings in Cultured Cells ................................ .............. 27 Induction of Rods and Rings in Cultured HEp 2 Cells ................................ ...... 28 I nduction of RR in Other Cancer and Primary Cells ................................ ......... 29 Induction of RR is Quantitatively Sensitive to the Level of Cellular IMPDH2 P rotein ................................ ................................ ................................ ........... 30 RR Expression in Embryonic Stem Cells (ESCs) ................................ ............. 32 Discussion ................................ ................................ ................................ .............. 33 RR Like Structures are Evolutionarily Conse rved ................................ ............ 34

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6 Embryonic Stem Cells Form RR in the Absence of Chemical Inducers ............ 36 3 CYTOPLASMIC RODS AND RINGS AUTOANTIBODIES DEVELOPED DURING PEGYLATED INTERFERON AND RIBAVIRIN THERAPY IN PATIENTS WITH CHRONIC HEPATITIS C ................................ ........................... 49 Background ................................ ................................ ................................ ............. 49 Methods ................................ ................................ ................................ .................. 50 Patients ................................ ................................ ................................ ............ 50 Cell Culture and Indirect Immunofluorescence Studies ................................ .... 51 Statistical Analysis ................................ ................................ ............................ 52 Results ................................ ................................ ................................ .................... 53 Discussion ................................ ................................ ................................ .............. 55 4 DIFFERENTIAL AUTOREACTIVITY TO IMPDH2 IN ANTI RODS/RINGS ANTIBODIES: CLUES TO UNRESPONSIVENESS TO INTERFERON ALPHA/RIBAVIRIN THERAPY IN US AND ITALIAN HCV PATIENTS ................... 63 Background ................................ ................................ ................................ ............. 63 Materials and Methods ................................ ................................ ............................ 65 Human Sera and Autoantibodies ................................ ................................ ...... 65 Indirect Immunofluorescence (IIF) ................................ ................................ .... 65 Immunoprecipitation (IP) ................................ ................................ .................. 66 Statistical Analysis ................................ ................................ ............................ 66 Results and Discussion ................................ ................................ ........................... 66 Aut oantibodies to Cytoplasmic Rods and Rings in the US and Italian Cohorts ................................ ................................ ................................ .......... 66 Prevalence of Anti IMPDH2 Antibody by IP ................................ ...................... 67 Comparison of IIF Titer Versus Anti IMPDH2 IP ................................ .............. 68 Correlation of Anti RR/IMPDH2 Prevalence and Titer with Therapeutic Outcome ................................ ................................ ................................ ........ 68 Conclusions ................................ ................................ ................................ ............ 70 5 GLUTAMINE DEPRIVATION INITIATES REVERSIBLE ASSEMBLY OF CYTOPLASMIC RODS AND RINGS ................................ ................................ ...... 75 Background ................................ ................................ ................................ ............. 75 Materials and Methods ................................ ................................ ............................ 77 Cell Culture ................................ ................................ ................................ ....... 77 Indirect Immunofluorescence ................................ ................................ ........... 77 Induction of RR Assembly by Glutamine Deprivation ................................ ....... 77 Induction of RR Formation Using CTPS or IMPDH Inhibitors ........................... 78 Induction of RR Formation Using Glutamine Transport and Glutamine Syn thetase Inhibitors ................................ ................................ ..................... 78 qRT PCR ................................ ................................ ................................ .......... 78 Western Blotting ................................ ................................ ............................... 79 Quantification and Statistical Analysis ................................ .............................. 79

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7 Results ................................ ................................ ................................ .................... 79 Glutamine Depriva tion Induces Rod and Ring Formation ................................ 79 Glutamine Add Back Disassembles RR Induced in Response to Glutamine Starvation ................................ ................................ ................................ ...... 81 Supplement of Guanosine or Cytosine Induces the Disassembly of RR Formed in Response to Glutamine Starvation ................................ ............... 82 MPA Induced RR Disassemble After Replenishing with Guanosine ................ 83 Inhibition of Glutamine Synthetase and Glutamine Transport Induce RR Formation ................................ ................................ ................................ ...... 83 Discussion ................................ ................................ ................................ .............. 84 6 COMPARISON OF RODS AND RINGS TO CYTOPLASMIC STRUCTURES COMPOSED OF CTPS OR IMPDH ................................ ................................ ........ 93 Rods and Rings in the Cytoplasm ................................ ................................ ........... 93 Nucleotide Biosynthesis Associated with RR ................................ .......................... 94 Assembly of Proteins for a Cellular Function ................................ .......................... 95 Rods/Rings Related Filament Like Structures ................................ ........................ 95 Conservation of RR Across Species ................................ ................................ ....... 99 Composition of RR ................................ ................................ ........................... 99 Formation of RR ................................ ................................ ............................. 100 Concluding Remarks ................................ ................................ ............................. 101 7 SUMMARY AND PERSPECTIVE ................................ ................................ ......... 107 Biological Function of RR Structure ................................ ................................ ...... 107 Rod Versus Ring C onfigurations ................................ ................................ .... 107 Induced Versus Native RR ................................ ................................ ............. 107 Attempts to Identify Additional Components in RR ................................ ......... 109 Cytoplasmic Versus Nuclear RR ................................ ................................ .... 111 Technical Difficulties in RR Detection ................................ ............................. 112 Clinical Implication of Anti RR ................................ ................................ ............... 112 Ribavirin as a Therap eutic Agent ................................ ................................ ... 112 MPA as a Therapeutic Agent ................................ ................................ .......... 113 Development of RR Antibodies and Resistance to Treatment ........................ 113 Conclusions ................................ ................................ ................................ .......... 116 LIST OF REFERENCES ................................ ................................ ............................. 126 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 143

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8 LIST OF TABLES Table page 2 1 Dose and time dependent induction of RR using different CTPS1 and IMPDH2 inhibitors. ................................ ................................ .............................. 48 3 1 Clinical, serological and immunological features of the patients at baseline, and response to treatment. ................................ ................................ ................. 61 3 2 Clinical and laboratory characteristics of the 15 patients wh o seroconverted to RR antigen during pegylated IFN 2a/ribavirin therapy. ................................ 62 6 1 Comparison of various reported RR like cytoplasmic filament structures. ........ 106 7 1 Methods of RR induct ion and quantification of rods vs rings. ........................... 124 7 2 Antibodies tested for RR detection by direct immunofluorescence. .................. 125

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9 LIST OF FIGURES Figure page 1 1 HCV genome and polyprotein cleavage products. ................................ .............. 18 2 1 The distribution of cytoplasmic rods and rings were independent of the Go lgi complex and centrosomes, and these structures were not enriched in tubulin or vimentin. ................................ ................................ ................................ ......... 38 2 2 Examples of various intermediate RR structures in HEp 2 cells stained with human serum 604 (g reen) and counterstained with DAPI (blue). ....................... 39 2 3 The expression of rods versus rings was not correlated with the cell cycle. ....... 40 2 4 CTPS1 and IMPDH2 were highly enriched in RR and human anti RR prototype serum It2006 recognized IMPDH2. ................................ ..................... 41 2 5 Inhibition of CTPS1 or IMPDH2 induced formation of rods and rings. ............... 42 2 6 Inhibition of CTPS1 induced formation of RR in several human cancer cell lines ................................ ................................ ................................ .................... 43 2 7 Inhibition of CTPS1 induced formation of RR in mouse primary cardiomyocytes, fibroblasts, and endothelial cells. ................................ ............. 44 2 8 Overexpression of IMPDH2 prevented the induction of RR. ............................... 45 2 9 RR formation becomes increasingly sensitive to ribavirin when IMPDH protein level is reduced by specific siRNA knockdown. ................................ ...... 46 2 10 Uninduced mouse embryonic stem cells (ESCs) expres sed predominantly cytoplasmic rings that were disassembled during retinoic acid (RA) induced differentiation and reassembled when treated with acivicin. ............................... 47 3 1 Cytoplasmic rods and rings struc ture stained by sera from HCV patients. ......... 59 3 2 Rod and ring structure can be induced with ribavirin. ................................ ......... 60 4 1 Antibody titer deter mined in two cohorts of HCV patients with autoantibodies ................................ ............... 71 4 2 Prevalence of anti 55kDa IMPDH2 in immunoprecipitation analysis of anti RR po sitive sera. ................................ ................................ ................................ 72 4 3 Correlation of anti RR IIF titer and anti 55kDa IMPDH2 IP intensity. .................. 73 4 4 Correlation of anti RR antibodie s and 55 kDa band IP intensity to IFN/R treatment outcome. ................................ ................................ ............................. 74

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10 5 1 Glutamine deprivation induces rods and rings. ................................ ................... 87 5 2 Longitudi nal analysis of RR formation during glutamine starvation. ................... 88 5 3 Glutamine add back disassembles RR formed in response to glutamine starvation. ................................ ................................ ................................ ........... 89 5 4 Supplement of guanosine or cytosine induces the disassembly of RR formed in response to glutamine starvation. ................................ ................................ ... 90 5 5 MPA induced RR disassemble after replenishing with g uanosine. ..................... 91 5 6 Inhibition of glutamine synthetase and glutamine transport induce RR formation. ................................ ................................ ................................ ............ 92 6 1 Rods and rings (RR) reco gnized by human autoantibodies. ............................ 103 6 2 A schematic of RR components CTPS1 and IMPDH2 along the purine and pyrimidine biosynthesis pathways. ................................ ................................ ... 104 6 3 Patient autoantibodies recognize multiple components of RR. ......................... 105 7 1 RR induction pathway showing hypothetical exchange between rods and rings. ................................ ................................ ................................ ................. 117 7 2 Schematic of RR assembly. Rods and rings formed from monomers after induction with CTPS/IMPDH inhibitors (red inserts). ................................ ........ 118 7 3 A schematic of putat ive RR components AICARFT and GARFT along the purine and pyrimidine biosynthesis pathways. ................................ .................. 119 7 4 Rods and rings located within the nucleus. ................................ ...................... 120 7 5 Hypothetical role of ENT1 contributing to resistance to ribavirin therapy and HCV viral persistence in vivo ................................ ................................ ........... 121 7 6 Experimental schematic to test whether ENT1 resistanc e is linked to viral persistence. ................................ ................................ ................................ ...... 122 7 7 Postulation of HCV antigens associated with RR. ................................ ............ 123

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11 Abstract of Dissertation Presented to the Graduate School of th e University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy HEPATITIS C PATIENTS TREATED WITH INTERFERON ALPHA / RIBAVIRIN THERAPY DEVELOP A UTOANTIBODIES TO DISTINCT CYTOPLASMIC ROD / RING STRUCTUR E S COMPOSED OF CTP/GTP NUCLEOTIDE BIOSYNTHETIC ENZYMES By Wendy Carcamo August 2012 Chair: Edward K. L. Chan Major: Medical Sciences Molecular Cell Biology Hepatitis C (HCV) is a pandemic disease affecting an estimated 180 million individuals worldwi de and infecting another 3 4 million people each year, making HCV a global public health issue. HCV is the main cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Despite significant improvements in antiviral intervention only approximat ely 50% of treated patients with HCV clear the virus after treatment. Human autoantibodies serve as useful disease specific markers for syste mic rheumatic and other autoimmune disorders. C ytoplasmic filamentous structure s referred to as rods and rings (RR ) w ere identified using human autoantibodies. Our data show that HCV patients who have been treated with p egylated interferon alpha and ribavirin (RBV) develop these antibodies The distinct cytoplasmic rods (~3 10 m in length) and rings (~2 5 m in diame ter) were visualized in HEp 2 cells by immunofluorescence using HCV patient sera. Co localization studies revealed that although RR had filament like features, they were not enriched in actin, tubulin, or vimentin, and were not associated with known cytopl asmic structures. T wo proteins, cytidine triphosphate synthetase (CTPS) and inosine mono phosphate dehydrogenase (IMPDH) were

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12 identified as components of RR. C TPS and IMPDH are important enzymes in the cytidine triphosphate (CTP) and guanosine triphosphate (GTP) biosynthesis p athways, respectively. Two CTPS inhibitors, 6 diazo 5 oxo L norleucine (DON) and acivicin were used to test the effects of CTPS inhibition. RR were formed in the presence of these inhibitors in a concentration dependent and time depende nt manner Similar results were observed when cells we re treated with RBV or mycophenolic acid (MPA) inhibitors o f IMPDH Additionally glutamine deprivation affects CTP/GTP levels which induced RR at a slower rate compared to CTP/GTP inhibitors. Thus, RR formation might be affected by reduction of intracellular CTP/ GTP levels Interestingly, RR were readily detected in the majority of untreated mouse embryonic stem cells (>95%). U pon retinoic acid induced differentiation, RR disassembled in these cells bu t reformed again when treated with acivicin. RR formation appears to represent a response to d ecrease in intracellular levels of CTP or GTP in cancer cell lines, mouse primary cells and mouse embryonic stem cells RR autoantibodies may develop in HCV pati ents from their immune system exposed to direct interaction of HCV and RR induced by pegylated interferon alpha and ribavirin therapy.

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13 CHAPTER 1 INTRODUCTION Autoantibodies in Rheumatic D iseases Autoantibodies directed against intracellular antigens are characteristic features of human autoimmune diseases such as systemic lupus erythematosus (SLE), scleroderma, and Sjgren's syndrome (SjS), certain malignancies, and paraneoplastic syndromes ( 1 5 ) Studies in systemic rheumatic diseases have provided strong evidence that autoantibodies are produced and maintained by antigen driven responses ( 3 6 7 ) and that autoantibodies can be reporters from the immune system revealing the identity of antigens involved in the autoimmune disease pathogenesis ( 1 ) Some of these autoantibodies serve as disease specific markers and are directed against intracellular m acromolecular complexes or particles such as nucleosomes, nucleoli, small nuclear ribonucleoproteins (snRNPs), centromere antigens, and Ro cytoplasmic RNPs (hY RNP) ( 1 3 8 ) In the last two decades there has been significant progress in identifying many intracellular autoantige ns. The study of human autoantibodies and their use as probes of cell structure and function has had an important impact on the disciplines of molecular and cell biology ( 2 ) First, the majori ty of autoantibodies studied have been shown to bind to highly conserved determinants on ubiquitous cellular proteins ( 1 3 ) Second, many of the autoantibodies associated with systemic rheumatic diseases are often directed to functional macromolecules rather than to structural components ( 1 3 ) These include, histones, DNA and HMG of the nucleosome, the snRNP complex, various centromere and kinetochore components (CENPs), components of the nucleolus and other subcellular structures such as Cajal bodies (a.k.a. coiled bodies) ( 9 ) and GW bodies

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14 (a.k.a. mammalian processing bodies or P bodies) ( 10 ) Third, in systems amenable to testing, autoantibodies have been shown capable of inhibiting the cellular functions served by the autoantigens ( 1 3 ) Examples include the inhibition of aminoacylation of transfer RNAs by anti tRNA synthetase antibodies, the relaxation of supercoiled DNA by anti topoisomerase I antibodies, inhi bition of precursor mRNA splicing by anti Sm/RNP antibodies, and the transcription o f RNA by anti RNA polymerases. Taken together, these observations suggest that the conserved epitopes recognized by human autoantibodies are often the functional or active sites of these intracellular proteins ( 1 3 ) For immunologists, one of the interesting objectives is the identificatio n of macromolecules and organelles, such as the nucleolus ( 11 12 ) Golgi complex ( 13 ) and Cajal bodies ( 9 ) as targets of autoimmune responses and how this may explain pathogenesis of autoimmune diseases. For cell biologists, attempts to unravel cellular events can be enhanced by the availability of specific aut oantibody probes. Hepatitis C Virus and D isease Hepatitis C virus (HCV) is a small positive sense single stranded RNA virus that causes acute and chronic hepatitis in humans ( 14 15 ) HCV is one of the major causative agents of liver disease worldwide, with more than 180 million people infected ( 15 16 ) It is estimated that 3 4 million people are newly infected each year ( 15 ) In the United States, HCV related chronic liver disease is a leadin g cause of liver transplantation and causes thousands of deaths annually ( 17 ) Although therapeutic options are improving, viral clearance fails in about 80% of infected patients, resulting in a chronic viral disease ( 18 ) In 4 20% of patients with chronic hepat itis, liver cirrhosis develops within 20 years, with 1 5% of these patients developing hepatocellular carcinoma (HCC). Persistent HCV infections are facilitated by the ability of virus to

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15 incorporate adaptive mutations in the host and exist as genetically distinct quasispecies. Moreover, the persistent infection may also result from the ability of the virus to disrupt host defense by blocking phosphorylation and function of interferon regulatory factor 3 (IRF 3), an antiviral signaling molecule ( 19 ) Unlike hepatitis A and B viruses there is no vaccine to prevent HCV infection; therefore, current treatment is a combination therapy of pegylated interferon alpha (IFN of serum HCV RNA. However, this treatment is only efficacious in approximately 50% of patients ( 20 22 ) Sever al host factors such as age, stage of liver fibrosis, body mass index (BMI), liver steatosis, insulin resistance, ethnicity and IL28B single nucleotide polymorphisms, as well as viral genotype can potentially influence the treatment outcome ( 23 24 ) For instance, patients with HCV genotypes 2 and 3 respond more favorably to treatment than patients with genotype 1 and 4 ( 25 ) Therefore, new antiviral compounds that are more efficacious and better tolerated need to be developed. One of the biggest challenges in developing and implementing therapy for HCV infection is finding the appropriate models to examine the translational capability. HCV and I ts Life Cycle HCV was originally referred to as non A non B Hepatitis. In 1989, a major breakthrough in HCV research was discovered in which the complete sequence of the viral genome was identified and cloned by Choo and collaborators ( 26 ) HCV is the only member of the Hepacivirus genus that belongs to the Flaviviridae family ( 27 28 ) Structural analysis of the virus revealed that the genetic material s is surrounded by a protective nucleocapsid, composed mainly of the protein core (C), and further protected by a lipid envelope ( 29 ) The lipid envelope contains two major glycoproteins, envelope protein 1 (E1) and E2, that are embedded in the envelope ( 30 ) The genome consi sts of

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16 a single open reading frame that is ~9,600 nucleotides long, which is made into a single polyprotein (3,010 or 3,033 amino acids) product ( Figure 1 2 ) ( 31 32 ) The HCV genome is flanked by two non translated reg ions which are essential in the replication and synthesis of viral proteins. Viral and cellular proteases mediate the processing of the polyprotein int o structural (core, E1, E2, and p7) and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B) as illustrated in Figure 1 2 ( 33 35 ) The HCV life cycle is ent irely cytoplasmic and replication occurs mainly in hepatocytes, but the virus may also replicate in peripheral blood mononuclear cells (PBMCs). The virus enters the host cells through a complex interaction between virions and cell surface molecules CD81, L DL receptor, scavenge r receptor class B type 1 Claudin 1 and Occludin ( 36 39 ) Additionally, Niemann Pick C1 like 1 cholesterol absorption receptor was identified as a new HCV entry factor ( 40 41 ) Once inside the cell, the virus takes over the intracellular machinery to repli cate ( 42 ) Due to its high mutation rate caused by the RNA dependent RNA polymerase activity ( 43 ) HCV is considered a quasispecies composed of 6 genotypes with several subtypes ( 25 ) The 6 genotypes have differences in geographic distribution, disease progression, and response to therapy. Genotypes 1, 2 and 3 are distributed worldwide, with genotypes 1a and 1b accounting for 60% of global infections. In the United States, genotypes 1a and 2b are more commonly encountered. HCV and A utoantibodies HCV autoimmune manife station s range from non organ specific a utoantibodies seropositivity and cryoglobulins to immunological diseases such as glomerulonephritis, vasculitis, and mixed cryoglobulinemia ( 44 46 ) Commonly found autoantibodies in the sera of HCV infected patients are organ and non organ specific. In chronic HCV

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17 infection, smooth muscles antibodies (SMA), which react with cytoskeleton antigens of smooth muscle cells, are found in 10 66% of cases. The prevalence of antinuclear antibodies (ANA) ranges between 6% and 22%, and they are usually presented at a low titer (1:40 1:80) ( 47 50 ) Anti liver kidney microsomal antibodies (LKM 1 ), antibodies against epitopes on cytochrome P450, are found in the cytoplasm of hepatocytes and proximal renal tubes. Anti LKM 1 was detected in up to 10% of chronic HCV patients ( 51 ) Other antibodies such as anti asialoglycoprotein receptor, anti liver membrane antigen, anti liver cytosol antigen, anti hepatocyte plasma membrane, anti thyroglobulin, anti thyroid peroxidase, anti thyroid microsome, anti phosph olipid, anti neutrophil cytoplasmic, and many other autoantibodies have also been described in patients with HCV i nfection ( 52 54 ) Although the roles of these antibodies are unclear, e ach antibody is directed against a certain intracellular antigen released during cell death and presented to the immune system. Most studies have not ob served significant difference in clinical and biochemical parameters between ch ronic HCV patients with or without positive serum autoantibodies ( 49 55 )

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18 Figure 1 1 HCV genome and polyp rotein cleavage products. A schematic representation of the HCV genome indicating the structural and non structural drawn within. The cleavage site and the corresponding protease are indicated (arrows). Schematic is modified from Bartenschlager and Lohmann ( 35 )

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19 CHAPTER 2 INDUCTION OF CYTOPLA SMIC RODS AND RINGS STRUCTURES BY INHIBI TION OF THE CTP AND GTP S YNTHETIC PATHWAY IN MAMMALIAN CELLS Back ground Over the past few decades many human autoantibodies have emerged as significant disease specific markers for systemic rheumatic diseases ( 2 ) In these diseases, many autoantibodies are dir ected against intracellular macromolecular complexes or particles, such as nucleosomes and small nuclear/cytoplasmic ribonucleoproteins ( 2 ) Thus human autoantibodies have also served as useful p robes for exploring subcellular structures and functions because of their unexpected specificity to novel self antigens. Examples of significant uses of human autoantibodies in further characterization of novel subcellular structures included the identifi cation of p80 coilin in Cajal bodies (formerly known as coiled bodies) ( 9 ) and GW182 in GW bodies ( 10 ) Within the past few years, our laboratories identified novel human autoantibodies that recognized unique cytoplasmic structures described provisionally as rods and rings (RR). The current study reports the identifica tion of cytidine triphosphate synthase 1 (CTPS1) and inosine 1,2 monophosphate dehydrogenase 2 (IMPDH2) as components associated with these mammalian RR. CTPS1 and IMPDH2 are key enzymes in the biosynthetic pathway for CTP and GTP, respectively. CTPS1 cata lyses the rate limiting step in generating cytidine triphosphate (CTP) from uridine triphosphate (UTP). CTP is involved in nucleic acid and phospholipid biosynthesis and plays an important role in controlling cellular proliferation ( 56 ) Two isoforms, CTPS1 and CTPS2, have been identified with 74% amino acid similarity. The 67 kDa CTPS1 is a target for antiviral, antineoplastic, and antiparasitic

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20 drug development. Currently available specific inhibitors of CTPS1 include acivicin and 6 diazo 5 oxo L norleucine (DON) ( 57 ) The oxidation of inosine monophosphate (IMP) to xantho sine monophosphate (XMP) is the rate limiting step in the de novo guanine synthetic pathway catalyzed by IMPDH2. IMPDH2 regulates cell proliferation. Due to its rate limiting property, it has been a major target for immunosuppressive, antiviral, and cancer chemotherapy. Two isoforms of IMPDH have been identified sharing 84% amino acid similarity, and both are 56 58 kDa proteins that function as tetramers ( 58 ) A study on the regulation of IMPDH with mycophenolic acid (MPA) showed that MPA binds to IMPDH and causes a conformational change resulting in the formation of inactive angular aggregates ( 59 ) IMPDH2 inhibitors such as MPA, ribavirin, and tiazofurin are currently used for various medical conditions ( 60 63 ) In the c urrent report, inhibiting either CTPS1 or IMPDH2 using different compounds demonstrated induction of RR in a variety of cell types. More importantly, RR were observed in mouse embryonic stem cells (ESCs) and changes in its expression were correlated with d ifferentiation. Materials and Methods Cell Culture HeLa (human cervical cancer), HEp 2 (human epidermoid larynx carcinoma), and HCT116 (human colon cancer) were cultured in DMEM containing 10% Fetal Bovine Serum (FBS). Human oral cancer cells CAL 27 were cultured in DMEM containing 10% FBS and 1.5 g/L sodium bicarbonate. THP 1, human monocytes, and K562, human erythroleukemia cells, were cultured in RPMI 1640 with 25mM HEPES, and 10% FBS. All cells were cultured in a 37C incubator with 5% CO2. Adherent cell lines were

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21 maintained at 50% confluence. Suspension cells were maintained at optimum concentration of 10 6 cells/mL. All media contained 100 I.U./ml penicillin and 100 g/ml streptomycin. Embryonic Stem Cell Culture and Differentiation The murine R1 E SCs were obtained from Dr. Andre Nagy (University of Toronto) and maintained according to Hamazaki et al. ( 64 ) ESCs were differentiated with 1 M retinoic acid for 4 consecutive days as described ( 65 ) Mous e Cardiomyocyte Collection and P reparation The mouse cardiomyocytes were harvested and prepared according to Chan et al ( 66 ) Autoantibody R eagents Autoantibodies with reactivity to RR were selected by authors GC and CAvM during routine screening anti nuclear antibody assay typically used to help define whether selected patients have developed autoimmune res ponse. All three human anti RR sera used in this study had chronic hepatitis C virus (HCV) infection and were de identified samples. These sera were selected largely related to their relative specificity in RR staining and in available quantity to allow fu rther analyses. Serum It2006 was provided by GC, and serum 604 and 609 provided by CAvM, are considered prototype sera for anti RR. The clinical characterization of these patients and others with anti RR will be summarized in a subsequent report. Immunopre cipitation Immunoprecipitation of [ 35 S] methionine labeled K562 cells for the analysis of the proteins recognized by human autoimmune sera was performed as described ( 67 )

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22 Indirect Immunofluorescence HEp 2 cell slides (INOVA Diagnostics, San Diego, CA) or various adherent cell types cultured in 8 well chamber slides (BD Falcon) were used for indirect immunofluorescence as described ( 68 69 ) The 8 well chamber slides were prepared using ~60,000 cells per well in 500L of media. Non adherent cells were prepared using the cytospin method to transfer cells onto glass slides as described ( 70 ) Fixation methods included acetone at 20C for 5 min, acetone/methanol (3:1) at 20C for 10 min, and 3% paraformaldehyde in PBS at room temperature for 10 min followed by 0.1% triton X/PBS for another 10 min. For co staining studies the following primary antibodies were used: rabbit anti giantin ( 71 ) anti pericentrin (a gift from Dr. Marvin Fritzler, University of Calgary), anti C TPS1 (a gift from Dr. Jim Wilhelm, University of California, San Diego), anti IMPDH2 (Abcam, Cambridge, MA: ab75790; Proteintech, Chicago, IL: 12948 1 AP), mouse anti actin, anti actinin (Sigma Aldrich, St. Louis, MO: A7811), anti tubulin, and anti vimenti n, and human anti RR serum It2006, 604, and 609. Induction of RR Formation Using CTPS or IMPDH I nhibitors Acivicin (Enzo Life Sciences, Ann Arbor, MI; BML El113 0010) and ribavirin (Sigma Aldrich; R9644) were solubilized in water to 100 mM and 50 mM stock concentration, respectively. 6 diazo 5 oxo L norleucine (DON, Sigma Aldrich; D2141) was solubilized in DMEM to a stock concentration of 100mM. Cells were seeded as monolayers and allowed to attach for 24 h. Subsequently, CTPS1 or IMPDH2 inhibitors were add ed to various final concentrations from 0.2 M to 2 mM and were kept in cell incubator for times ranging from 15 min to 48 h.

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23 siRNA Knockdown All siRNAs used in this study were obtained from Dharmacon RNA Technologies (Lafayette, CO). The siRNAs were disso lved in molecular biology grade water and the resulting 20 M stock was stored in aliquots at 80C before use. The two predesigned siRNAs were: ON TARGET plus SMART pool siRNA Human CTPS1 (target sequences: UAACAUUGAUGCAGGAACA, AGACUAAACCUACCCAGAA, GGUGUU AUAUCAGGAAUUG, and GUAUGCAGGUGCUCAAAUC), siGENOME SMARTpool siRNA IMPDH2 (target sequences: GGACAGACCUGAAGAAGAA, GCACGGCGCUUUGGUGUUC, GGAAAGUUGCCCAUUGUAA, and CUAAAGAAAUAUCGCGGUA). Transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, at a final concentration of 100 nM. Knockdown efficiency was determined by real time PCR analysis using CTPS primers (Applied Biosystems, Hs01041851_m1) or IMPDH2 primers (Applied Biosystems, Hs 00168418_m1) plus Western blot for protein expression. Transfection of GFP P lasmid HeLa cells were seeded in 8 well chambers at 50 70% confluency. Cells were transfected with 0.35 g GFP IMPDH2 (RG202977, OriGene, Rockville, MD) or GFP vector alone using 0 .53 g of Lipofectamine 2000. The transfected cells examined after 24 h directly as live cells or were processed for indirect immunofluorescence. Western B lotting Protein levels were analyzed as described ( 72 ) using rabbit anti CTPS (Abcam, 1:1000) or rabbit anti IMPDH (Santa Cruz Biotechnology, Paso Robles, CA, 1:1000). For IP Western, IgG was crosslinked to protein A Sepharose beads as described ( 73 )

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24 and human It2006 was used for immunoprecipitation and mouse monoclonal and rabbit anti IMPDH from Santa Cruz Biotechnology were used for detection. Mouse anti actin antibody 3 1G9 was used at 1:5,000 or ~1 g/ml as a normalizer to control for loading. Statistical A nalysis Cell counting was performed using Mayachitra Imago (Mayachitra, Santa Barbara, CA) to detect nuclei counterstained with DAPI. Unpaired, two test and analysis, Prism for Windows, version 5.0 (GraphPad Software, San Diego, CA) was used. Results Rods and Rings in the Cytoplasm A distinct cytoplasmic pattern was firs t identified in routine antinuclear antibody (ANA) clinical test using HEp 2 cell slides purchased from INOVA Diagnostics Inc. The structures recognized by a prototype human serum 604 were distinct cytoplasmic rods (~3 10 m in length) and rings (~2 5 m i n diameter, Figure 2 1). On average, there are one to two rods and/or rings per cell including some interesting apparent intermediate one end, as well as some that app eared to be transitioning from rods to this peculiar form (Figure 2 2). Figure 2 1A shows RR structure and location with respect to the nucleus and the Golgi complex. Some rods often align adjacent to the nucleus (short arrows, ~40% frequency) or perpendic ular to the nucleus (long arrows, ~40% frequency), and rings may be found in the cytoplasm (arrowheads, 5 10% frequency). RR are expressed in some mitotic cells; however, co staining studies with anti centromere protein F (CENP F), showed that the expressi on of either rods or rings were

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25 independent of the cell cycle, as they appear in all stages (Figure 2 3). CENP F is a cell cycle marker that has little or no expression at the G1 phase and with increasing level during the S phase, reaching highest expressi on during late S and G2 phases. To examine the relationship of RR and other known cytoplasmic organelles, co localization studies were performed using known markers for cytoplasmic structures. Co staining studies with antibodies to the Golgi complex showed that RR were independent of the Golgi complex (Figure 2 1A); although many rods appeared to be in the same vicinity as the Golgi complex, there are also many clear cut examples of rods unassociated with this subcellular structure. Cytoplasmic structures, infected cells ( 74 ) To determine whether RR are related to actin rockets, co staining wi th mouse anti actin antibody was performed to show that RR are not enriched in actin (data not shown). Furthermore, upon careful examination at higher magnification, the HEp 2 cells in our laboratory were not contaminated with bacteria and cell viability w as >99%. Since RR are filamentous, co staining experiments were also performed to determine whether tubulin or vimentin, was enriched in RR. Figure 2 1D is a merged image of RR (Figure 2 1B) and tubulin (Figure 2 1C), showing that RR are not enriched in tu bulin. Co staining of human prototype serum 604 and anti vimentin antibody ruled out enrichment of RR with enrichment of vimentin in RR. Figure 2 1G shows clear examples of RR with little or no vimentin staining. Another initial consideration was that pri mary cilia, which function as nonmotile sensory organelles and have a rod like appearance ( 75 ) could be a possible candidate. To rule out RR as primary cilia, a co staining study with anti pericentrin, which

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26 recognizes centrosomes, was conducted. Centrosomes are the precursors of primary cili a and are often located at the ends of the primary cilia. Figure 2 1J revealed that centrosomes were not located at the ends of rods recognized by human prototype serum 604, and therefore are not primary cilia. In fact, there is no noticeable association o f either rods or rings with centrosomes. Furthermore, upon closer examination, the fact that ring type structures have never been observed for primary cilia is consistent with the conclusion. These findings are highly reproducible using HEp 2 cell substra te from INOVA, as well as using home grown cells prepared with various commonly used fixatives in biological laboratories including acetone at 20C for 5 minutes, acetone/methanol for 10 minutes, or 3% paraformaldehyde and 0.1% triton X (after cells were treated with inhibitors see below). Co staining experiments with known subcellular markers showed no association with known cytoplasmic organelles such as GW bodies (data not shown). Identifica tion of CTPS1 and IMPDH2 as RR C omponents During the initial stage of characterization, personal consultation with Dr. Joseph G. Gall, Carnegie Institution of Washington, Baltimore, MD, led to the consideration that CTPS1 may be a candidate RR protein because similar rod like structures were observed in Drosophila e mbryos in their study of GFP insertional screening ( 76 ) This work has been recently published ( 77 ) Dr. Jim E. Wilhelm, University of California at San Diego, La Jolla, CA, kindly provided a rabbit anti CTPS1 serum produced to a peptide of the Drosophila CTPS1. Co staining studies with rabbit anti CTPS1 antibody showed localization of CTPS1 to RR, ident ified with prototype human anti RR serum It2006 (Figure 2 4A).

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27 A second candidate RR protein was postulated in part because of an isolated ring like structure in the cytoplasm presented in a poster at the 9th Dresden Symposium on Autoantibodies in Germany describing a single autoimmune serum recognizing the enzyme inosine monophosphate dehydrogenase 2 (IMPDH2) ( 78 ) and the consideration that CTPS1 and IMPDH2 are enzymes in closely linked pathways as discussed below. In the first experiment to demonstrate whether IMPDH2 was localized to RR, a commercially available rabbit anti IMPDH2 antibody was used to co stain HEp 2 cells. Figure 2 4B clearly shows anti IMPDH2 staining of both rods and rings (arrows), as detected by It2006. Immunoprecipitation analysis using an extract of [ 35 S] methionine labeled K562 cells showed that rabbit anti IMPDH2 and It2006 both pulled down a 55kDa protein (Figure 2 4C). Furthermore, IP Western analysis confirmed that the 55kDa protein immuno precipitated by It2006 was IMPDH2, since it was recognized by both mouse monoclonal anti IMPDH2 and rabbit anti IMPDH2 (Figure 2 4D). Expression of R ods and R ings in C ultured C ells HEp 2 cell slides from INOVA Diagnostics have been consistently positive f or RR, tested from lot to lot, since this study was initiated over four years ago. The same was not true for HEp 2 cell ANA slides from other manufacturers examined to date, with staining varying from completely negative to cytoplasmic granular to diffuse. This could be the explanation for why the subcellular staining observed by Blthner et al ( 78 ) was largely different from ours, except for showing one or two isolated ring structures. These differences among produc ts from several manufacturers remain a question not completely resolved, but may stem from differences in culture conditions or proprietary sample processing or both. Homegrown HEp 2 cells on coverslips or 8 chambered slides have not been generally positiv e for RR staining unless cells were kept in rapidly

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28 dividing log phase and under subconfluent cell density. Confluent cultures were generally negative for RR. Variability from experiment to experiment was clearly observed, and on average only 5 30% of cell s were positive for RR detectable by serum 604 or It2006. Cells were always co stained with both human anti RR and rabbit anti giantin (Golgi marker) antibodies to ensure that variables were not introduced in the fixation and immunostaining steps in these experiments. Exactly how INOVA Diagnostics can produce highly consistent HEp 2 cell slides with >95% cells positive for RR staining remains unresolved, but clearly the difference must stem from their unique manufacturing methods. Thus, HEp 2 cells do not h ave RR under normal culture conditions and cells that do not ordinarily have RR have a diffuse cytoplasmic pattern when stained with prototype human serum (Figure 2 5A). However, it was noted that cells recently thawed from liquid nitrogen form RR in about 15% of the cells. Treating cultured cells with 0.015 0.5% of DMSO overnight to simulate freezing medium condition (often with 10% DMSO) did not induce formation of RR. Induction of R ods and Rings in C ultured HEp 2 C ells When CTPS1 and IMPDH2 were identi fied as RR components, the next series of experiments focused on the effects of inhibiting these enzymes. Effects of each drug treatment were analyzed by immunofluorescence (Figure 2 5). The first applied was the CTPS1 inhibitor DON on HEp 2 cells culture using a 2 mM concentration, as reported in literature ( 57 79 ) A surprising finding was that both rods and rings wer e readily detected in >95% of cells after overnight incubation (Figure 2 5B) compared to 0% in untreated control cells cultured in parallel (Figure 2 5A). The RR detected in DON treated cells were identical in frequency as those observed in the INOVA HEp 2 slides. A second independent CTPS1 inhibitor acivicin ( 56 57 ) as well as the IMPDH2 inhibitor

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29 ribavirin, also showed the ability to induce RR formation under similar conditions (Figure 2 5C and D). Inhibition of CTPS1 or IMPDH2 induced RR formation in a concentration and time dependent manner. Table 2 1 shows the relative efficiencies of the three inhibitors in t he induction of RR formation in HEp 2 cells, with ribavirin inducing RR formation in the shortest time and at lowest concentration analyzed. On average, cells treated with DON or acivicin had two RR per cell, while cells treated with ribavirin had more tha n two RR per cell. An unrelated compound, enoxacin, used as an oral broad spectrum fluoroquinolone antibacterial agent observed to enhance miRNA activity ( 80 ) served as an additional negative control as it did not ind uce RR after 24 h treatment in various concentrations (10 100 nM). Thus, the inhibition of either CTPS1 or IMPDH2 appeared to be specific for the induction of RR in HEp 2 cells. Induction of RR in O ther C ancer and P rimary C ells To address whether the expre ssion of RR is conserved, multiple cell lines were analyzed, including other adherent monolayer cell lines like HeLa (Figure 2 6A), CAL 27 (Figure 2 6B), and HCT116 (Figure 2 6D), as well as a suspension monocytic cell line THP 1 (Figure 2 6C). Cells were incubated with DON for 24 h and co stained with serum It2006 and anti giantin. All cell lines expressed RR in high frequency, comparable to those observed in HEp 2 cells. Untreated cells normally did not show expression of RR, but isolated HeLa cells showe d some cytoplasmic granular staining and THP To determine whether the expression of RR could be observed in species other than human, mouse 3T3 fibroblasts and rat NRK cells were stained with It2006. Freshly defrosted 3T3 and NRK often showed expression of RR (~25% of cells) in the first one or two passages, but their expression disappeared completely in subsequent passages. Mouse primary cardiomyocytes, as

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30 well as the often co purified endothelial cells and fibroblasts during isolation from neonatal heart, did not form RR until induced with 2 mM DON in overnight culture (Figure 2 7). When counting all the cells, overall 88.4% had detectable RR, but cardiomyocytes detected by co staining of actinin had up to 92.6% showing induce d RR (Figure 2 7B). Thus, RR expression appeared to be conserved as they could be induced in multiple immortalized cells, as well as in primary cultured cells. Induction of RR is Quantitatively Sensitive to the Level of Cellular IMPDH2 P rotein To monitor t he formation of RR, the use of GFP fusion constructs was explored. HeLa cells were transfected with GFP IMPDH2 (Figure 2 8A C) or the GFP vector alone (Figure 2 8D F). Following transfection, cells were treated with 2 mM of ribavirin for 24 h, after which cells were fixed and stained with rabbit anti IMPDH2. GFP IMPDH2 transfected cells had brighter fluorescence compared to untransfected cells confirming the expression of recombinant IMPDH2 (Figure 2 8A). Surprisingly, GFP IMPDH2 transfected cells did not s how RR after ribavirin induction whereas neighboring untransfected cells showed RR as expected (Figure 2 8C). To rule out transfection induced artifacts interfering with the induction of RR, the induction of RR in control GFP vector transfected cells was n ot apparently affected by the transfection (Figure 2 8F). Therefore, the overexpression of IMPDH2 appeared to inhibit formation of RR even at high concentrations of ribavirin. Series of experiments were performed to explore whether the lowest expression of transfected GFP IMPDH2 could produce cells with GFP labeled RR by titrating the amount of transfected plasmid and by reducing the time of transfection. None of these experiment yielded live cells labeled with GFP RR. Since overexpression of IMPDH2 inhibit ed RR induction, the next series of experiments were to address whether knockdown of CTPS1 or IMPDH2 would affect

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31 the induction of RR. The strategy adopted was to examine cells transfected with either CTPS1 siRNA (siCTPS1) or IMPDH2 siRNA (siIMPDH2). Thus HeLa cells were either untransfected, transfected with siCTPS1, or siIMPDH2 for 44 h, followed by an additional 4 h of incubation with three different concentrations of ribavirin at 2 mM, 0.2 M, or 0.05 M, respectively. Based on the data presented in Tab le 2 1A, the 2 mM ribavirin concentration was selected as the highest concentration tested, and 0.2 M was selected as the lowest. Not shown in Table 2 1A for HEp 2 cells, 0.05 M ribavirin was tested in HeLa cells and shown to be not effective in the ind uction of RR. The induction of RR was monitored by double immunofluorescence using rabbit anti IMPDH2 antibody as well as It2006. Figure 2 9A shows the Western blot analysis after siIMPDH2 knockdown with reduction of IMPDH2 levels to 5 9% of untreated ce lls whereas siCTPS1 had little or no effect on the level of IMPDH2 as expected, although siCTPS1 was confirmed by real time PCR to be functional with 62% CTPS1 mRNA knockdown. Number of cells with RR were counted and plotted as percentage (Figure 2 9B). While siCTPS1 transfection did not affect the percentage of cells positive for RR in all three concentrations of ribavirin examined, knockdown of IMPDH2 showed significant increase in the percentage of cells with detectable RR formation at both 0.2 M and 0.05 M of ribavirin (p = 0.0001 for 0.2 M and 0.05 M compared to ribavirin treatment, although RR were induced in 100% of cells transfected with siIMPDH or siCTPS1 RR, kno ckdown of IMPDH2 showed a decrease in the size of RR (with the average length of rod at 4.6 m in siIMPDH2 transfected versus 6.9 m in untransfected) along with the number of RR per cell (4.5 RR/cell in siIMPDH2

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32 transfected versus 7.1 RR/cell in untransfe cted, p = 0.0024 by t test) when compared to untransfected cells at 2 mM ribavirin. At 0.2 M ribavirin, increased formation of RR was observed in cells with reduced levels of IMPDH2 (42.9% of cells in siIMPDH2 transfected vs 21.6% for siCTPS1, p = 0.0048 ). At 0.05 M ribavirin, siIMPDH2 transfected cells had 22.4% cells with shorter rods (Figure 2 9D) compared to siCTPS1 transfected cells with 3.6% (Figure 2 9C, p = 0.0001). Thus the data show that the formation of RR by ribavirin was sensitive to the to tal IMPDH2 level and excess intracellular IMPDH2 makes cells resistant to the formation of RR. RR Expression i n Embryonic Stem Cells (ESCs) From the data presented in the previous sections, except for unique case of the INOVA HEp 2 cells, various cultured cell types did not consistently form RR unless induced with one of the CTPS1/IMPDH2 inhibitors. The next question was whether certain cell types were uniquely capable of form ing RR in the absence of inducers. ESCs are highly proliferative cells that can self renew and are pluripotent. Undifferentiated, uninduced ESCs were found to form RR (Figure 2 10B) similar to those described in HEp 2 cells (Figure 2 1A) with 1 or 2 RR per cell. Interestingly, unlike HEp 2 cells or mouse 3T3 cells (Figure 2 10A), w here only ~10% of cells had rings, >90% of ESC expressed rings, and <5% of cells had rods (Figure 2 10B). In contrast, ESCs that were differentiated with retinoic acid (RA) for 4 days no longer expressed RR (Figure 2 10C) but those differentiated ESCs rem ained capable of RR formation upon treatment of 2 mM acivicin for 24 h and the RR phenotype was similar to the original undifferentiated ESC (Figure 2 10D). RR expression could be induced in multiple cancerous and primary cells, but RR were found to be ex pressed in untreated stems cells.

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33 Discussion In this study, a novel cytoplasmic structure was identified by human autoantibodies produced by HCV patients through ANA testing. Most subcellular organelles disassemble prior to mitosis and reform after cell division. Although some mitotic cells were devoid of RR, either RR disassembly was incomplete or it did not occur at all in other cells, since up to 50% of mitotic cells clearly retained RR. At least one autoantigen, IMPDH2, recognized by the human anti R R prototype serum It2006 was determined through IP Western analysis. Co localization studies determined CTPS1 to be a second component of RR. Inhibitors of the two enzymes induced formation of RR in cells that did not form RR during continuous culture. RR induction could be accomplished with low nanomolar inhibitor concentration. Overexpression of IMPDH2 prevented formation of RR while knockdown in IMPDH2 protein allowed for more sensitive induction of RR with ultralow concentrations of ribavirin that no rmally did not induce RR. Since CTPS1 and IMPDH2 are major enzymes controlling the biosynthetic pathway for CTP and GTP, respectively, the formation of RR using multiple independent inhibitors of these enzymes implies that this cytoplasmic structure was l inked to these cellular metabolic pathways. The observations that the formation of RR is sensitive to the concentration of IMPDH2 (Figure 2 9) and that RR are present in some cases in untreated cells suggest that the formation of RR is linked to the funct ional level of these enzymes in cellular regulation related to ribonucleotide synthesis. Although both CTPS1 and IMPDH2 were identified as components of RR, due to the large size of these structures, one may speculate that there are many other components involved in RR formation. Since our initial observation of RR over four years ago, morphologically

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34 similar structures were identified in two previous reports ( 59 81 ) as well as four recent reports ( 77 82 84 ) RR Li ke Structures a re Evolutionarily Conserved The first report of RR like structures may be the 1987 study by Willingham et al ( 81 ) antibody gener ated from a Balb/c mouse immunized with mouse tumor cells. These structures were identified using transmission electron microscopy and immuno electron by a lipid bil ayer membrane. Their structures were similar in appearance and number per cell to the RR in this study. The unknown antigen recognized by the monoclonal antibody, an unknown 58 kDa protein in immunoprecipitation of lysate of [ 35 S] methionine radiolabeled cells, was referred to as nematin (from nematodes). The identity of the antigen was not elucidated ( 81 ) Similar to our results, they reported that these structures were a) not related to tubulin or vimentin, and b) evolutiona rily conserved in human, mouse, and rat. It was also reported that the nematin structure occurred following thawing of stored frozen cells ( 81 ) such as HEp 2, 3T3, and NRK cells as also observed in our laboratory. The second s tudy reporting RR like structures was by Ji et al identifying IMPDH2 protein aggregates formed from inhibition of enzymatic activity through MPA treatment ( 59 ) The structures were identified through immunofluorescence and electron microscopy analysis, with data comparable to the earlier study ( 81 ) Further analyses revealed that the aggregates were reversible with the addition of guanosin e, guanine, guanosine monophosphate (GMP), guanosine diphosphate (GDP) and guanosine triphosphate (GTP). The structures in that study and in ours were induced with two

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35 independent IMPDH2 inhibitors, MPA versus ribavirin. Both inducers inhibit IMPDH2 but MPA is a noncompetitive and reversible inhibitor ( 85 ) while ribavirin is a nonreversible inhibitor. However, our study has ide ntified that the structures induced by IMPDH2 inhibition can be recognized by human autoantibodies. In addition, the RR structure was composed of CTPS1 and IMPDH2. Ingerson Mahar et al. demonstrated that CTPS formed filaments in C. crescentus ( 82 ) Identif ication of the structure was through electron cryotomography along the inner curvature of C. crescentus The metabolic enzyme CTPS has a bifunctional role, as a filament that regulates curvature and performs its catalytic function. In addition, Escherich ia coli CTPS homologue can also form filaments. A key difference was that treatment of DON in this study dissociated the CTPS filaments in bacteria, while DON induced the formation of RR in mammalian cells in our study. The most recent report identified RR like structure in both yeast and Drosophila Noree et al identified four novel filaments that were identified by screening yeast GFP strain collection ( 83 ) Through inhibition of CTPS, they determined that filaments were comprised of inhibited CTPS, similar to our results. In addition, they were able to visualize CTPS filaments in the Drosophila egg chamber, a subset of gut cells and neurons. Another report also identified an intracellular structure containing CTPS in Drosophila cells ( 77 ) The structures referred to as cytoophidia were identified in ovary and other tissues ( 86 ) The structure was recognized by an antibody against Cup protein, which was believed to have cross reactivity to an antigen in the cytoophidia. GFP protein trap fly stocks from the Carnegie Protein Trap Library identified CTPS as

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36 the compon ent of cytoophidia. RR and cytoophidia may be similar conserved structures among different species. Ramer et al. identified a large intracellular neuronal organelle that occurs in a subset of adult rat sympathetic ganglion neurons ( 84 ) The structure, identified by immunohistochemistry, plus confocal and electron microscopy, occurred mainly in a toroidal shape, but also twists or rods. The structures referred to as loukoumasomes, or donut like structures were recognized by a monoclonal antibody that was raised against neuron tubulin antibodies. One difference between this and our study was the increased toroidal (ring) structures in neurons, while we observed more rods than rings in all induced cancer cells, but more rings than rods in ESCs. Embryonic Stem Cells Form RR in the Absence o f Chemical Inducers Although RR may appear to be highly conserved as discussed above, our preliminary screening in mouse tissues including liver, kidney, and spleen, using indirect immunofluorescence did not detect these structures. Undifferentiated ESCs form RR without the use of inhibitors while differentiated cells do n ot form RR and, interestingly, differentiated cells were still capable of RR formation upon induction with a CTPS1 inhibitor; however, this should not be interpreted as de differentiation back to stem cells. A note of caution is needed as it is not clear whether ESCs RR are structurally and functionally the same as those of cancer cells treated with inhibitors. It is unclear whether the spontaneous formation of RR in ESCs is related to the functional activity of IMPDH2 or CTPS1. The ability to self renew and pluripotency are two unique properties of stem cells, and these properties may have an effect on IMPDH2 and CTPS1 activity. It is possible that once stem cells differentiate, the cellular regulation of

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37 CTPS1 or IMPDH2 may change and affect the format ion of RR. One biological function of RR is related to CTP/GTP synthetic pathway as demonstrated here. In summary, RR formation is regulated by CTPS1 and IMPDH2, important in purine and pyrimidine biosynthesis and critical for RNA and DNA synthesis. Stud ies have demonstrated that CTPS1 and IMPDH2 are aberrantly expressed in different cancers ( 56 87 ) CTPS1 and IMPD H2 are differentially expressed and, therefore, cells may regulate CTPS1 or IMPDH2 activity via the formation of RR. Further characterization and understanding of RR may lead to a new biomarker for diseases or novel markers for stem cells. T his work was p ublished: Carcamo W.C., Satoh M., Kasahara H., Terada N., Hamazaki T., Chan J.Y.F., Yao B., Tamayo S., Reeves W.H., Liu C., Covini G., von Mhlen C.A., and Chan E.K.L. (2011) Induction of cytoplasmic rods and rings structures by inhibition of the CTP and G TP synthetic pathway in mammalian cells. PLoS One E29690.

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38 Figure 2 1. The distribution of cytoplasmic rods and rings were independent of the Golgi complex and centrosomes, and these structures were not enr iched in tubulin or vimentin. A) Merged image of HEp 2 co stained with human anti RR prototype serum 604/Alexa 488 goat anti human Ig (green) and rabbit anti giantin (Golgi marker)/Alexa 568 goat anti rabbit Ig (red). Rods are often presented adjacent (short arrows) or perpendicular (long arrows) to the nucleus while rings (arrowheads) are found either 1 or 2 to a cell. M, mitotic cell. HEp 2 cells were also co stained with serum 604/Alexa 488 goat anti human Ig (green, B,E,H) and different cytoplasmic ma rkers using mouse anti tubulin C), anti vimen tin F), anti pericentrin I), followed by Alexa 568 goat anti mouse Ig (red). Nuclei were counterstained with DAPI (blue). Bar, 10

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39 Figure 2 2. Examples of various intermediate RR structures in HEp 2 cells stained with human serum 604 (green) and cou nterstained with DAP I (blue). A) structure (arrowhead) with a curved rod adjacent to the n uclear envelope; B) elongated ring; C) elongate d, twisted ring (short arrow); D) rod with a pin loop (long arrow); E) Fig F) another r od with pin loop (long arrow).

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4 0 Figure 2 3. The expression of rods versus rings was not correlated with the cell cycle. HEp 2 cells were co stained with rabbit anti CENP F/ Alexa 568 goat anti rabbit IgG A) and human anti RR ser um 604 /Alexa 488 goat anti human IgG B). G1 cells had little or no CENP F staining, whereas late S/G2 and mitotic (M) cells showed strong staining for CENP F. Nuclei counterstained with

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41 Figure 2 4. CTPS1 and IMPDH2 were highl y enriched in RR and human anti RR prototype serum I t2006 recognized IMPDH2. A) Enrichment of CTPS1, detected by rabbit anti CTPS1 (green), to RR (arrows) identified by It2006 (red). Nuclei were cou nterstained with DAPI (blue). B) IMPDH2 stained by rabbit anti IMPDH2 (red) localized to RR (arrows) detected by I t2006 (green). C) Immunoprecipitation (IP) analysis using an extract of [ 35 S] methionine labeled K562 cells and rabbit anti IMPDH2, It2006, and a second human anti RR serum 609. It2006 recognized a 55kDa protein band that co migrated with IMP DH2 immunoprecipitated by rabbit anti IMPDH2. Serum 609 did not immuno precipitate the 55kDa protein. D) IP Western blot demonstrated that serum It2006 immunoprecipitated IMPDH2, which was recognized by both mouse monoclonal and rabbit anti IMPDH2 antibodie s. NHS, control normal human serum.

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42 Figure 2 5. Inhibition of CTPS1 or IMPDH2 induced formation of rods and rings. Untreated HEp 2 cells A) and cells treated with inhibitors 2 mM DON (CTPS inhibitor, B), 2 mM acivicin (CTPS inhibitor, C), or 2 mM rib avirin (IMPDH2 inhibitor, D) for 24 h were co stained with human anti RR serum It2006 (green) and rabbit anti giantin (red). Nuclei were counterstained with DAPI (blue). The percentage of cells with RR displayed for each experiment is shown in the lower right corner with the total number of cells counted

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43 Figure 2 6. Inhibition of CTPS1 induced formation of RR in several human cancer cell lines. Induction of RR observed in human canc er cell lines HeLa A), CAL 27 B), THP 1 C), and HCT116 D) when treated with 2 mM DON in culture for 24 h, fixed, and co stained with human anti RR serum It2006 (green) and rabbit anti

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44 Figure 2 7. Inhibition of CTP S1 induced formation of RR in mouse primary cardiomyocytes, fibroblasts, and endothelial cells. Mouse primary cardiomyocytes preparation together with fibroblasts and endothelial cells, were treated with 2 mM DON and cultured for 24 h. Cells were co stain ed with human anti RR serum IT2006 (green) and mouse anti actinin monoclonal antibody (red). Nuclei counterstained with DAPI (blue). A ctinin positive cardiomyocytes A, B, red) as well as actinin negative f ibroblast or endothelial cells A, C) all show dist inct rods. The percentage of cells with RR displayed is shown in the lower right corner with the total number of cells co unted indicated in parentheses A, all cells; B, actinin positive cardiomyocytes only; C; actinin negative fibroblast and endothelial ce

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45 Figure 2 8. Overexpression of IMPDH2 prevented the induction of RR. HeLa cells were tra nsfected with either GFP IMPDH2 A C) or GFP vector D F) for 24 h and followed by 2 mM ribavirin incubation for 24 h. Cells were stained with rabbit anti IMPDH2 (red) and counterstained with DAPI (Blue). GFP IMPDH2 transfected cells (T) did not form RR while untransfected cells showed rods (arrows) and rings (arrowheads). RR were detected in both GFP vector transfected and untransfected cells. Nuclei

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46 Figure 2 9. RR formation becomes increasingly sensitive to ribavirin when IMPDH protein level is reduced by specific siRNA knockdown. HeLa cells were either untransfected, transfected with siCTPS1, or siIMPDH2 for 44 h and followed b y incubation of ribavirin at various concent rations for an additional 4 h. A) IMPDH2 expression was monitored by Western blot using rabbit anti IMPDH2 antibody. Serial dilutions of untreated HeLa cell lysates (100%, 50%, and 25%) were used to help to compa ratively quantitate the degree of protein knockdown. The level of actin was used as a loading control. Relative IMPDH2 protein levels were quantified using image J and normalized to the serial dilution standards. B) Percentage of cells with RR were quanti fied using Mayachitra Imago with a range of 131 to 298 cells counted per data point. Cells treated with siIMPDH2 had a significant increase in percentage of cells test. C D) Representati ve image of cells at the lowest concentration of ribavirin transfected with siCTPS1 or siIMPDH2. Arrows, rods; arrowhead, ring. Bar,

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47 Figure 2 10. Uninduced mouse embryonic stem cells (ESCs) expressed predominantly cytoplasmic rings that were dis assembled during retinoic acid (RA) induced differentiation and reassemble d when treated with acivicin. A) Mouse 3T3 cells for comparison shown with both rods (80 90%, arrowhea d) and rings (10 20%, arrows). B) undifferentiated mouse ESCs shown with ~90 % ri ngs (arrows) and few rods. C) ESCs treated wi th RA for 4 days showed no RR. D) RR were induced in these RA differentiated cells by treatment with 2 mM acivicin for 24 h. Cells were stained with human anti RR serum It2006 (green, A D) and co stained with ra bbit anti giantin (red, A, B only). Nuclei

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48 Table 2 1. Dose and time dependent induction of RR using different CTPS1 and IMPDH2 inhibitors. Concentration dependent induction of RR in HEp 2 cells after 24 h i ncubation by ribavirin, DON, and acivicin. Drug concentration Ribavirin DON Acivicin 2 mM +++ +++ +++ 1 mM +++ +++ +++ 0.5 mM +++ +++ +++ 0.25 mM +++ ++ +++ 125 M +++ ++ +++ 62.5 M +++ ++ +++ 31.3 M +++ ++ ++ 15.6 M +++ + + 7.81 M +++ 0 .20 M +++ Differential time requirements for RR induction in HEp 2 cells treated with 2 mM of the respective compound. Time after addition Ribavirin DON Acivicin 30 min + 1 h +++ + 2 h +++ + ++ 3 h +++ ++ ++ 24 h +++ +++ +++ 48 h +++ +++ +++ Legend indicates percent of cells with RR in (A) and (B): ( ) = 0%, (+) = <25%, (++) = 50%, (+++) = >95%.

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49 CHAPTER 3 CYTOPLASMIC RODS AND RINGS AUTOANTIBO DIES DEVELOPED DURIN G PEGYLATED INTERFERON AND RIBAVIRIN THERAP Y IN PATIENTS WITH CHRONIC HEP ATITIS C Background Serum organ and non organ specific autoantibodies are frequently detected in patients with chronic hepatitis C virus (HCV) infection, reflecting the wide spectrum of immune reactions associated with HCV ( 48 88 96 ) Experimental studies have in fact demonstrated the existence of an interaction between the HVR1 E2 region of the virus and CD81 rec eptors on the surface of B lymphocytes, which promotes B cell proliferation and activation, thereby stimulating antibody production ( 97 98 ) In addition, a significant homology motif between HCV polyproteins and tissue autoantigens has been postulate d to suggest role in priming specific autoantibodies in pat ients chronically infected by HCV ( 50 99 101 ) Finally, several clinical studies have reported deterioration of pre existing autoimmune diseases in patients receiving interferon (IFN) therapy for hepatitis C, as well as de novo induction of autoimmune phenomena in HCV infected patients on IFN monotherapy. In this setting, the most common phenomenon associated with IFN therapy is the de novo induction of serum tissue antibodies or boosting of the titer of pre existing autoantibodies that in some patients, ends with the development of an autoimmune disease ( 102 111 ) The immune properties of IFN, at least in part, rely on its ability to enhance the expression of HLA class I and II antigens on cell membranes, promote T cell activation and the subsequent release of cytokin es, mechanisms that could account for the onset of autoimmune reactions during IFN therapy in patients with chronic hepatitis C ( 112 )

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50 To date, there is littl e data on autoimmunity in HCV patients being treated with the combination (PEG) IFN plus ribavirin (RBV) ( 20 113 114 ) One main reason for this is that high titer of serum autoantibodies remains a widely accepted criteria against starting IFN therapy in patients with chronic hepatitis C, mainly as a con sequence of the strict criteria of exclusion adopted in the design of registration trials rather than for the evidence of adverse reactions in practice. In other studies, the presence of tissue autoantibodies was associated with an increased risk of treatm ent failure ( 115 118 ) W e describe a novel autoantibody developed in patients with chronic hepatitis C during therapy with PEG IFN and RBV that in immunofluores cence (IIF) studies appear like distinct rods and rings (RR) in the cytoplasm of HEp 2 cell slides, more commonly detected in patients who failed to respond to HCV therapy. In the present study, we determine the titer and prevalence of such anti RR antibod ies in HCV patients under treatment with PEG IFN and RBV and its correlation with the outcome of treatment Methods Patients From 2004 to 2007, 156 previously untreated patients with chronic hepatitis C were consecutively treated with PEG IFN/ RBV at the Liver Center, Istituto Clinico Humanitas, Rozzano, Italy. Seventy five patients with sera available in our laboratory for autoantibody tests at baseline and during treatment were investigated. All subjects had histologically proven, compensated liver disea se characterized by elevated serum alanine aminotransferase (ALT) activity, anti HCV antibodies and serum HCV RNA for at least six months. Excluded were patients with autoimmune diseases, infection with the HBV or the HIV and with general contraindication s to using either IFN or RBV ( 119 ) Patients were treated with subcutaneous injections of PEG IFN 2a (Pegasys;

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51 Roche, Basel, Switzerland) 180 g once weekly coupled with oral RBV (Copegus; Roche, Basel, Switzerland) at a dose of 1,000 mg per day for patients weighing 75 kg or less and 1,200 mg per day for those weighing more than 75 kg. All patients g ave their written informed consent to receive therapy and permission for use of their medical records. HCV 1 and HCV 4 infected patients were treated for 48 weeks. HCV 2 and HCV 3 patients were treated for 24 weeks. Patients were classified as non respon ders (NR) if HCV RNA was still detectable at week 24 of therapy, as relapsers (REL) if HCV RNA was detected after the end of treatment in patients with a virological response, and as sustained virological responders (SVR) if HCV RNA was undetectable in the 24 weeks after the completion of therapy. As controls, sera were obtained from 105 p rimary biliary cirrhosis patients, 43 with primary sclerosing cholangitis, 56 with autoimmune hepatitis, 100 untreated HBV related chronic active hepatitis, 100 with hepat ocellular carcinoma and 100 blood donors. This study meets and is in compliance with all ethical standards in medicine and information consent was obtained from all patients according to the Declaration of Helsinki. Cell Culture and Indirect Immunofluoresc ence Studies Different cell lines, such as normal human epidermoid larynx carcinoma HEp 2, human colon cancer cell line HCT116, human cervical cancer cell line HeLa, myelogenous leukaemia cell line K562, oral squamous cell carcinoma CAL 27 and rat kidney f ibroblasts NRK, were cultured and prepared with multiple fixation methods that included acetone, 20C, for 5 minutes, or 3% paraformaldehyde and 0.1% triton X fixation as commonly used in many standard cell biology laboratories. For the induction of RR, R BV (Sigma Aldrich St Louis, MO, USA ; R9644) was solubilized in water to a stock of 50 mM and was added to cultured cells seeded in monolayer at a final

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52 concentration of 2 mM for 24 h. RR were detected using patient sera and anti IMPDH2 antibodies (anti in osine monophosphate dehydrogenase 2; Proteintech, Chicago, IL, USA; 12948 1 AP). Anti nuclear (ANA), anti smooth muscle (SMA), anti mitochondrial (AMA) and anti liver kidney microsomal (LKM) antibodies were detected by IIF at a serum dilution of 1:40 or hi gher in PBS and incubated for 45 min. using as substrates commercial HEp 2 (INOVA Diagnostics, San Diego, CA,USA) for ANA, whereas cryostat sections of rat kidney were used to detect SMA, AMA and LKM antibodies. Slides were th en rinsed in PBS two times for 10 min. Goat anti human immunoglobulin G conjugated to fluorescein isothiocyanate (Cappel, ICN Biomedicals Inc., Aurora, Ohio) or Alexa 488 goat anti human Ig (Invitrogen, Carlsbad, CA USA) was used as secondary antibody diluted 1:100 in PBS and incubate d on slides for 30 min Slides were then rinsed again in PBS 2x for 10 min, mounted with cover slip and observed with a fluorescence microscope. IIF slides were read by a single investigator (GC) under standardized experimental conditions. Statistical A na lysis The following 18 biological and clinical variables were analyzed for statistical significance: sex, age, ALT, aspartate transaminase, gamma glutamyl transpeptidase, bilirubin level, prothrombin time, HCV genotype, HCV viremia level, presence of autoa ntibody at baseline (ANA, SMA, LKM and AMA), treatment outcome (long term response and relapse/non response), steatosis, cirrhosis, diabetes mellitus and HCC development during post treatment follow up. Data are expressed as number and percentage or mean a nd range, unless otherwise stated. The differences between test and Mann Whitney U test for 2 and Fisher's exact test for categorical data as appropriate. A P

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53 value of <0.05 was considered to be statistically significant. All analysis where made with Stata10 (StataCorp. College Station, TX, USA). Results A total of 75 HCV infected patients were treated with standard of care P EG IFN RBV regimens. Forty four patients (59%) were male with a mean age of 4911 years. Histological evidence of cirrhosis was present in 8 subjects (11%). The overall prevalence of serum autoantibodies at baseline was detected in 26 subjects (35%); the prevalence of ANA, SMA and anti LKM 1 was 24, 8 and 3% respectively. No ne of the patients were AMA positive. No patient met the criteria of probable or definite autoimmune hepatitis on the basis of the International Autoimmune Hepatitis Group score ( 120 ) Demographic, clinical and laboratory features of the patients are shown in Table 3 1. T he IIF study on HEp 2 cells substrate revealed the presence of antibodies reacting with a cytoplasmic structure in 15 patients ( 20%) with HCV who received PEG IFN/ RBV (Table 3 2) Human prototype serum It2006 recognized rods ranging approximately 3 rings ranging approximately 2 (Figure 3 were found in more than 95% of HEp 2 cells. Usually there were only one to two RR per HEp 2 cell. Rods may align either close to the nuclear envelope (Figure 3 1C) or extend perpendicularly from the nucleus (Figure 3 1B), while rings are found in low frequency (approximately 10% of all cells) only in the cytoplasm (Figure 3 1D). Co localization experiments with known markers showed no association with any known cytoplasmic organelles, including Golgi complex, mitochondria, centrosomes, and GW bodies which are cytoplasmic foci known to mediate gene expression via the

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54 small RNA guided translation silencing process ( 121 122 ) Table 3 2 summarizes the relevant clinical features of the 15 patien ts who tested positive for anti RR. Interestingly, anti RR autoantibodies were found in the sera collected in HCV patients during PEG IFN RBV treatment only (15/75, 20%) whereas it was never detected before antiviral therapy (0/75) or in any of the control groups listed in Methods Although the INOVA HEp 2 cell substrate was consistently positive (>95% of cells) for RR structures, h ome grown HEp 2 cells using standard conditions were mainly negative. In fact, among the few commercial ANA slides tested, none other than the INOVA slides showed >1% cells with RR, making these other products extremely difficult for the accurate detectio n of anti RR. Preliminary experiments were performed to determine whether IFN or RBV treatment would induce the formation of RR in in vitro culture. Several attempts of IFN treatment in HEp 2 or HeLa cells did not yield expression of RR when treated cells were stained by prototype anti RR serum It2006. In contrast, RBV treatment readily induced RR formation. Figure 3 2A shows HEp 2 cells that were untreated gave primarily diffuse cytoplasmic staining and no detection of RR; however after treating cells wit h 2 mM of RBV for 24 h, RBV appeared to induce the aggregation of the protein into RR (Figure 3 2B). Furthermore, different cell lines such as normal rat kidney fibroblasts NRK or immortalized human cancer cell line such as HCT116, HeLa, K 562, CAL 27 have been examined after RBV treatment and RR were induced in all cells stained with anti RR positive sera ( 122 ) Thus current data suggest that the RR staining pattern is not unique to HEp 2 cells but also detect ed in various immortalized human cell lines and also in cells derived from rodents

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55 The prevalence of anti RR antibody i s significantly higher in non responders/relapsers (10/30, 33%) than in responder patients (5/45, 11%; Fisher p value= 0.037; Table 1). In other words the prevalence of REL/NR in anti RR positive group (10/ 15 =67% ) is higher than that of anti RR negative group (20/60=33%; Fisher P=0.037). I nterestingly, seven anti RR positive patients had HCV genotype 2a/2c or 3 (five genotype 2 and two ge notype 3); of those seven easy to cure patients, three were non responders or relapsers. None of the anti RR positive patients had cirrhosis. Discussion This study describes a novel serum antibody (anti RR) reacting against a cytoplasmic structure that ca n be detected by IIF using HEp 2 cells as substrate in HCV patients treated with PEG IFN RBV. The overall prevalence of anti RR antibodies was 20% of HCV treated patients. RRs are the first autoantigen described in literature arising during this t reatment. In fact, only few drugs are reported in hepatology to induce autoantibodies to cytoplasmic antigens but all of them are organ specific like anti LKM 2 ( 123 ) anti gastric parietal cells ( 106 ) anti thyroid ( 91 124 ) anti pancreatic islet cells ( 107 ) anti glutamic acid decarboxylase ( 111 ) and anti adrenal cortex ( 107 ) Interestingly, anti RR antibodies were detected in PEG IFN/ RBV treated patients only. In fact baseline sera of HCV patients and of control s did not show any reactivity for anti RR antibodies, thus suggesting an important role of PEG IFN/ RBV in triggering RR seroconversion Although INOVA HEp 2 cells express RR and are suitable for the detection of anti RR antibodies, a few other pre made HEp 2 slides marketed for ANA testing are negative for RR staining using the same prototype serum and other anti RR positive sera (data not shown). Our novel data show that many cell lines treated with RBV in

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56 vitro induced the formation of RR (Figure 3 2B); the induction led to greater than 95% of the cells to expressing RR compared to untreate d cells not expressing RR. Induction of RR could happen also in vivo in human hepatocytes during antiviral therapy and these changes may induce the immune system resulting in the production of anti RR In general, the most common autoantibody encountered in HCV treated patients is ANA. Even if all published studies describe only fluorescence ANA patterns without focusing on their identity, it is reasonable to think that their specificities are heterogeneous like in HCC ( 96 ) being directed against different nuclear antigens, suggesting a nonspecific reaction of the immune system to nuclear antigens ( 114 125 ) By contrast, the production of anti RR antibodies in HCV sera is quite different than ANA. In fact, the most important difference is that ANA appears independently from HCV therapy although anti RR develop only during treatment and are apparently directed exclusively against a single antigenic structure; thus, anti RR appears to mimic reactivity of serum autoantibodies which develop in the course of sy stemic autoimmune disease where each disease is associated with a specific profile of autoantibody specificities ( 1 2 ) For example, systemic lupus erythematosus is characterized by a high prevalence of ANA directed to the same structures such as DNA and spliceosomal proteins. In this context, the high prevalence of anti RR antibodies that react to the same cytoplasm stru cture suggests their direct biological role in HCV treated patients. This hypothesis is supported by our data in which anti RR positivity seems to alter antiviral response with significantly higher prevalence of anti RR in non responders/relapsers than sus tained responders (33% versus 11%; P value = 0.037). This evidence is further enhanced in HCV genotype 2 and 3 patients where

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57 approximately half of anti RR positive patients failed to respond to therapy, further suggesting a correlation between anti RR ant ibodies and response to antiviral therapy. In our recent study ( 122 ) at least one autoantigen recognized by the prototype anti RR is the enzyme inosine monophosphate dehydrogenase 2 (IMPDH2), which catalyses the oxidation of inosine monophosphate to xanthosine monophosphate in the rate limiting step in the de novo guanine synthetic pathway responsible for the GTP production. It is intriguing that IMPDH2 is the enzyme known to be inhibited by RBV in vitro and a t the same time RBV induced the formation of RR in all cell lines examined to date, including primary mouse cells such as fibroblasts and endothelial cells. We speculate that RR can also form in vivo in the hepatocytes during RBV treatment; in some predisp osed patients, IMPDH2 condensation into RR may induce the immune system to develop antibodies. This observation is extremely important also for immunologists to understand the development of drug induced autoantibodies; in fact, this is the first study tha t elucidates a mechanistic action of a drug to induce autoantibodies through a topographic rearrangement of cytoplasmic proteins. As t his is a retrospective study, our sera were collected at different time s after starting treatment. For this reason we are not able to determinate the kinetic s of rising anti RR and we do not know whether these autoantibodies disappear after the end of therapy. W ith all the caveats of the limited sample size of the present study, one could speculate that anti RR reflect an imm une reactivity to cytoplasmic structure of the liver cells that interact with IFN/RBV therapy. Our current observations in HCV p atients undergoing antiviral therapy need to be expanded to clarify the association between anti RR and response to treatment.

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58 T his work was published: Covini G., Carcamo W.C. Bredi E., von Mhlen C.A., Colombo M., and Chan E.K. (2011) Cytoplasmic rods and rings autoantibodies developed during pegylated interferon and ribavirin therapy in patients with chronic hepatitis C. Antivir Ther. In Press.

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59 Figure 3 1 Cytoplasmic rods and rings structure stained by sera from HCV patients INOVA HEp 2 cells stained with human anti rods and rings (RR) serum It2 006/Alexa 488 goat anti human immunoglobulin G (green) Nuclei were counterstai ned with DAPI (blue). Arrows indicate rods or rings. A ) 20x magnification of HEp 2 cells; B) HEp 2 cell with rod perpendicular to the nucleus; C ) HEp 2 cell with rod adjacent to the nucleus; D ) HEp 2 cell with ring in cytoplasm. Scale bar 10 M.

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60 Figure 3 2 Rod and ring structure can be induced with ribavirin. HEp 2 cells were stained w ith human anti rods and rings (RR) serum It2006/ Alexa 488 goat anti human immunoglobulin G (green). HEp 2 cells are shown A) untreated or B) treated with 2 mM of ribav irin for 24 h Nuclei were counterstained with DAPI (blue). Scale bar 10 M. A) shows HEP 2 cells that were untreated gave primarily diffuse cytoplasmic staining a nd no detection of RR, however B) after treating cells with 2 mM of ribavirin for 24 h, ribav irin appeared to induce the aggregation of the protein into RR.

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61 Table 3 1. Clinical, serological and immunological features of the patients at baseline, and response to treatment Parameters Total Patients (n = 75) Patients with RR Ab (n = 15) Patients without RR Ab (n = 60) P value Patients, no. Male 44 (59) 9 (60) 35 (58) NS Female 31 (41) 6 (40) 25 (42) Age, years 49 11 48 11 49 12 NS ALT, UI/L 112 (23 408) 123 (32 408) 110 (23 389) NS Genotype NS HCV RNA ** NS ANA, no. 18 (24) 4 (27) 14 (23) NS ASMA, no. 6 (8) 3 (20) 3 (5) NS LKM, no. 2 (3) 1 (7) 1 (2) NS AMA, no. 0 (0) 0 (0) 0 (0) NS Steatosis, 16 (21) 2 (13) 14 (23) NS Cirrhosis, no. 8 (11) 0 (0) 8 (13) NS Diabetes, no. 5 (7) 0 (0) 5 (8) NS Treatm ent out come SVR 45 (60) 5 (33) 40 (67) 0.037 REL / NR 30 (40) 10 (67) 20 (33) Values presented as n (%) unless otherwise indicated. Patients were classified according to their genotype, but no statistica l correlation was found amo ng the two groups. ** Patients' viremia at the baseline were stratified as <400.000 UI/L, 400000 800000 UI/L, >800000 UI/L. These findings were not statistically relevant. Ab, antibody; ALT, alanine aminotransferase; AMA, anti mitochondrial antibody; ANA, anti nuclear antibody; ASMA, anti smooth muscle antibody; LKM, anti liver kidney microsomal; NS, Not statistically significant (P > 0.05); REL / NR, Relapsers / Non Responders; RR, rods and rings; SVR, Sustained Virological Response.

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62 Table 3 2 Clinical and laboratory characteristics of the 15 patients who seroconverted to RR ant igen during pegylated IFN 2a/ribavirin therap y. Sex Age Genotype HCV RNA SVR ANA ASMA AMA LKM anti RR Patients IU/L x 105 titer 1 M 59 1a 0,3 Yes Neg Neg Neg Neg 1:320 2 M 46 1b 4,0 No Neg Neg Neg Neg > 1:320 3 F 55 2a/2c 3,0 No 1:160* Neg Neg Neg 1:160 4 F 44 3a 7,0 No Neg Neg Neg Neg 1:320 5 F 45 3a 6,0 Yes Neg 1:160** Neg Neg 1:320 6 M 41 2a/2c 7,0 Yes Neg Neg Neg Neg 1:160 7 F 58 1b 5,7 No 1:160* Neg Neg Neg 1:320 8 M 36 1b 7,0 No Neg Neg Neg Neg > 1:320 9 M 41 1b 3,0 No Neg 1:160** Neg N eg 1:160 10 M 56 1b 7,0 No 1:80* Neg Neg Neg > 1:320 11 M 42 1b 6,0 No Neg Neg Neg Neg 1:160 12 F 55 2a/2c 7,0 Yes Neg 1:160** Neg Neg 1:320 13 F 60 1b 7,0 No Neg Neg Neg Neg > 1:320 14 M 47 2a/2c 6,0 No Neg Neg Neg Neg > 1:320 15 M 17 2a/2c 3,5 Yes 1:160* Neg Neg 1:160 > 1:320 Speckled. ** Vessel smooth muscle antibody. AMA, anti mitochondrial antibody; ANA, anti nuclear antibody; ASMA, anti smooth muscle antibody; F, female; LKM, antiliver kidney microsomal; M, male; Neg, negative; RR, rods and ri ngs; SVR, sustained virological responder.

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63 CHAPTER 4 DIFFERENTIAL AUTOREA CTIVITY TO IMPDH2 IN ANTI RODS / RINGS ANTIBODIES: CLUES TO UNRESPONSIV ENESS TO INTERFERON ALPHA/RIBAVIRIN THER APY IN US AND ITALIAN HC V PATIENTS Background Hepatitis C virus (HCV) is an enveloped positive single strand ed RNA virus classified in the genus Hepacivirus of the family Flaviviridae ( 126 ) It was identified in 1989 and is a major cause of chronic liver disease frequently lead ing to liver cirrhosis and eventually to hepatocellular carcinoma ( 14 26 127 ) An estimated 170 million people are infected worldwide and there is no vaccine available for this viral infection ( 128 ) The virus genome encod es a pol yprotein of approximately 3000 amino acids that is f coding regions ( 129 130 ) Translation of the polyprotein is mediated by an coding regions, and the individual viral proteins are produced upon cleavage of the polyprotein by host and viral proteases. These include three structural proteins (core, E1 and E2), the p7 protein and six non structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B), which are involved in virion assembly and viral RNA replication ( 129 130 ) The virus is classified into eleven different genotypes and more than a hundred subtypes, and it also manifests itself in sera or tissues of patient s with the appearance of quasi species ( 25 131 ) The current treatment of HCV is a combination of pegylated interferon alpha (IFN ) and ribavirin (RBV, IFN/R). Alt hough the addition of RBV to the IFN treatment has significantly improved the outcomes, only half of patients infected with HCV respond to therapy ( 132 ) For the ultimate goal in treatment is to achieve a sustained virological response (SVR; defined as undetectable HCV RNA for at least 24 weeks post therapy), new drugs have been approved for the treatment of HCV ( 133 )

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64 Patients with HCV may also develop aberrant immunological alterations such as autoantibody production, autoimmune thyroid disorders, B cell lymphomas and mixed cryoglobulinemia ( 134 136 ) HCV patients can produce non organ specific autoantibodies such as anti nuclear antibodies (ANA), anti smooth muscle antibod ies and anti LKM1 antibodies, with much higher prevalence th an healthy people ( 48 89 ) Since HCV enters hepatocytes through binding of its envelope protein E2 with CD81, also expressed in B lymphocytes Pileri et al. proposed that HCV binding to B lymphocytes lowers the B cell activation threshold and therefore facilitates the production of autoantibodies ( 97 ) Autoantibodies are commonly used for diagnos is of certain autoimmune conditions such as systemic lupus erythematosus scleroderma, and polymyositis ( 2 137 ) but data on the association of autoantibodies to the clinical and biochemical profile of chronic HCV hepatitis or response to treatment are still controversial ( 88 116 138 ) A novel autoantibody that recognizes distinct cytoplasmic r ods and rings (RR) structures was first identified in sera of HCV patients that were screened using commercial ANA slides ( 122 ) Localization of two specific enzymes, inosine monophosphate dehydrogenase 2 (IMP DH2) and cytidine triphosphate synthetase (CTPS1), to these structures was identified in our previous study ( 122 ) RR were induced in cultured cancer cells when exposed to IMPDH2 inhibitors RBV or acivicin, or CTPS1 inhibitor 6 diazo 5 oxo L norleucine (DON) ( 122 ) Anti RR antibodies were specific to HCV patients treated with IFN and RBV but not prior to treatment ( 139 ) The aim of the present study is to identify the clinical significance of anti RR autoantibodies developed during IFN/R therapy in HCV infected patients.

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65 Materials and Methods Human Sera and Autoantibodies One patient cohort consists of patients from the University of Florida (US), where samples (n=2000) were obtained from the Department of Pathology and tested for anti RR antibodies via indirect immunofluorescence (IIF) using HEp 2 slides (INOVA Diagnostics, San Diego, CA). US patients were classified as non responders (NR) or sustained vir ological response (SVR) according to the following criteria: NR show no decrease in viral load after completion of 24 months of treatment, while S VR have no viral RNA detection 6 months after completion of treatment. The second cohort consists of 48 sera prescreened positive for anti RR from an Italian cohort, obtained from three h ospital c linics in north east ern Italy. Serum samples were select ed a mong patients with HCV infection and those that displayed a positive anti RR pattern on HEp 2 cells. Th ese samples were screened by IIF again for the presence of anti RR antibodies using HEp 2 slides as described in the next paragraph, and 46 were confirme d as positive. Italian patients were classified as NR or SVR and an additional category of relapsers was included, to indicate previously SVR patients who tested positive for HCV RNA after 6 months of IFN/R treatment. Indirect Immunofluorescence (IIF) IIF analysis of autoantibodies in human sera was performed as described, using commercial HEp 2 ANA slides (INOVA Diagnostics, San Diego, CA) ( 122 ) To determine anti RR titer, samples were 2 fold serially dilute d in PBS containing 0.1% bovine serum albumin, with a starting dilution of 1:50 and ending dilution of 1:819,200. Titer analysis for anti RR was determined by two readers (WCC and SJC) and endpoint titer was defined by more than 50% of cells with detectabl e RR staining. Fluorescent

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66 images were captured with a Zeiss Axiovert 200M microscope fitted with a Zeiss AxioCam MRm camera using a 40x (0.75 NA) objective. The majority of anti RR positive samples had titer below 1:100,000. Titers over 1:100,000 were det ermined with the aid of a Zeiss AxioCam MRm camera. Immunoprecipitation (IP) IP of lysate from [ 35 S] methionine labeled K562 cells for the analysis of the proteins recognized by human autoimmune sera was performed as previously described ( 67 122 ) Statistical Analysis The statistical analysis was performed using the GraphPad Prism 5 software for Windows (La Jolla, CA). The one way analysis of variance (ANOVA) T test Mann Wh itney Test A p value of < 0.05 was considered statistically significant. Results and Discussion Autoantibodies to Cytoplasmi c Rods and Rings in the US and Italian Cohorts HCV patients from both US and Italian cohort s were confirmed to have antibodies to rods and rings using INOVA HEp 2 slides at 1:50 dilution of sera IIF staining of HEp 2 cells typically showed 1 to 2 distinct cytoplasmic rods (~3 rings (2 meter, Figure 1A ). Both US and Italian patients recognize the same RR as determined by costaining using a rabbit anti IMPDH2 antibody (data not shown). It was previously demonstrated that IMPDH 2 is a component of RR and rabbit anti IMPDH2 antibodies costain RR detected by sera of HCV patients ( 122 ) This confirms that the structures recognized by Italian and US patients are the same. Determination of the ultrahigh anti RR titers was accomplished with the aid of the Zeiss AxioCam

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67 camera, otherwise RR were visualized by eye at titers as high as 1:102,400. Figure 1 A C shows representative anti RR staining from HCV patient with titer at 1:400, 1:102,40 0, and 1:819,200, respectively. The highest dilution used was 1:819,200, and three patients in each cohort had titers higher than this upper limit. The US cohort had titers that ranged from 1:50 to >1:819,200 with a median titer of 1:1,200 while the Itali an cohort had titers that ranged from 1:200 to >1:819,200 with a median titer of 1:25,600 (Figure 1D) In the Italian cohort, a total of 18 females and 28 males had anti RR. Although the anti RR titer was slightly higher in females it was not significantly different when compared to anti RR titer in males. However, IMPDH2 IP intensity was significantly higher in females versus males (p=0.049). In the Italian cohort, the age range was 36 75 years with a median of 59.5. It is unclear why there is a big differ ence in the median titers between the two cohorts. Prevalence of A nti IMPDH2 Antibody by IP It was previously shown that IMPDH2 is a component of RR and some anti RR recognize IMPDH2 as autoantigen ( 122 ) altho ugh the prevalence of anti IMPDH2 has not been determined. Therefore, sera from the two cohorts were tested by IP for the prevalence of anti 55kDa IMPDH2. Figure 2 shows representative IP of 35 S methionine labeled K562 cell lysate using selected patients from both US and Italian cohorts. Not all patients immunoprecipitated IMPDH2 (arrow in Figure 2A,B). In the Italian cohort, 44 anti RR positive patients (9 6 %) immunoprecipitated IMPDH2 and only 2 patients with anti RR antibodies did not have the 55kDa band previously shown to be IMPDH2 ( 122 ) In addition, none of the patients without anti RR antibodies immunoprecipitated the IMPDH2 band (data not shown). However, in the US cohort, only 25 anti RR positive pati ents (53%) had the IMPDH2 band, while the other 22 anti RR positive patients did

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68 not immunoprecipitate the IMPDH2 band. Other antigens recognized by sera that are anti IMPDH2 negative but anti RR positive remain yet to be identified. Figure 2C shows the n umber of patients who were classified by IMPDH2 band intensity from 0 to 4 (weakest to strongest). Patients in the Italian cohort immunoprecipitate IMPDH2 with stronger intensity compared to patients in the US cohort. This difference may be due to the het erogeneity of the patients in the US cohort versus the Italian cohort ; IMPDH2 is the predominant RR autoantigen in the Italian cohort while the US cohort develops anti RR antibodies that may recognize additional components apart from IMPDH2, such as CTPS1, a previously identified component of RR, or other unknown components. Comparison of IIF Titer Versus Anti IMPDH2 IP Anti RR titer evaluated by IIF was correlated to the intensity of the IMPDH2 band in IP for both cohorts (Figure 3). Patients in the US an d Italian cohort with higher anti RR titers immunoprecipitated the IMPDH2 bands with higher intensity (p <0.0001 and 0.0034, respectively). This strong correlation validates the finding that anti 55kDa IMPDH2 is the predominant autoantibody in the anti RR response in these patients. This is clearly true in the Italian cohort with 9 6 % anti RR positive for the 55kDa IMPDH2 band. It is interesting that a significantly lower percent is seen in the US cohort. It remains to be determined what is the autoantige nic component(s) undetected in IP with those 47% who are negative for anti IMPDH2. C orrelation of A nti RR /IMPDH2 Prevalence and Titer with Therapeutic Outcome The aim of our study was to determine whether the production and titer of anti RR antibodies ar e different between US versus Italian patients and whether they are associated with the response to IFN/R therapy. A correlation was observed between anti RR titers and response to treatment in the US cohort, and in fact patients classified

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69 as NR had sign ificantly higher anti RR titers compared to SVR (mean=1:3,000 vs 1:100; p=0.0016, Fig. 4A). Patients in the Italian cohort were classified into three categories (Fig. 4B) and NR patients had lower titers than SVR, while relapsers had significantly higher t iter when compared to NR and SVR (p=0.0040 and p=0.015, respectively, Fig. 4B). When the intensity of the IMPDH2 band was compared between NR versus SVR in the US cohort, 8 out of 9 patients with strong (3+4+) IP band intensity were non responders (Fig. 4 C). For patients in the Italian cohort, relapsers had a significantly stronger 55 kDa band IP intensity compared to SVR (p = 0.031) or NR (p = 0.016) patients (Fig. 4D). The differences between the two cohorts could be due to the heterogeneity of the pat ients and to the different classification of patients for the US cohort (divided into two main groups, NR and SVR) and for the Italian cohort (divided instead in 3 groups: NR, SVR and relapsers). Covini et al demonstrated that anti RR antibodies were spec ific to HCV patients that had received the IFN/R therapy, and anti RR antibodies were more often detected in non responders/relapsers than in responder patients (33% vs 11%, p=0.037) ( 139 ) Although they observed the difference in prevalence of anti RR antibodies, they did not examine anti RR titers An additional recent study by Seelig et al on anti RR antibodies did not examine correlatio ns of antibodies with clinical and therapeutical response ( 140 ) The mechanism for the production of anti RR antibodies remain s unknown but we speculate that a subset of patients develop anti RR antibodies due to exposure of turnover of RR structures induced by RBV during IFN/R therapy. Pileri et al proposed that HCV binds to CD81 on B lymphocytes through its envelope protein E 2, thus lowering the activation threshold on the B cells and facilitating the

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70 production of autoantibodies ( 97 ) B cell activation in addition to RR formation by ribavirin may play a role in the development of anti RR antibodies. Conclusions Our results show for the first time that anti RR antibody titer has a strong correlation to treatment response from two independent HCV patient cohorts in the US and Italy. In terestingly, in the Italian cohort 9 6 % of anti RR sera identified by IIF immunoprecipitate the IMPDH2 band, whereas in the US cohort, only 53% anti RR positive by IIF showed the IMPDH2 IP band. Patients with high titer anti RR antibodies tend to be non res ponders or relapsers. Future studies are needed to examine the environmental or genetic element(s) that might contribute to the formation of anti RR against IMPDH2, and their potential application in HCV follow up.

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71 Figure 4 1. Antibody titer determine d in two cohorts of HCV patients with 2 ANA slide stained with HCV patient sera at various dilutions Representative images of rods and rings (RR, green) from sera diluted at 1:400 A), 1 :102,400 B), and 1:819,200 C) dilution. Nuclei were counterstained with DAPI (blue). Scale bar, 10 m D) Anti RR titers of US and Italian cohorts determined using 2 fold dilution of sera on HEp 2 ANA slides. Each dot represents a single positive anti RR p atient; patients that were negative for anti RR antibodies are not represented.

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72 Figure 4 2. Prevalence of anti 55kDa IMPDH2 in immunoprecipitation analysis of anti RR positive sera. Immunoprecipitation of 35 S methionine labeled K562 cell extract was performed with all anti RR positive patient sera from A) US and B) Italian cohorts. Numbers correspond to individual samples. The 55kDa band corresponding to IMPDH2 is indicated by arrows. C) Semi quantitative analysis of all patients from both cohorts, me asuring the intensity of the 55kDa band detected in IP using a scale from 0 (negative) to 4 (strongest). NHS, normal human serum.

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73 Figure 4 3. Correlation of anti RR IIF titer and anti 55kDa IMPDH2 IP intensity. The intensity of the 55kDa IP band was correlated to anti RR titer for each patient, as show n in A) for the US cohort and B) for the Italian cohort. Each dot represents one individual. Lines in each graph correspond to the median.

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74 Figure 4 4. Correlation of anti RR antibodies and 55 kDa b and IP intensity to IFN/R treatment outcome. Patients were divided according to their respon se to treatment. The US cohort A, C) was divided into two groups, NR and SVR, while the Italian cohort B,D) was divided into NR, SVR and relapsers. The intensity of the 55kDa IP band was visually graded from 0 (negative) to 4 (strongest). Lines in each graph represent the median. NR, non responders; SVR sustained virological responders; relapsers, responsive to therapy but after 6 months HCV RNA was detectable.

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75 CHAP TER 5 G LUTAMINE DEPRIVATION INITIATES REVERSIBLE ASSEMBLY OF CYTOPLASMIC ROD S AND RING S Background Cytoplasmic structures referred to as rods and rings (RR) were identified by indirect immunofluorescence with human autoantibodies in cancer cell lines and m ouse embryonic stem cells ( 122 ) Co staining experiments demonstrated that rods and rings are not part of nor do they colocalize with the Golgi complex or other known organelles. Although RR are filament like structures, there are not enriched in actin, tubulin or vimentin. Two proteins, cytidine triphosphate synthetase (CTPS) and inosine monophosphate dehydrogenase (IMPDH) have been identified as components of RR. Two CTPS1 inhibitors, 6 diazo 5 oxo L norleu cine (DON) and acivicin, and two IMPDH2 inhibitors, ribavirin and mycophenolic acid, induce RR formation which is dependent on concentration and treatment time of the respective inhibitors ( 122 ) T o date, t he four chemical inhibitors that can induce RR assembly share a common metabolic repression mechanism involving the inhibition of enzymes associated with the nucleotide biosynthesis pathway. CTPS1 and IMPDH2 are enzymes involved in the pyrimidine and purine biosynthesis pathways, respectively. CTPS1 is the rate limiting enzyme and catalyzes the conversion of uridine triphosphate into cytidine triphosphate (CTP), a key precursor of DNA, RNA, and phospholipids, with the use of glutamine as a nitrogen provider. CTPS exists as two isoforms, CTPS1 and CTPS2, with 74% identity in humans. CTPS1 is a 591 amino acid protein with a molecular weight of 67 kDa; it is composed of an N terminal synthetase domain and a C terminal glutaminase domain. Ammonium is cleaved off of glutamine by the glutaminase domain to form glutamate. T he synthetase

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76 domain uses the ammonium ion to generate CTP from ATP phosphorylated UTP. CTP synthetase activity is affected by all four ribonucleoside triphosphates in vitro ( 141 ) CTP acts as a feedback inhibitor by binding to the synthetase domain ( 142 143 ) while GTP is an allosteric activator ( 144 ) IMPDH2 is the committing enzyme involve d in the purine biosynthesis pathway and catalyz es the conversion of inosine monophosphate into xanthine monophosphate, which is subsequently converted to guanine monophosphate (GMP) ( 145 ) IMPDH is a key enzyme in the regulation of cell proliferation and differentiation ( 146 147 ) Human IMPDH also has two isoforms, IMPDH1 and IMPDH2, with 84% sequence identity and a molecular weight of 55 kDa. IMPDH activity can be affected by way of I MP site inhibitors, NAD site inhibitors, or allosteric site inhibitors ( 148 ) Glutamine is the most abundant free amino acid in the body and pl ays a role in many cell specific processes, such as metabolism, protein synthesis, and cell proliferation. Glutamine is also involved in changes in protein activity, gene and protein expression, and intracellular metabolite concentrations ( 149 ) Extracellular administration of glutamine can stimulate purine and pyrimidine synthesis. The first step in the de novo synthesis of purines is the conversion of ribose 5 pho sphate to glutamine phosphorybosylpyrophosphate by amidophosphorybosyltransferase. Glutamine, carbon dioxide, and ATP are required for the production of carbamyl phosphate in the de novo synthesis of pyrimidines ( 150 ) Extracellular glutamine level of 0.7 mM i s a requirement that is met by a large majority of standard cell culture media, as most common formulations contain between 1.0 and 4.5 mM ( 151 )

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77 Previous re ports identified CTPS1 and IMPDH2 as components of RR and that four inhibitors could induce the formation of RR in various cell lines ( 122 ) The current report investigates the effects of glutamine deprivat ion on RR assembly in human cancer cells. Materials and M ethods Cell Culture HeLa cells (human cervical cancer ATCC, Manassas, VA ) were cultured in DMEM containing 10% Fetal Bovine Serum (FBS) in a 37C incubator with 5% CO 2 Adherent cell lines were maintai ned at 50% confluence. All medium contained 100 I.U./ml penicillin and 100 g/ml streptomycin. Indirect Immunofluorescence HeLa cells were cultured in 8 well chamber slides (BD Falcon) and were used for indirect immunofluorescence as described ( 68 69 ) The 8 well chamber slides were prepared using ~50,000 cells per well in 500 L of medium Fixation method was 3% paraformaldehyde in PBS at room temperature for 10 min followed by 0. 5 % triton X/PBS for another 5 min. For co staining studies rabbit anti IMPDH2 (Proteintech, Chicago, IL: 12948 1 AP) and human anti RR prototype serum It2006 were used and followed by secondary antibodies Alexa 488 goat anti human Ig and Alexa 568 goat anti rabbit Ig. Induction o f RR Assembly by Glutamine Deprivation HeLa cells were cultured in 8 well chamber slides exactly as described above except in medium without glutamine (DMEM, Cellgro; 15 013 Cl). Glutamine minus medium was replaced each day for 2 day s. On the third day cells were either harvest ed or supplemented with L glutamine (Cellgro: 25 005 Cl), cytosine (Sigma Aldrich;

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78 C3506), guanosine (Sigma Aldrich; G6264) or thymidine (Sigma Aldrich; T1895) at designated concentrations ranging from 0.1 to 1 25 mM for another 24 h Induction of RR Formation Using CTPS or IMPDH I nhibitors Mycophenolic acid (MPA; Sigma Aldrich; M5255 ) was solubilized in ethanol to a 31 mM s tock concentration 6 diazo 5 oxo L norleucine (DON, Sigma Aldrich; D2141) was solubilized in DMEM to a stock concentration of 100 mM. Cells were seeded as monolayers and allowed to attach for 24 h. CTPS1 or IMPDH2 inhibitors were then added in various final concentrations ranging from 0.2 M to 2 mM and cells were kept for an additional 24 h. Induction of RR Formation Using Glutamine Transport and Glutamine Synthetase Inhibitors Cells were maintained in glutamine free medium and treated with methionine L glutamyl p nitroanilide (Fisher Scientific ; BP2656 ). Methio nine sulfoximide was solubilized in warm water to a 100mM stock concentration. L glutamyl p nitroanilide was solubilized in water to a 10mM stock concentration. Cells were treated for 24 h with either inhibitor. qRT PCR Total RNA samples were harvested u sing the mirVana T otal RNA I solation K it (Applied Biosystems) after 48 h in culture or at specific time point indicated Reverse transcription was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The relative mRNA leve ls of target genes were measured in duplicate s using TaqMan Fast Universal Master Mix (Applied Biosystems) with the corresponding TaqMan Gene Expression Assay (Applied Biosystems). GAPDH was used as an internal control and was run in duplex with the target s.

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79 Western B lotting The expression levels of CTPS and IMPDH were analyzed using rabbit anti CTPS (Abcam, 1:1000) and rabbit anti IMPDH (Santa Cruz Biotechnology, Paso Robles, CA, 1:1000) as described ( 72 ) The level of GAPDH was determined by r abbit anti GAPDH antibody ( Sigma, 1:2,000 ) as a normalizer to control for loading. Protein was quantitated using ImageJ and normalized to GAPDH. Quantification and Statistical A nalysis Cell counting was performed using Mayachitra Imago (Mayachitra, Santa Barbara, CA) to detect nuclei counterstained with DAPI. Quantification of the size of RR was accomplished using the threshold feature in Mayachitra Imago software. The intensity threshold was set to 50 (detects RR with total intensity pixels higher than 50) for the analysis of images. The size of the structures was defined by their area in pixels. Unpaired, two independent groups. Prism for Windows version 5.0 (GraphPad Software, San Diego, CA) was used for all statistical analysis Results Glutamine Deprivation Induces Rod a nd Ring Formation RR were previously identified as cytoplasmic structures composed of CTP S1 and IMPDH2 after using CTPS1 or IMPDH2 inhibitors ( 122 ) CTPS1 and IMPDH2 are involved in the purine and pyrimidine biosynthesis pathways, which both utilize glutamine extensively ( 57 ) In the pyrimidine biosynthesis pathway, CTPS1 utilizes glutamine to convert UTP into CTP. Due to the importance of glutamine in these pathways, our notion was to examine whether cells deprived of glutamine or serum can also lead to the formation of RR. Cells were cultured with or without glutamine at

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80 various concentrations of FBS: 10%, 5%, 1%, 0.1% and 0%. C ells maintained in medium containing 10% FBS but deprived of glutamine for 2 da ys form ed RR in an average of 48.7 % of cells after 2 days of starvation (Figure 5 1A) RR detect ion was confirmed by costaining using a rabbit anti IMPDH2 antibody and the prototype human anti RR ser um It2006 as previously described ( 122 ) In contrast, cells cultured in normal medium with glutamine showed diffused cytoplasmic staining and RR were not detected (Figure 5 1C). Cells cultured in 1% FBS without glutamine had RR in 10.7 % of cells (Figure 5 1B) and si gnificant cell death was noted In culture medium containing glutamine, a decrease in FBS from 10% (Figure 5 1C) to 1% induced only few RR (Figure 5 1D). When FBS concentration was lower ed than 1% there were too much cell death and no RR was detected Cha nging g lutamine levels from 0.5 to 15 mM was previously reported to have an effect on protein and gene expression ( 152 ) Messenger RNA (mRNA) levels of CTPS1 and I MPDH2 were elevated after cells were deprived of glutamine in the presence of 10% FBS when compared to cells maintained in medium with 10% FBS and glutamine (Figure 5 1E). In contrast to the ~2 fold increase in mRNA only a small increase was observed in C TPS1 and 56% increase in IMPDH2 protein levels in cells deprived of glutamine compared to cells in normal medium (Figure 5 1F). This data suggest that the formation of RR enriched in IMPDH2 is primarily resulted from the assembly of soluble cytoplasmic e nzyme pool and at the same time, there is increase synthesis of intracellular IMPDH2. The assembly of cytoplasmic RR in response to glutamine deprivation was further examined in cells treated for 1, 2, and 3 days ( Figure 5 2 ). RR were not detected in c ells maintained in medium containing glutamine A diffused cytoplasmic staining was

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81 observed when co stained with human serum It2006 and rabbit anti IMPDH2 antibodies (Figur e 5 2A C). H owever, when the medium was replaced with medium deprived in glutamine RR formation was observed clearly at day 2 (Figure 5 2G I) At day 1 short structures that resemble rods were detectable in the cytoplasm (Figure 5 2D F); these short rods were still detected among the long rods and rings at day 2 (Figure 5 2G I) At day 3 the majority of short rods disappeared and predominantly mature long RR remained (Figure 5 2J L) Counting of short and long RR showed the reduction of the short structures from 68.5% in day 1 to 10.1% at day 3 (Figure 5 2M). This is consistent with the h ypothesis that mature RR in later time points are assembled from shorter structures from earlier time points. Since RR formation was almost complete after 2 days of glutamine deprivation, all subsequent experiments were conducted with 2 days of glutamine d eprivation. Glutamine Add Back Disassembles RR Induced in Response t o Glutamine Starvation RR form ed in response to glutamine deprivation for 48 hours disassembled with in 24 h after the addition of 2.5 mM glutamine ( Figure 5 3 ). C ells with glutamine add ba ck had diffuse d cytoplasmic staining (Figure 5 3G I), as observed in cells in normal medium with glutamine (Figure 5 3A C). Normal culture medium containing 4 mM glutamine; therefore, the addition of 2.5 mM glutamine was below the normal concentration. Add ition of glutamine at concentrations as high as 125 mM all led to the disassembly of RR (data not shown). Addition of similar amount glutamine to cells maintained in normal culture medium did not lead to RR formation ( d ata not shown). It is i nteresting tha t the increase in CTPS1 and IMPDH2 mRNA levels in cells depleted of glutamine was reduced back to normal in cells that were given the glutamine add back

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82 (Figure 5 3 J). Since the RR induced in response to glutamine deprivation was co stained human prototype anti RR serum these RR are likely identical to previously reported RR induced by IMPDH2/CTPS inhibitors ( 122 ) Supplement of Guanosine or Cytosine Induces the Disassembly of RR Formed in Response to Glutami ne Starvation Glutamine is involved in several pathways, and its requirement in the purine and pyrimidine biosynthesis pathways is notable for this discussion T he steps in the se pathways that utilize glutamine are shown in Figure 5 4 A Glutamine is requir ed in the first and fourth step of the de novo pathway of purine biosynthesis. In addition, glutamine is also required in the second step of the synthesis of GMP from IMP. In the pyrimidine biosynthesis pathway glutamine is utilized by CTPS1 in the conver sion of UTP to CTP. Thus, depletion of glutamine prevents the formation of CTP and GTP. Intracellular levels of CTP and GTP can be increased by adding cytosine and guanosine, respectively to culture medium ( 153 154 ) RR induced in response to glutamine deprivation disassembled in a concentration dependent manner with the addition of cytosine and guanosine. H eLa cells that were deprived of glutamine for 48 h showed abundant RR (Figure 5 4B). Addition of 1 mM guanosine completely disassembled RR induced by glutamine deprivation (Figure 5 4C), while 1 mM of cytosine partially disassembly of RR into short cytopla smic punctuates (Figure 5 4D) A ddition of guanosine and cytosine at 0.1 mM showed no apparent change in RR (data not shown). Thymidine which is converted into thymidine triphosphate did not affect the RR induced by glutamine deprivation ( Figure 5 4E ).

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83 M PA Induced RR Disassemble After R eplenishing w ith Guanosine Mycophenolic acid ( MPA ) is a reversible inhibitor of IMPDH2 that can induce RR formation in a concentr ation dependent manner (Figure 5 5 A). MPA can induce RR in as little as 30 minutes and 2 M co ncentrations Addition of guanosine to cells treated with MPA for 24 h led to a decrease the siz e of RR induced by MPA (Figure 5 5 B). However, MPA induced RR in cells treated with glutamine or cytosine were not affected (Figure 5 5 C D). RR induced by MPA h ad longer RR and cells contained 1 or more long RR compared to RR disassembled after addition of guanosine (Figure 5 5E G; p value=0.0001) Longer rods were considered to have more than 70 px. Guanosine treated cells had on average 2 or more short rods per cell and only 1 out of 10 cells contained a long RR. In this analysis, rings were quantitated identical to linearized rods. I nhibition of Glutamine Synthetase a nd Glutamine Transport Induce RR Formation Intracellular level of glutamine is a summation from extracellular supplement glutamine, endogenous de novo synthesis, and the efficiency of transport from extracellular source. The next experiment was to determine whether inhibitors for de novo glutamine synthesis and glutamine transport would affect the f ormation of RR. DON functions as an inhibitor of CTPS1 and glutamine synthetase. It was previously shown that DON can induce RR formation in a concentration dependent manner ( 122 ) As expected, treatment with 1 mM of DON for 24 h induced RR formation (Figure 5 6 B). M ethionine sulfoximi n e, a glutamine synthetase specific inhibitor wa s used to determine if inhibition of glutamine synthetase could induce RR assembly. RR were induced with 1 mM methionine sulfoximi ne (Figure 5 6 C). Affecting intracellular

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84 glutamine levels by inhibiting glutamine transporter solute carrier family 1 member 5 in HeLa cells using 1 mM L glutamyl p nitroanilide induce d RR assembly (Figure 5 6 D). Discussion Previous studies on the formation of cytoplasmic structures composed of CTPS in yeast determined that nutrient deprivation could induce the formation of CTPS filaments. It was determ ined that glucose depletion is a potent inducer of CTPS filaments in yeast ( 83 ) Another study observed the formation of cytoophidia, also RR like filaments compos ed of CTPS, induced in human and Drosophila cells with glutamine analogs; such as DON or azaserine ( 155 ) Both DON and azaserine inhibit enzymes that require glutamine. The study in yeast supports that RR in our study and filament formation in yeast is in response to decrease in necessary factors for cellular division or proliferation. Additionally glucose deprivation in hamster fibroblast affected the levels (pool sizes) of intra cellular of UTP, GTP and CTP ( 156 ) Therefore, glucose deprivation and glutamine deprivation may both decrease in CTP and GTP causing formation of filaments in yeast and RR in HeLa cells. The u se of glutamine analogs to induce cytoophidia in Drosophila may suggest the importance of glutamine in preventing formation of cytoophidia or RR in HeLa cells. Glutamine serves many roles in different cellular process es and is essential for cell viability and growth ( 157 ) In this study, RR are formed in response to glutamine starvation. Glutamine starvation requires a minimum of 24 h to visualize the formation of short rods. Although visibly shorter (or immature) rods were observed at 24 h, these structures continue to aggregate and reach full size by 72 h after starvation is initiated. It is easy to visualize this progression of the structure: at 24 hours, a diffu sed cytoplasmic staining in conjunction with short, immature rods shows the beginning stages of

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85 aggregation. At 48 hours, there is notably less diffused staining, as more protein has been assimilated into the maturing RR. At 72 hours, when fully mature RR are formed, there is almost no diffused cytoplasmic staining visible, suggesting that all remaining IMPDH2 is incorporated into RR. Glutamine deprivation prevents the formation of CTP and GTP by affecting enzymes in the purine and pyrimidine biosynthesis pathways ( 149 158 ) RR formation is therefore in response to decreased intracellular levels of CTP and GTP. This notion is supported by the addition of extracellular guanosine and cytosine, converted into CTP and GTP after transport and entry into cells ( 153 154 ) in the present study could reverse the process as demonstrated by RR disassembly when cells are replenished with guanosine and cytosine. Thus, the assembly for RR from the polymerizing of IMPDH2 (and probably CTPS1 ) appears to be in response to decreased production of CTP and GTP after glutamine deprivation or by inhibition of enzymatic activity of CTPS1 or IMPDH2. Additionally, the requirement of glutamine in the induction of RR was supported by data from the glutamine synthetase inhibitors. The observed induction of RR by DON and methionine sulfoximide is therefore consistent with notion that decrease intracellular levels of glutamine cause RR assembly. A previous study also showed that MPA induced RR like filaments were disassembled by incubating cells with guanosine ( 59 ) Our data confirmed this finding with MPA induced RR disassembled when supplemented with guanosine but not with glutamine or cytosine. MPA inhibits IMPDH2 enzymatic activity, which prevents the formation of GTP. Replenishing GTP by the addition of guanosine after IMPDH2 inhibition can disassemble RR, while replenishing CTP levels after inhibiting IMPD H2

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86 did not. Additional data confirms that RR also form in response to changes in CTP levels caused by glutamine deprivation and that these structures can be disassembled with guanosine, cytosine or glutamine. Therefore, changes GTP and CTP levels can induc e RR formation. Genes that mediate the synthesis of necessary metabolites are tightly regulated by various cellular mechanisms. These cellular mechanisms must respond to internal and external factors in order to respond to cellular needs. RR may be a struc tural mechanism that forms in response to decreased glutamine, CTP, or GTP levels. This study demonstrates that restoring glutamine levels after glutamine deprivation can disassemble cytoplasmic RR. RR may also serve to regulate IMPDH and CTPS mRNA and pro tein levels in response to nutrient stresses.

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87 Figure 5 1. Glutamine deprivation induces rods and rings. HeLa cells maintained in DMEM medium with 10% FBS without glutamine A), DMEM medium with 1% FBS without glutamin e B) DMEM medium with 10% FBS C ), a nd DMEM medium with 1% FBS D) for 48 h were stained with rabbit anti IMPDH2 (red). Nuclei were counterstained with DAPI (blue). The average percentage of cells with RR are displayed in the lower right corner, with total number of cells counted in parenthes is. E) mRNA levels of CTPS1 and IMPDH2 are significantly elevated in cells depleted of glutamine. mRNA le vels were normalized to GAPDH. F) Western blot of CTPS1 and IMPDH2 protein levels using GAPDH as a loading control in HeLa cells maintained in 10% FBS medium with glutamine or without glutamine. There was an increase (156.4%) in IMPDH2 protein levels after glutamine deprivation in 10% FBS. Data are from three independent experiments (A D, mean; E, mean s.d.). *P < 0.05; (two tailed unpaired t test) com pared with cells maintained in DMEM medium with 10% FBS. Scale bar, 5

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88 Figure 5 2 Longitudinal analysis of RR formation during glutamine starvation. HeLa cells were maintained in n ormal DMEM medium with 10% FBS A C), or in glutamine depleted mediu m with 10% FBS for 1 day D F), 2 days G I), and 3 day s J L). Cells were co stained with human anti RR serum It2006 (green; C, F, I, L) and rabbit anti IMPDH2 (red; B, E, H, K). Merged images are shown in A, D, G, and J. Nuclei were counterstained with DAPI (blue). (M) Summary of the percentage of cells with short RR alone, short and long RR, and long RR alone for each day of glutamine starvation and control. Total number of cells counted are shown in parentheses.

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89 Figure 5 3. Glutamine add back disasse mbles RR formed in response to glutamine starvation. A C) RR were not detected in HeLa cells maintained in no rmal DMEM medium with 10% FBS. D F) RR formed in cells maintained in gluta mine deprived medium for 48 h. G I) Glutamine deprivation induced RR disa ssembled when cells were supplemented with 2.5 mM glutamine for 24 h. Scale bar, 5 J) mRNA levels of CTPS1 and IMPDH2 were elevated in glutamine deprived cells and returned to normal in cells supplemented with 2.5 mM glutamine. mRNA levels were normalized to GAPDH.

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90 Figure 5 4. Supplement of guanosine or cytosine induces the di sassembly of RR formed in res ponse to glutamine starvation. A) Purine and pyrimidine biosynthesis pathways with RR components shown in red and steps requiring glutamine in blue. IMPDH2 is an enzyme involved in the GTP biosynthesis pathway catalyzing the co nversion of IMP to GMP, which is a precursor to the production of GTP. CTPS1 is an enzyme involved in the CTP biosynthesis pathway and catalyzes the conversion of UTP to CTP. Arrows and numbers signify steps in the pathway. RR are detected in HeLa cells ma intained in medium with 10 % FBS minus glutamine for 48 h B ). Glutamine deprived cells are treated with 1 mM guanosine C ), 1 mM cytosine D), or 1 mM thymidine for 24 h E) The pathway schematic is adapted from Hofer et al. ( 57 )

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91 Figure 5 5. MPA induced RR disassemble after replenishing with guanosine. HeLa cells were treated with 1 mM MPA f or 24 h to induce RR formation A). Replicate wells we re treated with 1 mM guanosine B), 2.5 mM g lutamine C), or 1 mM cytosine (px) were quantitated by Mayachitra Imago and plotted for each condition (E, >300 cells analyzed for each). Px values for typical RR structure s are shown F). By defining short ro ds (S) as <70 px and long rods L) as >70 px, the number of short and long RR structures a re plotted for each conditions G).

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92 Figure 5 6. Inhibition of glutamine synthetase and glutamine transport induce RR formation. RR were not detected in HeLa cells maintain ed in DMEM medium with 10% FBS A). HeLa ce lls were treated with 1 mM DON B ), 1 mM methionine sulfoximine L glutamyl p nitroanilide D) for 24 hours induced RR formation. Scale bar, 5

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93 CHAPTER 6 COMPARISON OF RODS A ND RINGS TO CYTOPLAS MIC STRUCTURES COMPOSED OF CTPS OR IMPDH Rods and Rings in t he Cytoplasm We have recently reported interesting cytoplasmic structures, referred to as rods and rings (RR Figure 6 1), detected by using autoantibodies in specific patient sera ( 122 ) Many of the patients have hepatitis C (HCV) infection, and are producin g anti RR autoantibodies after treatment with a standard combination therapy of pegylated interferon and ribavirin ( 139 ) These structures contain cytidine triphosphate synthetase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2), proteins of 67kDa and 55kDa respectively ( 122 ) Staining by sera from HCV patients and rabbit anti IMPDH antibodies show s perfect colocalization to RR structures. CTPS inhibitors, 6 diazo 5 oxo L norleucine (DON) and acivicin, as well as the IMPDH2 inhibitor ribavirin induce formation of RR in a dose dependent manner. The i nduction of RR is confirmed in all c ancer cell lines and primary cells examined Interestingly, RR are visualized in undifferentiated mouse embryonic stem cells (ESCs) without the need of adding inhibitors, and were disassembled after differentiation with retinoic acid ( 122 ) Since RR are found in ESCs without the use of inhibitors, RR expression may be a very interesting biomarker for ESCs. Various other publications have described novel cytoplasmic filament like structures that are composed of enzymes involved in either cytidine triphosphate (CTP) or guanosine triphosphate (GTP) nucleotide biosynthesis. Here, we review all the relevant findings which allow us to speculate the main function of RR in enhanc ing production of CTP/GTP when their cell ular levels are lowered and the formation of RR is thus sensitive to lowered levels of GTP/CTP.

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94 Nucleotide Biosynthesi s Associated w ith RR The observation that two known components in RR are CTPS1 and IMPDH2 implicates these structures in CTP and GTP biosy nthesis. CTP and GTP are essential building blocks of DNA and RNA and are involved in a multitude of pathways in both prokaryotes and eukaryotes. They serve as energy carriers, participate in cellular signaling, and act as important cofactors in enzymatic reactions. Nucleotide biosynthesis can occur via de novo pathways or the salvage pathway and is tightly regulated by the cell. CTPS is a glutamine amido transferase enzyme that catalyzes the conversion of uridine triphosphate (UTP) to CTP and is a key enz yme in nucleic acid and phospholipid biosynthesis (Figure 6 2). Two human isoforms, CTPS1 and CTPS2, exist and are 74% identical at the amino acid level ( 56 ) The re are two CTPS isozymes in Saccharomyces cerevisiae URA7 and URA8, which correspond to the mammalian CTPS1 and CTPS2, respectively. The enzyme consists of the N terminal synthetase domain and the C terminal glutaminase domain. The latter catalyzes the de amination of glutamine. CTPS is an established target for antiviral, antineoplastic, and antiparasitic therapy ( 57 159 160 ) IMPDH is also tightly regulated and catalyzes the conversion of IMP into monophosphate, which is the rate limiting step in the de novo synthesis of guanine nucleo tides ( 146 161 ) IMPDH exists as two 55kDa isoforms IMPDHI and IMPDH2, with 84% amino acid sequence identity, and form s independent tetramers when active ( 162 ) IMPDH is an important regulator of cell proliferation and is a known target in the treatment of neoplastic and viral diseases. Mycophenolic acid (MPA), an IMPDH inhibitor, has been used as an immunosuppressive agent for organ transplants

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95 and autoimmune diseases ( 163 165 ) Another IMPDH inhibitor, ribavirin, is currently being used in the treatment of HCV ( 63 166 ) Assembly of Protein s for a Cellular Function Assembly of intercellular protein complexes can serve important functional purposes. GW Bodies is a cytoplasmic structure, assembled from miRNA/mRNA protein complexes, that plays a role in RNA interference and mRNA degradation ( 121 167 ) Previous studies have also identified purinosomes and U bodies as f unctional cytoplasmic structures ( 168 169 ) Various studies show that inhibition of certain enzyme activity by chemical inhibitors, or by altering nutrie nt levels, can induce formation of other filaments. Acetyl CoA involved in fatty acid synthesis has been shown to assemble into filament s in mammalian cells and yeast in response to citrate ( 170 171 ) Acetyl CoA filaments as well as glutamine synthetase filaments in yeast show enzyme activity and nutrient starvation play a role in filament polymerization ( 172 174 ) Nutrient deprivation plays a similar role in reducing nucleotide levels compared to using the nucleotide biosynthetic enzyme inhibitors as descr ibed above and, therefore, depleting nucleotide levels may be a direct cause for the formation of RR. Since CTPS1 and IMPDH2 are key RR components, it is speculated that formation of RR help s increase cellular production of GTP and CTP. Rods /Rings Related Filament Like Structures Upon scanning the literature, it appears that several reports have come across similar RR like structures. The earliest report is the work by Willingham et al. in 1987, who described a novel cytoplasmic filament named as nematin, practically identical to rods shown in Figure 6 1 ( 81 ) The structures were observed by immunofluorescence staining using a monoclonal antibody, referred to as anti nematin. The monoclonal

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96 antibody was derived from a BALB/c mous e that was immunized with SR BALB 3T3 cells and thus can be classified as an autoantibody as it was later shown positive for staining of mouse 3T3 cells. Most cells exhibited only one of these structures, but occasionally more than one were present. Some s tructures took on a distinct donut shaped structure, which appears indistinguishable from the ring shown in Figure 6 1. Through colocalization studies, the filaments were identified to be independent of tubulin or vimentin. The presence of these filaments was examined in nine different mammalian cell lines and they determined that rat and mouse cell s expressed the nematin structure at a higher frequency, while chick embryo cells contained virtually no such structures. Fixation methods affected the stability of the structures, with formaldehyde acting as the best fixative. Through the use of light and electron microscopy and microinjection, they identified this filament as a novel cytoplasmic structure without a lipid bilayer membrane. They concluded that the antigen recognized by the monoclonal antibody was a protein and that it may be a differentiation marker. Although the antigen was not identified, immunoprecipitation analysis revealed that the antigen was a 58kDa protein, very close in size to IMPDH. One additional common finding is that cells were found to have a higher frequency of RR right after thawing and that DMSO treatment does not induce RR formation. This study was not continued to determine the identity of the antigen, but these similarities sugg est that this may be the first publication to identify the novel cytoplasmic structure that we have described as RR The second report of a filament composed of a nucleotide biosynthetic enzyme was from Ji et al. who discovered that IMPDH inhibitors induc ed rods composed of

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97 IMPDH and that the structure was reversible with the addition of GTP ( 59 ) Cells treated with MPA induced the clustering of IMPDH in as little as 30 minutes, with full linear/rod and annular/ring observed after 24 hours. MPA treatment also caused a mobility shift of IMPDH band in SDS PAGE detectable in Western blotting. The band shift was preventable with the use of guanosine, which also caused the disassembly of the IMPDH aggregates. The aggregation of IMPDH was associated with the loss of enzymatic activity, as expected from the putative inhibitory action of MPA. They determined that GTP levels are important for the prevention and reversal of IMPDH aggregates. The structures identified by Ji et al. are the same as RR, although only IMPDH2 was identified. RR formation was also detected in HeLa cells treated with MPA using a rabbit anti IMPDH antibody as well as our patient autoantibody (unpublished). There are several recent studies reporting RR like structures in different systems. Ji Long Liu identified a RR like intracellular structure containing CTPS in Drosophila cells ( 77 ) The structu all major Drosophila cell types within the ovary and other tissues, such as brain, gut, trachea, testis, accessory gland, salivary gland, and lymph gland. The structure was recognized by an antibody against Cup protein that was believed to have cross reactivity. Using two GFP protein trap fly stocks from the Carnegie Protein Trap Library, CTPS was identified as a component of cytoophidia ( 77 ) Cytoophidia were also determined to be associated with the microtubular network but not associated with centrioles. Two types of structures were identified as macro and micro cytoophidium based on their length. In a follow up study, the sam e laboratory reported similar structures in human HeLa P4 cells using an anti CTPS antibody ( 155 ) Cytoophidia

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98 formation was increased by treatment with DON. Knocking down CTPS caused the cytoophidia to disappear however it is unknown if the disappearance is due to the requirement of CTPS or the decrease in protein. Azaserine, another CTPS inhibitor, showed e ffects similar to those of DON. Identification of cytoophidia in HeLa and clustering of CTPS suggests that this structure is identical to rods and rings. Cytoophidia and RR have clear similarities, as both structures have been determined to be unassociated with centrioles. Although the expression frequency of c ytoophidia varies, both structures can be induced with DON and both structures were detected with antibodies to CTPS. Another recent study from Noree et al. identified nine proteins that formed rod like filaments in S. cerevisiae ( 83 ) The filaments were identified using GFP tagged proteins; they also verified that the tag did not play a role in the formation of the filaments by changing the GFP tag to an HA epitope tag and observed the same filaments. Additionally the tagged version of each protein did not affect growth or viability of cells. The nine proteins are Glt1p (glutamate synthase), Psa1p (GDP mannose pyrophosphorylase), Ura7p/Ura8p (CTP synthase), Gcd2p (eIF2B GCD7p (eIF2B IF2B colocalized to form four distinct filaments. They observed that the Ura7p filaments were increased when cells were grown to saturation and concluded that this was due to a decrease in available carbon sources. In addition, treating cells with sodium azide to alter energy status caused an increase in Ura7p and Psa1p filaments. CTPS filaments were identified in all three cell types in the Drosophila egg chamber. They concluded that the different complexes form in response to environmental conditions either to

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99 promote or inhibit particular enzymatic processes, and not as a stress response. Additionally, CTPS filament assembly is caused by the regulation of enzyme activity by the binding of specific ligands such as CTP. The third recent report is from Ingerson Mahar et al. identifying a possible function of CTPS filaments in the fresh water bacterium Caulobacter crescentus ( 82 ) They demonstrated that CTPS formed filaments along the inner curve of the bacterium using electron cryotomography. This bacterial CTPS enzyme has a bifunctional role; as a filament, it regulates the curvature of C. crescentus and performs its catalytic function. They treated cells with the irreversible CTPS inhibitor DON and showed the d isruption of the CTPS filament. They determined via electron cryotomography that CTPS colocalizes with the filament and is required for its formation. In addition, Escherichia coli CTPS homologue was able to form filaments both in vivo and in vitro They c oncluded that CTPS is a negative regulator of cell curvature by mediating CreS. However, the mechanism of how CTPS controls cellular curvature remains unclear. Conservation o f RR Across Species RR like filaments have been identified in a wide range of spec ies as described above ( 86 ) RR like filaments have been identified in human, bovine, mouse, rat, Drosophila, yeast, and bacteria. Filament sizes range from <1 m to 50 m, with bacteria expressing the smallest structures a nd Drosophila expressing the largest ones (summarized in Table 6 1). The structures have also been identified in some cancer cell lines, primary cells, and stem cells as discussed above. Composition of RR Although CTPS1 and IMPDH2 are known RR components, immunoprecipitation of K562 cell extract using anti RR positive HCV patient sera shows some a nti RR

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100 antibody positive sera 604, 605, and 611 pull down a common 55kDa doublet ( Figure 6 3A, red dots), which corresponds to IMPDH1/2. Yet other sera with anti RR antibodies by immunofluorescence do not immunoprecipitate IMPDH protein (Figure 6 3A 603, 606, 608, 609, 610, and 612). Immunofluorescence analysis show serum 609 ( anti RR positive, 55kDa doublet negative) and serum 611 ( anti RR positive, 55kDa double t positive) both recognize the same structure costained with rabbit anti IMPDH2 (Figure 6 3B J). Amongst the studies reported to date, two identified IMPDH and five identified CTPS as component of the RR like filaments as summarized in Table 6 1 Liu specu lates that the se structures may be composed of additional components as seen in the yeast Ura7p and Ura8p filaments ( 86 ) Willingham et al. identified that nematin filaments were not enriched in vimentin or tubulin ( 81 ) Our study also concluded that RR are neither enriched in vimentin, tubulin, or actin, nor associated with any known cytoplasmic structures ( 122 ) In addition, we investigated the coloc alization of RR with centrioles and determined that RR are not primary cilia, because the structures are not attached to the centrioles. Formation of RR RR are not found in most mammalian culture cells in more than a few percent of cells. Inhibitors of CTP S and IMPDH are known to induce RR formation in 90 100% of cells detectable by both patient autoantibodies and rabbit anti IMPDH2 antibodies ( 122 ) Comparing IMPDH inhibitors ribavirin/MPA to CTPS inhibitors D ON/acivicin at relatively high concentration (2 mM) IMPDH inhibitors are consistently faster in induc ing RR in only 15 30 minutes versus CTPS inhibitors which require 2 3 hours to induce comparable levels of RR ( 122 ) M ost studies report that inhibition of these enzymes causes RR like filament formation with the only exception in C. crescentus when CTPS

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101 inhibitor DON disassembles CTPS filament as described earlier ( 82 ) Noree et al. specifically identified Ura7 p (CTPS) filaments as being affected by levels of nutrients ( 83 ) Some key differences between reports are summarized in Table 6 1. Concluding R emarks Recent stud ies show that cells can regulate nucleotide biosynthesis by clustering critical enzymes into RR although their mechanism of formation and functions are not completely understood. RR contain two nucleotide biosynthetic enzymes CTPS and IMPDH but we speculat e that these structures may be composed of other enzymes of the same pathways, based in part on its relatively large physical dimension and on the fact that there are many other anti RR autoantibodies that do not appear to recognize CTPS/IMPDH. Assembly of both CTPS and IMPDH may be due to the close interaction between both enzymes during nucleotide biosynthesis. Many studies suggest the possibility of a conserved RR structure composed of nucleotide biosynthetic enzymes and is a sensor to lowered levels of GTP/CTP. Our identification of abundant RR in mouse undifferentiated embryonic stem cells is intriguing, as it is the only native condition, to date; these structures are found without extrinsic manipulation. Chen et al. has described effects of DON on th e filaments and Drosophila oogenesis suggesting their biological function in development ( 155 ) We have also identified the fact that HCV patients treated with interferon alpha and ribavirin produce autoantibodie s against RR. Covini et al examined the prevalence of anti RR antibodies in other diseases and concluded that anti RR antibodies are highly specific to HCV patients after treatment, but not before treatment ( 139 ) Anti RR antibodies were determined to be more prevalent in patients that do not respond to therapy. Additional studies have also shown correlation of anti RR antibody production w ith HCV and that

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102 the antigen of anti RR antibodies is IMPDH2 ( 139 140 ) Since HCV patients are treated with ri bavirin and we have shown that ribavirin can induce RR formation in vitro ( 122 ) ribavirin treatment in HCV patients may cause RR formation, which leads to the induction of autoantibodies against these structu res when they are released to the host immune system during turnover. Studies thus far implicate novel clinical and biological significance of RR structures.

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103 Figure 6 1 Rods and rings (RR) recognized by human autoantibodies. HEp 2 cells are stained wi th human prototype anti RR serum It2006 followed by Alexa 488 goat anti human IgG (green) and nuclei counterstained with DAPI (Blue). Rods are sometimes presented perpendicular to the nucleus (long arrows) or adjacent to the nuclear envelope (short arrows) Rings (arrowheads) and other transition structures (double arrowhead) are found either 1 or 2 to a cell. Scale bar, 10 m.

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104 Figure 6 2 A schematic of RR components CTPS1 and IMPDH2 along the purine and pyrimidine biosynthesis pathways. RR comp onents are shown in red. IMPDH2 is an enzyme involved in the GTP biosynthesis pathway catalyzing the conversion of IMP to GMP that is a precursor to the production of GTP. CTPS1 is an enzyme involved in the CTP biosynthesis pathway and catalyzes the conver sion of UTP to CTP. The site of inhibition is indicated for DON, acivicin, ribavirin and MPA. Arrows signify a step in the pathway. Green arrows signify activation of pyrimidine and purine biosynthesis pathways by ATP and GTP. Red arrow signifies inhibitio n of pyrimidine biosynthesis pathway by end product CTP. The pathway schematic is adapted from Hofer et al ( 57 )

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105 Figure 6 3 Patient autoantibodies recogn ize multiple components of RR. A) Immunoprecipitation of K562 cell lysates using hepatitis C patient sera 602 612 and correlation with the presence of anti RR antibodies by immunofluorescence. Sera 604, 605, and 611 show common 55kDa doublet (red dots) corre sponding to IMPDH and they are also positive for anti RR antibodies. In contrast, sera 603, 606, 608, 609, 610, and 612 have anti RR antibodies but do not immunoprecipitate the 55kDa band. To confirm RR detected from patients was all the same structure, do uble staining of HEp 2 cells was per formed with rabbit anti IMPDH2 C, F, I) and serum 607(anti RR negative, B), serum 609 (anti RR positive but did not immunoprecipitate the 55kDa doublet, E), or serum 611 (anti RR positive and immunoprecipitated the 55kDa doublet, H). Since RR is recognized by both serum 611 and 609, serum 609 most likely recognizes other unidentified RR component(s). Nuclei are counterstained with DAPI. D, G, J, merged images. Scale bar, 10 m.

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106 Table 6 1 Comparison of various reported RR like cytoplasmic filament structures. Investigat or Rods/rings; Components Sizes Induction method Detection Species/cell types Carcamo et al. ( 122 ) both detected; CTPS1, IMPDH2 3 10 m rod length; 2 5 m ring diameter CTPS1 and IMPDH2 inhibitors: DON, acivicin, ribavirin IMDPH2 and CTPS1 antibodies and human autoantibodies Human: HeLa, HEp 2, K562, HCT116; mouse 3T3, primary cardiomyocytes, fibroblasts, ESCs Similarities vs. Carcamo et al. ( 122 ) Differences vs. Carcamo et al. ( 122 ) Other comments Willingha m et al. ( 81 ) both detected; nematin, 58kDa 0.5 3 m rod length not induced mous e IgG3 mAb human KB; bovine MDBK; rat NRK; mouse 3T3 Not enriched in vimentin or tubulin Prevalent in newly thawed cells Fixation methods affect structure mAb stains mouse skin and CNS Not abundant in human cell lines Ji et al. ( 59 ) both detected; IMPDH 0.1 >1 m in diameter IMPDH2 inhibitor MPA IMPDH antibody Human: MCF7, CEM, K66/B5 Untreated cells show IMPDH disperse in cytoplasm Aggregates appear 30 min after MP A treatment; mature rods by 24 h No association with other organelles Mobility shift of IMPDH after MPA treatment by Western Rods disassemble in cells added guanosine, guanine, GMP,GDP,GTP MPA causes aggregation of IMPDH on polylysine coated glass slides Liu ( 77 ) both detected as cytoophidia; CTPS <1 50 m rod length, depends on stages not induced three CTPS antibodies, GFP CTPS Drosophila follicle cells, nurse cells, oocytes Cytoophidia are not associated with centrioles CTPS functional in cytoophidium Associated with the microtubular network but do not overlap Chen et al. ( 155 ) both detected; CTPS 2 10 m rod length CTPS inhibitor DON CTPS antibody H uman HeLa P4 DON increases the formation of cytoophidia Only 40% RR can be induced at 4g/mL of DON DON induced apoptosis and induced filament formation in Drosophila ovaries Noree et al. ( 83 ) only rods; CTPS Ura7p 0.5 3 m length nutrient deprivation or inhibition of CTPS GFP protein in yeast S. cerevisiae, D. melanogaster Filament formation is due to regulation of enzymatic activity The tag on the protein does n ot affect the formation of the filament Kinase inhibitor staurosporine increased filament formation Ingerson Mahar et al. ( 82 ) only rods; CTPS 390 790 nm length not induced or CTPS over expression mcherry CTPS C. crescentus, E. coli DON disrupts rods Overe xpression increase length of rods CTPS interacts with CreS

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107 CHAPTER 7 SUMMARY AND PERSPECT IVE Biological Function of RR Structure Rod Versus Ring Configurations As described in Chapter 2, the cytoplasmic structure provisionally named rods and rings was identified using HCV patient sera. The rods and rings are distinct structures visualized via indirect immunofluore scences. It is unknown whether rods and rings have different function; however, it is reasonable to consider that there is interconversion be tween rods and rings This speculation is derived in part from the intermediate structures (Figure 2 2) that are observed via immunofluorescences. Although, rods and rings were induced in various cells lines, there are differences in size and the fractions of cells forming rods versus rings. HeLa cells were determined to have rods measuring 5 10 m while THP 1 cells formed shorter rods, measuring 2 3 m. Additional data suggests that the structures start off s hort and increase in size until reaching full /ma ture size of 5 10 m; this was described in Chapter 5 using glutamine deprivation to induce RR (Figure 5 2). Induced Versus Native RR As described in Chapter 2, RR can be induced in cancer cells and primary cells using IMPDH2 / CTPS1 inhibitors in as short as 15 minutes with IMPDH inhibitors and 3 hours with CTPS inhibitors Figure 7 1 summarizes the putative formation of RR using IMPDH2/CTPS1 inhibitors and the induction time required for the formation of m ature RR Additionally, RR can be induced in cancer cells via glutamine deprivation after 48 hours as illustrated in Chapter 5. However, RR are observed in almost 100% of cultured undifferentiated mouse embryonic stem cells without the addition of inhibitors or

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108 treatment to induce glutamine deprivation. Si nce mouse ESCs are highly active in cell division, this suggests different functional role for RR in these cells. RR structures induced with CTPS1/IMPDH2 inhibitors or by glutamine deprivation as well as RR in mESCs can be detected with patient anti RR ser a, anti IMPDH2, and anti CTPS1 antibodies, suggesting that induced RR versus native RR are similar structure s However, it is unclear whether they have the same molecular composition, due to observed differences in the ratio of rods to rings in cells that are induced versus native RR. Rods and rings, as independent structures, may hold different functions as 91% of ESCs form rings while cells that are treated with CTPS/IMPDH inhibitors or deprived of glutamine have 90% rods (Table 7 1). After induction of RR with CTPS1/IMP D H2 inhibitors proliferation is decreased, this is observed clearly with a decrease in number of mitotic cells after treatment with inhibitors compared to controls While ESCs that form RR have a higher percentage of rings (~91%) and have a high proliferation rate, IMPDH2 and CTPS1 associated with RR may be active as these enzymes are required for cell proliferation ( 56 163 ) A hypothetical schematic for RR formation is shown in Figure 7 2 RR that are formed after inhibition with CTPS1/IMPDH2 inhibitors are aggregated proteins that have the active sites blocked by the inhibitor shown in red (Fig ure 7 2) H owever, RR in ESCs may be composed of active enzymes because although the same structures are observed inhibitors are not present to block the active sites (Figure 7 2). Additionally, ESCs express more rings than rods suggesting that rings may be aggregates of active enzymes. RR induced via glutamine deprivation may also be composed of active enzymes (Figure 7 2) However, RR formed during glutamine deprivation could be in response to blocking of enzymatic activity of certain

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109 enzymes such as CTP S1, which requires glutamine to produce CTP. Since production of CTP and GTP is still required for cells to survive, some structures may be composed of active enzymes. Therefore, rods and rings induced by CTPS1 and IMPDH2 inhibitors may be composed of the inactive enzymes while rods and rings observed in untreated rapidly proliferating cells or induced in response to glutamine deprivation may be composed of active enzymes. Attempts to Identify Additional Components in RR As stated above, r ods and rings are composed of IMPDH2 and CTPS1, and likely to include other enzymes involved in purine and pyrimidine biosynthesis pathways. D ue to the large size of the RR structure, we speculate that RR may be composed of other additional components. For example, in GW bo dies which are known range from 0.1 to 0.3 m in diameter, more than 10 proteins have been identified ( 10 1 21 ) With rods being ~3 10 m in length and rings ~2 5 m in diameter it is very reasonable to postulate that RR are composed of addition al enzymes involved in nucleotide biosynthesis. There is experimental support for this consideration because RR for mation was observed in cells that were treated with 10 M pemetrexed for 24 hours Pemetrexed is a chemotherapeutic drug that inhibits thymidine synthetase, dihydrofolate reductase, glycinamide ribonucleotide formyltransferase (GARFT), and a minoimidazole c arboxamide r ibonucleotide formyltransferase (AICARFT). The fact that p emetrexed induced RR formation in HeLa c ells suggests that some of these enzymes are components of RR. Thymidine synthetase was an interesting candidate RR protein for obvious reason be cause it is the critical enzyme for the production of thymidine triphosphate (TTP). Additional thymidine synthetase inhibitors, 5 Fluorouracil and gemcitibane, were also tested but they did not induce RR formation. Also we were not

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110 able to detect RR using a mouse antibody to thymidine synthetase (TS antibody generously provided by Dr. Maria Zajac Kaye); however, this data cannot completely rule out TS may still be localized to RR. Similarily, rabbit anti dihydrofolate reducatase antibody (E18220, Spring B ioscience; Pleasanton, CA, USA) was unable to detect RR formed after treatment with pemetrexed. Thus, it remains necessary to examine whether RR may be composed of GARFT or AICARFT enzymes involved in purine biosynthesis pathway (Figure 7 3). In addition, these enzymes may also be affected by glutamine deprivation, although they do not require the use of glutamine, other steps before and after in the same purine biosynthetic pathway require glutamine (Figure 5 4). Interfering with enzymes within the purine biosynthesis pathways will likely have an effect on others enzymes within the pathway; therefore, glutamine might have an indirect effect on GARFT and AICARFT. RR that are induced by CTPS/IMPDH inhibitors are readily dissociated after cell lysis with deter gents, such as 0.3% Nonidet P40 and 0.5% triton X; therefore, attempts to purify RR for the identification of additional components using mass spectroscopy has not been feasible to date. However, crosslinking RR may help stabilize the se structures and allo w immunoaffinity purification and identification of additional components via mass spectroscopy. In order to isolate RR, we have use d a homobifu nctional cleavable crosslinker d dithiobispropionimidate2HCl (Cat# 20665; Thermo Scientific; Rockf ord, IL, USA). The cleavable crosslinker should allow us to isolate intact RR that can then be analyzed to identify novel components by mass spectroscopy If these components are the proposed enzymes as discussed above, they can be verified as RR component s by using inhibitors to those enzymes to

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111 detect whether RR are formed. M any antibodies of known RR components would not recognize RR, as shown in Table 7 2. This inability of certain antibodies to recognize RR structures may depend on the conformational c hanges of the individual component ; th erefore verification with an inhibitor, if the component is an enzyme or other antibodies is required. Cytoplasmic Versus Nuclear RR In addition to the cytoplasmic RR structures discuss so far, recent data show RR wer e detected within the nucleus. Nuclear RR are observed after treatment of CTPS1 or IMPDH2 inhibitors or glutamine deprivation; although they are located within the nucleus they may be similar to cytoplasmic RR. Multiple z stack images of a nucleus containi ng a short rod clearly demonstrated the structure within the nucleus (Figure 7 4); additional cells were observed with rings within the nucleus. These rod and ring structures were present within the nucleus and were co stained by both patient autoantibodie s and rabbit anti IMPDH2 antibody (data not shown). Nuclear rods were determined to range in size from 1.6 m to 4.5 m, while cytoplasmic rods w ere from 2.5 m to 16.8 m when counted from the same cell preparations. The mean size of cytoplasmic rods was 8.4 m and the mean size of nuclear rods was 3.2 m. This was determined by quantitating nuclear and cytoplasmic RR from 66 cells. Nuclear rods were substantially shorter than cytoplasmic rods. Although nuclear rods and cytoplasmic rods are highly similar structures, it is unknown whether they have the same function due to the difference in their subcellular locations. Studies suggest that IMPDH binds single stranded RNA/DNA and modulate localization or degradation of a set of mRNAs ( 175 176 )

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112 Technical Difficulties in RR Detection The study of RR formation and function has been limited by several obstacles. Fi rst, detection of RR antibodies in patient sera depends on the commercial ANA slides used. Most ANA slides do not give RR staining; for example ANA slides from INOVA diagnostics Inc. (San Diego, CA) consistently give RR staining with our prototype anti RR serum but ANA slides from Immuno Concepts (Sacramento, CA) are negative. After determining that RR can be induced with glutamine deprivation, we speculate that RR detected in INOVA slides may be due to techniques used to increase proliferation and therefo re may cause limitations in availability of nutrients in medium. Additionally, not all antibodies can detect the apparently conformational epitope(s) expressed on th e RR structures as compared to monomeric proteins in the cytoplasm Table 7 2 includes all the available antibodies we have tested for their ability to recognize RR on INOVA or home made slides to date. While all the antibodies can detect monomeric proteins by Western blot only one anti IMPDH2 antibody was able to detect RR structures. Liu detec ted RR like structures in Drosophila using a n anti CTPS1 antibody ( 77 ) However, the same anti CTPS1 antibody did not detect RR when tested in our laboratory and this was apparently because of lot to lot variation and that not all lots were able to detect RR. Therefore, inability of RR detection with an antibody does not rule out that the protein as being part of the RR structure. Clinical Implication o f Anti RR Ribavirin as a Therapeutic A gent R ib avirin is believed to be the cause for RR formation in HCV patients who have undergone months of therapeutic exposure, leading to the production of anti RR auto antibodies D ue to ribavirin induction of RR in vitro it is unknown whether ribavirin

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113 alon e or the co exposure to interferon in HCV treatment is required for the production of anti RR in selected patients Ribavirin is only approved for the treatment of HCV; however it is currently in trial for the treatment of other viral infections Although there are a few cases of patients with anti RR antibodies that do not have HCV, anti RR antibodies in HCV patients is highly correlated to the effects of ribavirin treatment, leading to RR formation and stimulation of the host immune system. MPA as a Therapeut ic Agent MPA is currently used for the treatment of systemic lupus erythematosus and to prevent organ rejection. Although MPA has been shown to induce RR formation in vitro lupus patients treated with MPA do not develop anti RR antibodies, with just one e xception to our knowledge. Interestingly, lupus patients are also often continuously exposed to type I interferon, the cytokine that is playing a major role in the disease pathogenesis ( 177 178 ) Thus, continuous exposure of interferon does not help induce anti RR antibodies in lupus patients undergoing MPA treatment. This suggests that the inhibitor of IMP DH alone is not sufficient to induce autoantibody formation and additional factors are likely required. Development of RR Antibodies and R esis tance to T reatment We have determined that a subset of patients that develop anti RR antibodies either are non res ponsive to therapy or relapse. It was previously determined that equilibrative nucleoside transporter 1 ( ENT1), the known transporter f or ribavirin from extracellular source into cytoplasm, b ecomes resistance after exposure to ribavirin and prevents furthe r transport of ribavirin into the cell ( 132 ) Studies u sing other IMPDH inhibitors have determined that ENT1 becomes resistant to transport after cells are treated with IMPDH inhibitors ( 179 ) ENT1 is sensitive to inactivation via IMPDH

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114 inhibitors. Since ribavirin induces formation in vitro in all cell types, we speculate that there is a linkage between RR and ENT1 that leads to patients becom ing non responders or relapse r s after therapy. After a patient is infected by HCV, cells may have varying levels of HCV (Figure 7 5A). When the patient is treated with interfero n alpha and ribavirin, this lead s to the formation of RR within some hepatocytes ribavirin may be utilized by the virus and may not be present at a high enough concentration to induce RR formation in infected cells (Figure 7 5B) RR may either interact directly with ENT1 or indirectly with activation proteins, such as protein kinase C ( 180 181 ) and thus inactivate further uptake of ribavirin. Additional studies have identified that ribavirin treatment can decrease the mRNA levels of protein kinase C, this decreas e due to ribavirin may cause the ENT1 resistance ( 182 ) For the cells that were infected and receive d ribavirin treatment some become virus free as to be expected (Figure 7 5C) After continuous treatment cells that have formed RR may b e resistant to further up take of ribavirin, allowing the infection to spread. The infection that has remained may then spread to all cells causing the patient to have a negative outcome to the interferon alpha and ribavirin therapy (Figure 7 5D). In other words, patients with more RR positive hepatocytes will like ly induce autoantibodies to RR and become at a later time resistant to viral clearance. This hypothesis can be tested by pretreating cells with ribavirin to induce RR formation and cause cellular resistance in the pretreated cells and then infecting with HCV as shown in Figure 7 6. Pretreating hepatocytes with ribavirin will help simulate the in vitro effect that not all cells are infected in HCV infected patients at time of treatment. Untreated ce lls ( Figure 7 6 A) and ribavirin treated cells are left overnight for the induction of RR ( Figure 7 6 B) and both will then be infected with HCV.

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115 I nfected cells will be treated with ribavirin If ribavirin causes ENT1 resistance due to presence of RR then v iral RNA will be detected at high levels in cell with RR after ribavirin treatment While cells that are not pretreated with ribavirin will clear the virus after ribavirin treatment. We also speculate that anti RR antibodies production is resulted from in teractions of HCV viral proteins/RNA with ribavirin induced RR. R ibavirin has a similar chemical structure as GTP ( Figure 7 7 A vs B). Since IMPDH2 is inhibited by ribavirin and can induce RR formation, we speculate that viral proteins may bind to IMPDH2 w ith or without ribavirin in RR (Figure 7 7C D). The viral proteins that may interact with RR are HCV structural and non structural proteins such as core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B. Our data has shown that approximately 20% of HCV pati ents develop anti RR antibodies ( 139 ) while the other 80% do not develop these specific antibod ies. This observation can be related to th e genetic background of individual patients. However, development of anti RR may be due to an intracellular level of IMPDH2. We previously determined that cells overexpressing IMPDH2 do not form RR (Figure 2 8). If overexpression of IMPDH2 prevents formati on of RR, it may also prevent formation of RR within patients and lead to the prevention of development of anti RR antibodies. Therefore, the heterogeneity of patients and levels of IMPDH2 may contribute to the variable development of anti RR antibodies. H CV viral protein/RNA RR complexes may be recognized by the host immune system and the productive immune response target the viral protein/RNA complex but also develop autoantibody to RR from epitope spreading or simply crossreaction.

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116 Conclusions Rods and r ings were identified using human autoantibodies produced by HCV patients treated with interferon alpha and ribavirin. HCV patients that do not respond to therapy or develop relapse tend to have higher anti RR antibody titers. RR were determined to be compo sed of nucleotide biosynthesis enzymes: CTPS1 and IMPDH2. We speculate that RR are composed of additional nucleotide biosynthetic enzymes, such as GARFT or AICARFT. RR currently form ed from the inhibition of CTPS1 or IMPDH2 or in response to depletion of g lutamine, are consistently correlated to a decrease in intracellular levels of CTP/GTP. Therefore, RR may represent polymerization of nucleotide biosynthetic enzymes in response to decrease in intracellular CTP or GTP levels in order to increase CTP/GTP le vels necessary for cellular function. This working hypothesis will require further experiments.

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117 Figure 7 1 RR induction pathway showing hypothetical exchange between rods and rings. This figure summarizes the overall timeline when IMPDH and CTPS inhib itors or glutamine deprivation induce the formation of RR in different cell lines. Some RR structures maybe i nterchangeable as indicated by double sided arrow s The timelines indicate induction time for RR using IMPDH inhibitors, ribavirin or mycophenolic acid (MPA), CTPS inhibitors 6 diazo 5 oxo L norleucine (DON) or acivicin, and by glutamine deprivation Representatives RR intermediate structures sewing pin A) B) are shown respectively as described in Carcamo et al ( 122 )

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118 Figure 7 2 Schematic of RR assembly. Rods and rings form ed from monomers after induction with CTPS/IMPDH inhibitors (red inserts). RR may also form from monomers without inhibitors as in ESCs or after glutamine d eprivation. These represent a detail view of rods and rings (green)

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119 Figure 7 3 A schematic of putative RR components AICARFT and GARFT along the purine and pyrimidine biosynthesis pathways. Putative RR components are shown in red. The site of inh ibition is indicated for pemetrexed Arrows signify a step in the pathway. The pathway schematic is adapted from Hofer et al. ( 57 )

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120 Figure 7 4 Rods and rin gs located with in the nucleus. HeLa cells treated with DON form RR detected by human It2006 (green). A I) A confocal z stack cutting s of rod with in nucleus 0.39 m thickness per section. Arrow signifies location of visible nuclear RR. Nuclei are countersta ined with DAPI. Scale bar 5 m

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121 Figure 7 5 Hypothetical role of ENT1 contributing to resistance to ribavirin therapy and HCV viral persistence in vivo A) After HCV infection some hepatocytes are infected with virus while others are not infected. B) Patient then receives pegylated interferon alpha and ribavirin therapy, causes RR formation in uninfected cells. C) Therapy is continued and clears virus from some cells and yet causes resistance t o ribavirin in cells with RR. D) Virus replicated and infec ts cells with RR, which are resistant to ribavirin and viral persistence occurs.

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122 Figure 7 6 Experimental schematic to test whether ENT1 resistance i s linked to viral persistence. A) The hepatocyte is not pre treated and then gets infected with HCV. Aft er viral infection it receives ribavirin treatment. After treatmen t viral infection is cleared. B) The hepatocyte is pre treated with ribavirin to induce RR and then gets infected with HCV. After viral infection it receives ribavirin treatment. After treat ment viral infection persists. Each column represents one cell, which represents multiple cells.

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123 Figure 7 7 Postulation of HCV antigens associ ated with RR. A) Structure of GTP and B) ribaviri n C) Schematic of HCV viral protein or viral RNA (yellow spi kes) interacting with rod or ring (b lue) induced to form from exposure to ribavirin (red)

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124 Table 7 1 Methods of RR induction and quantification of rods vs rings. Induction Method Cell Types Rods vs rings percentage IMPDH/CTPS inhibitors Canc er and primary cell lines 90% rods Glutamine Deprivation HeLa 90% rods Uninduced ESCs 91% rings

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125 Table 7 2 Antibodies tested for RR detection by direct immunofluorescence Investigator Rods/rings; Components Sizes Induction method Detection IMPDH2 R abbit Abcam Ab75790 NO IMPDH2 Rabbit Proteintech 12948 1 AP YES 1:100 CTPS 1 Rabbit Abcam Ab65045 NO CTPS1 Rabbit Proteintech 15914 1 AP NO CTPS1 Mouse Sigma Aldrich WH0001503M1 NO CTPS1 Rabbit Santa Cruz Sc 101925 NO (lot specific*) TS 106 Mouse No t a commercial antibody NO DHFR Rabbit Spring Bioscience E18220 NO UMPS Mouse Abgent AT4467a NO PCYT1A Mouse Abgent AT3244a NO *CTPS1 antibody from Santa Cruz was tested 3 times due to inconsistent reports ; Liu reported that this antibody detect ed RR ( 77 ) However, we determined that detection of RR with this antibody was lot specific and only one lot was able to detect RR while another lot could only detect RR at 1:50 and were very faint requi red co staining to visualize RR.

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143 BIOGRAPHICAL SKETCH Wendy Cristina Carcamo was born in Los Angeles, California. She graduated from Piper High School in 2004 and then attended the University of Florida. During the four laboratory. In 2008, she graduated with a Bachelor in Science in microbiology with a minor in chemistry and plant molecular and cellular biology. With the support of Dr. Chan and his graduate students she applied to the Interdisciplinary Program in Biomedi cal Sciences (IDP) at the University of Florida In 2008, she joined the molecular cell biology concentration, and decided to continue in the laboratory of Dr. Edward Chan. Here, her research focused o n a novel cytoplasmic structure that were detected with human autoantibodies. Her research focused on identifying the components, the function of the structure, and the correlation to the disease. After her doctorate work, Wendy plans on entering medical school where she hopes to specialize as an obstetrician/ gynecologist.