NONSEGMENTED NEGATIVE STRAND RNA VIRUSES: VIRAL RNA CAP METHYLATION AND POTENTIAL APPLICATIONS AS AN ANTICANCER THERAPY by Andrea Mary Murphy A dissertation submitted to the faculty of The University of North Carolina at Charlotte in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology Charlotte 2012 Approved by: ______________________________ Dr. Valery Grdzelishvili ______________________________ Dr. Ian Marriott ______________________________ Dr. Pinku Mukherjee ______________________________ Dr. Laura Schrum ______________________________ Dr. Michael Turner ii © 2012 Andrea Mary Murphy ALL RIGHTS RESERVED iii ABSTRACT ANDREA MARY MURPHY. Nonsegmented negative strand RNA viruses: viral RNA cap methylation and potential applications as an anticancer therapy. (Under the direction of DR. VALERY GRDZELISHVILI) The viruses of the order Mononegavirales include important human, animal, and plant pathogens and additionally, have great potential as vaccine, oncolytic and gene therapy vectors. This dissertation focuses on two prototypic Mononegavirales, vesicular stomatitis virus (VSV) and Sendai virus (SeV), their virus-encoded cap methylation function, and potential applications as an anticancer therapy. The L protein of Mononegavirales has six conserved domains postulated to constitute the specific enzymatic activities of this multifunctional protein. We conducted a comprehensive mutational analysis by targeting the entire SeV L protein domain VI, creating twenty-four infectious L mutants. Our analysis identified several residues required for successful cap methylation and virus replication. This study confirms structural and functional similarity of this domain across different families of the order Mononegavirales. Additionally, the oncolytic potential of VSV was analyzed for the first time in a panel of human pancreatic ductal adenocarcinoma (PDA) cell lines and compared to other oncolytic viruses. VSV showed superior oncolytic abilities; however, cells were heterogeneous in their susceptibility to virus-induced oncolysis and several cell lines were resistant to all tested viruses. Four cell lines that varied in their permissiveness to VSV were tested in mice, and in vivo results closely mimicked those in vitro. While our results demonstrate VSV is a promising oncolytic agent against PDA, further studies are needed to better understand the molecular mechanisms of resistance to oncolytic virotherapy. iv ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Valery Grdzelishvili for his faith in me as a graduate student and his neverending support. I would like to thank Dr. Ian Marriott, Dr. Pinku Mukherjee, Dr. Laura Schrum, and Dr. Michael Turner for serving on my committee and providing encouragement and feedback. I would like to thank the members of the Grdzelishvili lab, Megan Moerdyk-Shauwecker, Nirav Shah, Eric Hastie, Natascha Moestl, and Carlos Molestino, for their support and friendship throughout my graduate career. I would also like to thank the Mukherjee lab for all of their help and input. I would like to thank the UNCC Graduate School for the Graduate Assistant Support Award for funding my graduate education. I would also like to thank the Lucille P. and Edward C. Giles Foundation for awarding me a generous dissertation-year fellowship. Lastly, I would like to thank my husband, Frank, my parents, Jim and Laura, and all my family and friends who encouraged and supported me while I pursued my graduate degree. v TABLE OF CONTENTS LIST OF FIGURES vi LIST OF TABLES viii LIST OF ABBREVIATIONS ix CHAPTER 1: INTRODUCTION 1 CHAPTER 2: SEQUENCE-FUNCTION ANALYSIS OF THE SENDAI 26 VIRUS L PROTEIN DOMAIN VI 2.1 Objective of the study 26 2.2 Materials and Methods 29 2.3 Results 40 2.4 Conclusions 62 2.5 Figures 76 2.6 Tables 88 CHAPTER 3: VESICULAR STOMATITIS VIRUS AS AN ONCOLYTIC 92 AGENT AGAINST PANCREATIC DUCTAL ADENOCARCINOMA 3.1 Objective of the study 92 3.2 Materials and Methods 96 3.3 Results 104 3.4 Conclusions 114 3.5 Figures 121 3.6 Tables 133 CHAPTER 4: DISSERTATION SUMMARY 136 REFERENCES 148 vi LIST OF FIGURES FIGURE 1: Proposed 5’ capping mechanisms for eukaryotes and viruses of 11 Mononegavirales. FIGURE 2: Recognition of viral RNA by RIG-I and MDA5 results in 16 production of IFN-β. FIGURE 3: IFN-αβ is recognized by IFNAR and a subsequent signaling 17 cascade leads to the production of ISGs. FIGURE 4: Schematic of oncolytic virus (OV) therapy. 20 FIGURE 5: SeV L protein mutants generated and analyzed in this study. 76 FIGURE 6: In vitro mRNA synthesis and genome replication with SeV mutant 78 L proteins using a VVT7 expression system. FIGURE 7: Host range and temperature sensitivity analysis of SeV mutants. 79 FIGURE 8: Multistep growth kinetics of wt and recombinant SeV in Vero and 80 HEp-2 cells at 34°C. FIGURE 9: Growth analysis of recombinant SeV mutants in Vero cells. 81 FIGURE 10: One-step growth kinetics of SeV mutants in Vero cells. 82 FIGURE 11: In vitro mRNA synthesis with purified mutant SeV. 83 FIGURE 12: Cap methylation analysis using tobacco acid pyrophosphotase. 84 FIGURE 13: Superinfection of Vero cells with SeV mutants. 85 FIGURE 14: SeV infectivity of MEFs. 86 FIGURE 15: Western blot analysis of MEFs infected with SeV mutants. 87 FIGURE 16: Viruses used in the PDA study. 121 FIGURE 17: PDA cell viability following infection with viruses. 122 FIGURE 18: Kinetics of cytopathogenicity of VSV-∆M51-GFP in PDA cells. 123 FIGURE 19: Permissiveness of PDA cell lines to different viruses. 124 vii FIGURE 20: Permissiveness of selected PDA cell lines to virus infection. 125 FIGURE 21: Surface expression of adenovirus CAR receptor. 126 FIGURE 22: Analysis of viral protein accumulation in cells at 16 h p.i. 127 FIGURE 23: Early viral RNA levels in infected cells. 128 FIGURE 24: One-step growth kinetics of VSVs in PDA cell lines. 129 FIGURE 25: Type I interferon sensitivity of PDA cell lines. 130 FIGURE 26: Type I interferon production by PDA cell lines. 131 FIGURE 27: Efficacy of VSV-∆M51-GFP and CRAd-dl1520 in nude mice 132 bearing human PDA tumors. viii LIST OF TABLES TABLE 1: Sequences of primers used in this study to generate SeV mutant L genes 88 TABLE 2: Comparative titers of recombinant Sendai viruses in Vero or HEp2 90 cells at 34°C or 40°C. TABLE 3: Cap methylation analysis using SeV detergent-activated purified 91 virions. TABLE 4: PDA cell lines used in this study. 133 TABLE 5: Early viral RNA synthesis in cells infected with VSV-∆M51-GFP. 134 TABLE 6: Correlation between IFN sensitivity, production and resistance 135 of PDA cells to VSV. ix LIST OF ABBREVIATIONS AND SYMBOLS 4 CN 4-chloro-1-napthol aa amino acid AdoHcy S-adenosylhomocysteine AdoMet S-adenosylmethionine BSA bovine serum albumin CBC cap binding complex CE capping enzyme CIU cell infectious units CPE cytopathic effects CRAd conditionally replicative adenovirus CTD C-terminal domain DAB 3,3’-diaminobenzidine tetrahydrochloride hydrate DI defective-interfering DNA deoxyribonucleic acid DMEM Dulbecco’s modified Eagle’s medium ECL Enhanced Chemiluminescence eIF eukaryotic initiation factor F fusion protein FBS fetal bovine serum G glycoprotein GDP guanosine diphosphate GFP green fluorescent protein x GMP guanosine monophosphate GTP guanosine triphosphate GTPase guanosine 5’-triphosphatase H3 tritium HN hemagglutanin/neuraminidase h p.i. hours post infection hr host range HRP horseradish peroxidase hTERT human telomerase reverse transcriptase IACUC Institutional Animal Care and Use Committee ICC immunocytochemistry IF immunofluorescence IFN interferon IFNAR IFN-αβ receptor ISG interferon-stimulated gene IT intratumoral KO knock out L large polymerase protein M matrix protein MDA5 melanoma differentiated-associated gene 5 MDSC myloid-derived suppressor cells MEFs mouse embryonic fibroblasts MEM modified Eagle’s medium xi MOI multiplicity of infection mRNA messenger RNA MTase methyltransferase MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide N nucleoprotein NIH National Institutes of Health NNS nonsegmented, negative strand NTP nucleotide triphosphate ORF open reading frame OV oncolytic virus P phosphoprotein PanIN pancreatic intraepithelial neoplasias PBS phosphate-buffered saline PDA Pancreatic ductal adenocarcinoma PFA paraformaldehyde PRNTase polyribonucleotidyltransferase PVDF polyvinylidene difluoride RdRp RNA-dependent RNA polymerase RIG-I retinoic-acid inducible gene I RNA ribonucleic acid RNGTT RNA guanylyltransferase and 5’ triphosphatase RNMT RNA guanine-7 methyltransferase RNP ribonucleoprotein xii RPMI Roswell Park Institute medium-1640 SDS-PAGE sodium-dodecyl sulfate polyacrylamide gel electrophoresis SeV Sendai virus SFM serum-free media STAT signal transducers and activators Treg regulatory T cells TAM tumor-associated macrophages TAP tobacco acid pyrophosphatase TBST Tris-buffered saline with Tween-20 ts temperature sensitive VSV Vesicular stomatitis virus VV Vaccinia virus wt wild type CHAPTER 1: INTRODUCTION The nonsegmented negative strand (NNS) RNA viruses of the order Mononegavirales include many important human, animal, and plant pathogens such as rabies virus, Ebola and Marburg viruses, measles, mumps and respiratory syncytial virus. Most of our current understanding of the biology of Mononegavirales comes from studying two prototypic members of this order, vesicular stomatitis virus (VSV, Family Rhabdoviridae) and Sendai virus (SeV, Family Paramyxoviridae) (Lamb and Parks 2007; Lyles 2007). There are several advantages to using VSV and SeV as research models