The Pennsylvania State University The Graduate School Department of Biochemistry and Molecular Biology VIRAL POLYMERASE MECHANISM-BASED STRATEGIES FOR VIRAL ATTENUATION AND VACCINE DEVELOPMENT A Dissertation in Biochemistry, Microbiology and Molecular Biology by Cheri Annette Lee © 2015 Cheri Annette Lee Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2015 The dissertation of Cheri Annette Lee was reviewed and approved* by the following: Craig E. Cameron Professor and Eberly Chair in Biochemistry and Molecular Biology Dissertation Advisor Chair of Committee Avery August Professor and Chair of Immunology, Cornell University Special Member Richard Frisque Professor and Associate Department Head in Biochemistry and Molecular Biology Andrea Mastro Professor of Biochemistry and Molecular Biology Anthony Schmitt Associate Professor and Director in Pathobiology Scott B. Selleck Professor of Biochemistry and Molecular Biology Head of the Department of Biochemistry and Molecular Biology *Signatures are on file in the Graduate School iii ABSTRACT RNA viruses replicate as a heterogenous population of mutants dubbed ‘quasispecies.’ Some of the most life-threatening diseases today are caused by RNA viruses including, Ebola, hepatitis c virus, dengue, west nile and influenza. While some progress has been made over the years, at this time no effective vaccines exist for these pathogens, and demand is high. The RNA dependent RNA polymerase (RdRp) is responsible for viral genetic diversity. Although a high mutation rate can lead to deleterious changes in the genome, this offers an advantage to the virus: the ability to quickly adapt to a complex host environment, and evade barriers to establishing disease. This adaptability poses a unique challenge, for example, when it comes to developing antiviral drugs and vaccines. In this dissertation, studies on altering the fidelity of the poliovirus (PV) polymerase, 3Dpol, are described using biological evaluation of four 3Dpol derivatives: Ser-64 (G64S), Arg-273 (H273R), Arg-359 (K359R) and Arg-273,Arg-359 (H273R- K359R). Previous biochemical and biological studies performed on PV RdRp have shown that G64S is a high fidelity polymerase and that functions at WT levels and creates less genomic diversity relative to the WT polymerase. In the PV transgenic animal model (cPVR) G64S was attenuated. From this discovery, it was hypothesized, that lack of diversity in the viral population leads to attenuation. However, in our hands the G64S is virulent. At the same time we performed studies with the low fidelity polymerase, H273R. H273R has a 2-3 fold increase in ribonucleotide misincorportation frequency relative to WT, which translates into a 2-3 fold mutation frequency in cell culture. Quantitative increase in mutation frequency, measured by CirSeq, confirmed the mutator phenotype and also suggested RdRp-independent factors may contribute to the iv generation of the viral quasispecies. H273R was attenuated in the cPVR model and induced neutralizing antibodies. This result suggested that the WT PV RdRp is at the optimal fidelity for virulence. Lys-359 is a conserved residue in the active site that serves as a general acid catalyst. With the K359R mutant we observe an increase in polymerase fidelity and decrease in replication kinetics relative to WT PV. This resulted in attenuation in mice and generated a protective immune response. Given that this residue is conserved in many RdRps, this study suggests that a universal mechanism-based strategy for vaccine development is possible for RNA viruses. We reasoned that if fidelity is the primary cause of the attenuated phenotype, then a double fidelity mutant combining both a high and low fidelity mutant will have restored virulence. Double polymerase mutants G64S-K359R, G64S-H273R and H273R-K359R were created using existing fidelity mutants. G64S-K359R did not produce any virus by infectious center assay (ICA) and G64S-H273R was unstable after transfection into HeLa cells. The serine-64 reverted back to glycine leaving a H273R PV. H273R-K359R was stable over 4 passages at a low MOI. This double mutant has faster replication kinetics compared to K359R alone, although it still has decreased replication kinetics relative to WT and H273R. We see a restoration of WT fidelity in vitro but not in vivo. H273R-K359R is attenuated in the cPVR transgenic mouse model despite having similar WT fidelity. Attenuation of H273R-K359R in vivo suggests viral replication kinetics plays an integral role in viral virulence. Together, these studies confirm that fidelity, as well as replication speed, are integral to viral virulence and provide insight on rational strategies for vaccine development. v TABLE OF CONTENTS LIST OF FIGURES .................................................................................................... vii LIST OF TABLES ...................................................................................................... ix ABBREVIATIONS ...................................................................................................... x ACKNOWLEDGEMENTS .......................................................................................... xii Chapter 1 Background and introduction ................................................................... 1 Pathogenesis ...................................................................................................... 2 Quasispecies theory ........................................................................................... 3 Population genetics theory ................................................................................. 5 Poliovirus as a model ......................................................................................... 6 Genome organization ......................................................................................... 9 3Dpol .................................................................................................................... 11 Poliovirus receptor .............................................................................................. 11 Tissue tropism .................................................................................................... 12 cPVR: transgenic mouse model ......................................................................... 14 Poliovirus Pathogenesis ..................................................................................... 15 Figures ................................................................................................................ 17 References ......................................................................................................... 24 Chapter 2 A Polymerase Mechanism-based Strategy for Viral Attenuation and Vaccine Development ......................................................................................... 31 Summary ............................................................................................................ 31 Introduction ......................................................................................................... 32 Materials and Methods ....................................................................................... 33 Results and Discussion ...................................................................................... 36 Figures ................................................................................................................ 42 References ......................................................................................................... 45 Chapter 3 RNA Virus Population Diversity: An Optimum for Maximal Fitness and Virulence ............................................................................................................. 48 Abstract .............................................................................................................. 48 Introduction ......................................................................................................... 49 Materials and Methods ....................................................................................... 51 Results ................................................................................................................ 63 The H273R substitution in PV RdRp produces a mutator phenotype in vitro ....................................................................................................... 63 Replication characteristics of H273R PV in cell culture ............................... 64 H273R PV expresses a mutator phenotype in cell culture .......................... 65 H273R PV exhibits reduced fitness in cell culture relative to WT PV .......... 68 vi H273R is attenuated in vivo ......................................................................... 70 H273R elicits a protective immune response .............................................. 71 H273R phenotype is unique ........................................................................ 72 Discussion .......................................................................................................... 72 Figures ................................................................................................................ 77 References ......................................................................................................... 88 Chapter 4 Polymerase speed and fidelity: Determinants of viral virulence ............... 90 Abstract .............................................................................................................