Host and Viral Determinants of Reovirus Oncolysis by Adil Mohamed A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Virology Department of Medical Microbiology and Immunology University of Alberta © Adil Mohamed, 2018 ABSTRACT Mammalian orthoreovirus (reovirus) is a non-pathogenic virus that naturally infects through the enteric and respiratory routes. Reoviruses are non-enveloped and have a 10-segment double stranded RNA (dsRNA) genome encased in 2 concentric capsid shells. Representative isolates of each of the 3 reovirus serotypes include [(Type 1 Lang (T1L), Type 2 Jones (T2J), Type 3 Dearing (T3D) and Type 3 Abney (T3A)]. A proprietary variant of T3D (REOLYSIN®) is currently being evaluated as an oncolytic virotherapy in Phase I/II/III clinical trials for a multitude of cancers. However, in most clinical trials, REOLYSIN® treatment does not improve efficacy over standard of care. Therefore, in order to enhance REOLYSIN® efficacy as an oncolytic virus, there needs to be an improved understanding of mechanisms of reovirus replication both from a virus and host cell perspective. The first chapter of our studies include an in-depth phenotypic and genotypic characterization of laboratory T3D strains. We demonstrate for the first time that oncolytic properties differ between T3D laboratory strains. Specifically, compared to T3DKC and T3DTD, T3DPL had enhanced replication in cancer cell lines and demonstrated improved in-vivo tumor regression. Reassortment analysis and site-directed mutagenesis identified 3 genes (S4, M1, L3) and specific polymorphisms that account for replication differences between T3DTD and T3DPL. Gene-function analysis determined that accelerated T3DPL replication kinetics was linked to i) M1 gene modulating superior inherent viral core transcriptase activity and filamentous virus factory morphology, ii) S4, M1 and L3 gene impacting suppression of RIG-I/MDA5 dependent signaling, iii) S4 gene mediating a heightened RIG-I/MDA5 independent signaling and iv) L3 gene governing large viral factories. An extensive phylogenetic comparison identified that current T3D laboratory strains have diverged from the original clinical T3D isolate, and laboratory specific virus propagation methodologies likely contributed to this divergence. Finally, the proprietary REOLYSIN® isolate was resolved to be identical in amino acid sequence to our laboratory T3D strain (T3DPL), making our findings from this study directly applicable to improving our understanding of REOLYSIN®. The second chapter involved functional characterization of host cell p38 MAPK signaling during reovirus replication. It was previously determined that the ras-p38 MAPK-IFN axis affected reovirus replication during multiple rounds of replication but the role of p38 MAPK during initial reovirus replication was unknown. We demonstrated that initial reovirus replication is diminished following p38 MAPK signaling inhibition using a panel of specific p38 MAPK inhibitors. Specifically, the efficiency of reovirus outer capsid cleavage was diminished following p38 MAPK inhibitor treatment and was specifically linked to reduced i) virion endocytosis and ii) endosome to lysosome transition. Additionally, we identified a beneficial role of p38 MAPK signaling during secondary reovirus amplification. Therefore, an active p38 MAPK signaling pathway enhanced various reovirus replication steps to facilitate productive infection. By assessing reovirus replication in a panel of breast cancer cell lines and subsequent microarray analysis, we identified MAPK11 (p38β) as a potential prognostic marker for reovirus ii uncoating. Moreover, we propose the utilization of methodical prognostic markers specific for reovirus replication steps, rather than global prognostic markers as is currently performed. Overall, our studies uncover novel virus and host cell determinants of reovirus replication and not only increases our appreciation of inter-laboratory reovirus diversity but also directly impacts our current understating of REOLYSIN®. iii TABLE OF CONTENTS CHAPTER 1: INTRODUCTION .................................................................................................. 1 1.1 Cancer burden ............................................................................................................... 1 1.2 Cancer treatment ........................................................................................................... 1 1.2.1 Conventional cancer therapies ................................................................................. 2 1.2.2 Biological cancer therapies ...................................................................................... 3 1.2.3 Oncolytic viruses ..................................................................................................... 4 1.2.3.1 Clinically approved oncolytic viruses .................................................................. 5 1.2.3.2 T-VEC® ............................................................................................................ 6 1.2.3.3 REOLYSIN® .................................................................................................... 7 1.3 Reovirus .......................................................................................................................... 8 1.3.1 Background .............................................................................................................. 8 1.3.2 Disease burden ......................................................................................................... 9 1.3.3 Genome composition ............................................................................................. 11 1.3.4 Genome packaging ................................................................................................ 12 1.3.5 Virion composition ................................................................................................ 13 1.3.6 Replication ............................................................................................................. 14 1.3.6.1 Cell binding .................................................................................................... 14 1.3.6.1.1 Sialic acid (low affinity binding receptor) ............................................. 14 1.3.6.1.2 Junction adhesion molecule-A (JAM-A) (high affinity binding receptor) ............................................................................................................................... 16 1.3.6.1.3 Nogo receptor (NgR1) (neuron targeting receptor) ................................ 17 1.3.6.2 Endocytosis .................................................................................................... 18 1.3.6.2.1 β1 integrin (endocytosis trigger) ............................................................ 18 1.3.6.2.2 Non-polarized cell entry ......................................................................... 19 1.3.6.2.3 Polarized cell entry ................................................................................. 21 1.3.6.2.4 Intestinal M cell entry ............................................................................ 22 1.3.6.2.5 Apical vs basolateral surface entry ......................................................... 25 1.3.6.2.6 Serotype specific entry ........................................................................... 25 1.3.6.3 Capsid uncoating ............................................................................................ 26 iv 1.3.6.3.1 Intestinal uncoating ................................................................................ 28 1.3.6.3.2 Respiratory uncoating ............................................................................ 29 1.3.6.3.3 Intracellular uncoating ............................................................................ 30 1.3.6.4 Membrane perturbation .................................................................................. 31 1.3.6.5 Viral mRNA transcription .............................................................................. 33 1.3.6.6 Virion assembly ............................................................................................. 34 1.3.6.7 Viral factories ................................................................................................. 35 1.3.6.7.1 µ2 – viral factory scaffold ...................................................................... 35 1.3.6.7.2 µNS – viral core shield ........................................................................... 36 1.4. Project Objectives ....................................................................................................... 49 MATERIALS AND METHODS ................................................................................................. 50 Cell lines ......................................................................................................................... 50 Reovirus stocks ............................................................................................................... 51 Reovirus plaque assays ................................................................................................... 54 Immunocytochemistry .................................................................................................... 56 Colorimetric infectivity assay: ................................................................................... 56 Fluorescence infectivity assay: .................................................................................