Sequences at the 5' end of the nascent RNA are known to be important as signals for encapsidation of the genome of vesicular stomatitis virus. In order to define the specific sequences involved in this process and to develop an in vitro encapsidation system, in vitro transcription from SP64-based plasmids was used to synthesize RNA molecules corresponding to various portions of the viral 5' plus strand sequence. Some of these RNAs were tested for their ability to bind the capsid N protein in vitro. N protein in this assay was provided either from VSV mRNA programmed reticulocyte lysates or from infected cell extracts or, in collaboration with Dr. Sue Moyer (Gainesville, Florida), purified from viral nucleocapsids. This thesis describes the construction of the SP64-based plasmids and the use of their RNA transcription products in the experiments described above. I also constructed a series of plasmids that could direct the synthesis of RNA molecules which have many of the features of VSV defective particle genomes. Two of the constructs generate a defective-like RNA carrying a reporter gene capable of expressing the bacterial lac Z protein. These RNAs have the potential, after in vitro encapsidation and transfection into mammalian cells, of producing readily detectable helper-dependent virions. / Thesis / Master of Science (MS)

La réponse interféron est une réponse antivirale complexe qui, après la détection de pathogènes par des PRR (récepteurs de motifs associés aux pathogènes), conduit à l’induction de centaines de gènes appelés ISG (gènes stimulés par l’interféron). Dans la littérature, il existe plusieurs ISG capables de s’opposer à l’infection virale ; cependant le rôle antiviral précis d’un grand nombre d’entre eux reste inconnu ou mal caractérisé. Pendant ma thèse, je me suis concentré sur la caractérisation d’ISG20 pendant la réplication de deux virus, VSV et le VIH-1. La protéine ISG20 a été décrite au préalable comme une exonucléase 3’-5’ antivirale en agissant sur la dégradation directe du génome viral. Cependant, la diminution de la quantité d’ARN viraux liée à ISG20 était controversée.Afin de mieux comprendre le mécanisme par lequel ISG20 interfère avec la réplication virale, j’ai construit plusieurs mutants d’ISG20. Les résultats obtenus indiquent que l’activité antivirale d’ISG20 ne repose pas uniquement sur sa capacité à dégrader l’ARN, puisque plusieurs mutants ont perdu leurs propriétés antivirales malgré une robuste activité RNase in vitro.Mes résultats montrent qu’ISG20 peut bloquer la réplication virale en bloquant la traduction. Dans les cellules exprimant ISG20, ce blocage intervient à la fois pendant l’infection virale et lors de l’expression ectopique de gènes rapporteurs. Les résultats que nous avons obtenus indiquent que la protéine ISG20 affecte la traduction qu’elle soit cap- ou IRES-dépendant. Cette inhibition de la traduction est très probablement indépendante de l’initiation.Afin d’étayer le rôle antiviral d’ISG20 pendant l’infection virale, des souris invalidées pour isg20 (-/-) ont été générées et leur capacité à supporter l’infection par VSV in vivo a été analysée. Les résultats obtenus impliquent clairement ISG20 dans le contrôle naturel de la propagation virale in vivo, confirmant nos données ex vivo.Dans l’ensemble, les données obtenues pendant ma thèse indiquent qu’ISG20 est un important facteur antiviral et mettent en évidence un nouveau mécanisme d’inhibition virale où ISG20 interfère avec la traduction d’ARNm viral. / Interferons specify a complex antiviral response that upon the detection of pathogens through various cellular pattern-recognition receptors (PRRs) lead to the induction of hundreds of genes named interferon-stimulated genes (ISGs). Several ISGs have been reported to restrict viral infection, however the antiviral role/s of many of them remains either unknown or poorly characterized. During my thesis I have focused on the characterization of ISG20 during the replication of two viruses, VSV and HIV-1. ISG20 had been previously identified as an antiviral 3’-5’ exonuclease and was thought to act by directly degrading viral genomes. However, the decrease in viral RNAs specified by ISG20 was controversial. To gather further insights into the mechanism with which ISG20 interfered with viral replication, I constructed several mutants of ISG20. The results we have obtained indicated that the antiviral activity of ISG20 does not solely rely on it's the ability of ISG20 to degrade RNA, as several mutants were identified that lost their antiviral properties despite a robust RNase capacity in vitro.We have found here that ISG20 could block viral replication through a block in translation. This block occurred both during viral infection as well as during the ectopic expression of reporter genes in ISG20-expressing cells. The results we have obtained indicate that ISG20 affects both cap- and IRES-mediated translation in a manner that is very likely independent from translation initiation.To substantiate the antiviral role of ISG20 during viral infection, knock-out isg20 -/- mice were generated and then analyzed for their ability to support VSV infection in vivo. The results obtained, clearly implicate ISG20 in the natural control of viral spread in vivo, strongly supporting our data ex vivo.Overall, the data obtained during my thesis indicate that ISG20 is an important antiviral factor and shed light on a novel mechanism of viral inhibition whereby ISG20 interferes with viral mRNA translation.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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In polarized epithelial cells, vectorial protein trafficking is important for transporting specific membrane proteins to generate distinct apical and basolateral membrane protein compositions. The Exocyst is a conserved hetero-octameric protein complex, which regulates different aspects of protein trafficking, including tethering of the Golgi-derived vesicles to target membranes. Two of the Exocyst subunits, Sec5 and Exo84, competitively bind to the small GTPases, RalA and RalB, in a GTP-dependent manner. Although Ral GTPases have been proposed to mediate assembly of Exocyst holocomplexes, we hypothesize that they actually serve to allosterically regulate Exocyst functions by promoting association or disassociation of additional factors. Previous studies have shown that active RalA, but not RalB, accelerated basolateral exocytosis of E-cadherin. In contrast, knockdown of RalB, but not RalA, disrupts endocytosis of E-cadherin. However, mechanisms by which association of Ral GTPases with Sec5 and Exo84 regulate basolateral protein trafficking remain unclear. Here we investigate roles of Ral GTPases and the Exocyst in regulating basolateral protein trafficking using Madin Darby canine kidney (MDCK) cells and RNA interference (RNAi) technology. We show that RalA, but not RalB, is required for basolateral exocytosis of vesicular stomatitis virus glycoprotein (VSV-G) in the MDCK cells. We combined immunofluorescent labeling and surface biotinylation assays to demonstrate that RalA regulates VSV-G trafficking through the distinct interactions with Sec5 and Exo84. We also show that a Ral-uncoupled Sec5 mutant, but not a Ral-uncoupled Exo84 mutant, inhibits E-cadherin exocytosis. These results suggested that RalA and the Exocyst are required for basolateral exocytosis, and that RalA-Sec5 and RalA-Exo84 interactions play different roles during this process. Our study may provide new insights into mechanisms regulating protein trafficking in epithelial cells, and potentially lead to development of new therapeutic targets for the treatment of diseases in which exocytosis is impaired, such as Polycystic kidney disease and diabetes.

The use of oncolytic viruses (OVs) to selectively destroy cancer cells is poised to make a major impact in the clinic and potentially revolutionize cancer therapy. Pre-clinical and clinical studies have shown that OV therapy is safe, well-tolerated and effective in a broad range of cancers. Still, resistance due to tumour heterogeneity highlights areas for improvement in OV based therapeutics. Combining OVs and small molecules is a promising strategy to selectively enhance OV-mediated anti-tumour effects. To this end, we have previously identified the synthetic compound Viral Sensitizer 1 (VSe1) that enhances the spread of oncolytic vesicular stomatitis virus (VSVΔ51) in resistant cancer cell lines up to 1000-fold, resulting in synergistic cell killing and improved efficacy in vitro and in vivo. The electrophilic nature of VSe1 prompted us to investigate the scaffold to identify active analogs with more favourable physiochemical properties and explore structure-activity relationships (SAR). In vitro assays and a rational approach in the design of VSe1 analogs allowed us to identify functional groups that can be modified without hampering activity. Lead compounds created in this study based on a pyrrole scaffold increase OV growth up to 2000-fold in vitro and demonstrate remarkable selectivity for cancer cells over normal tissue ex vivo and in vivo. Compared to the parental VSe1, these small molecules also possess enhanced stability with reduced electrophilicity and are well-tolerated in animals, leading to reduced tumour burden and prolonged survival in vivo when used in combination with VSVΔ51. It was known from previous studies that VSe1 suppresses the type I interferon response generated by cancer cells to defend against viral infection. In this study, further investigation revealed that VSe1 and its analogs inhibit the nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), resulting in dampened transcriptional expression and secretion of IFN-β and interferon stimulated genes, thereby increasing viral replication and spread. While these findings further elucidated the effect these compounds have on the innate antiviral response, the molecular mechanisms leading to NFκB inhibition remained unclear. We used the newly generated VSe1 analogs to perform ligand-based affinity capture studies leading to the identification of glutathione-s-transferases as interacting proteins, catalytically inhibited by VSe1 and to a lesser extent by its pyrrole analogs. Further inquiry revealed that VSe1 and its analogs cause an imbalance in cellular glutathione homeostasis and increase oxidative stress, which is associated with inhibition of the nuclear translocation of NFκB. However, further studies are required to assess whether these phenomena are directly or indirectly linked. Overall, this study highlights a novel approach to improving OV therapy by using a previously uncharacterized class of compounds that ultimately alter the innate cellular antiviral response through inhibition of NFκB.

Oncolytic viruses promise to significantly improve current cancer treatments through their tumour-selective replication and multimodal attack against cancer cells. However, one of the biggest setbacks for oncolytic virus therapies is the intravenous delivery of the virus, as it can be cleared by neutralizing antibodies (nAbs) from the bloodstream before it reaches the tumour cells. In our group, we have succeeded in developing aptamers to vesicular stomatitis virus (VSV), as well as to rabbit anti-VSV polyclonal neutralizing antibodies (nAbs). We tested these aptamers’ biological activity with a cell-based plaque forming assay and found that the aptamers prevented in vitro neutralization of VSV by nAbs and increased the virus infection rate of transformed cells up to 77%. In line with this approach, we enhanced the delivery of oncolytic viruses by selecting aptamers to the CT26 colon carcinoma cell line. The binding of aptamer pools has been tested on flow cytometry and the best pools were subjected to high throughput sequencing. Selected aptamers were linked to anti-VSV aptamers and applied for target delivery of the virus to cancer cells. Development of this aptamer-based technology aims to improve viral anti-cancer therapies, with a potential to be applied as treatment for patients affected with cancer. Finally, in collaboration with a group from Erlangen University, we performed an aptamer selection using capillary electrophoresis and cell-SELEX. The target, the extracellular domain of human CD83, is a maturation marker for dendritic cells and is involved in the regulation of the immune system. Selected aptamer sequences bound selectively to mature dendritic cells, in comparison to immature dendritic cells, and thus hold promise to be applied for further studies leading to a better understanding of CD83’s mechanism of action.

With the explosion of protein tools as popular platforms for discovery and therapeutics, we see greater need for regulator systems that work congruently within these frameworks, especially safe and effective tools that can be implemented in humans. To this end, we endeavor to create orthogonal, precise and flexible protein modulators that can be easily employed to control protein tools with little need to iterate design for novel contexts. Hepatitis C NS3 protease is employed as a stabilizable linker between protein domains, enabling control over protein localization with FDA approved anti-viral drugs. The power of this tool is demonstrated by controlling gene expression through the controlled tether and release of a transcription factor. Inteins have already been employed to modulate proteins in synthetic contexts, however we observe that natural systems lack the avenues of control necessary to make them indispensable. We employ existing protein tools to construct a system of modular protein association, as well as drug and light inducible schema that reveal gaps in our knowledge of how to repurpose inteins in vivo. Despite this, we use inteins in the construction of a novel cargo delivery platform based on the fusogenic properties of the viral envelope glycoprotein from Vesicular stomatitis virus (VSV-G). We confirm reduced tropism of cargo delivery based on an intein lock-and-key mechanism that has implications for both biosafety as well as targeted delivery in vivo of natively folded proteins to target cells. / 2024-05-23T00:00:00Z

<p>Autophagy is a cellular process in which cytoplasmic material is lysosomally degraded into its basic components. The primary functions of this process are cellular recycling and stress mitigation however it also has roles in both viral pathogenesis and tumourigenesis. Beclin1 is a key mediator of autophagy and is involved in its initiation. In an attempt to examine the effects of enhanced autophagy in the context of oncolytic VSV infection, a VSV mutant (ΔM51) expressing Beclin1 was constructed and characterized. It was determined through western blot analysis of autophagy marker LC3, that while VSV infection enhanced autophagy in infected cells, Beclin1 expression resulted in a transient increase in autophagy followed by markedly reduced levels of autophagy at mid to late time points. Still, Beclin1expression, either directly or possibly through altering the kinetics of VSV induced autophagy, enhanced the pathogenesis of VSV<em> </em>in some cell lines <em>in vitro</em>. However examination of the <em>in vivo</em> pathogenesis of VSV-Belcin1 elicited no differences from that of the parental virus. Despite enhanced pathogenesis in CT26 cells <em>in vitro</em>, VSV-Beclin1 displayed no improvement in the oncolysis of CT26 tumours <em>in vivo</em>, compared to VSV-GFP. It is hoped that the conclusions drawn from this study will help direct future research aimed at exploring the relationship between autophagy and VSV pathogenesis as well as future attempts to arm VSV with the intent of augmenting its oncolytic potential.</p> / Master of Science (MSc)