Oncolytic Viruses (OVs) are often attenuated to increase their safety profile, however this can lead to reduced efficacy in heterogeneous malignancies and result in resistance to OV therapy. Our group utilizes small molecule enhancers of OV therapy termed viral sensitizers. These small molecules have been shown to enhance the replication and spread of oncolytic rhabdovirus VSVΔ51 in vitro and prolong survival in tumour-bearing mice. In this study, we evaluate the ef-fect of these viral sensitizers on the innate antiviral response in order to identify the mechanism of action responsible for their viral-sensitizing properties. Our previous data suggest that VSe1 and its structural analogues affect the type I IFN antiviral response and have the potential to af-fect cellular redox homeostasis. We hypothesized that VSe1 and its structural analogues potenti-ate VSV∆51 activity by inhibiting the type I IFN response via redox-mediated dysregulation. In this study, we demonstrate that the viral sensitizers inhibit the nuclear translocation and transcrip-tional activity of NFκB, which in turn dampens the expression of antiviral cytokines IFN-, TNFα and IL-6. We also provide evidence supporting the possibility that the NFκB inhibition may be a result of the formation of ROS intermediates by the viral sensitizers, which leads to re-duced nuclear translocation of NFκB subunits, thereby preventing NFκB-mediated cytokine production. Overall, this work contributes to the identification of the mechanism of action of our viral sensitizers and highlights the finding that oncolytic VSV infection can be enhanced through redox-mediated modulation of the innate antiviral response.

Les cellules épithéliales pulmonaires constituent la première ligne de défense face aux virus respiratoires via la sécrétion de mucus, de peptides, de cytokines et chimiokines qui déterminent l'élimination ou la progression de l’infection. Les principales cytokines antivirales produites par les cellules épithéliales alvéolaires (AEC) sont les interférons (IFN) type I (α/β) et III (λ). La liaison d’IFNβ à son récepteur induit une voie antivirale bien caractérisée qui aboutit à l’activation du complexe ISGF3 (STAT1, STAT2 et IRF9) qui permet la transcription de multiples gènes codant pour des protéines à activité antivirale et immunorégulatrice. Il a récemment été démontré que la costimulation des cellules épithéliales pulmonaires par l’IFNβ et le Tumor Necrosis Factor α (TNFα), également produit lors d’une infection, synergisent pour induire un état antiviral tardif distinct. D’autre part, il a été montré que la synergie entre le TNFα et l'IFNβ induit une voie de signalisation impliquant STAT2 et IRF9, mais indépendante de STAT1 permettant l’expression du gène DUOX2. Notre but est de déterminer l’importance de cette nouvelle voie de signalisation induite par la costimulation du TNFα+IFNβ, impliquant STAT2 et IRF9 indépendamment de STAT1 dans la régulation d’un programme transcriptionnel antiviral et immunorégulateur tardif. Notre premier objectif est de déterminer si des gènes antiviraux et immunorégulateurs qui sont induits par la costimulation par TNFα+IFNβ sont dépendants de la voie STAT2/IRF9, indépendamment de STAT1. En utilisant la technique de qRT-PCR, nous avons identifié 3 gènes immunorégulateurs, CXCL10, IDO et APOBEC3G, induits de manière synergique en réponse à TNFα+IFNβ dans les cellules A549, un modèle de cellules épithéliales pulmonaires. Afin de confirmer que ces gènes sont induits indépendamment de STAT1, nous avons validé leur expression dans la lignée cellulaire U3A déficiente en STAT1. Par l'utilisation d'ARN interférants (ARNi) dirigés contre STAT2 et IRF9, nous avons confirmé que l’induction de ces gènes est dépendante de STAT2 et IRF9. Finalement, l’analyse de l’activité du promoteur de CXCL10 en réponse à TNFα+IFNβ par des essais rapporteurs luciférases a permis de montrer que la régulation se fait au niveau transcriptionnel. Notre deuxième objectif, est de déterminer si STAT6 pourrait remplacer STAT1 dans la voie de signalisation induite par TNFα+IFNβ. En effet, STAT6 est un inducteur connu de l’expression de DUOX2 en réponse à IL4+IL13. Contrairement à notre hypothèse, l’inhibition de STAT6 par ARNi augmente l’expression de DUOX2 en réponse à TNFα+IFNβ suggérant que STAT6 est un régulateur négatif. Nos résultats ont permis de comprendre de manière plus détaillée les mécanismes mis en place dans le développement d’une réponse antivirale. D’autre part, l’étude de l’effet de l’IFNβ et du TNFα est également pertinente pour les maladies chroniques inflammatoires et autoimmunes. De plus, nos résultats illustrent un nouveau paradigme concernant les mécanismes de signalisation cellulaire impliqués dans la synergie entre deux cytokines qui pourrait être applicable à des combinaisons de cytokines autres que TNFα+IFNβ. / Lung epithelial cells are the first line of defense against respiratory viruses via mucus secretion, peptides, cytokines and chemokines that determine the progression of the infection. The main antiviral cytokines produced by alveolar epithelial cells (AEC) are the interferons (IFN) type I (α / β) and III (λ). IFNβ binding to its receptor induces an antiviral pathway that is well characterized and leads to activation of the ISGF3 complex (STAT1, STAT2 and IRF9) which allows the transcription of multiple genes encoding proteins with antiviral and immunoregulatory activity. It has recently been shown that the costimulation of lung epithelial cells by IFNβ and Tumor Necrosis Factor α (TNFα), also produced during infection, induces a separate and late antiviral state, through synergy. On the other hand, it has been shown that the synergy between IFNβ+TNFα induces a signaling pathway involving STAT2 and IRF9 independently of STAT1 permitting the expression of the DUOX2 gene. Our goal is to determine the importance of this new signaling pathway induced by costimulation of TNFα+IFNβ involving STAT2 and IRF9 regardless of STAT1 in regulating the antiviral immunoregulatory and late transcriptional program. Our first objective is to determine whether antiviral and immunomodulatory genes that are induced by costimulation TNFα+IFNβ are dependent on the STAT2/IRF9 way, indenpant of STAT1. Using the technique of qRT-PCR, we identified 3 immunoregulatory genes, CXCL10, IDO and APOBEC3G, synergistically induced in response to TNFα+IFNβ in A549 cells, a model of pulmonary epithelial cells. To confirm that these genes are induced independantly of STAT1, we validated their expression in the STAT1 deficient cell line, U3A. By the use of interfering RNA (siRNA) directed against STAT2 and IRF9, we confirmed that the induction of these genes is dependent STAT2 and IRF9. Finally, the analysis of the activity of CXCL10 promoter in response to TNFα+IFNβ by luciferase reporter assays has shown that the regulation is at the transcriptional level. Our second objective is to determine whether STAT6 could replace the STAT1 in the signaling pathway induced by TNFα+IFNβ. Indeed, STAT6 is a known inducer of the expression of DUOX2 in response to IL4+IL13. Contrarily to our hypothesis, inhibition of STAT6 by RNAi increases the expression of DUOX2 in response to TNFα+IFNβ suggesting that STAT6 is a negative regulator. Our results allow the understanding of the mechanisms in the development of an antiviral response in more detail. On the other hand, the study of the effect of IFNβ and TNFα is also relevant for chronic inflammatory and autoimmune diseases. In addition, our results illustrate a new paradigm for cell signaling mechanisms involved in the synergy between two cytokines that may be applicable to combinations of cytokines other than TNFα+IFNβ.

L’adénovirus (AdV) est un virus non enveloppé à ADN double brin qui entre dans la cellule par endocytose. Dans l’endosome un désassemblage partiel de la capside permet la libération d’une protéine interne de la capside, la protéine VI (PVI). Cette protéine code une hélice amphipathique qui va permettre la rupture de l’endosome. Des travaux antérieurs du laboratoire ont montré que le transport des particules virales vers le noyau nécessite la présence du motif conservé PPxY dans la PVI qui permet le recrutement d’ubiquitines ligases de la famille des Nedd4 (telles que Nedd4.1 et Nedd4.2). Il a précédemment été montré que la rupture des membranes induite lors d’infections bactériennes activait l’autophagie afin d’éliminer le pathogène intracellulaire via une dégradation lysosomale. Nos résultats démontrent que l’AdV induit également l’autophagie lors de son entrée dans la cellule. L’utilisation de différents AdV mutants nous a permis de démontrer que la rupture de l’endosome était responsable de l’induction de l’autophagie. De plus nos résultats montrent que le virus sauvage est capable d’éviter sa dégradation en contrôlant l’autophagie grâce au recrutement de la ligase Nedd4.2 via le motif PPxY de la PVI. Au contraire, un virus mutant dépourvu du motif PPxY et donc incapable de recruter la Nedd4.2 est séquestré dans les vésicules autophagiques puis dégradé par la fusion de ces vésicules avec les lysosomes. Ainsi le motif PPxY constitue un déterminant moléculaire permettant au virus de contourner les défenses cellulaires antivirales. / Adenoviruses (AdV) are linear ds-DNA containing, non-enveloped viruses that enter cells by receptor-mediated endocytosis. Once in the endosome it occurs a partial disassembly of the capsid allowing the releases of the membrane lytic capsid protein VI, which encodes an N-terminal amphipathic helix responsible for the endosome rupture. Our previous work showed that transport to the nucleus requires a conserved PPxY motif in PVI, which recruits ubiquitin ligases of the Nedd4 family (e.g. Nedd4.1 and 4.2). Previous work has shown that membrane damage induced by invasive bacteria elicits selective cellular autophagy to eliminate the pathogen via lysosomal degradation. In our current work we show that Adv also induce autophagy upon entry. Using a set of mutant AdV’s attenuated at each step of the membrane penetration process we show that indeed the membrane damage induced by the virus is causative for autophagy induction. Moreover the data show that wildtype AdV limit the level of autophagy induction and evade autophagic degradation by using a Nedd4.2 dependent process. In contrast, mutant viruses mutated for its PPxY and that fail to recruit Nedd4.2 are subject to autophagic degradation. Our data suggest that the presence of the PPxY motif in the virus subverts the autophagic process and thus identify the PPxY motif as an integral part of the virus to undermine cellular antiviral mechanism.

Translation control is crucial during virus-host interaction, in which the host relies on the translation machinery to mount an antiviral response or induce the inflammation response to reduce virus spread, while the virus aims to take control of this system to thwart the host defense while producing viral progeny. The field of oncolytic virus (OV) therapy relies on replicating, engineered viruses that preferentially infect tumor cells to induce direct oncolysis or promote an antitumor immune response. Despite the importance of translation control in virus-host interaction, not much has been described on the interaction at the translation level between OV and cancer cells. I propose that this knowledge gap could reveal significant improvements in OV efficacy in treating cancer. In my first study, I set out to characterize the translatome of an infection-resistant breast cancer cell line infected by three clinically advanced OVs to identify residual antiviral activity in cancer cells regulated by translation control. I found the inositol phosphatase Inpp5e to be a novel antiviral gene that is translationally induced during infection via a transcript variant shift. Mechanistically, I showed that the majority of Inpp5e transcripts in uninfected cells contain a long 5’ UTR that harbor four translationally inhibitory upstream reading frames (uORF). Yet, OV infection induced the expression of a shorter 5’ UTR with a spliced intron that removes three uORFs, derepressing the translation of Inpp5e mRNA. CRISPR-Cas9 knockout of Inpp5e also enhanced the infectivity of oncolytic HSV1 and VSV. My study suggests the existence of a class of translationally regulated antiviral genes in cancer cells. In my second study, I sought to adapt the translation of transgenes to the unique translation condition imposed by the infecting virus via the incorporation of a viral 5’UTR. I identified HSV1 5’UTRs by locating the transcription start site of most HSV1 genes using RNA-seq data, then determined the 5’UTR of US11 as a potent translation enhancer during HSV1 infection. Incorporation of this 5’UTR into the transgene expression cassette inserted into the HSV1 genome enhanced transgene expression significantly at the translation level. In my third study, I set out to explore the mechanism of miR-223 mediated inflammation inhibition. miR-223 is a protective miRNA in the context of atherogenesis via suppressing inflammatory signaling. Using transcriptome and translatome profiling (RNA-seq and Ribo-seq), I found that the inhibitory effect of miR-223 on inflammation occurs primarily at the translation level. Overall, my work highlights the importance of translation control in OV-cancer cells interaction, as well as in inflammation-related diseases.

The classical description of the innate antiviral response involves the production of type I interferon (IFN) and the subsequent expression of hundreds of interferon stimulated genes (ISGs), which cooperatively repress viral replication and spread. More recently, an IFN-independent antiviral response has also been described, in which the entry of an enveloped virus induces a subset of ISGs without requiring the production of IFN, although the details of this response remain unclear. In this work, multiple approaches were used to further characterize antiviral signaling pathways. Initially, the potential involvement in the IFN-independent response of the small GTPase Rac1, which has been implicated in both viral entry and antiviral signaling, was investigated. Here, Rac1 was shown to have a possible function in the negative regulation of ISG expression, although technical complications prevented definitive conclusions. As an alternative strategy to identify novel aspects of antiviral signaling, the mechanism of action of ICP0, a herpes simplex virus (HSV) protein involved in innate immune evasion, was investigated. Although ICP0 is generally thought to perform its actions in the nucleus, by tagging proteins for proteasome-mediated degradation via the E3 ubiquitin ligase activity of its RING finger domain, here it was shown that not only does cytoplasmic ICP0 have a RING-dependent but proteasome-independent ability to block antiviral signaling, but also that ICP0 has a previously unknown RING-independent function in the promotion of viral replication in the cytoplasm. To further investigate the cytoplasmic activities of ICP0, proteins interacting with ICP0 in the cytoplasm were identified using quantitative mass spectrometry. This revealed several intriguing binding partners for ICP0, including WDR11, a poorly-characterized cellular protein which was shown to undergo a dramatic relocation during HSV infection, although it was not required for viral replication in cultured cells. Therefore, this study has uncovered several new and unexpected insights into ICP0 behavior. / Thesis / Doctor of Philosophy (PhD)

Les LT CD8 mémoires constituent une population hétérogène. Une modulation des différents signaux d’activation des LT CD8 naïfs influe sur la diversité des LT CD8 effecteurs et mémoires générés. A coté des sous populations classiquement décrites de LT CD8 mémoires développés dans des conditions infectieuses, il existe une population de LT CD8 mémoires générés dans des conditions d’inflammation stérile, c’est-à-dire en absence de pathogènes et de signaux moléculaires dérivés de pathogènes : les TIM pour « T-inflammatory memory cells ». Au cours de cette thèse j’ai eu pour objectif : 1) de mieux caractériser la population de TIM 2) d’évaluer leur capacité à répondre à une activation par un pathogène viral et ainsi leur plasticité et 3) d’étudier leur recrutement au site d’une infection locale respiratoire.J’ai ainsi pu identifier de nouvelles propriétés des TIM et montrer que les TIM sont recrutés dans une réponse secondaire à un virus exprimant la même spécificité antigénique, en générant des LT CD8 mémoires secondaires aux fonctions améliorées. De plus, les LT CD8 mémoires secondaires générés présentent un avantage fonctionnel par rapport aux LT CD8 mémoires primaires dans leur capacité de production de TNF-α et de la chimiokine XCL1. Cette dernière propriété pourrait leur conférer un avantage pour la réponse à des antigènes cross présentés. J’ai par ailleurs montré la capacité des TIM à être recrutés au niveau du poumon et des voies aériennes au cours d’une infection respiratoire virale. Ces résultats montrent que les LT CD8 mémoires générés dans des conditions d’activation inflammatoires stériles peuvent prendre part au contingent de cellules immunitaires impliquées dans des réponses à des infections. Ces résultats ouvrent un champ d’investigation intéressant concernant la plasticité des LT CD8 mémoires. Ils pourraient avoir une incidence sur certaines pathologies inflammatoires, dans le cas d’une ré-activation des TIM par un virus cross réactif. / Memory CD8 T cells represent a heterogeneous population. A modulation of the activation signals during naïve CD8 T cells activation influences the diversity of the Effector and Memory CD8 T cells generated. Besides the classically described subsets of Memory CD8 T cells, generated under infectious conditions, are T inflammatory memory CD8 T cells (TIM). TIM are generated under sterile priming conditions that are devoid of pathogens and pathogen-derived danger signals.During this thesis, I had the latter objectives: 1) a better characterisation of TIM, 2) to evaluate whether they could be recruited in an immune response directed against a virus and thus investigate their plasticity, 3) to examine their recruitment to the site of a respiratory local infection.I have identified new features of TIM and shown that TIM can take part to the immune response triggered by a virus expressing their cognate antigen and further differentiate into secondary memory cells with improved functions. The secondary memory CD8 T cells they generate display a functional advantage over primary memory cells in their capacity to produce TNF-α and the XCL1 chemokine. This last result could give them an advantage against cross-presented antigen. Furthermore, I have shown that TIM cells can be recruited in lung and airways during a respiratory viral infection. These results show that memory CD8 T cells generated under sterile priming conditions can take part to the contingent of immune cells involved in responses to infection. They open an interesting field of investigation of the plasticity of memory CD8 T cells. They could have an impact in inflammatory diseases, in the case of re-activation of TIM by a cross-reactive virus.

The molecular mechanisms underlying susceptibility to severe respiratory syncytial virus (RSV) infection remain poorly understood. Herein, we report on the role of osteopontin (OPN) in regulation of RSV infection in human epithelial cells and how interleukin-1 beta (IL-1β), a cytokine secreted soon after RSV infection, when persistently expressed can induce OPN expression leading to increased viral infection. We first compared OPN expression in two human epithelial cell lines: HEK-293 and HEp-2. In contrast to HEp-2, HEK-293 expresses low levels of pro-caspase-1 resulting in decreased IL-1β expression in response to RSV infection. We found a correlation between low IL-1β levels and a delay in induction of OPN expression in RSV-infected HEK-293 cells compared to HEp-2. This phenomenon could partially explain the high susceptibility of HEp-2 cells to RSV infection versus the moderate susceptibility of HEK-293 cells. Also, HEK-293 cells expressing low levels of pro-caspase-1 exhibit decreased IL-1β expression and delayed OPN expression in response to RSV infection. HEK-293 cells incubated with human rIL-1β showed a dose-dependent increase in OPN expression upon RSV infection. Also, incubation with rOPN increased RSV viral load. Moreover, HEp-2 cells or mice infected with a mucogenic RSV strain RSV-L19F showed elevated levels of OPN in contrast to mice infected with the laboratory RSV strain rA2. This correlated with elevated levels of OPN following infection with RSV-L19F compared to rA2. Together, these results demonstrate that increased OPN expression is regulated in part by IL-1β, and the interplay between IL-1β and OPN signaling has a pivotal role in the spread of RSV infection.

No description available.

No description available.

Les cellules dendritiques plasmacytoides (pDCs), spécialisées dans la réponse antivirale, produisent de fortes quantités d’interféron (IFN) lorsqu’elles sont en contact avec des cellules infectées par des virus. Pourtant, les pDCs sont réfractaires à l’infection. Ce mécanisme d’activation de la réponse antivirale par le contact physique avec les cellules infectées, nouvellement découvert, constituerait un aspect général des voies de défense de l’hôte contre les virus.En utilisant le virus de l’Hépatite C et de la Dengue comme modèle viral, nous avons observé une réorganisation moléculaire au niveau des contacts entre les pDCs et les cellules infectées. La polarisation d’éléments cellulaires, notamment de régulateurs du cytosquelette d’actine et de molécules de la machinerie d’endocytose en direction du contact favoriserait son établissement et/ou sa stabilisation ainsi qu’une transmission efficace d’éléments viraux, ensuite reconnus par les pDCs. Nous avons également démontré que les pDCs effectuent des contacts plus stables et présentent une polarisation plus importante d’éléments cellulaires aux contacts avec des cellules infectées qu’avec des cellules non infectées. Ces interactions présentent des similarités avec les synapses, contacts cellulaires organisés impliqués dans la communication cellulaire. Notamment, les synapses immunologiques jouent un rôle important dans l’activation de la réponse immunitaire adaptative. Nous proposons donc de nommer ces contacts activateurs de pDCs des « synapses immunologiques innées ». Ce mécanisme représenterait un processus de reconnaissance des infections par les pDCs généralisable à différents types de virus, par « scan » du statut infectieux des cellules par contact. Nos résultats suggèrent également que des éléments viraux s’accumulent au niveau de ces contacts. Ces éléments diffèrent en fonction du type d’infection. Notamment, nous avons mis en évidence dans un contexte d’infection par le virus de la Dengue que des structures virales non canoniques et non infectieuses, différentes des particules virales infectieuses dites « classiques », jouent un rôle important dans l’activation de la réponse antivirale. Notre travail apporte un nouvel angle d’analyse de l’activation des pDCs et des stratégies de détection des infections virales par l’hôte. / Plasmacytoid dendritic cells (pDCs), specialized in the antiviral response, are important producers of interferons (IFN) after cell-cell contacts with virally infected cells. Nonetheless, they are poorly permissive to the majority of viral infections. This newly uncovered mechanism of the activation of an antiviral response by physical cell-cell contacts with infected cells could constitute a general aspect of the host defense against viral infections.Using Hepatitis C virus and Dengue virus as models, we observed a molecular reorganization of the contacts between pDCs and infected cells. The polarization toward contacts of cellular elements, such as regulators of the actin cytoskeleton and components of the endocytic machinery could favor their establishment and/or their stabilization, as well as the efficient transmission of viral elements that are recognized by pDCs. We also demonstrated that pDCs contacts with infected cells are more stable and present a higher polarization of cellular components than contacts with uninfected cells. These interactions present similarities with synapses, a type of organized contact involved in cell-to-cell communication. Notably, immunological synapses are known to play an important role in the activation of the adaptive immune response. We thus propose to call these pDC-activating contacts « innate immunological synapses ». This mechanism could represent a general process of recognition of viral infections by pDCs, by « scanning » the infectious status of the cells by cell-cell contacts. Our results also suggest that viral elements cluster at the level of contacts. These elements differ depending on the type of viral infection. Notably, we observed in the context of Dengue virus infection that non-infectious non-canonical viral structures, that differ from the « classical » viral infectious particles, play an important role in the activation of the antiviral response. Our work brings a new light in the mechanisms of pDC activation and in the host defense strategies against viral infection.