Le polyomavirus humain BK (virus BK) établit une infection persistante asymptomatique dans les voies rénales de 80% de la population humaine. Chez les patients transplantés, la réactivation du virus BK est à l'origine de néphropathies et de cystites hémorragiques. L'augmentation des pathologies associées au virus BK en même temps que l'utilisation de traitements immunosuppresseurs de plus en plus puissants souligne un lien étroit entre la réponse immunitaire de l'hôte et la réactivation virale. Cependant la réponse immune à l'infection par le virus BK, en particulier le rôle des cytokines antivirales dans le contrôle de l'infection est peu documentée. Ici, nous avons étudié l'efficacité antivirale des interférons (IFN) sur la multiplication du virus BK. Nous avons testé les IFN-alpha, lambda et gamma sur la souche Dunlop du virus BK dans les cellules Véro et MRC 5. L'IFN-gamma inhibe de façon dose-dépendante la transcription virale de la région précoce et de la région tardive ainsi que l'expression de la protéine virale VP1. Un moindre effet antiviral a été observé avec l'IFN-alpha et l'IFN lambda. Ces résultats sont associés à une phosphorylation prolongée de STAT 1 avec l'IFN-gamma, non retrouvée avec l'IFN-alpha et lambda. La différence d'efficacité entre ces trois types d'IFN suggère que certaines protéines induites seulement par l'IFN-gamma ont un effet antiviral dans l'infection par le virus BK. L'analyse transcriptionnelle révèle neuf protéines qui pourraient être impliquées dans cet effet antiviral spécifique. Parmi elles, nous avons étudié l'effet antiviral de l'indoleamine 2,3-dioxygénase (IDO) et les protéines de liaison au guanylate (GBP ou guanylate binding protéines), GBP1 et GBP2, sur le virus BK. Nos résultats montrent que GBP1 et GBP2 mais pas IDO contribuent à l'activité antivirale de l'IFN-gamma sur le virus BK. Trouver le mécanisme d'action de ces protéines antivirales induites par l'IFN pourrait nous aider à développer une stratégie thérapeutique / The human polyomavirus BK (BK virus) establishes an asymptomatic persistent infection in the urinary tract of 80% of the human population. In transplant recipients, reactivation of the BK virus infection is the cause of nephropathy and hemorrhagic cystitis. Diseases associated with BK virus infections are increasing at the same time as potent immunosuppressive therapies are developing. This highlights the importance of components of the immune system in controlling viral reactivation. However, the immune response to the BK virus, particularly the role of antiviral cytokines in infection control, is poorly documented. Here, we investigated the antiviral efficacy of interferons (IFN) on the BK virus multiplication. We tested IFN-alpha, lambda and gamma on the Dunlop strain of BK virus in Vero cells and MRC 5 cells. Treatment with IFN-gamma inhibited the expression of the viral protein VP1 in a dose dependent manner and decreased the expression of the early and late viral transcripts. A weaker antiviral effect was observed with IFN-alpha and IFN-lambda. These results are associated with a prolonged STAT1 phosphorylation with IFN-gamma but not with IFN-alpha and lambda. The difference of efficacy between these three types of interferon suggests that some interferon induced proteins only produced by IFN-gamma had an antiviral effect on BK virus infection. Transcriptomic analysis reveals that nine proteins could be involved in this specific antiviral effect. Among them, we selected and investigated the antiviral effect of indoleamine 2,3-dioxygenase (IDO) and guanylate binding protein 1 and 2 (GBP1 and GBP2) on the BK virus. Our results suggest that GBP1 and GBP2 but not IDO contribute to the antiviral activity of IFN-gamma on the BK virus. Finding the action mechanism of these IFN gamma induced antiviral proteins could help to develop a therapeutic strategy

<p>The Immunity-Related GTPases (IRGs) are a family of dynamin-like proteins found in vertebrates that play critical roles in cell-autonomous resistance to bacteria and protozoa. The IRGs are divided into two subfamilies, with the GMS IRGs exerting a regulatory function over the GKS IRGs, affecting GKS IRG expression, localization, and ultimately function. The profound loss of host resistance seen in mice lacking the GMS protein Irgm1 suggests that GMS IRGs may additionally have broader functions beyond the regulation of GKS IRGs, though the nature of these functions remains poorly understood. In this dissertation, we address the regulatory functions of GMS IRGs in mouse cells.</p><p>We first addressed regulation of GKS IRGs (Irga6 and Irgb6) by GMS IRGs (Irgm1 and Irgm3). We found that in both fibroblasts and macrophages lacking these GMS IRGs, that the GKS IRGs relocalized to form punctate structures that were ubiquitin-, p62-, and LC3-positive. A biochemical analysis indicated that the GKS IRGs were directly ubiquitinated through K63 linkages. Collectively, these results suggested that GMS IRGs regulate aggregation of GKS IRGs and their transfer to autophagosomes through one of at least two possible mechanisms -- by the direct association of GMS IRGs with GKS IRGs to block their aggregation that subsequently leads to autophagic removal, and/or by directly promoting autophagic removal of spontaneously forming GKS aggregates. The latter hypothesis was addressed using a series of complementary assays, which ultimately showed that absence of Irgm1 has no effect on the maturation of autophagosomes in fibroblasts, and only a very small and statistically insignificant effect in macrophages. Thus, we conclude that the major mechanism through which GMS IRGs regulate GKS IRGs is by directly inhibiting their aggregation, rather than through general effects on autophagic initiation or maturation of GKS IRG-containing autophagosomes.</p><p>We also addressed the possibility of broad regulatory functions of GMS IRGs beyond the regulation of GKS IRGs by examining whether GMS IRGs can affect another family of dynamin-like GTPases, the guanylate-binding proteins (GBPs). Despite no previous evidence of interactions between these two protein families, we found that the absence of GMS IRGs had striking effects on the localization of the murine Gbp2, leading it to colocalize with GKS IRG aggregates formed as a consequence of GMS IRG deficiency. We further demonstrated that unlike the GKS IRGs, Gbp2 was not tagged with K63-linked ubiquitin chains, which might have targeted it for specific macroautophagy, implying that Gbp2 is not aggregating in the absence of Irgm1. We then showed both that Gbp2 forms puncta in the presence of generic protein aggregates, and that guanylate-binding proteins including Gbp2 promote the degradation of GKS IRG protein aggregates. These findings suggest that GMS IRGs do not exert direct control over GBPs, but rather that GBPs are involved in the macroautophagic degradation of protein aggregates as a primary function, and are thus influenced indirectly by GMS IRGs.</p><p>In total, our experiments contribute to the understanding of regulatory interactions among GMS IRGs, GKS IRGs, and GBPs. These results will be important in establishing the mechanisms through which these important families of proteins influence eradication of bacterial and protozoan pathogens through key innate immune mechanisms.</p> / Dissertation