Yes / Virus-encoded miRNAs are emerging as key regulators of persistent infection and host-cell immune evasion. Merkel cell polyomavirus (MCPyV), the predominant aetiological agent of Merkel cell carcinoma (MCC), encodes a single miRNA, MCV-miR-M1, which targets the oncogenic MCPyV large T antigen (LT). MCV-miR-M1 has previously been shown to play an important role in establishment of long-term infection, however, the underlying mechanism is not fully understood. A key unanswered question is whether, in addition to auto-regulating LT, MCV-miR-M1 also targets cellular transcripts to orchestrate an environment conducive for persistent infection. To address this, we adopted an RNA-Seq-based approach to identify cellular targets of MCV-miR-M1. Intriguingly, bioinformatics analysis of transcripts that are differentially expressed in cells expressing MCV-miR-M1 revealed several genes implicated in immune evasion. Subsequent target validation led to the identification of the innate immunity protein, SP100, as a direct target of MCV-miR-M1. Moreover, MCV-miR-M1-mediated modulation of SP100 was associated with a significant decrease in CXCL8 secretion, resulting in the attenuation of neutrophil chemotaxis towards Merkel cells harbouring synthetic MCPyV. Based on these observations we propose that MCV-miR-M1 targets key immune response regulators to help facilitate persistent infection, which is a pre-requisite for cellular transformation in MCC. / Funded in part by a University of Bradford studentship to PA and a Royal Society research award to JRB.

Les polyomavirus sont des virus très prévalents dans la population générale. Chez les sujets immunodéprimés, quatre polyomavirus sont associés à des pathologies. Parmi ces virus, le MCPyV est quant à lui responsable du carcinome à cellules de Merkel. Son implication dans un cancer humain a conduit à un regain d’intérêt pour la famille des Polyomaviridae. Au cours de ma thèse, nous nous somme intéressés à l’épidémiologie de six nouveaux polyomavirus humains (MCPyV, HPyV6, HPyV7, TSPyV, HPyV9 et MWPyV). Ces études ont montré que ces virus sont très répandus dans la population générale et que les infections surviennent dès l’enfance. Par ailleurs, nous nous sommes intéressés aux réactivités croisées entre polyomavirus humains et simiens phylogénétiquement proches. Nous avons montré qu’il existe des cross réactions entre le LPyV et l’HPyV9, entre le MCPyV et deux virus de chimpanzé (PtvPyV1 et PtvPyV2). Ces polyomavirus simiens ne circulent donc pas chez l’Homme. D’autre part, afin de mieux comprendre le cycle du MCPyV, nous avons initié l’identification des partenaires cellulaires de l’ensemble de ses protéines. Ce travail a tout d’abord été réalisé sur les protéines mineures de capside VP2/VP3. Des cribles en double hybride en levure ont permis d’identifier les partenaires cellulaires des VP2/3. L’interaction effective entre les protéines virales et cellulaires a ensuite été validée en cellules de mammifères par une approche de complémentation de la luciférase Gaussia princeps. Les partenaires cellulaires des protéines VP2/VP3 identifiés sont impliqués dans les voies de prolifération cellulaire, d’apoptose, NF?B et dans le transport intracellulaire du virus. / Polyomaviruses are ubiquitous in the general population and in immunocompromised patients, and four are associated with disease. 0f these viruses, MCPyV is responsible for Merkel ceil carcinoma, and its involvement in human cancer has led to a renewed interest in the Polyomaviridae family. My thesis work has focused on the epidemiology of six new human polyomaviruses (MCPyV, HPyV6, HPyV7, TSPyV, HPyV9 and MWPyV). These studies have shown that these viruses are widespread in the genera population and that infection occurs in early childhood. We have also focused on cross-reactivity between phylogenetically closely related human and simian polyomaviruses. We have shown that there is cross-reactivity between sirnian virus LPyV and HPyV9 and between MCPyV and two chimpanzee viruses (PtvPyVl and PtvPyV2). However, these simian polyomaviruses do not circulate in humans. Moreover, in order to improve understanding of the cycle of MCPyV, we set out to identify the cellular partners of its proteins. This work was initially performed on minor capsid proteins VP2 and VP3. Screening in yeast two-hybrid identified cellular partners of VP2 and VP3. Interactions between viral and cellular proteins were then validated in mammalian celis by complementation assay using Gaussia princeps luciferase. Cellular patners of VP2 and VP3 are involved in ceil proliferation, apoptosis, NFkB and intracellular transport of the virus.

Yes / Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high propensity for recurrence and metastasis. Merkel cell polyomavirus (MCPyV) is recognised as the causative factor in the majority of MCC cases. The MCPyV small tumour antigen (ST) is considered to be the main viral transforming factor, however potential mechanisms linking ST expression to the highly metastatic nature of MCC are yet to be fully elucidated. Metastasis is a complex process, with several discrete steps required for the formation of secondary tumour sites. One essential trait that underpins the ability of cancer cells to metastasise is how they interact with adjoining tumour cells and the surrounding extracellular matrix. Here we demonstrate that MCPyV ST expression disrupts the integrity of cell-cell junctions, thereby enhancing cell dissociation and implicate the cellular sheddases, A disintegrin and metalloproteinase (ADAM) 10 and 17 proteins in this process. Inhibition of ADAM 10 and 17 activity reduced MCPyV ST-induced cell dissociation and motility, attributing their function as critical to the MCPyV-induced metastatic processes. Consistent with these data, we confirm that ADAM 10 and 17 are upregulated in MCPyV-positive primary MCC tumours. These novel findings implicate cellular sheddases as key host cell factors contributing to virus-mediated cellular transformation and metastasis. Notably, ADAM protein expression may be a novel biomarker of MCC prognosis and given the current interest in cellular sheddase inhibitors for cancer therapeutics, it highlights ADAM 10 and 17 activity as a novel opportunity for targeted interventions for disseminated MCC. / In parts by the Medical Research Council (95505126) to AW, Royal Society (UF100419) to JM and Biotechnology and Biological Sciences Research Council (BB/R000352/1) to GEB and AW.

Merkel cell polyomavirus (MCPyV) is a recently discovered human virus, having it's genome often integrated in a genome of Merkel carcinoma cells. Although this type of carcinoma is not so usual, it is very aggressive and it's incidence has been rising in last few years. It is not surprising that this virus is nowadays in the centre of scientific interest, as well as other pathogens and mechanisms affecting human life. Because the virus was discovered not so long ago, its research has been at the whole beginning. This diploma thesisaims to contribute to the study of this virus from the molecular-virology point of view. A neutralizing monoclonal antibody, type IgG2a, targeted against the main capsid protein of MCPyV, VP1, and recognizing its conformational epitote was prepared. This antibody was then used for a pilot study of VP1 VLPs MCPyV movement in mammalian cells. Results showed that the studied virus, at least particularly, utilizes caveolin-1-carrying vesicles for its movement in cells (colocalisation of VP1 VLPs and caveolin-1 was observedColocalisation with EEA1 marker of early endosomes, LamP2 marker of endolysosomal compartments or with BiP marker of endoplasmic reticulum was sporadic but significant. These preliminary results suggest that MCPyV might utilise an endocytic pathway leading...

La recherche de facteurs génétiques de susceptibilité aux infections virale dans des populations générales exhaustives endémiques est une approche originale en épidémiologie génétique. Nos travaux de thèse nous ont permis d’établir, dans une population endémique pour deux virus oncogènes MCPyV et HHV-8 au Cameroun et dans une population endémique pour le VHC en Egypte, plusieurs arguments forts en faveur d’une susceptibilité génétique aux infections par les virus oncogènes humains définies par la séropositivité/ séronégativité vis-à-vis du virus impliqué. Concernant l’infection par le MCPyV, dont les modes de transmission sont peu connus, nous avons mis en évidence l’existence de fortes corrélations familiales mère-enfant et entre enfants pour la séropositivité au virus, en faveur d’une transmission virale par contacts proches. Ces résultats sont similaires à ceux observés pour l’HHV-8, dans la même population, virus pour lequel la transmission par voie salivaire est l’hypothèse la plus forte. Concernant l’infection par l’HHV-8, nous avons identifié un locus majeur de prédisposition à l’infection par une analyse de ségrégation mettant un gène majeur mendélien autosomique récessif prédisposant à l’infection, suivie d’une analyse de liaison paramétrique utilisant le modèle de l’analyse de ségrégation. Concernant l’infection par le VHC, nous avons identifié par une analyse de liaison génétique un locus majeur de prédisposition à l’infection. Nous avons ensuite identifié, par une analyse d’association en génome entier sur une grande cohorte de plus de 6500 individus, trois signaux associés avec l’infection par le VHC. Par ailleurs, nous avons également réalisé une étude fine des variants du locus du gène IL28B, associés à la clairance du VHC, cohérente avec les résultats publiés au cours de nos travaux. L’identification de facteurs génétiques impliqués dans la susceptibilité aux infections virales oncogènes et aux cancers associés permettra de mieux comprendre la physiopathologie de la réponse à ces infections et les mécanismes intervenant depuis l’exposition virale jusqu’au développement de cancers. / The identification of genetic variants predisposing to viral infection in highly endemic general populations is an original approach in genetic epidemiology. Our work suggests a genetic control of the susceptibility to human oncogenic viruses infection, in a population in Cameroon in which MCPyV and HHV-8 are highly endemic and in an Egyptian population in which HCV is endemic. MCPyV is thought to be the etiological agent of Merkel cell carcinoma, but little is known about its distribution and modes of transmission. We provided evidence for familial aggregation of MCPyV infection status suggesting that MCPyV infection is acquired through close contact, possibly involving saliva and/or the skin, especially between young siblings and between mothers and their children. Infection with HHV-8 has been shown to display strong familial aggregation, in countries in which HHV-8 infection is endemic. Our segregation analysis provided strong evidence for a recessive major gene conferring predisposition to HHV-8 infection. The following linkage analysis identified a single region on chromosome 3p22 significantly linked to HHV-8 infection. This study provides the first evidence that HHV-8 infection in children in endemic areas has a strong genetic basis. Concerning HCV infection, we performed a linkage analysis that mapped a major locus predisposing to HCV infection in an Egyptian cohort. We then performed a genome-wide association study in more than 6500 individuals, identifying three signals associated with HCV infection. Finally we investigated the role of several IL28B SNPs in HCV spontaneous clearance in an Egyptian population. The results confirm the major role of IL28B variants in the spontaneous clearance of HCV genotype 4 infection in an Egyptian population. The identification of genetic variants predisposing to viral infection should greatly improve our understanding of the molecular mechanisms involved in the response to these infections and may also unravel new pathways for investigation in viruses-associated diseases, such as cancer.

Le polyomavirus à cellules de Merkel est aujourd’hui reconnu comme l’agent étiologique du carcinome à cellules de Merkel (CCM). Le cycle viral et les mécanismes de l’oncogenèse viro-induite sont peu connus et les connaissances se basent essentiellement sur les études menées notamment sur le polyomavirus SV40. L’objectif des travaux de thèse était d’identifier les interactions entre les protéines virales et les protéines cellulaires lors de l’infection ou dans le contexte du carcinome à cellules de Merkel (CCM). Pour identifier ces interactions, nous avons réalisé des cribles double hybride en levures sur les oncogènes du MCPyV et du BKPyV. Afin valider les interactions obtenues en levures, nous avons utilisé une méthode orthogonale de validation par complémentation en cellules de mammifères reposant sur la restauration de la luciférase de Gaussia princeps. La combinaison de ses deux techniques nous a permis de valider des interactions avec des partenaires cellulaires impliqués dans la régulation du cycle cellulaire ou encore de la voie Akt-mTOR. Les précédents travaux du laboratoire, qui portaient sur l’interactome des protéines mineures de capsides VP2/VP3, avaient également permis d’identifier des interactions avec des protéines de la voie NF-kB. Nous avons alors testé les interactions entre les oncogènes et la protéine mineure de capside VP2 avec des protéines cellulaires impliquées dans cette voie. Ces travaux nous ont conduits à tester l’activation de la voie, l’expression des gènes sous le contrôle de NF-kB et la régulation de l’apoptose. Les résultats obtenus montrent une action de la protéine VP2 sur l’activation de la voie NF-kB et une induction de l’apoptose. / The Merkel cell polyomavirus is now recognized as the etiologic agent of Merkel cell carcinoma (MCC). The viral cycle and viro-induced oncogenesis mechanisms are not fully understood and the knowledge is mainly based on the studies carried out particularly on the SV40 polyomavirus. The aim of our work is to identify interactions between viral proteins and cellular proteins during productive infection or in MCC context. To identify these interactions, we performed yeast two hybrid screens on MCPyV and BKPyV oncogenes, as control. To validate the interactions obtained in yeasts, we used an orthogonal method of validation by complementation in mammalian cells based on the restoration of Gaussia princeps luciferase. The combination of these two orthogonal techniques allowed us to validate interactions with cellular partners involved in cell cycle regulation or Akt-mTOR pathway. Previous lab work on VP2/VP3 minor capsid proteins allowed the identification of interactions with NF-kB pathway involved proteins. We examined the interactions between oncogenes, VP2, with the cellular proteins involved in this pathway. This work led us to evaluate pathway activation, genes expression under the control of NF-kB and apoptosis regulation. These results evidenced an action of the VP2 protein on the activation of NF-kB pathway and an induction of apoptosis.