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National audience; Les vésicules extracellulaires (VEs) émises par les cellules constituent un nouveau mode de communication intercellulaire. Ces VEs (exosomes et microvésicules) permettent la transmission de cellule à cellule de molécules bioactives telles que des protéines, de l'ADN, de l'ARNm et/ou des microARN entrainant ainsi un échange d'information entre cellules et une éventuelle « reprogrammation » des cellules receveuses. Les cellules tumorales sont capables de libérer une quantité importante de VEs qui peuvent influer sur le développement des tumeurs, la croissance tumorale, les processus métastatiques et la résistance aux thérapies anti-cancéreuses. De plus, les VEs produites par les cellules tumorales véhiculeraient des signaux aux cellules immunitaires et contribueraient ainsi à l’échappement à la réponse immune des cellules cancéreuses et à l’établissement d’un micro-environnement immunosuppresseur facilitant la formation des tumeurs. L’herpesvirus de la maladie de Marek (MDV) est à l’origine du développement de lymphomes T chez la poule, son hôte naturel. Malgré l’identification de molécules virales pro-oncogènes, les processus conduisant à la tumorigenèse induite par le MDV restent mal connus. Nous avons cherché à déterminer le rôle des VEs produites par des lymphocytes transformés par le MDV dans les mécanismes néoplasiques en précisant notamment leur impact sur la prolifération cellulaire et sur l’établissement d’un environnement immunosuppresseur propice au développement tumoral. La caractérisation moléculaire des VEs isolées à partir de ces lymphocytes tumoraux a été réalisée par western blot et par analyse protéomique. Ces techniques nous ont permis de valider la présence de marqueurs spécifiques des VEs, d’identifier des biomolécules enrichies dans ces vésicules et de montrer que ces VEs ne contiennent pas de protéines virales. Par ailleurs, nous avons pu observer que ces VEs sont rapidement internalisées par les cellules receveuses et qu’elles présentent des propriétés modulatrices de la prolifération cellulaire sur des cellules immunes de poule. Ces VEs ont un effet proprolifératif sur les lymphocytes T spléniques et un effet anti-prolifératif sur les lymphocytes B de bourse de Fabricius. De plus, ces VEs n’ont pas d’effet sur la prolifération des macrophages. Ces résultats préliminaires indiquent que les VEs sont capables de transférer des signaux opposés aux lymphocytes B et T non-infectées, affectant la prolifération cellulaire. Nous recherchons actuellement si ces VEs peuvent également influer sur l’état d’activation de ces cellules.

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International audience; VP22 is a tegument protein specific of alpha-herpesviruses encoded by the UL49 gene. Unlike most species from this subfamily, VP22 is essential for Marek’s disease virus (MDV) and Varicella-Zoster virus (VZV) replication. Several properties have been associated to MDV VP22, including virus cell-to-cell spread, DNA binding and cell cycle arrest. This 249aa-long protein contains three regions: a disordered N-terminus, a central conserved domain and a disordered C-terminus. To decipher the structure-function of VP22, we generated three series of recombinant MDV genomes harboring (i) orthologous UL49, from Varicelloviruses (VZV and PRV) and infectious laryngotracheitis virus (ILTV), in place of UL49 MDV, (ii) chimeric genes between UL49 from MDV and ILTV, by substituting one of the three regions of MDV by ILTV (r22IMM, r22MIM, r22MMI), or (iii) mutated MDV UL49 encoding truncated C-term VP22. All mutant viruses were assessed for spread in primary avian fibroblasts. The WT and mutated VP22 were also tested for their ability to arrest the cell cycle in S-phase, their sub-cellular location and their histones association after transient transfection in an avian cell line. We found that VP22 proteins and domains are not functionally homologous between alphaherpesviruses in term of MDV spread and cell cycle modulation, and this independently that the % in AA identity in the central domain. We showed that these two VP22 functions are supported by the N-terminus associated to the central domain and independent of the C-terminus from AA190. Interestingly, the region between AA173 and AA190 is essential for cell cycle arrest and MDV spread. All VP22 which cis-complemented MDV for spread were able to efficiently arrest the cell cycle and showed a nuclear localization and histones association. The relationship between cell cycle arrest and MDV spread will be discussed.

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National audience; Le virus de la maladie de Marek (MDV) est un herpesvirus à l’origine du développement de lymphomes chez les volailles. A ce jour, les mécanismes moléculaires conduisant à la tumorigenèse viro-induite sont encore méconnus malgré l’identification d’oncogènes viraux. Récemment nous avons montré que l’infection par le MDV induit une dérégulation du cycle cellulaire se traduisant par un arrêt prolifératif en phase-S et que la protéine virale VP22 jouerait un rôle majeur dans ce processus. La surexpression de VP22 entraine un arrêt drastique intra-S mais elle engendre également de nombreuses cassures double-brins (CDB) dans l’ADN. Aussi, au vu de l’implication des dommages à l’ADN dans les processus néoplasiques, nous avons initié un projet visant à préciser l’impact de l’infection par le MDV sur l’intégrité génomique. Nous avons ainsi pu mettre en évidence in vivo et in vitro que la réplication du MDV est associée à une accumulation de cassures (double-brin) dans l’ADN des cellules infectés et que l’induction de ces dommages et/ou la réponse cellulaire engendrée seraient favorables à la réplication du virus. De plus, l’apparition de ces lésions serait également étroitement liée au pouvoir oncogène du MDV. En effet, les lymphocytes de poulets infectés par un virus atténué présentent un taux de dommage significativement plus faible que celui détecté à partir de lymphocytes de poulets infectés par une souche oncogène. Par conséquent, les lésions générées dans l’ADN cellulaire lors de l’infection par le MDV pourraient non seulement favoriser la réplication du virus, mais également participer à l’induction d’une instabilité génomique à l’origine de la tumorigenèse viro-induite.

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VP22 is a major tegument protein of alphaherpesviruses encoded by the UL49 gene. Two properties of VP22 were discovered by studying Marek&rsquo, s disease virus (MDV), the Mardivirus prototype, it has a major role in virus cell-to-cell spread and in cell cycle modulation. This 249 AA-long protein contains three regions including a conserved central domain. To decipher the functional VP22 domains and their relationships, we generated three series of recombinant MDV genomes harboring a modified UL49 gene and assessed their effect on virus spread. Mutated VP22 were also tested for their ability to arrest the cell cycle, subcellular location and histones copurification after overexpression in cells. We demonstrated that the N-terminus of VP22 associated with its central domain is essential for virus spread and cell cycle modulation. Strikingly, we demonstrated that AAs 174-190 of MDV VP22 containing the end of a putative extended alpha-3 helix are essential for both functions and that AAs 159&ndash, 162 located in the putative beta-strand of the central domain are mandatory for cell cycle modulation. Despite being non-essential, the 59 C-terminal AAs play a role in virus spread efficiency. Interestingly, a positive correlation was observed between cell cycle modulation and VP22 histones association, but none with MDV spread.

Marek's disease virus (MDV) is a highly contagious alphaherpesvirus that infects chickens and causes a deadly neoplastic disease. We previously demonstrated that MDV infection arrests cells in S phase and that the tegument protein VP22 plays a major role in this process. In addition, expression of VP22 induces double-strand breaks (DSBs) in the cellular DNA, suggesting that DNA damage and the associated cellular response might be favorable for the MDV life cycle. Here, we addressed the role of DNA damage in MDV replication and pathogenesis. We demonstrated that MDV induces DSBs during lytic infection in vitro and in the peripheral blood mononuclear cells of infected animals. Intriguingly, we did not observe DNA damage in latently infected MDV-induced lymphoblastoid cells, while MDV reactivation resulted in the onset of DNA lesions, suggesting that DNA damage and/or the resulting DNA damage response might be required for efficient MDV replication and reactivation. In addition, reactivation was significantly enhanced by the induction of DNA damage using a number of chemicals. Finally, we used recombinant viruses to show that VP22 is required for the induction of DNA damage in vivo and that this likely contributes to viral oncogenesis. IMPORTANCE Marek's disease virus is an oncogenic alphaherpesvirus that causes fatal T-cell lymphomas in chickens. MDV causes substantial losses in the poultry industry and is also used in small-animal models for virus-induced tumor formation. DNA damage not only is implicated in tumor development but also aids in the life cycle of several viruses; however, its role in MDV replication, latency, and reactivation remains elusive. Here, we demonstrate that MDV induces DNA lesions during lytic replication in vitro and in vivo . DNA damage was not observed in latently infected cells; however, it was reinitiated during reactivation. Reactivation was significantly enhanced by the induction of DNA damage. Recombinant viruses that lacked the ability to induce DNA damage were defective in their ability to induce tumors, suggesting that DNA damage might also contribute to cellular transformation processes leading to MDV lymphomagenesis.

International audience; Marek's disease is one of the most common viral diseases of poultry affecting chicken flocks worldwide. The disease is caused by an alphaherpesvirus, the Marek's disease virus (MDV), and is characterized by the rapid onset of multifocal aggressive T-cell lymphoma in the chicken host. Although several viral oncogenes have been identified, the detailed mechanisms underlying MDV-induced lymphomagenesis are still poorly understood. Many viruses modulate cell cycle progression to enhance their replication and persistence in the host cell, in the case of some oncogenic viruses ultimately leading to cellular transformation and oncogenesis. In the present study, we found that MDV, like other viruses, is able to subvert the cell cycle progression by triggering the proliferation of low proliferating chicken cells and a subsequent delay of the cell cycle progression into S-phase. We further identified the tegument protein VP22 (pUL49) as a major MDV-encoded cell cycle regulator, as its vector-driven overexpression in cells lead to a dramatic cell cycle arrest in S-phase. This striking functional feature of VP22 appears to depend on its ability to associate with histones in the nucleus. Finally, we established that VP22 expression triggers the induction of massive and severe DNA damages in cells, which might cause the observed intra S-phase arrest. Taken together, our results provide the first evidence for a hitherto unknown function of the VP22 tegument protein in herpesviral reprogramming of the cell cycle of the host cell and its potential implication in the generation of DNA damages.

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