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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.

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.