Recent studies demonstrated that the structural maintenance of chromosomes complex 5/6 (SMC5/6) acts as an important antiviral restriction factor that silences episomal DNA. Consequently, viruses have evolved strategies to antagonize this defence system. Interestingly, it was shown that SMC5/6-mediated silencing of extrachromosomal DNA depends on its recruitment to PML nuclear bodies which is mediated by a protein termed SMC5/6 localization factor 2 (SLF2). Since human cytomegalovirus (HCMV) dissociates PML nuclear bodies (PML-NBs) during the first hours of infection, we asked for the fate of SMC5/6 components during the HCMV replicative cycle. We first investigated the expression levels of SMC5/6 core components at the onset of HCMV infection and found that they were not downregulated by HCMV. Instead, we observed a distinct decrease of SLF2 protein levels which correlated with a complete dispersal of the SMC5/6 complex from PML-NBs. This was also observed after infection with UV inactivated HCMV and could not be blocked by cycloheximide treatment suggesting the involvement of an imported structural component of the virion. While we could exclude a role of the HCMV tegument protein pp71 (UL82), a targeted screen identified the tegument protein UL35 as being required and sufficient for proteasomal degradation of SLF2. In line with these results, SLF2 levels remained stable after infection with a recombinant HCMV harbouring a stop codon within the UL35 gene region and this correlated with a defect in the onset of HCMV immediate early gene expression. Furthermore, we observed an enhanced recruitment of SMC5/6 to PML-NBs and an overall increase in the association of parental viral genomes with SMC5/6 containing PML-NBs in the absence of UL35. Congruently, siRNA mediated depletion of SLF2 resulted in a reversal of this phenotype. This strongly suggests that UL35 serves as a novel viral SLF2 antagonist to prevent the initial silencing of incoming viral genomes by SMC5/6.
Montagner, F., Scherer, M., Stegmann, C., Hahn, F., Ploil, C., Einsiedler, C. S., Maeki, T., Ramani, R., Andree-Busch, N., Brinkmann, M. M., Stamminger, T.
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