Merkel Cell Polyomavirus DNA Replication Induces Senescence in Human Dermal Fibroblasts in a Kap1/Trim28-Dependent Manner

Abstract

Merkel cell polyomavirus (MCPyV) is the only polyomavirus known to be associated with tumorigenesis in humans. Similarly to other polyomaviruses, MCPyV expresses a large tumor antigen (LT-Ag) that, together with a small tumor antigen (sT-Ag), contributes to cellular transformation and that is of critical importance for the initiation of the viral DNA replication. Understanding the cellular protein network regulated by MCPyV early proteins will significantly contribute to our understanding of the natural MCPyV life cycle as well as of the mechanisms by which the virus contributes to cellular transformation. We here describe KRAB-associated protein 1 (Kap1), a chromatin remodeling factor involved in cotranscriptional regulation, as a novel protein interaction partner of MCPyV T antigens sT and LT. Kap1 knockout results in a significant increase in the level of viral DNA replication that is highly suggestive of Kap1 being an important host restriction factor during MCPyV infection. Differently from other DNA viruses, MCPyV gene expression is unaffected in the absence of Kap1 and Kap1 does not associate with the viral genome. Instead, we show that in primary normal human dermal fibroblast (nHDF) cells, MCPyV DNA replication, but not T antigen expression alone, induces ataxia telangiectasia mutated (ATM) kinase-dependent Kap1 S824 phosphorylation, a mechanism that typically facilitates repair of double-strand breaks in heterochromatin by arresting the cells in G2. We show that MCPyV-induced inhibition of cell proliferation is mainly conferred by residues within the origin binding domain and thereby by viral DNA replication. Our data suggest that phosphorylation of Kap1 and subsequent Kap1-dependent G2 arrest/senescence represent host defense mechanisms against MCPyV replication in nHDF cells. IMPORTANCE We here describe Kap1 as a restriction factor in MCPyV infection. We report a novel, indirect mechanism by which Kap1 affects MCPyV replication. In contrast with from other DNA viruses, Kap1 does not associate with the viral genome in MCPyV infection and has no impact on viral gene expression. In MCPyV-infected nHDF cells, Kap1 phosphorylation (pKap1 S824) accumulates because of genomic stress mainly induced by viral DNA replication. In contrast, ectopic expression of LT or LT MCPyV mutants, previously shown to be important for induction of genotoxic stress, does not result in a similar extent of pKap1 accumulation. We show that cells actively replicating MCPyV accumulate pKap1 (in a manner dependent on the presence of ATM) and display a senescence phenotype reflected by G2 arrest. These results are supported by transcriptome analyses showing that LT antigen, in a manner dependent on the presence of Kap1, induces expression of secreted factors, which is known as the senescence-associated secretory phenotype (SASP).

Senescence and Host–Pathogen Interactions

“…The biological relevance of this finding has been validated in an in vivo model, where the induction of genotoxicity and consequent senescence by the administration of bleomycin reduces viral recovery from the lungs of VSV-infected mice 6 days post-infection [85]. Similarly, the infection of human primary dermal fibroblasts with the double stranded DNA merkel cell polyoma virus (MCPyV) activates the ATM-dependent DDR response and KAP1-dependent senescence, resulting in a significant reduction of the viral replication rate [86].…”

The Interaction of Viruses with the Cellular Senescence Response

“…Expression of the large T antigen of MCPyV has been demonstrated to induce DSB, the activation of ataxia–telangiectasia mutated (ATM) protein kinase and the phosphorylation of p53 and KRAB-associated protein 1 (KAP-1). The accumulation of phosphorylated KAP-1 has been associated with a G2 phase arrest, a mechanism that may avoid viral DNA replication [ 24 , 25 ].…”

“…Finally, a negative role of senescence on MCPyV infection has been reported. Siebels et al (2020) demonstrated that KAP-1 is a restriction factor for MCPyV infection and that replication of the virus induces the phosphorylation of KAP-1 and the subsequent cellular senescence [ 25 ]. Therefore, it has been proposed that senescence is a host defense mechanism against MCPyV [ 25 ].…”

Cellular senescence preserves viral genome maintenance

“…MCPyV was able to exploit these senescent cells by maintaining its viral genome for long-term survival (Figure 6f). MCPyV-induced senescence was recently reported by Siebels et al 45 . Although their MCPyV LT interaction study revealed an interaction with KRAB-associated protein 1 (KAP1), a senescence-related factor 46 in primary normal human dermal fibroblasts (nHDF), it was not clear if LT expression alone is sufficient to induce host cellular senescence.…”