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...

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Autores principales:	Svenja Siebels, Manja Czech-Sioli, Michael Spohn, Claudia Schmidt, Juliane Theiss, Daniela Indenbirken, Thomas Günther, Adam Grundhoff, Nicole Fischer
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	Kap1/Trim28 Merkel cell polyomavirus senescence viral DNA replication viral restriction factor Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/a6a16d03140547a4ae7f58173e582679
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spelling	oai:doaj.org-article:a6a16d03140547a4ae7f58173e5826792021-11-15T15:57:02ZMerkel Cell Polyomavirus DNA Replication Induces Senescence in Human Dermal Fibroblasts in a Kap1/Trim28-Dependent Manner10.1128/mBio.00142-202150-7511https://doaj.org/article/a6a16d03140547a4ae7f58173e5826792020-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00142-20https://doaj.org/toc/2150-7511ABSTRACT 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).Svenja SiebelsManja Czech-SioliMichael SpohnClaudia SchmidtJuliane TheissDaniela IndenbirkenThomas GüntherAdam GrundhoffNicole FischerAmerican Society for MicrobiologyarticleKap1/Trim28Merkel cell polyomavirussenescenceviral DNA replicationviral restriction factorMicrobiologyQR1-502ENmBio, Vol 11, Iss 2 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Kap1/Trim28 Merkel cell polyomavirus senescence viral DNA replication viral restriction factor Microbiology QR1-502
spellingShingle	Kap1/Trim28 Merkel cell polyomavirus senescence viral DNA replication viral restriction factor Microbiology QR1-502 Svenja Siebels Manja Czech-Sioli Michael Spohn Claudia Schmidt Juliane Theiss Daniela Indenbirken Thomas Günther Adam Grundhoff Nicole Fischer Merkel Cell Polyomavirus DNA Replication Induces Senescence in Human Dermal Fibroblasts in a Kap1/Trim28-Dependent Manner
description	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).
format	article
author	Svenja Siebels Manja Czech-Sioli Michael Spohn Claudia Schmidt Juliane Theiss Daniela Indenbirken Thomas Günther Adam Grundhoff Nicole Fischer
author_facet	Svenja Siebels Manja Czech-Sioli Michael Spohn Claudia Schmidt Juliane Theiss Daniela Indenbirken Thomas Günther Adam Grundhoff Nicole Fischer
author_sort	Svenja Siebels
title	Merkel Cell Polyomavirus DNA Replication Induces Senescence in Human Dermal Fibroblasts in a Kap1/Trim28-Dependent Manner
title_short	Merkel Cell Polyomavirus DNA Replication Induces Senescence in Human Dermal Fibroblasts in a Kap1/Trim28-Dependent Manner
title_full	Merkel Cell Polyomavirus DNA Replication Induces Senescence in Human Dermal Fibroblasts in a Kap1/Trim28-Dependent Manner
title_fullStr	Merkel Cell Polyomavirus DNA Replication Induces Senescence in Human Dermal Fibroblasts in a Kap1/Trim28-Dependent Manner
title_full_unstemmed	Merkel Cell Polyomavirus DNA Replication Induces Senescence in Human Dermal Fibroblasts in a Kap1/Trim28-Dependent Manner
title_sort	merkel cell polyomavirus dna replication induces senescence in human dermal fibroblasts in a kap1/trim28-dependent manner
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/a6a16d03140547a4ae7f58173e582679
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Merkel Cell Polyomavirus DNA Replication Induces Senescence in Human Dermal Fibroblasts in a Kap1/Trim28-Dependent Manner

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