The Ubiquitin specific Protease Usp7, a novel Merkel cell polyomavirus Large T-Antigen interaction partner, modulates viral DNA replication

TY - JOUR

T1 - The Ubiquitin specific Protease Usp7, a novel Merkel cell polyomavirus Large T-Antigen interaction partner, modulates viral DNA replication

AU - Czech-Sioli, Manja

AU - Siebels, Svenja

AU - Radau, Sonja

AU - Zahedi, René P

AU - Schmidt, Claudia

AU - Dobner, Thomas

AU - Grundhoff, Adam

AU - Fischer, Nicole

N1 - Copyright © 2020 American Society for Microbiology.

PY - 2020/2/14

Y1 - 2020/2/14

N2 - Merkel cell polyomavirus (MCPyV) is the major cause for Merkel cell carcinoma (MCC), a rare but highly aggressive skin cancer predominantly found in elderly and immunosuppressed patients. The early viral gene products large T-antigen (LT) and small T-antigen (sT) are important for efficient viral DNA replication, and both contribute to transformation processes. These functions are executed mainly through interactions with host factors. Here, we identify the cellular ubiquitin-specific processing protease 7 (Usp7) as a new interaction partner of the MCPyV LT. Using glutathione S-transferase pulldown experiments, we show that MCPyV LT directly binds to Usp7 and that N- as well as C-terminal regions of LT bind to the TRAF (tumor necrosis factor receptor-associated) domain of Usp7. We demonstrate that endogenous Usp7 coprecipitates with MCPyV T-antigens and relocalizes to viral DNA replication centers in cells actively replicating MCPyV genomes. We show that Usp7 does not alter ubiquitination levels of the T-antigens; however, Usp7 binding increases the binding affinity of LT to the origin of replication, thereby negatively regulating viral DNA replication. Together, these data identify Usp7 as a restriction factor of MCPyV replication. In contrast to other DNA viruses, Usp7 does not affect MCPyV gene expression via its ubiquitination activity but influences MCPyV DNA replication solely via a novel mechanism that modulates binding of LT to viral DNA.IMPORTANCE MCPyV is the only human polyomavirus that is associated with cancer; the majority of Merkel cell cancers have a viral etiology. While much emphasis was placed on investigations to understand the transformation process by MCPyV oncoproteins and cellular factors, we have only limited knowledge of cellular factors participating in the MCPyV life cycle. Here, we describe Usp7, a cellular deubiquitination enzyme, as a new factor involved in MCPyV replication. Usp7 is known in the context of large DNA tumor viruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus, to restrict viral replication. Similar to EBV, where Usp7 binding to EBNA1 increases EBNA1 binding affinity to viral DNA, we find MCPyV LT binding to the origin of replication to be increased in the presence of Usp7, resulting in restriction of viral DNA replication. However, Usp7-induced restriction of MCPyV replication is independent of its enzymatic activity, thereby constituting a novel mechanism of Usp7-induced restriction of viral replication.

AB - Merkel cell polyomavirus (MCPyV) is the major cause for Merkel cell carcinoma (MCC), a rare but highly aggressive skin cancer predominantly found in elderly and immunosuppressed patients. The early viral gene products large T-antigen (LT) and small T-antigen (sT) are important for efficient viral DNA replication, and both contribute to transformation processes. These functions are executed mainly through interactions with host factors. Here, we identify the cellular ubiquitin-specific processing protease 7 (Usp7) as a new interaction partner of the MCPyV LT. Using glutathione S-transferase pulldown experiments, we show that MCPyV LT directly binds to Usp7 and that N- as well as C-terminal regions of LT bind to the TRAF (tumor necrosis factor receptor-associated) domain of Usp7. We demonstrate that endogenous Usp7 coprecipitates with MCPyV T-antigens and relocalizes to viral DNA replication centers in cells actively replicating MCPyV genomes. We show that Usp7 does not alter ubiquitination levels of the T-antigens; however, Usp7 binding increases the binding affinity of LT to the origin of replication, thereby negatively regulating viral DNA replication. Together, these data identify Usp7 as a restriction factor of MCPyV replication. In contrast to other DNA viruses, Usp7 does not affect MCPyV gene expression via its ubiquitination activity but influences MCPyV DNA replication solely via a novel mechanism that modulates binding of LT to viral DNA.IMPORTANCE MCPyV is the only human polyomavirus that is associated with cancer; the majority of Merkel cell cancers have a viral etiology. While much emphasis was placed on investigations to understand the transformation process by MCPyV oncoproteins and cellular factors, we have only limited knowledge of cellular factors participating in the MCPyV life cycle. Here, we describe Usp7, a cellular deubiquitination enzyme, as a new factor involved in MCPyV replication. Usp7 is known in the context of large DNA tumor viruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus, to restrict viral replication. Similar to EBV, where Usp7 binding to EBNA1 increases EBNA1 binding affinity to viral DNA, we find MCPyV LT binding to the origin of replication to be increased in the presence of Usp7, resulting in restriction of viral DNA replication. However, Usp7-induced restriction of MCPyV replication is independent of its enzymatic activity, thereby constituting a novel mechanism of Usp7-induced restriction of viral replication.

KW - Antigens, Viral, Tumor/metabolism

KW - Carcinoma, Merkel Cell/virology

KW - Cell Line

KW - Cell Proliferation

KW - DNA, Viral/metabolism

KW - HEK293 Cells

KW - Humans

KW - Merkel cell polyomavirus/genetics

KW - Polyomavirus Infections/virology

KW - Protein Binding

KW - Protein Interaction Domains and Motifs

KW - Tumor Virus Infections/virology

KW - Ubiquitin-Specific Peptidase 7/metabolism

KW - Virus Replication/physiology

U2 - 10.1128/JVI.01638-19

DO - 10.1128/JVI.01638-19

M3 - SCORING: Journal article

C2 - 31801860

VL - 94

JO - J VIROL

JF - J VIROL

SN - 0022-538X

IS - 5

ER -