Role of Merkel cell polyomavirus small T antigen in persistent infection

/ABSTRACT Merkel cell polyomavirus (MCV) is a common virus that establishes life-long persistent infection in human skin. Yet, in elderly and immunocompromised patients, MCV can cause the aggressive skin cancer Merkel cell carcinoma (MCC). MCV replicates as a circular episomal DNA in infected cells, however, viral DNA is clonally integrated in MCC tumors, which allows stable expression of viral T antigens that drive malignant transformation. Serological and genetic evidence show that MCV replication precedes viral integration and transformation, indicating that inhibition of virus replication is a promising strategy to prevent MCV-associated MCC. How MCV gene expression regulates virus replication is poorly defined but is believed to be controlled by two bidirectional early and late promoters located in the non-coding control region. Critical MCV promoter elements and their regulation by host transcription factors and epigenetic regulators remain unknown. The MCV T antigen gene encodes two conserved replication proteins: large T (LT) and small T (ST). While LT is a well-established replication helicase that directly binds to the viral replication origin, the functional role of ST as a replication accessory factor is poorly understood. We generated a ST deletion MCV mutant (MCVΔsT) virus and found that while MCVΔsT initiates viral DNA replication similar to wild-type MCV, it cannot complete genome replication and encapsidation. Viral early and late mRNA expression is largely turned off in MCVΔST, which can be rescued by trans expression of ST. Consistent with ST being a viral transcriptional regulator, an unbiased proximity-based ST-host protein interaction study (together with confirmatory immunoprecipitation) identified multiple transcriptional regulators Cux1, c-Jun, BRD9, and CBP histone acetyltransferase interacting with ST. Of these regulators, CBP inhibition significantly attenuated ST-mediated viral early and late mRNA activation, suggesting that CBP histone acetyltransferase mediates viral transcriptional control by ST. Our preliminary results show that inhibition of CBP suppresses not only viral mRNA expression in replicating MCV, but also viral T antigen mRNA expression in MCV-transformed MCC. We hypothesize that, together with the MCV LT replicative enzyme, ST controls viral gene expression by altering the epigenetic state of viral chromatin through interaction with CBP and transcription factors. The goals of this proposal are: (1) to define MCV early and late promoter sequences, (2) to investigate the impact of ST on viral chromatinization and the epigenetic state of the virus genome focusing on histone marks affected by CBP; and to identify transcription factors that activate the viral promoters in the context of ST and CBP, and (3) to directly visualize single-molecule binding of ST, LT, and CBP to the viral DNA through C-Trap laser capture confocal microscopy. Defining the transcriptional functions of MCV ST on viral gene expression will lead to strategies to disrupt MCV replication for the prevention and control of Merkel cell carcinogenesis. Project Number: 1R01AI181892-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Masahiro Shuda | Institution: UNIVERSITY OF PITTSBURGH AT PITTSBURGH, PITTSBURGH, PA | Award Amount: $634,981 | Activity Code: R01 | Study Section: Viral Pathogenesis and Immunity Study Section [VPI] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01AI18189201A1