Person:

Yoon, Rosa

The small genome of polyomaviruses encodes a limited number of proteins that are highly dependent on interactions with host cell proteins for efficient viral replication. The SV40 large T antigen (LT) contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle. In addition, the LT C-terminal region contains the host range and adenovirus helper functions required for lytic infection in certain restrictive cell types. To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling. LT perturbed the expression of p53 target genes and subsets of cell-cycle dependent genes regulated by the DREAM and the B-Myb-MuvB complexes. Affinity purification of LT followed by mass spectrometry revealed a specific interaction between the LT C-terminal region and FAM111A, a previously uncharacterized protein. Depletion of FAM111A recapitulated the effects of heterologous expression of the LT C-terminal region, including increased viral gene expression and lytic infection of SV40 host range mutants and adenovirus replication in restrictive cells. FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT.

Merkel cell polyomavirus (MCPyV) small T antigen (ST) is expressed in the majority of Merkel cell carcinomas (MCC), a highly lethal and aggressive cancer of the skin. Since the discovery of MCPyV in 2008, the role of ST in the context of the virus and MCC has been under intense investigation. Much of our knowledge of polyomavirus ST comes from research on other polyomaviruses, including mouse polyomavirus (MPyV) and simian virus 40 (SV40). Both MPyV and SV40 ST contribute to transformation in part by binding to and inhibiting the cellular phosphatase PP2A. Likewise, MCPyV ST interacts with PP2A, although mutants that are reported to abolish this interaction still transform cells, suggesting that MCPyV ST has PP2A-independent functions. Understanding the unique cellular perturbations induced by MCPyV ST will thus be important for understanding the tumorigenesis of MCC. In this dissertation, we sought to understand the manipulation of cellular functions by MCPyV ST. We began by characterizing the MCPyV ST protein itself, starting with structural and functional comparisons with other well characterized polyomaviruses and identifying the interaction of MCPyV ST with cellular proteins. We observed that MCPyV ST uniquely interacts with the TIP60 cellular complex, which contains an ATPase and an acetyltransferase and is involved in histone modifications and DNA damage repair. Through predictions of the structure, we identified a surface-exposed region of ST, loop 4, and observed that regions in this loop were important for regulating the binding of ST to the TIP60 complex. Functionally, we investigated the role of MCPyV ST in the DNA damage response because of its interaction with TIP60 and because DNA damaging agents are used to treat MCCs. In addition, overcoming checkpoint regulation in the p53 pathway is an open question in MCPyV infection. We determined that ST increased sensitivity to DNA damage by γ-irradiation and etoposide and that expression of ST caused persistence of double strand DNA breaks (DSB) after damage, suggesting that DSB repair was delayed in ST expressing cells. Specifically, we observed that ST expression inhibits repair of breaks by nonhomologous end joining (NHEJ) but does not inhibit repair by homologous recombination (HR). These effects on the DNA damage response are explained in part by a less robust phosphorylation of DNA-PKcs at serine residue 2056, which is important for regulating end processing and repair by NHEJ. Taken together, these results indicate that MCPyV ST disrupts the cellular DNA damage response, which has implications on the viral life cycle and the initiation and treatment of MCC.