Date of Award

2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Jianxin You

Abstract

Due to its association with Merkel cell carcinoma (MCC), a substantial effort has been made to better understand how Merkel cell polyomavirus (MCPyV) proteins drive oncogenesis; however, our understanding of the early steps of MCPyV infection remains poor. The polyomavirus Large Tumor antigen (LT) is a highly multi-functional protein with a wide range of activities, including: stimulation of cellular proliferation through its interaction with retinoblastoma protein and DnaJ heatshock protein family members; arrest of the cell cycle through a poorly understood activity localized to the C-terminal region; and regulation of the initiation of viral DNA replication. LT proteins also play important roles in regulating viral transcription. How these various functions are regulated to ensure an orderly progression of events conducive for the viral life cycle has not been well established.

In this study, I show how phosphorylation of MCPyV LT plays an important role in regulating its many functions. I identify threonines 297 and 299 as key phospho-sites which regulate LT's ability to initiate replication. T297 phosphorylation inhibits LT binding to the viral origin of replication and acts as an "off" switch, while phosphorylation of T299 is required to stimulate LT-mediated replication of viral genomes. This study was the first to identify phosphorylation sites of LT and link them to important protein functions.

Cross-reactivity to a phospho-specific antibody revealed yet another phosphorylation site on MCPyV LT as S816. We discovered that this phosphorylation event is mediated by ATM kinase, and may play a role in the MCPyV LT C-terminal domain's ability to arrest the cell cycle. This study helps to further elucidate MCPyV's association with the host DNA Damage Response (DDR) and provides some rational for the recruitment of these factors to viral replication centers.

Finally, studies of the viral non-coding control region (NCCR) reveal a surprising interaction between LT and sT on the late promoter. MCPyV LT is able to robustly stimulate the late promoter only in the context of an intact Ori and sT co-expression. Using phosphomutant LTs and mutant Ori sequences, I highlight the importance of LT binding to the Ori and stimulation of replication as key factors in LT-mediated activation of the late promoter in the context of sT co-expression. LT alone actually represses the late promoter and requires sT coexpression to efficiently stimulate the late promoter after replication. This study therefore reveals an important dependence on sT expression for the regulation of transcription that has not yet been reported with other polyomaviruses.

In sum, this study demonstrates multiple mechanisms of regulation including protein phosphorylation, protein-DNA interactions, and co-expression of key viral proteins as regulators of LT function. These studies may help elucidate critical factors required for establishing a robust cellular infection system which is greatly needed to further our understanding of the basic virology of this important human tumor virus.

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