Analysis of Murine Polyomavirus DNA Replication:  Large T-Antigen Interaction with Host DNA Damage Repair Protein RPA During Cellular Infection

Undergraduate Honors Thesis

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/6w924c42p

Abstract

Murine Polyomaviruses are dsDNA viruses that hijack the host’s DNA damage response (DDR) pathway to replicate their own genomes (Heiser et al., 2016), offering a model for human polyomavirus replication. The viral protein large T-antigen (LT) is essential for viral replication and can interact with a variety of DDR proteins in viral DNA replication centers (Brodsky & Pipas, 1998). Replication protein A (RPA) is a DDR protein complex made up of three subunits, RPA70, RPA32, and RPA14. RPA70 and RPA32 have been shown to directly interact with LT in cells over-expressing each protein, either individually or together (Banerjee et al., 2013). Individual point mutations within LT could disrupt the LT-RPA interaction (Banerjee et al., 2013). However, the LT–RPA interaction has yet to be explored in the context of viral infection. This study aimed to evaluate mCherry-tagged RPA32 binding with wild-type LT and previously characterized point-mutants E320A and K308E during infection of mouse fibroblasts (Banerjee et al., 2013). We show that, vDNA was replicated from genomes encoding both wild-type and mutant LT proteins. However, we did not observe an interaction between wild-type LT and mCherry-tagged mouse RPA32 (mCh-muRPA32).

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