Date of Award

Spring 1-1-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Robert L. Garcea

Second Advisor

Bradley Olwin

Third Advisor

Tin Tin Su

Fourth Advisor

James Orth

Fifth Advisor

Linda van Dyk

Abstract

Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which may detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. I investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, ATM, RPA, MRN, and DNAPK. I found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling. ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. Additionally, by creating a mutant virus (E320A), I found large T-antigen binding to RPA was required for appropriate viral genome resolution. The E320A mutant virus produced approximately 5-fold less infectious virus than WT and replicated viral DNA that was concatemerized. Taken together, these data suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. Specifically, these results demonstrate the requirement for DDR proteins in appropriate resolution and packaging of the viral genome. Finally, I utilized an optimized set-up for Stochastic Optical Reconstruction Microscopy (STORM) to interrogate the sub-nuclear domains of MuPyV replication and assembly. I observed an exclusionary relationship between T-antigen and the host cell protein, PML. I was also able to visualize MuPyV “factories” and virions formed by viral protein, VP1 for the first time using a light microscopy based technique. This technique will greatly enhance our ability to observe and study the composition of these sub-nuclear structures. These findings expanded to our understanding of the mechanisms of PyV replication and assembly.

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