Date of Completion

8-27-2015

Embargo Period

8-19-2016

Major Advisor

Sandra K. Weller

Associate Advisor

Stephen King

Associate Advisor

Bing Hao

Associate Advisor

Peter Setlow

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

This thesis describes three separate but related projects exploring how herpes simplex virus 1 (HSV-1) induces and copes with a pro-oxidant environment during infection. We found that the UL6 portal ring, the conduit responsible for the uptake and release of viral DNA, required disulfide bonds for ring formation and stability. Mutagenesis studies revealed that these disulfide bonds might be mediated by cysteines 166 and 254. We next examined the protein UL32 as a potential virally encoded oxidoreductase. UL32 contains three C-X-X-C motifs reminiscent of proteins that regulate disulfide bond formation. Mutation of two of these C-X-X-C motifs abrogated protein function. We used a thiol-alkylation assay to show that viral capsid proteins, including UL6, had altered disulfide bond profiles in the absence of UL32, suggesting that UL32 influences disulfide bond formation in capsid proteins during assembly and maturation. We then confirmed that HSV induces oxidation using a redox-sensitive GFP probe. We also found that treating HSV-infected cells with the antioxidant glutathione (GSH) inhibited viral growth. This growth defect was likely due to delayed viral protein expression and replication compartment formation. We further investigated the role of ROS generating NADPH-oxidases (NOXs), which are important for the growth of several viruses. Using a pan-NOX inhibitor, we found similar defects in viral growth, protein expression and replication compartment formation as observed with GSH treatment, suggesting that NOX-derived ROS might be important for HSV infection. Collectively, we propose that HSV has evolved to use and manipulate the intracellular redox-balance for its own benefit.

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