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Authors: Rowles, Daniell Lynn
Advisors: Cristea, Ileana M
Contributors: Molecular Biology Department
Keywords: DNMT3A
mass spectrometry
Subjects: Molecular biology
Issue Date: 2013
Publisher: Princeton, NJ : Princeton University
Abstract: Herpes simplex virus type 1 (HSV-1) is an infectious pathogen imposing a substantial societal burden, while also having therapeutic potential. Throughout the course of infection, viral and host proteins undergo changes in their expression levels, localizations, and binding partners, changes that are required for effective viral replication and spread. This project focused on characterizing the HSV-1 small capsid protein VP26 with the ultimate goal to better understand its roles in modulation of host functions and assembly of the mature virion. In these studies, I have developed and utilized proteomic-based approaches to examine these virus-virus and virus-host interactions during the progression of HSV-1 infection. Here, I used fluorescently tagged HSV-1 strains to examine VP26 interaction dynamics at different time points during infection by immunoaffinity purification and quantitative mass spectrometry. Stringent and mild lysis conditions were used to generate networks of relatively stronger and weaker viral protein associations during virion assembly. Previously unreported VP26 interactions with viral proteins pUS10 and pUL3 were identified. Additionally, temporal changes in VP26 binding to pUL26, pUL31, pUL47, and pUL36 between 8 and 22 hpi were highlighted. Furthermore, I identified novel phosphorylation sites on VP26 at S107 and T108, as well as the first acetylation observed on any HSV-1 protein at the K1218 residue on the major capsid protein VP5. These results indicate that VP26 has a complex role in the infectious life cycle of HSV-1 that is characterized by dynamic interactions and modification profiles. Next, I sought to identify interactions of VP26 with host factors that may be necessary for efficient viral replication. I have identified the host DNA methyltransferase DNMT3A to associate with VP26 during the late stages of infection in the host nucleus. I explored the functional significance of the interaction using shRNA- and siRNA-mediated knockdowns and treatment with a small molecule inhibitor. I have demonstrated that reduction in both DNMT3A levels and enzymatic activity reduces the efficiency of HSV-1 replication. I propose that VP26, either directly or indirectly via a member of its associated viral protein complex, recruits DNMT3A to optimize the host environment for HSV-1 production.
Alternate format: The Mudd Manuscript Library retains one bound copy of each dissertation. Search for these copies in the library's main catalog
Type of Material: Academic dissertations (Ph.D.)
Language: en
Appears in Collections:Molecular Biology

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