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Cellular Response to Adenovirus and Adeno- Associated Virus Coinfection

by Bevington, Joyce M.

Abstract (Summary)
Adeno-associated virus (AAV) is a small single-stranded linear DNA virus that requires a helper-virus to efficiently replicate inside a host cell. The cellular effects of an AAV coinfection with its helper-virus, adenovirus (Ad), were assessed. Affinity column chromatography and crosslinking experiments identified an interaction between the cellular nucleolar protein, nucleophosmin (NPM, B23), with the AAV replicative proteins, Rep68 and Rep78. Increased substrate binding and nicking by Rep68/78 in the presence of NPM was observed using Rep protein functional assays with bacterially expressed proteins. Immunoflourescence studies demonstrated extensive co-localization between NPM and AAV capsid proteins at nucleoli and at small foci within the nucleus. Co-localization was minimal between Rep proteins and NPM during early infection occurring mostly in and around the edge of the nucleoli. Capsid proteins were pulled out with Rep and NPM proteins in crosslinking experiments indicating either a direct or indirect interaction between capsid proteins and NPM. With AAV and Ad having potentially antagonizing effects on the cell cycle, the cellular effects of an AAV and Ad coinfection were examined. The preliminary results indicated that there were no statistically significant changes in protein levels for the cell cycle and cell cycle-related proteins that were examined when comparing Ad infection to Ad and AAV coinfections. There was an increase in the amount of phosphorylated Sp1 in an Ad and AAV coinfection compared to Ad infection. Whether this is related to Sp1 activation of AAV promoters is unknown. With AAV infection affecting expression of Ad proteins involved in disrupting the cellular DSB response, the effects of an AAV and Ad coinfection on the DSB response were examined. Increased amounts of the active phosphorylated form of the DSB response proteins, NBS1, ATM, and Chk2, were observed in an AAV and Ad coinfection compared to Ad infection. This activation was induced by the presence of intact AAV virus and not due to increased viral DNA. Concatamerization of the Ad viral DNA was not detected during an AAV and Ad coinfection, which although unexpected does not preclude the possibility that AAV activation of the DSB response contributes to decreased Ad viral replication.
Bibliographical Information:

Advisor:

School:University of Toledo Health Science Campus

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:adeno associated virus nucleophosmin cell cycle adenovirus dna damage repair response host interaction

ISBN:

Date of Publication:07/14/2009

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