Cellular Response to Adenovirus and Adeno- Associated Virus Coinfection
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.
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
Date of Publication:07/14/2009