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Widespread Phosphorylation of H2AX by Adenovirus Requires Viral Genome Replication and the E1B-55k or E4orf3 Proteins

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abstract
Adenovirus infection activates cellular DNA damage response and repair pathways. The viral E1B-55K and E4orf3 proteins prevent recognition of viral genomes as a substrate for DNA repair by targeting members of the sensor complex composed of Mre11/Rad50/NBS1 for degradation and relocalization, as well as targeting the effector protein DNA ligase IV. Despite inactivation of these cellular sensor and effector proteins, infection results in high levels of histone 2AX phosphorylation, or γH2AX. Although phosphorylated H2AX is a characteristic marker of double-stranded DNA breaks, this modification was widely distributed throughout the nucleus of infected cells and was coincident with the bulk of cellular DNA. H2AX phosphorylation occurred after the onset of viral DNA replication and independently of Mre11 and NBS1. Each of the serine-threonine kinases ataxia telangiectasia mutated (ATM), AT- and Rad3-related (ATR), and DNA protein kinase (DNA-PK) likely contribute to H2AX phosphorylation, although ATR may be the largest contributor. Viral DNA replication appears to be the stimulus for this phosphorylation event, since infection with a nonreplicating virus did not elicit phosphorylation of H2AX. Infected cells also responded to high levels of input viral DNA by localized phosphorylation of H2AX. These results are consistent with a model in which adenovirus-infected cells sense and respond to both incoming viral DNA and viral DNA replication. Infection with a double-mutant virus defective in both E1B-55K and E4orf3, and thus unable to prevent concatenation of the viral genome, results in the phosphorylation of H2AX at the periphery of viral replication centers. ATR and cellular RPA32 localize to single-stranded DNA accumulation sites of both wild-type and double-mutant virus replication centers, while activated ATM localizes to the periphery of double-mutant virus replication but is dispersed throughout the nucleus during wild-type virus infection. Individual inactivation of ATM, ATR, or DNA-PK did not alter the pattern of H2AX phosphorylation during infection with either virus. Intriguingly, H2AX was incorporated evenly into cellular DNA during wild-type virus infection, but was localized to viral replication centers during double-mutant virus infection. Thus, the activation of pathways responsible for H2AX phosphorylation requires viral genome replication, and the E1B-55K and E4orf3 proteins facilitate widespread phosphorylation by preventing the localization of H2AX to viral replication centers.
subject
Virology
Research Subject Categories::NATURAL SCIENCES::Biology::Cell and molecular biology::Molecular biology
contributor
Nichols, Gena (author)
Akman, Steven (committee chair)
Ornelles, David (committee member)
Lyles, Douglas (committee member)
Parks, Griffith (committee member)
Schwartz, Elizabeth (committee member)
date
2009-08-13T17:54:15Z (accessioned)
2010-06-18T18:59:21Z (accessioned)
2009-08-13T17:54:15Z (available)
2010-06-18T18:59:21Z (available)
2009-08-13T17:54:15Z (issued)
degree
Microbiology & Immunology (discipline)
identifier
http://hdl.handle.net/10339/14845 (uri)
language
en_US (iso)
publisher
Wake Forest University
rights
Release the entire work immediately for access worldwide. (accessRights)
title
Widespread Phosphorylation of H2AX by Adenovirus Requires Viral Genome Replication and the E1B-55k or E4orf3 Proteins
type
Thesis

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