An intact sequence-specific DNA-binding domain is required for human cytomegalovirus-mediated sequestration of p53 and may promote in vivo binding to the viral genome during infection

Kyle Rosenke; Melanie A Samuel; Eric T McDowell; Melissa A Toerne; Elizabeth A Fortunato

doi:10.1016/j.virol.2005.12.013

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An intact sequence-specific DNA-binding domain is required for human cytomegalovirus-mediated sequestration of p53 and may promote in vivo binding to the viral genome during infection

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An intact sequence-specific DNA-binding domain is required for human cytomegalovirus-mediated sequestration of p53 and may promote in vivo binding to the viral genome during infection

Kyle Rosenke, Melanie A Samuel, Eric T McDowell, Melissa A Toerne and Elizabeth A Fortunato

Virology (New York, N.Y.), Vol.348(1), pp.19-34

2006

DOI: https://doi.org/10.1016/j.virol.2005.12.013

Handle:

https://hdl.handle.net/2376/107838

PMID: 16455125

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Abstract

DNA-binding domain

In vivo binding assays

Sequestration

p53

Human cytomegalovirus

The p53 protein is stabilized during infection of primary human fibroblasts with human cytomegalovirus (HCMV). However, the p53 in HCMV-infected cells is unable to activate its downstream targets. HCMV accomplishes this inactivation, at least in part, by sequestering p53 into viral replication centers within the cell's nucleus soon after they are established. In order to better understand the interplay between HCMV and p53 and the mechanism of sequestration, we constructed a panel of mutant p53-GFP fusion constructs for use in transfection/infection experiments. These mutants affected several post-translational modification sites and several sites within the central sequence-specific DNA-binding domain of the protein. Two categories of p53 sequestration were observed when the mutant constructs were transfected into primary fibroblasts and then infected at either high or low multiplicity. The first category, including all of the post-translational modification mutants, showed sequestration comparable to a wild-type (wt) control, while the second category, mutants affecting the DNA-binding core, were not specifically sequestered above control GFP levels. This suggested that the DNA-binding ability of the protein was required for sequestration. When the HCMV genome was analyzed for p53 consensus binding sites, 21 matches were found, which localized either to the promoters or the coding regions of viral proteins involved in DNA replication and processing as well as structural proteins. An analysis of in vivo binding to these identified sites via chromatin immunoprecipitation assays revealed differential binding to several of the sites over the course of infection.

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Title: An intact sequence-specific DNA-binding domain is required for human cytomegalovirus-mediated sequestration of p53 and may promote in vivo binding to the viral genome during infection
Creators: Kyle Rosenke
Melanie A Samuel
Eric T McDowell
Melissa A Toerne
Elizabeth A Fortunato
Publication Details: Virology (New York, N.Y.), Vol.348(1), pp.19-34
Academic Unit: UNKNOWN
Publisher: Elsevier Inc
Identifiers: 99900546706201842
Language: English
Resource Type: Journal article