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

Abstract

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.

Transient increases in p53-responsible gene expression at early stages of Epstein-Barr virus productive replication

“…33 Interestingly, HCMV genome has 21 potential p53 responsible sites. 35 HCMV genome-bound p53 molecules were most influential on HCMV gene expression at immediateearly and early stages of postinfection, implying a mechanism for the reduced and delayed production of virions in p53 -/-cells. Similarly, potential p53 recognition sequences were also present on EBV genome (Murata T, et al unpublished results).…”

Human Cytomegalovirus pUL29/28 and pUL38 Repression of p53-Regulated p21CIP1 and Caspase 1 Promoters during Infection

“…Many of these pathways and the associated proteins are also altered in cancers and are conserved targets among diverse herpesviruses. Examples include DAXX (death domain-associated protein) (3)(4)(5)(6), PML (promyelocytic leukemia protein) (7)(8)(9)(10)(11), IFI16 (interferoninducible protein 16) (12, 13), Tip60 (Tat-interactive protein, 60 kDa) (14,15), and p53 (16)(17)(18)(19)(20)(21)(22)(23)(24). Upon infection, delivery of the HCMV tegument protein pp71 (UL82) results in the degradation of cellular DAXX and disruption of an intrinsic antiviral response (3)(4)(5)(6).…”

Stimulation of Homology-Directed Repair at I-SceI-Induced DNA Breaks during the Permissive Life Cycle of Human Cytomegalovirus

“…Primary HFFs (propagated as described previously [33]) were electroporated with pDRGFP in a Bio-Rad Gene Pulser II using the following conditions: 300 V/cm, 75 ⍀, 2,500 F, 2-mm gap cuvettes, and 1 ϫ 10 6 cells suspended in 400 l of tissue culture medium containing 100 mM HEPES (pH 7.4) and 10 g pDRGFP. Two individual stable clones were generated through continued growth in puromycin (1 g/ml).…”