The Presence of p53 Influences the Expression of Multiple Human Cytomegalovirus Genes at Early Times Postinfection

Abstract

Human cytomegalovirus (HCMV) is a common cause of morbidity and mortality in immunocompromised and immunosuppressed individuals. During infection, HCMV is known to employ host transcription factors to facilitate viral gene expression. To further understand the previously observed delay in viral replication and protein expression in p53 knockout cells, we conducted microarray analyses of p53 ؉/؉ and p53 ؊/؊ immortalized fibroblast cell lines. At a multiplicity of infection (MOI) of 1 at 24 h postinfection (p.i.), the expression of 22 viral genes was affected by the absence of p53. Eleven of these 22 genes (group 1) were examined by real-time reverse transcriptase, or quantitative, PCR (q-PCR). Additionally, five genes previously determined to have p53 bound to their nearest p53-responsive elements (group 2) and three control genes without p53 binding sites in their upstream sequences (group 3) were also examined. At an MOI of 1, >3-fold regulation was found for five group 1 genes. The expression of group 2 and 3 genes was not changed. At an MOI of 5, all genes from group 1 and four of five genes from group 2 were found to be regulated. The expression of control genes from group 3 remained unchanged. A q-PCR time course of four genes revealed that p53 influences viral gene expression most at immediate-early and early times p.i., suggesting a mechanism for the reduced and delayed production of virions in p53 ؊/؊ cells.

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).…”

Human Cytomegalovirus IE1-72 Protein Interacts with p53 and Inhibits p53-Dependent Transactivation by a Mechanism Different from That of IE2-86 Protein

“…Cellular protein p53 has been known as a tumor suppressor gene product (17,31,42) and cell cycle inhibitor (48). It is known to regulate transcription of a variety of genes, including genes involved in cell cycle regulation, DNA repair, and programmed cell death (28,52,62). Elevated levels of p53 were observed in cell lines transformed by a variety of agents, including DNA and RNA tumor viruses, and chemical carcino-gens (for reviews, see reference 61).…”

Mutation of Glutamine to Arginine at Position 548 of IE2 86 in Human Cytomegalovirus Leads to Decreased Expression of IE2 40, IE2 60, UL83, and UL84 and Increased Transcription of US8-9 and US29-32

“…The custom design for this array was generously donated by E. Fortunato and was described previously (11). Each Combimatrix slide contains four identical arrays with 2,240 features, of which 305 are control features, 1,265 are cellular gene features, and 670 are viral gene features.…”

“…In order to gain a greater understanding of the Q548R IE2 mutant, we examined the expression levels of viral and cellular RNA corresponding to approximately 2,000 RNA oligonucleotides via a custom microarray. The design for this array, which contains 1,265 cellular features and 670 viral features, was generously donated by E. Fortunato (11). For this experiment, G 0 -synchronized HFFs were mock infected or infected with WT C-F HB5 or Q548R IE2 C-F HB5 virus (MOI ϭ 2).…”

“…This set of infections was carried out three times to create three biological replicates. RNA was extracted, amplified, and Cy5 labeled as previously described (11).…”