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. 1998 Mar;72(3):1814-25.
doi: 10.1128/JVI.72.3.1814-1825.1998.

Identification of positive and negative regulatory regions involved in regulating expression of the human cytomegalovirus UL94 late promoter: role of IE2-86 and cellular p53 in mediating negative regulatory function

B A Wing  1 , R A Johnson, E S Huang
Affiliations

Identification of positive and negative regulatory regions involved in regulating expression of the human cytomegalovirus UL94 late promoter: role of IE2-86 and cellular p53 in mediating negative regulatory function

B A Wing et al. J Virol. 1998 Mar.

Abstract

The human cytomegalovirus (HCMV) UL94 gene product is a herpesvirus-common virion protein that is expressed with true late kinetics. To identify the important cis- and trans-acting factors which contribute to UL94 transcriptional regulation, we have cloned, sequenced, and analyzed UL94 promoter function by transient transfection analysis. Transfection of UL94 promoter-reporter gene constructs into permissive human fibroblasts or U373(MG) cells indicated that promoter activity was detected following infection with HCMV. Point mutations within a TATA-like element located upstream of the RNA start site significantly reduced UL94 promoter activity. Deletion mutagenesis of the promoter indicated that a positive regulatory element (PRE) was likely to exist downstream of the UL94 mRNA start site, while a negative regulatory element (NRE) was present upstream of the TATA box. At late times of infection, the PRE appeared to have a dominant effect over the NRE to stimulate maximum levels of UL94 promoter activity, while at earlier times of infection, no activity associated with the PRE could be detected. The NRE, however, appeared to cause constitutive down-regulation of UL94 promoter activity. Binding sites for the cellular p53 protein located within the NRE appeared to contribute to NRE function, and NRE function could be recapitulated in cotransfection assays by concomitant expression of p53 and HCMV IE2-86 protein. Our results suggest a novel mechanism by which the cellular protein p53, which is involved in both transcriptional regulation and progression of cellular DNA synthesis, plays a central role in the regulation of a viral promoter which is not activated prior the onset of viral DNA replication.

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Figures

FIG. 1
FIG. 1
Late-specific utilization of the mRNA start site immediately upstream of the UL94 ORF, determined by primer extension analysis of UL94 mRNA from HCMV (Towne)-infected fibroblasts using primers UL94-3 (A), located 208 nt upstream of the UL94 ORF, and primer UL94-2 (B), which overlaps the ATG for UL94 ORF. The relative positions of the two probes with respect to the UL94 ORF are shown. The concentration of DHPG was 10 μM.
FIG. 2
FIG. 2
DNA sequence analysis of the UL94 promoter region from HCMV (Towne). All bases are identical to the reported sequence for the same region of HCMV (Ad169), except where indicated, below the Towne sequence. Also shown are the putative UL94 TATA box and RNA start site (+1), as well as consensus binding sites (boxed) for cellular transcription factors.
FIG. 3
FIG. 3
Transient transfection analysis of UL94 promoter activity from reporter construct 9412CAT, containing the full-length UL94 promoter region. UL94 promoter-CAT (9412CAT) or the parent vector (pCATBasic), lacking enhancer-promoter sequences, was transfected into HEL (A) or U373(MG) (B) cells. Twenty-four hours later, cells were infected (72 hpi) or mock-infected (mock) with HCMV (Towne). Cell lysates were prepared at 72 hpi and assayed for CAT activity. Shown is the mean percent acetylation from triplicate samples for each time point and plasmid. Samples were standardized for transfection efficiency by determining β-Gal activity from a cotransfected RSV–β-Gal plasmid.
FIG. 4
FIG. 4
Deletion and point mutation analysis suggests that three important regulatory elements located within 130 bp of the UL94 mRNA start site affect UL94 promoter activity: a 3′ PRE, a 5′ NRE, and a TATA box. (A) Progressive deletions in downstream sequences (with respect to the RNA start site); (B) progressive deletions in the upstream region, as well as point mutations in the TATA box. All constructs were generated by subcloning the regions shown in the diagram into pCATBasic. Plasmids were transfected into U373(MG) cells and subsequently infected with HCMV (Towne) or mock infected. The relative activity determined for triplicate samples of each construct following standardization by β-Gal activity is shown.
FIG. 5
FIG. 5
(A) The putative downstream PRE is dominant to the upstream NRE at late times of infection. (B) The dominant effect of the PRE over the NRE is not obviously observed prior to the onset of viral DNA replication (24 hpi) or if viral DNA replication is inhibited (72 hpi + DHPG [10 μM]). Shown are the relative CAT activities of triplicate samples for each plasmid construct standardized by β-Gal activity. Transfection-infection was carried out in U373(MG) cells, and cells were harvested at 24 or 72 hpi as indicated. (C) Constructs containing the UL94 TATA box as well as the PRE (9447CAT), the NRE (9413CAT), both elements (9414CAT), or neither element (9437CAT).
FIG. 6
FIG. 6
Deletion or mutation of the consensus p53-binding sites in the putative NRE region leads to increased UL94 promoter activity at late times of infection or when viral DNA replication is inhibited. (A) CAT activities at 72 hpi for UL94 promoter constructs containing both p53 sites (9413CAT) or with deletions in the perfect consensus p53 site (9438CAT), both (9436CAT) p53 sites, or all sequences upstream of the TATA box; (B) CAT activities of the same constructs at 72 hpi with DHPG (10 μM); (C) CAT activities at 72 hpi from constructs containing both (9439CAT) or neither (9437CAT) p53 site, or point mutations in the first perfect consensus p53 site (94313CAT). The mean relative activity of triplicate samples of each construct, standardized by β-Gal activity, is shown. All constructs were transfected into U373(MG) cells, infected with HCMV 24 h later, and harvested for CAT activity at 72 hpi.
FIG. 7
FIG. 7
Stimulation of UL94 promoter activity by p53 in Saos-2 cells. (A) Saos-2 cells were cotransfected with UL94 promoter construct 9439CAT, containing both p53 sites, or pCATBasic, along with expression plasmids for wild-type (wt) or mutant (mut) p53. (B) UL94 promoter constructs containing both p53 sites (9415CAT, 9439CAT, and 9413CAT), neither p53 site (9437CAT and 9436CAT), the downstream nonconsensus p53 site (9438CAT), or point mutations in the upstream perfect consensus p53 site (94313CAT) were cotransfected with expression plasmid for wild-type p53. For both panels, the relative mean activity for triplicate samples of each construct and/or condition is shown.
FIG. 8
FIG. 8
Binding of baculovirus-expressed p53 to the UL94 promoter p53 sites by EMSA. (A) Free, labeled, wild-type (wt) (lane 1) or mutant (mut) (lane 2) oligonucleotide probes, or wild-type (lane 3) and mutant (lane 4) probes incubated with purified, histidine-tagged p53; (B) p53 binding to labeled wild-type p53 probe containing both p53 sites of UL94 promoter (lane 10, W3C3) compared to only the upstream consensus p53 site (lane 9 and 11, W2C2) as well as both probes alone (lane 5 and 6, respectively), and supershift/stabilization of p53 binding to UL94 promoter p53 sites with p53-specific antibodies 421 (lanes 7) and DO-1 (lane 8), respectively.
FIG. 9
FIG. 9
IE2-mediated inhibition of p53 transactivation of UL94 promoter constructs, with or without the PRE region, recapitulates NRE function. (A) Cotransfection of UL94 promoter construct 9439CAT, which lacks the PRE, with expression vectors for p53, p53 plus IE2-86, or the empty expression vector (pCDNA); (B) transfection-infection experiment demonstrating that UL94 promoter construct 94115CAT, which contains an additional deletion in the PRE region, has levels of activity comparable to those of 9414CAT at late times of infection; (C) IE2-86-mediated inhibition of p53 transactivation of UL94 promoter construct 94115CAT, containing the reduced PRE region. The relative mean activity of triplicate samples for each condition is shown in each case.
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