Human cytomegalovirus US3 and UL36-38 immediate-early proteins regulate gene expression

Abstract

We have established the ability of the human cytomegalovirus (HCMV) UL36-38 and US3 immediate-early (IE) gene products to alter gene expression in human cells by using transient transfection assays. The cellular heat shock protein 70 (hsp70) promoter was transactivated following cotransfection with the HCMV IE regions in nonpermissive HeLa cells by UL36-38, US3, or [El and in permissive human diploid fibroblasts (HFF) by IEl or IE2. Moreover, hsp70 expression was synergistically increased in HeLa cells cotransfected with US3 and UL36, with US3 and UL37, or with US3 and UL37xl. The synergistic transactivation of hsp7O expression by US3 and UL36-38 was not observed in HFF cells. Synergy was also not observed in HeLa cells between US3 and UL38, an early gene product encoded by the UL36-38 IE locus. Synergistic transactivation of hsp70 expression in HeLa-cells required the syntheses of UL36-38 and US3 IE proteins, since nonsense mutants were not functional. hsp70 expression increased with increasing amounts of transfected US3 and UL37 DNA and occurred at the level of stable hsp70-promoted RNA. In contrast to the broad hsp70 response, promoters from the HCMV UL112 early gene and another cellular gene, brain creatine kinase, both responded strongly only to singly transfected IE2 in HeLa cells. Nevertheless, IE2 transactivation of the UL112 promoter was further stimulated by cotransfection of [El or of UL36-38 in both HeLa and HFF cells. Thus, different patterns of promoter transactivation and interactions between HCMV IE gene products in transactivation were found in HFF cells and in HeLa cells. These results establish the ability of the HCMV US3 and UL36-38 proteins to alter cellular and viral gene expression and are consistent with involvement of cellular transcription factors in HCMV IE regulation of gene expression.

Four of eleven loci required for transient complementation of human cytomegalovirus DNA replication cooperate to activate expression of replication genes

“…The next most potent activator in our experiments was the pZP8 plasmid, which spans the UL36-38 region. Activation by this region has been shown to be both promoter and cell type dependent (11). Our results are consistent with previous findings and additionally provide new evidence that this locus contributes significantly to regulation of viral early-gene transcription.…”

“…1B). Consistent with results of Colberg-Poley et al (11), UL36-38 subtraction had a lesser effect on UL112-113 promoter expression than on most other promoters, producing only a marginally statistically significant twofold reduction (compare Fig. 3F with A through E).…”

“…Individually it was generally the most potent activator, and within the context of our assay its subtraction produced the greatest drop in overall expression. It is not surprising that the combination of immediate early loci spanning UL36-38, MIE, and IRS1/TRS1 can cooperatively promote expression of viral early promoters, because these have been shown individually to activate expression of viral early (10,40,48,60) and cellular (11) genes; moreover, both UL36-38 and IRS1/TRS1 have been shown to cooperate with MIE proteins in activating selected targets (11,60). The MIE region expresses several proteins via differential splicing, the most abundant and best understood of which are IE1-72kDa and IE2-86kDa (61).…”

“…Like MIE, the UL36-38 region is complex in that it expresses at least four different proteins from overlapping transcripts produced by differential splicing and promoter usage (63,64). Three of those, UL36, UL37, and UL37x1, were shown by Colberg-Poley et al (11) to cooperate individually with US3 to activate expression of the hsp70 promoter in HeLa cells. It remains to be determined which of these contributed to the cooperative activation of viral early promoters observed in our experiments.…”

“…It will be of interest to revisit previous findings with those targets in the light of these data. For example, UL36-38 proteins have been found to cooperate with MIE to activate expression of certain cellular genes (11). Do UL112-113 and IRS1/TRS1 proteins participate in those regulatory activities as well?…”