Multiple regulatory events influence human cytomegalovirus DNA polymerase (UL54) expression during viral infection

Abstract

The human cytomegalovirus (HCMV) DNA polymerase gene (UL54; also called pol) is a prototypical early gene in that expression is mandatory for viral DNA replication. Recently, we have identified the major regulatory element in the UL54 promoter responsive to the major immediate early (MIE) proteins (UL122 and UL123) (J. A. Kerry, M. A. Priddy, and R. M. Stenberg, J. Virol. 68:4167-4176, 1994). Mutation of this element, inverted repeat sequence 1 (IR1), abrogates binding of cellular proteins to the UL54 promoter and reduces promoter activity in response to viral proteins in transient-transfection assays. To extend our studies on the UL54 promoter, we aimed to examine the role of IR1 in UL54 regulation throughout the course of infection. These studies show that viral proteins in addition to the MIE proteins can activate the UL54 promoter. Proteins from UL112-113 and IRS1/TRS1, recently identified as essential loci for transient complementation of HCMV oriLyt-dependent DNA replication, were found to function as transactivators of the UL54 promoter in association with MIE proteins. UL112-113 enhanced UL54 promoter activation by MIE proteins three-to fourfold. Constitutive expression of UL112-113 demonstrated that the MIE protein dependence of UL112-113 transactivational activity was not related to activation of cognate promoter sequences, suggesting that UL112-113 proteins function in cooperation with the MIE proteins. Mutation of IR1 was found to abrogate stimulation of the UL54 promoter by UL112-113, suggesting that this element is also involved in UL112-113 stimulatory activity. These results demonstrate that additional viral proteins influence UL54 promoter expression in transient-transfection assays via the IR1 element. To confirm the biological relevance of IR1 in regulating UL54 promoter activity during viral infection, a recombinant virus construct containing the UL54 promoter with a mutated IR1 element regulating expression of the chloramphenicol acetyltransferase (CAT) reporter gene (RVIRmCAT) was generated. Analysis of RVIRmCAT revealed that mutation of IR1 dramatically reduces UL54 promoter activity at early times after infection. However, at late times after infection CAT expression by RVIRmCAT, as assessed by RNA and protein levels, was approximately equivalent to expression by wild-type RVpolCAT. These data demonstrate IR1-independent regulation of the UL54 promoter at late times after infection. Together these results show that multiple regulatory events affect UL54 promoter expression during the course of infection.

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

“…Moreover, either or both of these could occur at any step in the series of events controlling protein accumulation. In this regard, we note that UL112-113-mediated activation of the UL54 promoter depends upon activation by MIE proteins mediated through the IR1 element, whether or not the UL112-113 proteins are expressed constitutively (30).…”

“…Thus, we might have overlooked the apparent role of this region in regulating early gene expression if we had used simpler experimental designs; these results underscore the power of transient complementation in dissecting complex processes. However, UL112-113 did activate the UL54 promoter in combination with MIE when transfected under slightly different conditions (30).…”

“…Several lines of evidence argue that the cooperation between these effectors observed in our transient-transfection assays reflects interactions important to viral replication. First, although some aspects of viral gene expression are not accurately reproduced by transient-transfection assays, basic findings of transient-transfection analyses have been validated by using recombinant viruses (30,32), and it is likely that results of transient-transfection experiments reflect at least a subset of the regulatory events that occur in the natural context of the viral genome. Therefore, similar interactions are predicted to occur during viral infection of permissive cells.…”

Growth phenotypes of cytomegalovirus isolates do not correlate with glycoprotein B, major immediate early genotypes or antiviral sensitivity

“…This protein is synthesized only in the presence of viral IE antigens, and has been shown to increase the activity of viral DNA polymerase that is the major beta cascade protein [Ertl and Powell, 1992]. It has been shown that multiple regulatory events affect DNA polymerase expression and is dependent upon the co-operative regulatory interactions of viral and cellular proteins Kerry et al, 1996]. When agarose was layered on cultures infected with a low virus dose, the production of all antigens at Day 7 was reduced.…”

Characterization of the human cytomegalovirus irs1 and trs1 genes: a second immediate-early transcription unit within irs1 whose product antagonizes transcriptional activation

“…Studies of pIRS1 (846 amino acids [aa], 91 kDa) and pTRS1 (788 aa, 84 kDa) function have demonstrated that one of the two open reading frames is required for transient complementation of oriLyt-dependent viral DNA synthesis (21,22) and that the irs1 region is not essential for viral replication, as a deletion of this region did not appreciably change the pattern of viral growth in permissive cell lines (13). Transactivating properties of pIRS1 and pTRS1 were investigated in cotransfection experiments (11,14,26). Plasmids containing the irs1 and trs1 genes upregulated the activity of the late ICP36 promoter in the presence of the viral IE1 and IE2 proteins but did not transactivate it when transfected alone (26).…”