Separate DNA Elements Containing ATF/CREB and IE86 Binding Sites Differentially Regulate the Human Cytomegalovirus UL112-113 Promoter at Early and Late Times in the Infection

Human cytomegalovirus (HCMV), a member of the betaherpesvirus family, is an important opportunistic pathogen in immunocompromised individuals and is recognized as a major viral cause of birth defects in newborns (2). As for other herpesviruses, HCMV genes are expressed in a temporal pattern upon infection (5, 18, 29, 34, 35). Members of the immediate-early (IE) class of genes are expressed upon viral infection and do not require de novo protein synthesis for production of their RNAs. Several of the IE proteins are transactivators of the products of the next class of genes, the early genes. Concomitant with viral DNA replication, there is expression of the late class of viral genes, whose protein products are generally involved in viral genome packaging and virion maturation. Since several of the early gene protein products are essential for viral DNA replication, understanding the control of early viral gene expression is important for designing therapeutic strategies to inhibit viral replication. In order to understand the mechanisms that control early viral gene expression, several laboratories have studied a number of different early viral promoters by various types of transcription assays (for reviews, see references 20 and 26). To date, the most common assay to study viral promoter activity has been the transient expression assay. In this assay, a plasmid containing a reporter gene under control of an early viral promoter is transfected into cells along with expression plasmids encoding HCMV transcriptional regulatory proteins. These assays have proven useful in determining which promoter sequences are important for gene expression as well as which HCMV proteins behave as transcriptional regulators. In fact, these assays have identified the HCMV IE2 86-kDa protein (IE86) and the IE1 72-kDa protein (IE72) as the major transregulators of early viral gene expression (for reviews, see references 20 and 26). However, transient expression assays are limited in that they do not provide accurate information about the regulatory events that occur during a normal viral infection. Several labs have attempted to circumvent this issue by transfecting cells with plasmid and subsequently superinfecting with virus (3, 10, 25, 27, 32). Although this allows one to study gene expression in infected cells, there remain template-specific differences between plasmid DNA and viral DNA. In particular, the late induction of the 1.2-kb RNA promoter, normally observed in infected cells, does not occur when the promoter is located on a plasmid in transfection-infection assays unless an HCMV origin of replication, oriLyt, is also included on the plasmid (33). In addition, it was shown that at least two genes, UL83 and UL99, with early-late and true late kinetics, respectively, were activated earlier and to higher levels in transfection-infection assays than observed with the endogenous genes during a normal infection (14). In an attempt to study early and late viral gene expression without the inaccuracies associated with transfection assays, Kohler et al. have used a gene replacement strategy based on the finding that a portion of the unique short region of the HCMV genome is dispensable for viral growth (14). To this end, they have constructed recombinant viruses in which a viral promoter linked to a reporter gene is inserted between the US9 and US10 genes and have shown that the inserted promoters exhibit kinetics similar to those of the endogenous viral promoters. As a model for early HCMV gene expression, our laboratory has studied the expression of the 2.2-kb class of RNAs (open reading frame UL112-113) which encode a family of nuclear phosphoproteins (13, 27, 28, 36, 37). Although the function of these proteins is unclear, there is evidence that they behave as transcriptional activators and are required for viral DNA replication (7, 10, 21). By transient expression analysis, our laboratory has shown that activation of the UL112-113 promoter is mediated through a major regulatory domain between nucleotides −113 and −59 (23, 24). This region contains a weak binding site for the HCMV transactivator IE86 (−113 to −85) and a consensus ATF/CREB binding site (−71 to −66) (15, 23, 24). In addition, the UL112-113 promoter contains three other binding sites for IE86 (−286 to −257, −248 to −218, and −148 to −120) (1, 24). Results of mutational analyses of the UL112-113 promoter suggest that in transfection assays, the ATF/CREB site is the major regulatory element upstream of the TATA box, whereas the IE86 binding sites play an accessory role (1, 15, 23, 24). The goal of this study was to determine the functions of various sequence elements within the UL112-113 promoter at different times during HCMV infection. To this end, we have further defined the roles of the sequences containing the ATF/CREB binding site and the weak IE86 binding site within the UL112-113 promoter at early and late times during infection. We have constructed recombinant viruses in which various mutations of the UL112-113 promoter, linked to the chloramphenicol acetyltransferase (CAT) gene, were inserted between the US9 and US10 genes in the viral genome and have analyzed UL112-113 promoter–CAT activity at different times during the infection. We find that the kinetics of expression of the UL112-113 promoter from the US9/10 locus are identical to that of the endogenous viral promoter, including the switch to a different RNA start site late in infection (28). Consistent with transient expression data, our results demonstrate that the sequences containing the ATF/CREB site play a major role in UL112-113 promoter activity early during viral infection, whereas the region containing the weak IE86 binding site has a moderate effect on transcription. However, at late times in the infection, we find that the ATF/CREB site plays little if any role in expression, but the sequences between −113 and −85, which include the weak IE86 binding site, are required for transcription from the late RNA start site within the UL112-113 promoter. Furthermore, replacement of the region between nucleotides −108 and −95 with the HCMV cis-repression signal (CRS) indicated that the sequences between −108 and −95 play a role in distance-dependent, and possibly orientation-dependent, late transcription initiation from the UL112-113 promoter. These data underscore the value of using recombinant viruses to study viral promoter activity and demonstrate that different promoter regulatory elements are employed at early and late times in the infection.