Multiple 5′ ends of human cytomegalovirus UL57 transcripts identify a complex, cycloheximide-resistant promoter region that activates oriLyt

Abstract

The human cytomegalovirus (HCMV) UL57 gene lies adjacent to HCMV oriLyt, from which it is separated by an organizationally conserved, mostly noncoding region that is thought to both regulate UL57 expression and activate oriLyt function. However, the UL57 promoter has not been studied. We determined the 5' ends of UL57 transcripts toward an understanding of the potential relationship between UL57 expression and oriLyt activation. The results presented here identified three distinct 5' ends spread over 800 bp, at nt 90302, 90530, and 91138; use of these sites exhibited differential sensitivity to phosphonoformic acid treatment. Interestingly, a 10-kb UL57 transcript accumulated in cycloheximide-treated infected cells, even though other early transcripts were not detectable. However, the 10-kb transcript did not accumulate in cells treated with the more stringent translation inhibitor anisomycin. Consistent with the notion that the identified 5' ends arise from distinct transcription start sites, the sequences upstream of sites I and II functioned as promoters responsive to HCMV infection in transient assays. However, the origin-proximal promoter region III required downstream sequences for transcriptional activity. Mutation of candidate core promoter elements suggested that promoter III is regulated by an initiator region (Inr) and a downstream promoter element. Finally, a 42-bp sequence containing the candidate Inr activated a minimal oriLyt core construct in transient replication assays. Thus, these studies showed that a large, complex promoter region with novel features controls UL57 expression, and identified a sequence that regulates both UL57 transcription and oriLyt activation.

Epstein-Barr Virus Immediate-Early Protein Zta Co-Opts Mitochondrial Single-Stranded DNA Binding Protein To Promote Viral and Inhibit Mitochondrial DNA Replication

“…In studies with the related gammaherpesvirus, Kaposi's sarcoma-associated herpesvirus, RNA transcription initiation within OriLyt was found to be essential for efficient lytic replication (52). Studies with cytomegalovirus have found that RNA transcripts and RNA hybrids exist at the lytic origin, and these structures may reflect complex transcription-related events during the initiation of lytic replication (25,40). Our experiments do not address whether mtSSB plays a similar role in replication initiation at EBV OriLyt as it does at mitochondrial DNA origins or whether RNA transcripts contribute to EBV replication.…”

Human Cytomegalovirus DNA Replication Requires Transcriptional Activation via an IE2- and UL84-Responsive Bidirectional Promoter Element within ori Lyt

“…Other transcripts were also identified within the promoter region (22). Recent evidence has indicated that another promoter, outside of the core region, activates oriLyt (28). This promoter is upstream of the oriLyt promoter described in this report and comprises the cis regulatory region for the UL57 gene.…”

Analysis of Human Cytomegalovirus ori Lyt Sequence Requirements in the Context of the Viral Genome

“…HCMV oriLyt contains numerous reiterated and inverted sequence elements, including known consensus transcription factor recognition sites. Although the analysis of these sequence elements in either oriLyt function or local transcription regulation has just begun (33), their distribution resembles the organization of some complex transcriptional enhancers, and the existence of these binding sites suggests the presence of putative promoter regions. Transcriptional control elements and enhancers are indeed common components of many viral and eukaryotic origins of DNA replication (21,42,48,50,51).…”

“…These findings also strongly suggest that the mutants were not impaired in the expression of transcripts that are essential for the initiation and progression of viral DNA replication, e.g., the smallest replicator transcript (SRT), v-RNA-2, or the UL57 RNA encoding the single-stranded DNA binding protein. The 5Ј ends of the UL57 transcripts were precisely mapped in a recent study (33). Although the UL57 promoter may extend considerably upstream, it is extremely unlikely that the growth defect of the genomes with a deletion of minimal oriLyt was caused by an impairment of UL57 expression, especially since pHG-⌬1.5 could be rescued by ectopic insertion of the complete oriLyt.…”

“…Although the UL57 promoter may extend considerably upstream, it is extremely unlikely that the growth defect of the genomes with a deletion of minimal oriLyt was caused by an impairment of UL57 expression, especially since pHG-⌬1.5 could be rescued by ectopic insertion of the complete oriLyt. Since the left boundary (nt 91165) of the complete oriLyt as used in this study and the most distal transcription start site of the UL57 RNA (nt 91138), as well as the candidate promoter sequences (33), are located in close proximity, one cannot completely rule out an effect on UL57 expression in the mutants carrying a deletion of complete oriLyt. However, given the pivotal role of the UL57 protein (single-stranded DNA binding protein) for replication of HCMV DNA, impaired UL57 expression would hardly be compatible with the unaltered DNA synthesis displayed by the mutants RV-pHG-6 and RV-pHG-7.…”