Summary auto-generated
This 1997 study investigated the transcription patterns of the human cytomegalovirus (HCMV) glycoprotein B (gB) gene and adjacent genes in the gB/DNA polymerase gene cluster (ORFs UL54–57). Using Northern blotting with gene-specific antisense RNA probes on infected human foreskin fibroblast and astrocytoma cells, researchers identified seven distinct transcripts: monocistronic transcripts of 5 kb and 3.7 kb for DNA polymerase and gB genes respectively, plus five polycistronic transcripts ranging from 6 to 14 kb. Mapping of transcription start and termination sites confirmed that initiation occurred at expected distances from TATA promoter elements and termination occurred at two polyadenylation signals. Time-course analysis revealed that most transcripts appeared early (24 h post-infection), but notably, the monocistronic 3.7 kb gB-specific transcript was only detected late (36–48 h). However, immunological studies demonstrated that gB protein was produced early after infection even without viral DNA synthesis, suggesting an early-expressed bicistronic 8 kb transcript encoding both gB and DNA polymerase genes supported early gB synthesis. The gB gene product appears to be classified as a γ2 (true late) gene based on strict dependence on viral DNA synthesis for the monocistronic transcript.
Key findings
- At least seven distinct transcripts are produced from the HCMV gB/POL gene cluster, including three pairs of 5'-coterminal polycistronic mRNAs and monocistronic transcripts
- The monocistronic gB-specific transcript (3.7 kb) is a late gene product synthesized only after 36-48 hours post-infection in a DNA synthesis-dependent manner
- An early-expressed bicistronic 8 kb transcript (UL55-UL54) likely accounts for early gB protein synthesis detected in the absence of viral DNA replication
- Transcription initiates at sites 23-32 nucleotides downstream of TATA elements and terminates 16 nucleotides beyond two polyadenylation motifs
- Early polycistronic transcripts appear at 24 hours post-infection while late gene-specific transcripts emerge after 36-48 hours, demonstrating temporal regulation of the gene cluster
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Abstract
Northern hybridizations were carried out using mRNA preparations of human cytomegalovirus (HCMV)-infected cultures and gene-specific antisense RNA probes for transcriptional analysis of the gene cluster composed of genes for DNA polymerase, glycoprotein B (gB), herpes simplex virus-infected cell protein 18.5 homologue p130 and a major DNA- binding protein corresponding to open reading frames (ORFs) UL54-UL57, respectively. Monocistronic transcripts of 5 kb and 3.7 kb were found for ORFs UL54 and UL55, respectively, and five additional high molecular mass overlapping transcripts of 14 kb, 10 kb, 10 kb, 8 kb and 6 kb were found. Mapping of 5' ends showed that transcription was initiated at the expected distance downstream of predicted TATA elements; in the case of a UL56-specific transcript two potential initiation sites were identified. Transcription was found to terminate at the expected distance downstream of either of two prominent polyadenylation consensus motifs in the region of UL54. All transcripts were identified early in the infectious cycle, except for the UL55 (gB)- specific transcript of 3.7 kb which was not synthesized until late post- infection. However, specific immunoreactions demonstrated the presence of a gB-specific polypeptide early after infection in the absence of viral DNA synthesis. It is suggested that a bicistronic transcript of 8 kb encoded by ORFs UL55 and UL54 is involved in biosynthesis of early HCMV gB.