The human cytomegalovirus UL98 gene transcription unit overlaps with the pp28 true late gene (UL99) and encodes a 58-kilodalton early protein

Abstract

A murine monoclonal antibody (I2) reacts strongly with the nucleus of human cytomegalovirus (HCMV)infected human fibroblasts. Western blot (immunoblot) analysis using I2 demonstrated that a protein with an apparent molecular mass of 58-kDa (E58) was expressed at 5 h after infection, and levels increased through 72 h. Immunoblot screening of an early cDNA expression library resulted in a positive clone which hybridized to the right end of the XbaI C fragment of the HCMV Towne strain. Further analysis demonstrated that the E58-specific clone was homologous to the putative UL98 open reading frame, which has been proposed to encode the viral alkaline exonuclease homolog. RNA analysis demonstrated a 3.0-kb RNA which is expressed at early times after infection, as well as in the absence of viral DNA replication, and which is 3 coterminal with the pp28 (UL99) gene region. Insertion of the UL98 genomic sequence into a eucaryotic expression vector and subsequent Western blot analysis using I2 demonstrated that the expressed protein comigrated with E58 from infected cells. E58 also reacts specifically with a previously described antibody, anti-P 2-1 , which was proposed to recognize a putative late 58-kDa protein. E58 comigrates with the putative late 58-kDa protein, indicating that these two proteins are likely the same. Analysis of the UL98 promoter revealed a TATATAA sequence located at nucleotide 142525. Insertion of the putative promoter 5 to a reporter gene demonstrated that the UL98 promoter was activated in cotransfection experiments with IE1 and IE2 proteins. These studies demonstrate that UL98 is a bona fide early gene, which is consistent with its probable role as the viral alkaline exonuclease gene.

Transcription of True Late (γ2) Cytomegalovirus Genes Requires UL92 Function That Is Conserved among Beta- and Gammaherpesviruses

“…Like other herpesviruses, HCMV encodes distinct categories of late genes, commonly referred to as leaky late (or ␥ 1 ) and true late (or ␥ 2 ); the former are expressed independent of viral DNA synthesis, and the latter are not expressed at all when viral DNA synthesis is blocked by specific inhibitors (1,24). Genes in the true late category include UL99 (encoding pp28) (25,26), UL94 (27), UL75 and UL115 (encoding gH/gL) (28)(29)(30), UL32 (encoding pp150) (31,32), the middle transcription start site of UL44 (33)(34)(35), and a start site located within UL122 (IE2-p86) encoding IE2-p40 (also called L40) (10,36). Many other viral transcripts are detected at late times (37).…”

Translational regulation of the human cytomegalovirus pp28 (UL99) late gene

“…The first begins 177 bp downstream of the CAP site and consists of the carboxy-terminal 120 amino acids of the alkaline exonuclease homolog (UL98) (1). However, no evidence of this truncated carboxy-terminal protein has been observed in either infected cells or cells transfected with a genomic construct of this region (1). The second ORF of this 1.6-kb mRNA encodes the pp28 tegument protein.…”

“…This mRNA is of high abundance and contains two potential ORFs. The AUG codon of the first ORF is located 177 bp downstream of the CAP site and corresponds to the carboxy terminus of the UL98 ORF, which encodes the viral alkaline exonuclease enzyme (1). The pp28 ORF begins 476 bp downstream of the CAP site of the 1.6-kb mRNA.…”

Characterization of 3′ termini of human cytomegalovirus UL138-UL145 transcripts in a clinical strain

“…The structurally polycistronic transcripts similar to those found in other genomic regions of HCMV appeared to be a general feature of the HCMV genome (7, 15–17), although most of polycistronic transcripts existed in prokaryotes (18, 19). The multiple uses of polyadenylation signals contributed HCMV to utilizing genetic information (20, 21), which indirectly implied that transcription of HCMV genes might be regulated mostly by their promoters and sequences in 5′ ends.…”