trans-activation and autoregulation of gene expression by the immediate-early region 2 gene products of human cytomegalovirus

Abstract

The maior immediate-early (IE) gene region mapping at coordinates 0.71 to 0.74 in the genome of human cytomegalovirus (HCMV) gives rise to a series of overlapping spliced IE mRNAs that are all under the transcriptional control of the complex IE68 promoter-enhancer region. We show here that one of the phosphorylated nuclear proteins encoded by this region behaves as a powerful but nonspecific trans-activator of gene expression. In transient chloramphenicol acetyltransferase (CAT) assay experiments with Vero cells all relatively weak heterologous target promoters tested, including those of herpes simplex virus IE175 and delayed-early genes, adenovirus E3, the enhancerless simian virus 40 early gene, and the human beta interferon gene, were stimulated between 30and 800-fold by cotransfection with the HindIII C fragment of HCMV (Towne) DNA. In contrast, expression of the homologous HCMV IE68-CAT gene but not SV2-CAT was specifically repressed. Inactivation mapping studies of the effector DNA, together with dose-response comparisons with subclones from the region, revealed that an intact 7. 1-kilobase sequence encompassing both the IE1 and 1E2 coding regions (exons 1 to 5) in the major IE transcription complex was required for both the nonspecific trans-activation and autoregulatory responses. The IEl coding region alone (exons 1 to 4) was inactive, but both functions were restored by insertion of the IE2 coding region (exon 5) in the correct orientation downstream from the IE1 coding region. Internal deletions or inserted terminator codons in IEI (exon 4) still gave efficient trans-activation and autoregulation, whereas the insertion of terminator codons in IE2 (exon 5) abolished both activities. Finally, IE2 (exon 5) sequences only (under the direct transcriptional control of the strong simian CMV IE94 promoter) were still able to specifically down regulate IE68-CAT expression but failed to exhibit trans-activation properties. Therefore, the IE2 gene product(s) of HCMV appear likely to be key control proteins involved in gene regulation during HCMV infection. All of the major classes of DNA viruses that replicate in mammalian cell nuclei encode immediate-early (IE) gene products that behave as trans-activators of subsequent viral gene expression and are themselves under the control of complex upstream protnoters and regulatory or enhancer regions. The large T antigens of papovaviruses, the ElA gene products of adenoviruses, the IE175 (ICP4) and IE110 (ICPO) proteins of herpes simplex virus (HSV), the E2 proteins of papillomaviruses, and the rep gene products of parvoviruses all appear to fit into this classification. They also all share the characteristic of being hydrophilic nuclear phosphoproteins whose mRNAs are synthesized immediately after infection and in the absence of de novo protein synthesis. Most of these proteins are needed within their own systems for efficient synthesis of subsequent viral genes (usually acting at the transcriptional level), but they may also show various degrees of relaxed specificity...

Direct correlation between a negative autoregulatory response element at the cap site of the herpes simplex virus type 1 IE175 (alpha 4) promoter and a specific binding site for the IE175 (ICP4) protein

“…Specificity of the down-regulation by IE175. We have recently described a similar phenomenon of specific inhibition of expression from the major IE68 promoter of HCMV by a powerful trans-activator protein encoded by the HCMV IE2 gene (48). This effect is not exerted on the simian virus 40 early promoter, but does act to inhibit the closely related IE94 promoter from SCMV(Colburn).…”

“…MATERIALS AND METHODS Construction of IE110-CAT and IE68-CAT gene plasmids. The hybrid HSV-1 IE target genes IE175(-1900/+30)-CAT and IE175(-380/+30)-CAT, HSV-2 DE38(-420/+50)-CAT, and human cytomegalovirus (HCMV) IE68(-760/+ 10)-CAT in plasmids pPOH2, pPOH13, pPOH1, and pCAT760wt, respectively, have been described previously (40,43,48). To make an HSV-1 IE110-CAT hybrid test gene containing the intact HSV-1(KOS) IE110 promoter, the pIGA15 effector plasmid was digested with BAL 31 from the BamHI site in the large intron in the IE110 coding region, and BamHI linkers were added.…”

Prolonged activation of NF-κB by human cytomegalovirus promotes efficient viral replication and late gene expression

“…The function of the second (later) phase of NF-κB activation in the HCMV life cycle remains unknown. Transcription from the MIEP can be inhibited by viral proteins early after infection (Cherrington, Khoury, and Mocarski, 1991;Hermiston, Malone, and Stinski, 1990;Liu, Hermiston, and Stinski, 1991;Pizzorno and Hayward, 1990;Pizzorno et al, 1988;Stenberg et al, 1990), and, although IE1-72 protein is very stable, new IE1-72 protein synthesis does not occur after 12 hpi (Poma et al, 1996). Therefore, it is unlikely that the prolonged activation of NF-κB induced by HCMV functions to transactivate this promoter at later times of infection.…”

The 86-kilodalton IE-2 protein of human cytomegalovirus is a sequence-specific DNA-binding protein that interacts directly with the negative autoregulatory response element located near the cap site of the IE-1/2 enhancer-promoter

“…This appears to be regulated at the posttranscriptional level via alternative splicing or alternative polyadenylation (36). At later stages during the infectious cycle, both transcripts are downregulated, which has been attributed to the autoregulatory function of IE-2 proteins (7,15,31). At late times after infection, an additional RNA of 1.5 kb is transcribed from the IE-2 gene region under the control of a true late promoter and encodes a protein of 40 kDa in molecular mass corresponding in sequence to the carboxy-terminal part of the 86-kDa IE-2 polypeptide (33,37).…”