Effect of a modulator deletion on transcription of the human cytomegalovirus major immediate-early genes in infected undifferentiated and differentiated cells (original) (raw)
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Journal of Virology
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
Journal of virology, 1990
The 82-kDa IE2 protein of human cytomegalovirus (HCMV) acts as both a powerful nonspecific trans activator of heterologous promoters and a negative autoregulator of HCMV immediate-early gene expression in transient assays. We show here that the highly specific down-regulation effect occurs in permissive diploid human fibroblast cells as well as in nonpermissive Vero cells and that the target sequences are conserved within the major immediate-early promoters of both HCMV and simian cytomegalovirus. The response sequences were localized between -67 and +30 in the simian cytomegalovirus IE94 promoter and upstream of position +9 in the HCMV IE68 promoter. Deletion of sequences downstream of -14 in a target IE68-CAT gene abolished the negative phenotype and resulted in a reporter gene that was stimulated instead of inhibited by cotransfection with IE2 effector DNA. Insertion of an oligonucleotide containing sequences from between -17 and +9 into the IE68-CAT deletion construction restore...
Journal of virology, 1994
We have utilized a number of well-defined, simple, synthetic promoters (upstream factor binding sites and TATA elements) to analyze the activation mechanisms of the human cytomegalovirus immediate-early (IE) proteins. We found that the 86-kDa IE protein (known as IEP86, IE2(559aa), or ppUL122a) can recognize and activate a variety of simple promoters, in agreement with the observation that it is a promiscuous activator. However, in the comparison of otherwise identical promoters IEP86 does have preferences for specific TATA elements (hsp70 > adenovirus E2 > simian virus 40 early) and specific upstream transcription factor binding sites (CAAT > SP1 approximately Tef-1 > ATF; no activation with AP1 or OCT). In contrast, the 72-kDa IE protein (known as IEP72, IE1(491aa), or ppUL123) alone did not significantly activate the simple promoters under our experimental conditions. However, each promoter activated by IEP86 was synergistically affected by the addition of IEP72. In a...
Journal of General Virology, 1988
In this paper we describe stably transfected rat cell lines which harbour either the human cytomegalovirus (HCMV) immediate early (IE) gene encoding the 72K IE nuclear antigen (IEA) or the bacterial chloramphenicol acetyltransferase (CAT) gene both under transcriptional control of the HCMV IE enhancer-promoter (-484 to-19 relative to the IE cap site, + 1). In these cell lines IE gene or CAT gene expression is repressed but can be induced by heat-shock, by sodium arsenite and by inhibitors of protein synthesis such as cycloheximide (CH). In addition, we present evidence suggesting that CH-mediated activation is cell cycle-dependent Thus CH-mediated induction of the 72K IEA as well as CAT gene expression was impaired and accumulation of mRNAs did not occur when cellular DNA synthesis was inhibited. Activation of IE genes by CH occurred almost exclusively in those cells which were in S-phase. In contrast, activation of gene expression by sodium arsenite occurred independently of cellular DNA synthesis and was not restricted to cells in S-phase. The data are consistent with, but not proof of, the hypothesis that the activation of IE transcription, brought about by inhibition of protein synthesis, resulted from a disturbed chromatin conformation due to DNA synthesis continuing in the absence of a supply of chromatin-organizing proteins. The possible relevance of these observations with regard to HCMV latency and reactivation is discussed.
Transcription of the Human Cytomegalovirus Genome in Productively Infected Cells
Journal of General Virology, 1981
Nuclear and cytoplasmic RNAs, synthesized in cells productively infected with human cytomegalovirus (HCMV) were analysed at various times after infection by liquid and filter DNA-RNA hybridization. Results of these experiments have revealed that: (i) the fraction of the genome transcribed increased as infection progressed. In the nucleus, transcripts represented approx. 20% of the virus DNA sequences at both 2 and 4 h post-infection (p.i.) and 36 % of the virus DNA at 40 h p.i.; (ii) the increase in virus sequences among nuclear transcripts at late times was prevented by the DNA synthesis inhibitor, 2'-deoxyfluorouridine; (iii) early virus RNA transcripts were a subset of those represented in late RNA; (iv) two classes of early RNA were identified by competition hybridization; (v) approx. 10% of the late nuclear transcripts were symmetrical. Results of filter hybridization at DNA excess indicated that virus-specific RNA represented 0.6 % of RNA labelled from 0 to 2 h p.i., and 1.8 % of RNA labelled from 28 to 30 h. Polyadenylated RNA isolated from cytoplasm represented 1.2% and 10% of labelled mRNA at 2 h and 30 h respectively. Our data show that during productive infection of human cells by HCMV, gene expression is under temporal, quantitative and post-transcriptional control.
Journal of virology, 1984
The replication of murine cytomegalovirus strain Smith in murine embryonic fibroblasts was investigated at immediate early, early, and late times after infection. Cloned subgenomic HindIII fragments of murine cytomegalovirus DNA served to define the regions of transcription. At immediate early times viral RNA classes ranging in size from 5.1 to 1.05 kilobases (kb) were transcribed mainly from the fragments HindIII-K and -L, whereas low levels of transcription were detected from the two termini HindIII-E and HindIII-N. A characteristic pattern of proteins could be translated from immediate early RNA in vitro. At early and late times after infection transcription from all HindIII fragments occurred, but different patterns of transcripts and proteins could be identified. Inhibitors of DNA synthesis induced differences in the late transcription pattern, located in the HindIII-F fragment. The coding region for abundant immediate early transcription could be located at between 0.769 and 0...
Virology, 1992
The major immediate-early promoter (MIEP) of human cytomegalovirus directs the expression of several differentially spliced and polyadenylated mRNAs. These mRNAs encode nuclear phosphorproteins (IE55, IE72, and IE86), which consist of common and unique amino acid sequences. To date, very little is known of the functional role of the 55-kDa (IE55) protein. Here we present evidence that the IE55 protein is a positive activator of the MIEP. In human fibroblast cells IE55 protein activated the MIEP between IO-and 30-fold. Fusion of IE55 to the GAL4 DNA binding domain resulted in a chimeric protein capable of trans-activating a reporter with GAL4 recognition sequences. These results strongly suggest that IE55 is a bona fide transcriptional activator protein. In addition, the IE55 protein was found not to act synergistically with the IE72 activator protein. The IE55 protein shares the same amino acid sequence as IE86 except for a 154-amino-acid deletion at the C-terminal end of the protein. These proteins were functionally antagonistic;
Journal of virology, 1997
We have characterized the irs1 and trs1 genes of human cytomegalovirus. The previously identified mRNAs as well as their corresponding protein products pIRS1 and pTRS1 could be detected during all phases of the viral replication cycle. The proteins were present in the nucleus and cytoplasm during the immediate-early and early phases of the viral growth cycle but were predominantly cytoplasmic late after infection. Although pIRS1 and pTRS1 exhibited little transcriptional activation potential on their own, both cooperated with the IE1 and IE2 proteins to enhance expression from a variety of viral promoters. We have also identified a previously undescribed immediate-early gene product encoded within the irs1 gene that we have termed pIRS1(263). This new protein is encoded within the 3' end of the irs1 gene and is in the same reading frame as the large pIRS1 protein. Expression of the irs1(263) gene is controlled by a promoter that resides within the irs1 open reading frame in the ...
Negative Regulation of a Heterologous Promoter by Human Cytomegalovirus Immediate-Early Protein IE2
Virology, 1997
The HCMV IE2 protein promiscuously activates transcription of many viral and cellular genes. IE2 also negatively autoregulates its own expression by binding to a strategically positioned IE2 binding site, called CRS, located immediately downstream of the TATA box of the HCMV major IE promoter. Here we show that IE2 is able to repress transcription driven by a heterologous promoter, RSV LTR. Repression of RSV LTR by IE2 is completely dependent of DNA sequences downstream of the TATA box of RSV LTR. A DNA sequence, 5-CGATACAATAAACG-3, evidently matching the proposed CRS consensus sequence, is located between nucleotides 013 and /1 (relative to the transcription start site) of RSV LTR. Three lines of evidence support the notion that this RSV CRS element is involved in the IE2-mediated repression of RSV LTR. First, introduction of mutation to the RSV CRS element renders to the mutant RSV LTR resistance to IE2-mediated repression. Second, a mutant IE2 defective in DNA binding cannot downregulate transcription from RSV LTR. Third, IE2 specifically binds to the wild-type, but not the mutant, RSV CRS element in vitro. These data, in conjunction with previous works, demonstrate that IE2 can passively repress transcription of homologous and heterologous promoters that contain a CRS element.