A long and complex enhancer activates transcription of the gene coding for the highly abundant immediate early mRNA in murine cytomegalovirus (original) (raw)
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Multiple sequence-specific transcription factors modulate cytomegalovirus enhancer activity in vitro
Molecular and Cellular Biology, 1988
The possibility of DNA-binding proteins interacting in vitro with the polymerase H transcriptional machinery was explored by using a competition assay with individual target sequences for enhancer-binding factors. Transcription factors binding to at least five specific enhancer sequences mediate the activity of the human cytomegalovirus immediate-early 1 gene in vitro. Furthermore, our data suggest that individual DNA-bound enhancer factors can interact with the promoter transcription complex.
Immediate-early genes of murine cytomegalovirus: location, transcripts, and translation products
Journal of virology, 1987
Cloned genomic fragments from the region (0.769 to 0.818 map units) coding for immediate-early (IE) transcripts of murine cytomegalovirus (MCMV) were used to analyze the physical organization of this region, the direction of transcription, and the proteins synthesized in vitro. Three IE transcription units could be identified. From IE coding region 1 (ie1; 0.781 to 0.796 map units) a dominant 2.75-kilobase (kb) RNA was transcribed from right to left on the prototype arrangement of the MCMV genome which directed the synthesis of an 89,000-molecular-weight polypeptide (89K polypeptide), the major IE protein. This phosphoprotein (pp89) has been shown to be active in the regulation of transcription. Upstream of ie1 and separated by the MCMV enhancer sequence was a second IE coding region, ie2, which was mapped at 0.803 to 0.817 map units. From ie2 a 1.75-kb RNA of moderate abundance was transcribed in the direction opposite to that of the ie1 RNA. After hybrid selection of the ie2 trans...
Journal of virology, 1990
The regulation of murine cytomegalovirus early (E) gene expression was studied in the cell line B25, which is stably transfected with the immediate-early ie1/ie3 gene complex. Infection of B25 cells in the presence of the protein synthesis inhibitor cycloheximide resulted in the expression of some E genes, whereas for the expression of other E genes prior protein synthesis was still mandatory, thus showing differences in the expression requirements of individual E genes. Transcription unit e1, a member of the E genes induced by immediate-early products of the ie1/ie3 gene complex, was characterized. It is located between map units 0.709 and 0.721 of the genome of murine cytomegalovirus strain Smith. A 2.6-kilobase RNA specified in this region is spliced from three exons of 912, 177, and 1,007 or 1,020 nucleotides, which are separated by introns of 93 and 326 nucleotides. The second AUG located in the first exon 119 nucleotides downstream of the 5' cap site is followed by an open...
Journal of Virology, 2002
We have shown previously that the human cytomegalovirus (HCMV) major immediate-early (MIE) distal enhancer is needed for MIE promoter-dependent transcription and viral replication at low multiplicities of infection (MOI). To understand how this region works, we constructed and analyzed a series of HCMVs with various distal enhancer mutations. We show that the distal enhancer is composed of at least two parts that function independently to coordinately activate MIE promoter-dependent transcription and viral replication. One such part is contained in a 47-bp segment that has consensus binding sites for CREB/ATF, SP1, and YY1. At low MOI, these working parts likely function in cis to directly activate MIE gene expression, thus allowing viral replication to ensue. Three findings support the view that these working parts are likely cis-acting elements.
Journal of virology, 1997
Differentiation-dependent expression of the human cytomegalovirus (HCMV) major immediate-early (MIE) genes, encoding IE1 and IE2, may partly govern virus replication in monocytic THP-1 and embryonal carcinoma (Tera-2) cells. The modulator of the MIE promoter was shown previously in transient transfection assays to repress transcription from promoter segments in undifferentiated THP-1 and Tera-2 cells but not in differentiated cells. To determine the biological importance of these findings, we constructed a recombinant HCMV (r delta MSVgpt) without a modulator. In comparison to wild-type (WT) virus, r delta MSVgpt exhibits a slight delay in growth in human fibroblasts, but there is no appreciable change in IE1 and IE2 transcription. Moreover, there is no appreciable change in the early/late kinetics of transcription of RNAs colinear with the predicted UL128 coding region, which is adjacent to the modulator, although the size distribution and abundance of these RNAs are altered. In in...
Archives of Virology, 1990
The stimulatory effects of the 13S adenovirus E1A gene product on the human cytomegalovirus (HCMV) major immediate early (IE) enhancer were examined. Chimeric plasmids containing cloned portions of the HCMV major IE enhancer-promoter positioned upstream of the chloramphenicol acetyltransferase gene (cat) were cotransfected into HeLa cells with the plasmid p13S-wt which contained a cDNA encoding the adenovirus 13S E1A gene product. CAT expression from chimeric plasmids containing at least one copy of the HCMV 19 base pair (bp) repetitive motif was stimulated 10-fold in the presence of p13S-wt. The 19-bp motif contains a potential binding site for the cellular transcription factor ATF/CREB. Deletion analysis indicated that the ATF/CREB site was crucial for E1A-mediated stimulation. Insertion of a synthetic oligonucleotide homologous to a 19-bp motif and containing an ATF/CREB binding site into an HCMV chimera lacking ATF/CREB motifs conferred E1A responsivity on HCMV promoter-mediated CAT expression whereas insertion of a similar oligonucleotide containing a change of two bases in the sequence of the ATF/CREB site did not. Measurement of CAT-specific RNA verified the results of the CAT enzyme experiments. The ATF/CREB motif may be a target for stimulation of HCMV gene expression through either viral or cellular transcription factors.
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, 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 virology, 1998
The cytomegalovirus (CMV) enhancer is a highly complex regulatory region containing multiple elements that interact with a variety of host-encoded transcription factors. Many of these sequence elements are conserved among the different species strains of CMV, although the arrangement of the various elements and overall sequence composition of the CMV enhancers differ remarkably. To delineate the importance of this region to a productive infection and to explore the possibility of generating a murine CMV (MCMV) under the control of human CMV (HCMV) genetic elements, the MCMV enhancer was resected and replaced either with nonregulatory sequences or with paralogous sequences from HCMV. The effects of these various deletions and substitutions on viral growth in transfected or infected tissue-culture cells were evaluated. We found that mutations in MCMV that eliminate or substitute for the enhancer with nonregulatory sequences showed a severe deficiency in virus synthesis. This growth de...
Transcription of the immediate early genes of human cytomegalovirus strain AD169
Virus Research, 1984
Cloned sub-genomic fragments of human cytomegalovirus strain AD169 were used to analyse immediate-early (IE) transcription in virus-infected cells. Transcriptionally active regions of the HCMV genome were identified by hybridising cytoplasmic IE poly(A)+-RNA with dot blots and Southern transfers of restriction endonuclease digests of recombinant plasmids. The size. number and, in some cases, the orientation of transcription of IE RNA species were determined. The most abundant IE mRNA (IE-1.95) was mapped at 0.0764-0.086.5 map units. The transcription of two middle abundant (1.7 and 2.15 kb) IE RNAs was initiated immediately downstream, and in the same orientation as the IE-1.95 gene. A second transcriptionally active area was identified at 0.593-0.619 map units. Three mRNA species (IE-1.75, IE-3.8 and IE-4.8) were derived from this region. Additional minor IE transcription was also observed from other regions of the HCMV genome. Hybrid-selected translation was used to identify the polypeptides encoded by the major IE RNA species.