Human Cytomegalovirus Gene Expression Research Articles

Translational control of human cytomegalovirus gp48 expression.

Posttranscriptional controls modulate the expression of several human cytomegalovirus genes. Previous studies have shown that one cytomegalovirus gene transcript leader contains AUG codons which inhibit translation of a downstream reading frame. However, two other cytomegalovirus gene transcript leaders of similar structure do not inhibit translation. We have extended these studies to the analysis of the structural glycoprotein gp48, whose predominant transcript contains three upstream AUG codons. The 5' leader of this transcript strongly inhibits downstream translation in fibroblasts. Analyses of deletions and point mutations identify the second upstream AUG codon as an essential component of the inhibitory signal. Other leader sequences, but neither the first nor the third AUG codon, are also required. Intriguingly, the inhibitory signal appears also to depend on the amino acid coding information of the short reading frame associated with the second AUG codon. Insights derived from these studies are germane to understanding the translational regulation of other viral and cellular genes of similar structure.

Effect of human cytomegalovirus on replication of SV40 origin and the expression of T antigen

Human cytomegalovirus (HCMV) induces replication of the cloned SV40 origin of DNA replication in both productive and nonproductive infections. HCMV-induced replication of the SV40 DNA origin required the presence of T antigen. Human embryonic lung (HEL) cells were found to be fully permissive for SV40 origin replication only in the presence of HCMV gene expression. In addition, expression of plasmid encoded SV40 T antigen in HEL cells was only induced in the presence of HCMV. Cotransfection of the SV40-cloned origin of replication and seven overlapping cosmids containing the entire HCMV genome is capable of stimulating replication of the SV40 origin. We conclude that induction of the SV40 origin of replication by HCMV is (i) not due to any component of the HCMV virion, (ii) has a requirement for T antigen expression, (iii) occurs in an SV40 nonproductive cell, when T antigen expression is induced by HCMV gene expression.

Translational inhibition by cytomegalovirus transcript leaders

The regulation of human cytomegalovirus gene expression depends on both transcriptional and post-transcriptional controls. Previous studies revealed that either of two AUG codons contained in the 5′ leader of a β gene (2.7β) transcript inhibited translation of a downstream reading frame. We investigated the regulatory effects of 5′ leader sequences from the cytomegalovirus DNA polymerase and pp150 genes, each of which also contains upstream AUG codons. Surprisingly, these two leaders did not affect expression of the downstream open reading frame. Detailed analyses were carried out to examine the role of the AUG codons within the pp150 leader. These upstream AUG codons allowed efficient downstream translation, despite the predictions of the scanning model of eukaryotic translation. Further studies of the 2.7β leader revealed that an upstream AUG codon, although necessary, was not sufficient to inhibit downstream translation. These results reveal that translational inhibition by CMV transcript leaders requires an AUG codon and additional leader sequences.

The human cytomegalovirus immediate early enhancer-promoter is responsive to activation by the adenovirus-5 13S E1A gene

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.

Expression of Oncogenic ras in Human Teratocarcinoma Cells Induces Partial Differentiation and Permissiveness for Human Cytomegalovirus Infection

Retinoic acid (RA)-induced differentiation of human teratocarcinoma (T2) cells results in a change from a normally non-permissive phenotype for human cytomegalovirus (HCMV) infection to cells which are fully permissive. We have used this system to analyse factors associated with differentiation which may regulate HCMV gene expression. Differentiation of T2 cells results in an increase of c-ras expression. Consequently, we have introduced ras expression vectors into T2 cells. We find that, as with RA induction, transfection of T2 cells with oncogenic human Ha-ras results in cells which are permissive for HCMV infection and gene expression. However, unlike RA which induces a cessation of cell proliferation and terminal differentiation, ras transfection only appears to result in changes associated with early events in RA-induced differentiation of T2 cells.

Repression of human cytomegalovirus gene expression asscoiated with a novel immediate early regulatory region binding factor

A major determinant controlling reactivation of persistent viruses is likely to be the level of cellular factors which regulate virus transcription. Human cytomegalovirus (the largest human herpesvirus) does not replicate in human teratocarcinoma (T2) cells due to a block in transcription of immediate early (IE) gene expression, but these cells become permissive upon retinoic acid induced differentiation. We have analysed changes in DNA binding factors to the major IE promoter/regulatory region of HCMV that occur during differentiation of T2 cells to a permissive phenotype. We show that undifferentiated T2 cells contain a specific nuclear factor that binds to a far upstream region of the major IE regulatory region. Differentiation of T2 cells is associated with a major decrease in this factor and deletion of its specific binding site from IE expression vectors also results in increased levels of expression in undifferentiated cells. Consequently, this novel factor present in undifferentiated cells is a candidate for a differentiation specific negative regulator of HCMV IE gene expression which binds to an element upstream of the major IE enhancer.

Correlation between stimulation of host cell DNA synthesis by human cytomegalovirus and lack of expression of a subset of early virus genes

The stimulation of host cell DNA synthesis was studied in permissive human embryonic lung (HEL) cells and in nonpermissive rabbit kidney (RK) cells infected with human cytomegalovirus (HCMV). Host cell DNA synthesis was induced by HCMV infection in resting cells of both types. In permissive cultures the stimulation of cellular DNA synthesis was detectable mainly in those cells which had not become productively infected and in which virus antigens were not detectable. In abortively infected RK cells, on the other hand, stimulation of host cell DNA synthesis and the expression of virus antigens were detected in the same cells. Infection of actively growing permissive HEL cells resulted in a shutdown of cellular DNA synthesis beginning approximately 10 hr postinfection. Shutdown of cellular DNA synthesis also occurred when the infected cells were treated with phosphonoacetic acid and was thus classified as an “early” virus function. In actively growing, abortively infected RK cells, on the other hand, host cell DNA synthesis was not affected, indicating that the early virus function(s) responsible for inhibition of cellular DNA synthesis was not expressed in these cells. Virus-encoded DNA polymerase activity, another early virus gene function, was also not detected in these abortively infected cultures. In RK cells the cellular DNA synthesized as a result of infection was capable of undergoing at least one further round of replication, indicating that the HCMV gene expression which occurred in abortively infected RK cells was not lethal for these cells.

Organization and expression of the immediate early genes of human cytomegalovirus.

The immediate early genes of human cytomegalovirus were characterized according to map location, RNA transcripts, and translation products. Three regions in the large unique component (0.709 to 0.751 map units) were transcribed in the presence of an inhibitor of protein synthesis (anisomycin). A single size class of polyadenylated mRNA, 1.95 kilobases (kb), was transcribed abundantly relative to the other size classes. The predominant 1.95-kb viral RNA was transcribed from right to left on the prototype arrangement of the viral genome and spanned a region of approximately 2.8 kb (0.739 to 0.751 map units). This mRNA codes for a 75,000-dalton protein that represents the predominant immediate early protein detected in infected cells. Immunoprecipitation of viral proteins synthesized in vitro as well as in vivo demonstrated that the predominant immediate early protein is synthesized as a protein of 75,000 daltons, but is presumably modified in vivo, resulting in a broad banding pattern ranging from 75,000 to 68,000 daltons. A different immediate early viral gene (0.732 to 0.739 map units) is transcribed from left to right at relatively low levels. The 3' ends of the above viral RNAs terminate at approximately 230 base pairs apart in the region of approximately 0.739 map units. Five RNA size classes ranging from 2.25 to 1.10 kb were detected, but the 1.75-kb and 1.40-kb RNA size classes were more abundant from this region. At least four minor proteins are coded by these mRNAs, with apparent molecular weights ranging from 56,000 to 16,500. Last, a 1.95-kb mRNA was transcribed from a third region (0.709 to 0.728 map units). This viral mRNA was present at relatively low concentration and coded for a minor protein of 68,000 daltons. Since immediate early gene expression of human cytomegalovirus is dominated by the synthesis of an mRNA from the region of 0.739 to 0.751 map units that codes for the predominant immediate early protein found in the infected cell, we hypothesize that this protein is the major regulatory protein influencing the switch from restricted to extensive transcription.

Post-transcriptional control of human cytomegalovirus gene expression

During the immediate-early, early, and late phases of human cytomegalovirus infection in human fibroblasts, transcripts accumulated, respectively, from approximately 20, 75, and 90% of the sequences on the genome. Not all of the sequences which accumulated during the immediate-early and early phases were represented on polysomes, however. Four transcripts synthesized in cycloheximide-treated cells were studied in detail. A 2.2-kb transcript (713–733 map units) represented 95% of the polysome-associated RNA in cycloheximide-treated cells and was the first to be detected on polysomes at 2 hr postinfection in untreated cells. A second, less abundant, transcript of 5.2 kb (670–733 map units) was also found on polysomes in cycloheximide-treated cells, and preliminary evidence suggested that this transcript may be spliced during processing. A 3.25-kb transcript (190–217 map units) was identified also as a minor polysome-associated species of RNA. One transcript of 4.8 kb (630–670) remained associated with the nucleus and was not processed into mRNA in cycloheximide-treated cells. Differential stability between the various transcripts was observed, the 2.2-kb transcript being the most stable. The results showed that in human cytomegalovirus-infected cells controls exist at the level of transcript accumulation, transport into the cytoplasm, preferential association with polysomes, and relative stability of RNAs.