Characterization of the major immediate-early polypeptides encoded by murine cytomegalovirus.

The immediate-early (IE) infected cell proteins induced by the murine cytomegalovirus (Smith strain) were studied. These polypeptides were identified as IE proteins by their synthesis in the presence of actinomycin D after removal from a protein synthesis block mediated by cycloheximide. By using a murine antiserum against murine cytomegalovirus, three abundant polypeptides of 89, 84, and 76 kilodaltons (kd) were immunoprecipitated. The three major proteins are phosphorylated but not glycosylated and share antigenic determinants recognized by monoclonal antibodies. The 84 and 76-kd polypeptides represent post-translational modification products of the 89-kd protein. Accordingly, in vitro translation of IE infected cell RNA revealed only the 89-kd polypeptide. The viral origin of the RNA species directing the synthesis of the major 89-kd IE polypeptide was verified by hybrid selection of IE RNA with DNA fragments representing the region from 0.769 to 0.815 map units of the murine cytomegalovirus genome. IE polypeptides were found to be located in the nuclei and the cytoplasm of infected cells. Studies on the kinetics of IE polypeptide synthesis revealed negative regulatory effects on IE gene expression correlated with the synthesis of early proteins.     

Human cytomegalovirus immediate-early two protein region involved in negative regulation of the major immediate-early promoter.

The immediate-early two (IE2) gene products of human cytomegalovirus negatively regulate gene expression from the major immediate-early promoter in permissive human fibroblasts. A mutational analysis of the IE2 proteins indicated that the carboxyl-terminal region is required for negative regulation. The IE2 proteins that lack amino acid residues 365 to 519, or the carboxyl-terminal amino acids failed to negatively regulate. Most of the amino-terminal portion of the IE2 protein was not required for negative regulation. A possible explanation of the negative effect on downstream expression by the IE2 proteins is discussed.     

Characterization of the bovine herpesvirus 4 major immediate-early transcript.

The major immediate-early (IE) RNA of bovine herpesvirus 4 (BHV-4) has been identified and characterized by analyzing cytoplasmic polyadenylated RNA isolated from Madin-Darby bovine kidney cells infected with BHV-4(DN-599) in the presence of cycloheximide. Hybridization of cDNA to Southern blots of viral DNA, Northern (RNA) blot analysis, and S1 nuclease analyses showed that the major BHV-4 IE RNA is a spliced, 1.7-kb RNA, which is transcribed from right to left on the restriction map of the BHV-4 genome from DNA contained in the 8.3-kb HindIII fragment E. The major IE RNA contains three small exons at its 5' end, spliced to a 1.3-kb 3' exon. This RNA is present in much-reduced amounts when cells are infected in the absence of cycloheximide. However, late in infection, the major IE RNA gene region encodes abundant RNAs which differ in structure from the major IE RNA. Nucleotide sequence analysis of the gene encoding the major IE RNA revealed an open reading frame encoding 284 amino acids. A homology search of amino acid sequence data bases showed that a 141-amino-acid region near the amino terminus of the predicted amino acid sequence is similar to sequences near the amino terminus of herpes simplex virus type 1 IE110. This region of homology includes CXXC pairs, which could be involved in zinc finger structures. The region encoding this putative zinc finger domain is also found in RNAs transcribed from this IE region late in infection, but it is spliced to different sequences than those used in IE RNA. Thus, the major IE region of the BHV-4 genome could encode a family of proteins sharing a zinc finger domain.     

Alternative splicing of the human cytomegalovirus major immediate-early genes affects infectious-virus replication and control of cellular cyclin-dependent kinase

The major immediate-early (MIE) gene locus of human cytomegalovirus (HCMV) is the master switch that determines the outcomes of both lytic and latent infections. Here, we provide evidence that alteration in the splicing of HCMV (Towne strain) MIE genes affects infectious-virus replication, movement through the cell cycle, and cyclin-dependent kinase activity. Mutation of a conserved 24-nucleotide region in MIE exon 4 increased the abundance of IE1-p38 mRNA and decreased the abundance of IE1-p72 and IE2-p86 mRNAs. An increase in IE1-p38 protein was accompanied by a slight decrease in IE1-p72 protein and a significant decrease in IE2-p86 protein. The mutant virus had growth defects, which could not be complemented by wild-type IE1-p72 protein in trans. The phenotype of the mutant virus could not be explained by an increase in IE1-p38 protein, but prevention of the alternate splice returned the recombinant virus to the wild-type phenotype. The lower levels of IE1-p72 and IE2-p86 proteins correlated with a delay in early and late viral gene expression and movement into the S phase of the cell cycle. Mutant virus-infected cells had significantly higher levels of cdk-1 expression and enzymatic activity than cells infected with wild-type virus. The mutant virus induced a round-cell phenotype that accumulated in the G(2)/M compartment of the cell cycle with condensation and fragmentation of the chromatin. An inhibitor of viral DNA synthesis increased the round-cell phenotype. The round cells were characteristic of an abortive viral infection.     

Isolation and characterization of a low-abundance splice variant from the human cytomegalovirus major immediate-early gene region.

The major immediate-early (IE) gene region of human cytomegalovirus (HCMV) encodes several proteins as a result of differential RNA splicing events. By expression vector cloning of HCMV IE mRNA, we isolated and characterized a cDNA for a novel splice variant from the major IE gene region. The RNA product is a derivative of the IE55 mRNA and contains an additional splice from nucleotides 170,635 to 170,307 in the IE2 gene region (UL122), resulting in a 1.4-kb mRNA. The predicted open reading frame codes for a 164-amino-acid protein with a calculated molecular mass of 18 kDa (IE18). Mung bean nuclease analysis and PCR were used to characterize expression of IE18 mRNA in HCMV-infected cells. While the 1.4-kb mRNA was detected in infected human fibroblasts in the presence of a protein synthesis inhibitor, it was not detectable during a normal infection. However, the 1.4-kb mRNA was readily detected in infected human monocyte-derived macrophages at IE times. These results suggest that the novel IE18 mRNA exhibits cell type-specific expression indicating differential regulation of the major IE gene region in different permissive cell types.     

The human cytomegalovirus major immediate-early distal enhancer region is required for efficient viral replication and immediate-early gene expression

The human cytomegalovirus (HCMV) major immediate-early (MIE) genes, encoding IE1 p72 and IE2 p86, are activated by a complex enhancer region (base positions -65 to -550) that operates in a cell type- and differentiation-dependent manner. The expression of MIE genes is required for HCMV replication. Previous studies analyzing functions of MIE promoter-enhancer segments suggest that the distal enhancer region variably modifies MIE promoter activity, depending on cell type, stimuli, or state of differentiation. To further understand the mechanism by which the MIE promoter is regulated, we constructed and analyzed several different recombinant HCMVs that lack the distal enhancer region (-300 to -582, -640, or -1108). In human fibroblasts, the HCMVs without the distal enhancer replicate normally at high multiplicity of infection (MOI) but replicate poorly at low MOI in comparison to wild-type virus (WT) or HCMVs that lack the neighboring upstream unique region and modulator (-582 or -640 to -1108). The growth aberrancy was normalized after restoring the distal enhancer in a virus lacking this region. For HCMVs without a distal enhancer, the impairment in replication at low MOI corresponds to a deficiency in production of MIE RNAs compared to WT or virus lacking the unique region and modulator. An underproduction of viral US3 RNA was also evident at low MOI. Whether lower production of IE1 p72 and IE2 p86 causes a reduction in expression of the immediate-early (IE) class US3 gene remains to be determined. We conclude that the MIE distal enhancer region possesses a mechanism for augmenting viral IE gene expression and genome replication at low MOI, but this regulatory function is unnecessary at high MOI.     

The enhancer domain of the human cytomegalovirus major immediate-early promoter determines cell type-specific expression in transgenic mice.

The human cytomegalovirus (HCMV) major immediate-early promoter (MIEP) is one of the first promoters to activate upon infection. To examine HCMV MIEP tissue-specific expression, transgenic mice were established containing the lacZ gene regulated by the MIEP (nucleotides -670 to +54). In the transgenic mice, lacZ expression was demonstrated in 19 of 29 tissues tested by histochemical and immunochemical analyses. These tissues included brain, eye, spinal cord, esophagus, stomach, pancreas, kidney, bladder, testis, ovary, spleen, salivary gland, thymus, bone marrow, skin, cartilage, and cardiac, striated and smooth muscles. Although expression was observed in multiple organs, promoter activity was restricted to specific cell types. The cell types which demonstrated HCMV MIEP expression included retinal cells of the eye, ductile cells of the salivary gland, exocrine cells of the pancreas, mucosal cells of the stomach and intestine, neuronal cells of the brain, muscle fibers, thecal cells of the corpus luteum, and Leydig and sperm cells of the testis. These observations indicate that the HCMV MIEP is not a pan-specific promoter and that the majority of expressing tissues correlate with tissues naturally infected by the virus in the human host.