Regulation of cytomegalovirus gene expression: alpha and beta promoters are trans activated by viral functions in permissive human fibroblasts.

We have fused immediate (alpha) and delayed (beta) early promoter-regulatory sequences taken from the cytomegalovirus (CMV) genome to Escherichia coli lacZ (beta-galactosidase) as an indicator gene to study regulated expression of these promoters. After transfection of human fibroblast cells with plasmid constructs carrying beta-galactosidase fusions, and subsequent infection with CMV, we have demonstrated that viral trans-acting functions up-regulate the expression of these genes in a temporally authentic manner. The alpha promoter is activated even when de novo protein synthesis is blocked and when UV-inactivated virus is used, suggesting that, as for herpes simplex virus type 1 (HSV-1), a virion structural protein is responsible for its up-regulation. We have found that HSV-1, as well as CMV, is capable of trans activating the CMV alpha promoter. The beta promoter is activated by CMV but is completely unresponsive to HSV-1 infection. The temporal synthesis of the alpha and beta promoters in the transient expression system conforms with their natural regulation during viral replication. The beta-galactosidase fusions we describe provide a most exquisitely sensitive indicator system for the study of cis- and trans-acting viral regulatory functions.     

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.     

Identification and characterization of the human cytomegalovirus immediate-early region 2 gene that stimulates gene expression from an inducible promoter.

The human cytomegalovirus (HCMV) XbaI E cloned DNA fragment of approximately 20 kilobases can complement an adenovirus mutant (dl312) defective in the E1a viral gene product (D. J. Spector and M. J. Tevethia, Virology 151:329-338, 1986). This viral DNA fragment contains three immediate-early (IE) genes between 0.709 and 0.751 map units (M. F. Stinski, D. R. Thomsen, R. M. Stenberg, and L. C. Goldstein, J. Virol. 46:1-14, 1983). Two of the IE genes, IE1 and IE2, were isolated and tested for a role in regulating viral gene expression. Since HCMV early and late promoters require additional characterization, the chloramphenicol acetyl transferase (cat) gene, driven by the adenovirus E2 promoter, was used as an indicator of gene expression. cat expression from this heterologous viral promoter was shown to be stimulated by HCMV at early times after infection. The IE1 gene product did not function independently in activating this promoter. The IE2 gene products could independently stimulate the expression of a plasmid of a plasmid when the cat gene was placed downstream of the inducible E2 promoter (E2CAT). Five proteins of different sizes have been predicted to originate from IE2, depending on mRNA splicing. The protein products specified by the IE2 gene were characterized with an antibody to a synthetic peptide according to the open reading frame of exon 2. Three of the five proteins are encoded by exon 2. Three viral proteins of 82, 54, and 28 kilodaltons (kDa) were detected. The exons contained in the region designated as IE2a have open reading frames that could code for two of the smaller proteins of 27 and 30 kDa. This region, when driven by the HCMV enhancer, could independently stimulate gene expression from E2CAT to a high level. A plasmid with the HCMV enhancer upstream of exons, that could code for the HCMV IE2 proteins of 48 and 51 kDa, as well as 27- and 30-kDa proteins, also stimulated E2CAT expression but at a lower level. The activity of this plasmid was augmented by the IE1 gene product, despite the fact that the latter gene product alone was inactive. It is proposed that the HCMV IE region 2 gene products are involved in the regulation of viral or host cell promoters either independently or in combination with other HCMV IE proteins.