The human cytomegalovirus (HCMV) IE2 86-kDa protein is an essential transactivator of viral and cellular gene expression. Additional proteins of 60 and 40 kDa are expressed from the IE2 gene at late times postinfection and are identical to the C terminus of IE2 86. We have constructed HCMV recombinants that express wild-type full-length IE2 86 but do not express the IE2 40-and 60-kDa proteins. Each of these recombinants is viable, indicating that neither the 60-kDa nor the 40-kDa protein is required for virus replication, either alone or in combination. Cells infected with the IE2 60 and IE2 40 deletion mutants, however, exhibit decreased expression of selected viral genes at late times. In particular, expression of the viral DNA replication factor UL84 is affected by the deletion of IE2 40, and expression of the tegument protein pp65 (ppUL83) is affected by the deletion of both IE2 40 and IE2 60. IE2 60 and IE2 40 are also required for the production of normal levels of infectious virus. Finally, IE2 40 appears to function as a repressor of major immediate-early transcription in the infected cell. These results begin to define functions for the IE2 60-and IE2 40-kDa proteins and indicate that these products contribute both to the expression of selected viral genes and to the overall progression of the infection.Human cytomegalovirus (HCMV) gene expression, like that of all herpesviruses, occurs as a tightly controlled series of events beginning with expression of the immediate-early (IE) genes. These go on to activate expression of early viral genes, allowing replication of the viral genome and subsequent transcription of late, primarily structural, genes. Two important IE genes, the UL122 and UL123 genes, comprise the major IE region. This segment of the HCMV genome encodes two predominant products, the IE1 72-kDa (ppUL123) and IE2 86-kDa (ppUL122) proteins. These are expressed from alternatively spliced forms of a five-exon transcript; the IE1 72 mRNA consists of exons 1 to 4, and the IE2 86 mRNA contains exons 1 to 3 and 5. Translation of both proteins begins in exon 2, so that IE1 72 and IE2 86 have identical 85-amino-acid (aa) N termini and unique C-terminal domains. Both proteins have been characterized extensively, most recently in studies that have used recombinant viruses containing deletions in the major IE region to elucidate the functions of IE1 72 and IE2 86 in the HCMV-infected cell. These studies indicate that the IE1 72-kDa protein contributes to virus replication during lowmultiplicity, but not high-multiplicity, infections and is therefore nonessential (10,11,28). In contrast, IE2 86 is strictly required for HCMV replication, and even small deletions or changes to the sequence of the protein can result in a virus that does not replicate (26,46). Both IE1 72 and IE2 86 are transcriptional activators, and their ability to promote viral gene expression has been studied in a number of transient-transfection assays. IE2 86-mediated transactivation of the 1.2-and 2.7-kb RNA and UL112-113 (2.2-kb RNA...

The major immediate-early (IE) region of human cytomegalovirus encodes two IE proteins, IE1 72 and IE2 86, that are translated from alternatively spliced transcripts that differ in their 3 ends. Two other proteins that correspond to the C-terminal region of IE2 86, IE2 60 and IE2 40, are expressed at late times. In this study, we used IE2 mutant viruses to examine the mechanism by which IE2 86, IE2 60, and IE2 40 affect the expression of a viral DNA replication factor, UL84. Deletion of amino acids (aa) 136 to 290 of IE2 86 results in a significant decrease in UL84 protein during the infection. This loss of UL84 is both proteasome and calpain independent, and the stability of the protein in the context of infection with the mutant remains unaffected. The RNA for UL84 is expressed to normal levels in the mutant virus-infected cells, as are the RNAs for two other proteins encoded by this region, UL85 and UL86. Moreover, nuclear-to-cytoplasmic transport and the distribution of the UL84 mRNA on polysomes are unaffected. A region between aa 290 and 369 of IE2 86 contributes to the UL84-IE2 86 interaction in vivo and in vitro. IE2 86, IE2 60, and IE2 40 are each able to interact with UL84 in the mutant-infected cells, suggesting that these interactions may be important for the roles of UL84 and the IE2 proteins. Thus, these data have defined the contribution of IE2 86, IE2 60, and IE2 40 to the efficient expression of UL84 throughout the infection.Human cytomegalovirus (HCMV) is the major viral cause of birth defects and poses a severe threat to immunocompromised and transplant patients (for review, see reference 33). Gene expression has been classified into three major groups, referred to as the immediate-early (IE), early, and late genes, which are temporally regulated throughout the infection. The two major IE (MIE) genes, IE1 72 and IE2 86 (encoded by UL122 and UL123), are of particular interest for understanding the various regulatory mechanisms that govern a productive viral infection. They can transactivate viral early promoters, serve in viral promoter repression, and alter the expression of many host cellular genes in order to make the environment favorable for viral replication (for reviews, see references 14 and 33). Both MIE proteins arise from a single transcript that consists of five exons that are differentially spliced to produce IE1 72 (exons 1 to 4) and IE2 86 (exons 1 to 3 and 5) (54-56). While IE1 72 is dispensable for infection at a high multiplicity of infection (MOI), IE2 86 is essential (16,18,31,32,36,37,59).At late times in infection, transcripts that arise from within exon 5 of the UL122 gene encode the late IE2 60 and IE2 40 proteins, which correspond to the C-terminal region of IE2 86 (41). The IE2 60 protein is expressed from an initiator methionine at amino acid (aa) 170, with the putative TATAA region occurring in the intron between exons 4 and 5. The IE2 40 protein is expressed from a 1.5-kb RNA, with translation initiating at methionine 242, and a putative TATAA box just upstream of t...