Analysis of Splice Variants of the Immediate-Early 1 Region of Human Cytomegalovirus

Abstract

The major immediate-early (MIE) gene of human cytomegalovirus (HCMV) is a complex region consisting of a promoter, five exons, and two poly(A) signals (Fig. 1). Alternative splicing and differential usage of the two polyadenylation signals give rise to at least five reported transcripts (23, 26). The two major transcripts encode the most studied MIE proteins (MIEPs), immediate-early 72 (IE72; IE1, IE1 491aa , or ppUL123) and IE86 (IE2, IE2 579aa , or ppUL122a). These proteins have been extensively studied for their effects on the transcriptional activities of viral and cellular promoters and their role in controlling the temporal expression of the HCMV genes (2, 6-10, 12, 14-17, 21, 22, 25, 27, 29). They have also been shown to regulate cellular processes such as cell cycle control, metabolism, and apoptosis (13,28,30).In addition to the transcripts for IE72 and IE86, other MIE gene splice variants have been identified: IE19 (from the IE1 region) (20) and IE55 (IE2 425aa or ppUL122b) (1) and IE18 from the IE2 region (11). IE19 has been reported to function in synergy with IE72 to transcriptionally coactivate the HsOrc1 promoter (20). The functions of IE55 and IE18 have not been extensively studied; however, available data suggest that IE55 can suppress transcriptional activation mediated by IE72 and IE86 (5,12,15,25).While some of the alternative MIE gene transcripts give rise to well-characterized proteins, the products of others have not been well studied (23,26). In addition, it is quite likely that transcripts remain to be discovered, as few studies have been initiated using present technologies to carefully evaluate the variety of MIE transcripts.In this study, we used reverse transcriptase PCR (RT-PCR) to identify splice variants from the IE1 region. In addition to IE72 and the recently reported IE19 transcript (20), we detected and characterized two other heretofore-uncharacterized splice variants. Interestingly, each of the IE1 variants arises from utilization of alternative splice sites, not exon skipping. Based on the molecular mass of their predicted translation products, we named the two transcripts IE17.5 and IE9. Nuclease protection analyses demonstrated that the IE17.5 and IE9 transcripts are expressed with immediate-early kinetics during HCMV infection, similar to those of IE72 and IE19. Despite the presence of IE19, IE17.5, and IE9 transcripts, we did not detect their protein products during HCMV infection in infected human foreskin fibroblast (HFF), HEL, and U373MG cells. Our observation that IE19 is not detectable in HCMV-infected cells differs from a previous study of this protein (20).When cells were transfected with cDNAs encoding the IE1 transcripts, we readily detected protein expression of IE19 and IE17.5; however, we did not detect IE9. Expression of IE19 or IE17.5 in transfected cells was found to repress the MIE promoter and result in reduced levels of the MIEPs IE72 and IE86. In HCMV-infected U373MG cells transfected with IE19 cDNA, we also observed a reduction in the level of IE72...

Detection of human cytomegalovirus in medulloblastomas reveals a potential therapeutic target

“…The CH160 clone from Abcam recognizes both IE72 and IE86, while the Chemicon antibody recognizes a 68- to 72-kDa protein, which likely explains the different staining pattern of IE protein expression revealed by direct immunofluorescence and immunohistochemistry. We and others have noted that IE protein is expressed in both the cytoplasm and the nucleus in tumor cells, a pattern that may depend on different IE splicing variants encoded by the IE gene (43).…”

Multiple Transcripts Encode Full-Length Human Cytomegalovirus IE1 and IE2 Proteins during Lytic Infection

“…Several spliced and alternatively spliced transcripts are transcribed from the MIE locus (16)(17)(18)(19). Many of the alternative IE1 and IE2 mRNAs encode truncated versions of the IE1 and IE2 proteins that have distinct roles in regulating HCMV gene expression (18,19).…”

“…Several IE1 and IE2 splice variants have been identified. For example, IE1 transcript variants arise from alternative splice donor and acceptor usage in exons 3 and 4, respectively (16)(17)(18)(19). Similarly, IE2 transcript variants arise from alternative splicing within exon 5 (17).…”

Alteration of cellular RNA splicing and polyadenylation machineries during productive human cytomegalovirus infection

“…Alternatively spliced IE1 and IE2 RNAs appear earlier than UL37 spliced RNAs during HCMV infection of hTERT-RPEs. Whether the complexity of IE1/2 pre-mRNA processing (Awasthi et al, 2004) also increases when hypo-phosphorylated SF2 is decreased requires further studies.…”