An isoform variant of the cytomegalovirus immediate-early auto repressor functions as a transcriptional activator

Baracchini, E; Glezer, Emilia; Fish, Kenneth N.; Stenberg, R M; Nelson, Jay A.; Ghazal, Peter

doi:10.1016/0042-6822(92)90506-k

Cited by 46 publications

(52 citation statements)

References 51 publications

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“…It must be noted, however, that a second, 55-kDa IE-2 polypeptide is also substantially encoded by UL122 sequences (37). Very little is known about this minor IE-2 species; however, Baracchini et al have demonstrated transactivation by this protein (3). Since the T-BAC⌬122 deletion is also expected to affect the 55-kDa species, we can not presently ascribe the observed phenotype unambiguously to the 86-kDa IE-2.…”

Section: Discussionmentioning

confidence: 57%

Human Cytomegalovirus with IE-2 (UL122) Deleted Fails To Express Early Lytic Genes

Marchini

Hai-qing

Zhu

2001

J Virol

313

324

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Much evidence suggests that the major immediate-early (IE) transactivator of human cytomegalovirus (HCMV), IE-2, is likely to be critical for efficient viral replication; however, the lack of an IE-2 mutant HCMV has precluded an experimental test of this hypothesis. As an initial step toward characterizing an IE-2 mutant, we first cloned the HCMV Towne genome as a bacterial artificial chromosome (BAC) and analyzed the ability of transfected Towne-BAC DNA (T-BACwt) to produce plaques following introduction into permissive human fibroblasts. Like Towne viral DNA, transfected T-BACwt DNA was infectious in permissive cells, and the resulting virus stocks were indistinguishable from Towne virus. We then used homologous recombination in Escherichia coli to delete the majority of UL122, the open reading frame encoding the unique portion of IE-2, from T-BACwt. From this deleted BAC, a third BAC clone in which the deletion was repaired with wild-type UL122 was created. In numerous transfections of permissive human foreskin fibroblast cells with these three BAC DNA clones, the rescued BAC and T-BACwt consistently yielded plaques, while the UL122 mutant BAC never generated plaques, even after 4 weeks. Protein and mRNA of other IE genes were readily detected from transfected UL122 mutant BAC DNA; however, reverse transcription-PCR failed to detect mRNA expression from any of five early genes examined. The generalized failure of this mutant to express early genes is consistent with expectations from in vitro assays which have demonstrated that IE-2 transactivates most HCMV promoters. These experiments provide the first direct demonstration that IE-2 is required for successful HCMV infection and indicate that virus lacking IE-2 arrests early in the replication cycle.

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Section: Discussionmentioning

confidence: 57%

Human Cytomegalovirus with IE-2 (UL122) Deleted Fails To Express Early Lytic Genes

Marchini

Hai-qing

Zhu

2001

J Virol

313

324

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“…Precedence for low expression of MIEP variants comes from IE55 (Fig. 1), which is expressed at such low levels in HCMV-infected cells that detection requires cycloheximide treatment and removal (1). We have performed similar cycloheximide experiments aimed at detecting IE19, IE17.5, and IE9 but have found none (data not shown).…”

Section: Discussionmentioning

confidence: 99%

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

Awasthi

Isler

Alwine

2004

J Virol

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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...

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“…Pizzorno et al (62) reported that this protein is present in infected cells only after protein synthesis inhibitors are removed, and thus it is possible that it does not play a physiological role in the viral infection but is an artifact of the experimental conditions. Although there are two reports that this protein can activate the HCMV major IE promoter, the human immunodeficiency virus long terminal repeat, and the SV40 promoter to a low level (3,23) Crude Drosophila embryo extracts with and without histone Hi have also been used for in vitro transcription assays with a variety of eukaryotic promoters (27,36,45). In our experiments with Drosophila extracts in the absence of histone Hi, the basal activity of the HCMV early promoter was much higher than that seen in the U373MG or HeLa cell nuclear extracts.…”

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confidence: 99%

In vivo and in vitro analysis of transcriptional activation mediated by the human cytomegalovirus major immediate-early proteins.

et al. 1993

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To define mechanistically how the human cytomegalovirus (HCMV) major immediate-early (IE) Human cytomegalovirus (HCMV), a member of the herpesvirus family, is an important pathogen implicated in a variety of diseases in newborn and immunocompromised individuals (for reviews, see references 2 and 59). Like other members of the herpesvirus family, the genome of HCMV is temporally expressed during the viral life cycle (16, 56,81,88,89 (16,17,29,33,34, 56,64,79,80,82,88,89,91). At least three IE RNAs have been found to be transcribed from these regions (Fig. 1B) (62,66,77,80).In previous studies, we and others have used transientexpression assays to demonstrate that HCMV early promoters as well as heterologous viral and cellular promoters can be activated by the region of the genome specifying the IE1 and IE2 gene products (4,5,9,12,15,18,20,23,26,29,39,53,60,63,67,75,85,87). Recent studies suggest that the IE2 86-kDa protein plays a major role in activating HCMV early promoters as well as in repressing its own promoter (the major IE promoter), while the IE1 72-kDa protein acts to enhance the activity of the major IE promoter and may augment the stimulatory effect of the IE2 86-kDa protein (10-12, 26, 28, 50, 53, 61-63, 69, 78).Any consideration of the mechanism by which the HCMV IE1 and IE2 proteins affect transcription must take into account the fact that multiple viral and cellular promoters 1238 on April 27, 2019 by guest

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An isoform variant of the cytomegalovirus immediate-early auto repressor functions as a transcriptional activator

Cited by 46 publications

References 51 publications

Human Cytomegalovirus with IE-2 (UL122) Deleted Fails To Express Early Lytic Genes

Human Cytomegalovirus with IE-2 (UL122) Deleted Fails To Express Early Lytic Genes

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

In vivo and in vitro analysis of transcriptional activation mediated by the human cytomegalovirus major immediate-early proteins.

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