Small Internal Deletions in the Human Cytomegalovirus IE2 Gene Result in Nonviable Recombinant Viruses with Differential Defects in Viral Gene Expression

Abstract

Human cytomegalovirus (HCMV), a betaherpesvirus, has a double-stranded DNA genome of approximately 230 kbp that encodes at least 150 open reading frames (17). Infection with HCMV has serious consequences for immunocompromised patients and is the leading viral cause of birth defects (for a review, see reference 48). HCMV gene expression is separated into three temporal categories (for a review, see reference 46). Immediate-early (IE) genes are the first to be activated and do not require de novo host or viral protein synthesis for their expression. Early genes represent a broadly defined class whose transcription is typically regulated by the interaction of IE gene products with cellular factors. Viral DNA replication follows early gene expression. Finally, late viral genes, many of which encode structural proteins, are expressed.Since IE gene expression begins this cascade of events, the regulation and products of IE genes have been studied extensively. The major immediate-early (MIE) gene, made up of open reading frames UL122 and UL123, is a region of particular interest. It consists of five exons that are transcribed, differentially spliced, and translated to give two predominant products: the IE1 72-kDa protein (exons 1 to 4) and the IE2 86-kDa protein (exons 1 to 3 and 5). The translation of each transcript initiates in exon 2, and the two proteins share 85 amino acids (aa) at their amino termini (66-68; reviewed in reference 20). The IE2 region also encodes an additional product that is expressed later in the infection and a splice variant that is present in infected human monocyte-derived macrophages (32,36,53,64). IE1 72 is the more abundant product at both the mRNA and protein levels and has modest transactivating effects, including the ability to transactivate the MIE promoter (MIEP) (for reviews, see references 20 and 46). Numerous in vitro and in vivo studies have shown that IE2 86 is a strong transactivator and also represses its own promoter (13,14,29,40,50,52,65). Other IE genes include IRS1, TRS1, the UL36 to -38 genes, and US3. Many of these, including US3, UL36, and IRS1, are not required for HCMV replication in cultured cells (8,10,33,49). A virus lacking TRS1 exhibits normal gene expression during IE and early times postinfection but is defective in late stages of replication (8). An IE1 mutant virus is viable but shows growth defects during a lowmultiplicity infection (22,24,47). In contrast, the failure of a virus lacking most of the IE2 gene to support early gene expression and to replicate indicates that IE2 86 is essential for productive infection (44).Significant efforts have been directed towards defining the elements that provide IE2 86 with its strong regulatory capabilities, and IE2 86 is thought to transactivate and repress via protein-protein and protein-DNA interactions. IE2 86 binds to the product of the viral UL84 gene and to multiple cellular proteins. These host factors include components of the basal transcription complex TBP and TFIIB, numerous cellular transcription factors...

Granzyme M targets host cell hnRNP K that is essential for human cytomegalovirus replication

“…In view of the importance of IE2 as an absolutely indispensable protein in HCMV replication, 26,27 we further examined the mechanism by which hnRNP K regulates IE2 protein levels. First, we investigated the transcriptional activity of hnRNP K towards the MIEP, which regulates IE2 gene (UL122) expression.…”

“…IE2 is absolutely indispensable for HCMV replication. 26,27 Therefore, we explored how hnRNP K regulates IE2 protein levels. As a multifunctional protein, hnRNP K is involved in many biological processes, including transcription, mRNA splicing, translation, and protein signaling.…”

Identification and function of human cytomegalovirus microRNAs

“…The most abundant of these proteins are the major trans-activators IE72 and IE86, which are expressed from 5 exons within the major immediate early region (figure 2) (Stenberg et al, 1989;Stenberg et al, 1984;Stenberg et al, 1985). Both proteins are thought to be important in promoting early and late viral transcription, although only IE86 is essential for virus replication (Greaves and Mocarski, 1998;Mocarski et al, 1996;Stenberg et al, 1990;White et al, 2004;White et al, 2007). Disruption of IE72 expression does result in a significant attenuation of viral replication following low multiplicity infections (Mocarski et al, 1996).…”

Proteasome Subunits Relocalize during Human Cytomegalovirus Infection, and Proteasome Activity Is Necessary for Efficient Viral Gene Transcription

“…Quantitative real-time PCR (qPCR) was performed with the ABI Prism 7000 sequence detection system (Applied Biosystems) using the TaqMan Universal PCR master mix (Applied Biosystems) along with oligonucleotide primers and TaqMan dually labeled (5Ј-6-carboxyfluorescein and 3Ј-black hole quencher-1) probes (Integrated DNA Technologies) with 40 ng DNA per reaction mixture. Probes were targeted to an unspliced region of HCMV UL77, IE2 (UL122), and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, which was used for normalization of input DNA, as previously described (60). Standard curves were generated using DNA isolated from cells harvested at 48 h p.i.…”