Cytomegalovirus UL91 Is Essential for Transcription of Viral True Late (γ2) Genes

119

During their productive cycle, herpesviruses exhibit a strictly regulated temporal cascade of gene expression that has three general stages: immediate early (IE), early (E), and late (L). Promoter complexity differs strikingly between IE/E genes and L genes. IE and E promoters contain cis-regulating sequences upstream of a TATA box, whereas L promoters comprise a unique cis element. In the case of the gammaherpesviruses, this element is usually a TATT motif found in the position where the consensus TATA box of eukaryotic promoters is typically found. Epstein-Barr virus (EBV) encodes a protein, called BcRF1, which has structural homology with the TATA-binding protein and interacts specifically with the TATT box. However, although necessary for the expression of the L genes, BcRF1 is not sufficient, suggesting that other viral proteins are also required. Here, we present the identification and characterization of a viral protein complex necessary and sufficient for the expression of the late viral genes. This viral complex is composed of five different proteins in addition to BcRF1 and interacts with cellular RNA polymerase II. During the viral productive cycle, this complex, which we call the vPIC (for viral preinitiation complex), works in concert with the viral DNA replication machinery to activate expression of the late viral genes. The EBV vPIC components have homologs in beta-and gammaherpesviruses but not in alphaherpesviruses. Our results not only reveal that beta-and gammaherpesviruses encode their own transcription preinitiation complex responsible for the expression of the late viral genes but also indicate the close evolutionary history of these viruses. IMPORTANCEControl of late gene transcription in DNA viruses is a major unsolved question in virology. In eukaryotes, the first step in transcriptional activation is the formation of a permissive chromatin, which allows assembly of the preinitiation complex (PIC) at the core promoter. Fixation of the TATA box-binding protein (TBP) is a key rate-limiting step in this process. This study provides evidence that EBV encodes a complex composed of six proteins necessary for the expression of the late viral genes. This complex is formed around a viral TBP-like protein and interacts with cellular RNA polymerase II, suggesting that it is directly involved in the assembly of a virus-specific PIC (vPIC). Herpesviruses are enveloped viruses containing relatively large, double-stranded DNA genomes. They are divided into three subfamilies (alpha-, beta-, and gammaherpesviruses) according to sequence homology, cellular tropism, and productive cycle behavior under laboratory culture conditions. Nine herpesviruses have been identified in humans. Herpes simplex virus 1 (HSV-1) and 2 (HSV-2) and varicella-zoster virus (VZV) are alphaherpesviruses that due to neurotropism cause recurrent skin lesions, meningitis, and rare but very serious encephalitis in the case of HSV-1; human cytomegalovirus (HCMV), human herpesviruses 6A and 6B (HHV-6A and HHV-6B), and human he...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Identification Of Viral Proteins Required For Late Gene Exprmentioning

confidence: 99%

See 1 more Smart Citation

Epstein-Barr Virus Late Gene Transcription Depends on the Assembly of a Virus-Specific Preinitiation Complex

Aubry

Mure

Mariamé

et al. 2014

119

“…These five viral ORFs play an important role in activating viral late gene promoters, and it has also been shown that ORF30 and -34 are critical for recruiting RNA polymerase II (Pol II). More recently, studies in cytomegalovirus (CMV), a betaherpesvirus, have demonstrated that the UL79, -87, -91, -92, and -95 genes, homologous to MHV-68 ORF18, -24, -30, -31, and -34, respectively, are also essential for viral late gene expression (18)(19)(20)(21)(22). The evidence strongly suggests that beta-and gammaherpesviruses share a similar mechanism to regulate late gene expression.…”

mentioning

confidence: 94%

Kaposi's Sarcoma-Associated Herpesvirus ORF18 and ORF30 Are Essential for Late Gene Expression during Lytic Replication

Gong

Xie

et al. 2014

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several human malignances. As saliva is likely the major vehicle for KSHV transmission, we studied in vitro KSHV infection of oral epithelial cells. Through infection of two types of oral epithelial cells, normal human oral keratinocytes (NHOKs) and papilloma-immortalized human oral keratinocyte (HOK16B) cells, we found that KSHV can undergo robust lytic replication in oral epithelial cells. By employing de novo lytic infection of HOK16B cells, we studied the functions of two previously uncharacterized genes, ORF18 and ORF30, during the KSHV lytic cycle. For this purpose, an ORF18-deficient virus and an ORF30-deficient virus were generated using a mutagenesis strategy based on bacterial artificial chromosome (BAC) technology. We found that neither ORF18 nor ORF30 is required for immediately early or early gene expression or viral DNA replication, but each is essential for late gene expression during both de novo lytic replication and reactivation. This critical role of ORF18 and ORF30 in late gene expression was also observed during KSHV reactivation. In addition, global analysis of viral transcripts by RNA sequencing indicated that ORF18 and ORF30 control the same set of viral genes. Therefore, we suggest that these two viral ORFs are involved in the same mechanism or pathway that coregulates the viral late genes as a group. IMPORTANCEWhile KSHV can infect multiple cell types in vitro, only a few can support a full lytic replication cycle with progeny virions produced. Consequently, KSHV lytic replication is mostly studied through reactivation, which requires chemicals to induce the lytic cycle or overexpression of the viral transcriptional activator, RTA. In this study, we present a robust de novo lytic infection system based on oral epithelial cells. Using this system, we demonstrate the role of two viral ORFs, ORF18 and ORF30, in regulating viral gene expression during KSHV lytic replication. As the major route of KSHV transmission is thought to be via saliva, this new KSHV lytic replication system will have important utility in the field.

“…Precisely how these proteins coordinate to direct late gene expression, including whether they interact with the late gene promoter, remains under investigation. Interestingly, studies on human cytomegalovirus (HCMV), a betaherpesvirus, have shown that UL79, UL87, UL91, UL92, and UL95 (the homologues of ORF18, ORF24, ORF30, ORF31, and ORF34, respectively) are also essential for late gene transcription (13)(14)(15)(16).…”

mentioning

confidence: 99%

Unconventional Sequence Requirement for Viral Late Gene Core Promoters of Murine Gammaherpesvirus 68

Wong-Ho

Davis

et al. 2014

f Infection with the human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), is associated with several cancers. During lytic replication of herpesviruses, viral genes are expressed in an ordered cascade. However, the mechanism by which late gene expression is regulated has not been well characterized in gammaherpesviruses. In this study, we have investigated the cis element that mediates late gene expression during de novo lytic infection with murine gammaherpesvirus 68 (MHV-68). A reporter system was established and used to assess the activity of viral late gene promoters upon infection with MHV-68. It was found that the viral origin of lytic replication, orilyt, must be on the reporter plasmid to support activation of the late gene promoter. Furthermore, the DNA sequence required for the activation of late gene promoters was mapped to a core element containing a distinct TATT box and its neighboring sequences. The critical nucleotides of the TATT box region were determined by systematic mutagenesis in the reporter system, and the significance of these nucleotides was confirmed in the context of the viral genome. In addition, EBV and KSHV late gene core promoters could be activated by MHV-68 lytic replication, indicating that the mechanisms controlling late gene expression are conserved among gammaherpesviruses. Therefore, our results on MHV-68 establish a solid foundation for mechanistic studies of late gene regulation.