Murine Cytomegalovirus Protein pM92 Is a Conserved Regulator of Viral Late Gene Expression

117

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: Discussionsupporting

confidence: 81%

Section: Discussionmentioning

confidence: 80%

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

117

“…While WT and revertant iSLK cell lines yielded readily detectable infectious virus, iSLK-31S cells failed to yield detectable progeny virus (Fig. 1B), consistent with the described role of its orthologs in other herpesviruses (7,13,16). Immunoblot analysis showed that, compared to WT and revertant viruses, ORF31-deficient virus was specifically defective in the expression of the late gene ORF8 but not in the expression of latent (LANA) or E (K3) genes or in replication of viral DNA (Fig.…”

supporting

confidence: 83%

Association of Kaposi's Sarcoma-Associated Herpesvirus ORF31 with ORF34 and ORF24 Is Critical for Late Gene Expression

Brulois

Wong

Lee

et al. 2015

bTranscription of herpesvirus late genes depends on several virus-encoded proteins whose function is not completely understood. Here, we identify a viral trimeric complex of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 31 (ORF31), ORF24, and ORF34 that is required for late gene expression but not viral DNA replication. We found that (i) ORF34 bridges the interaction between ORF31 and ORF24, (ii) the amino-terminal cysteine-rich and carboxyl-terminal basic domains of ORF31 mediate the ORF31-ORF34 interaction required for late gene expression, and (iii) a complex consisting of ORF24, ORF31, and ORF34 specifically binds to the K8.1 late promoter. Together, our results support the model that a subset of lytic viral proteins assembles into a transcriptional activator complex to induce expression of late genes. Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) belongs to the gammaherpesvirus subfamily, which also includes Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV-68). KSHV is etiologically linked to the AIDS-associated malignancy Kaposi's sarcoma, as well as two rare lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD) (1). During the latent phase of infection, the KSHV genome is maintained as a circular episome in the nucleus (2), where only a few genes are transcribed as part of the latency program (3, 4). The lytic phase begins with the induction of immediate early (IE) genes, which in turn promotes the expression of early (E) genes (5). The transcription of late genes depends on lytic replication of viral DNA, a process carried out by the core DNA replication machinery composed of KSHV-encoded proteins (6). Chemical inhibitors of the herpesvirus-encoded DNA polymerase (e.g., phosphonoacetic acid [PAA]) efficiently prevent expression of late genes but do not affect genes of the other kinetic classes (5).Several studies have begun to shed light on the regulation of late gene expression in beta-and gammaherpesviruses. Genetic studies of MHV-68 and EBV identified six gene products (open reading frame 18 [ORF18], -24, -30, -31, -34, and -66 in the case of KSHV), each of which is required for the transcription of late genes but dispensable for viral DNA replication and the expression of IE and E genes (7-11). Many of these evolutionarily conserved genes of the beta-and gammaherpesviruses (␤-␥ genes) have since been shown to have similar effects on the gene expression of murine cytomegalovirus (MCMV) (12, 13), human CMV (HCMV) (14-16), and KSHV (17, 18). Of note, none of these six ␤-␥ genes are found in the alphaherpesvirus subfamily, suggesting that a mechanistically distinct process controls late gene expression in the alpha subfamily. Recent studies suggest that the ␤-␥ gene products assemble into a viral preinitiation complex (vPIC) that also includes RNA polymerase II (RNAPII) (11,18). One of the vPIC components is ORF24, which was identified as a viral protein distantly related to the cellular TATA ...

“…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: 99%

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.