Transcription of True Late (γ2) Cytomegalovirus Genes Requires UL92 Function That Is Conserved among Beta- and Gammaherpesviruses

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: Identification Of Viral Proteins Required For Late Gene Exprmentioning

confidence: 99%

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

Aubry

Mure

Mariamé

et al. 2014

117

“…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.

“…EBV BAC DNAs were digested with BamHI or EcoRI and separated in an agarose gel. 5. Immunoprecipitation (IP) was carried out using anti-Flag antibody, and the results were detected by Western blotting using anti-Myc, anti-HA, and anti-Flag antibodies.…”

Section: Acknowledgmentsmentioning

confidence: 99%

The Epstein-Barr Virus BDLF4 Gene Is Required for Efficient Expression of Viral Late Lytic Genes

Watanabe

Narita

Yoshida

et al. 2015

Epstein-Barr virus (EBV) is a gammaherpesvirus, associated with infectious mononucleosis and various types of malignancy. We focused here on the BDLF4 gene of EBV and identified it as a lytic gene, expressed with early kinetics. Viral late gene expression of the BDLF4 knockout strain was severely restricted; this could be restored by an exogenous supply of BDLF4. These results indicate that BDLF4 is important for the EBV lytic replication cycle, especially in late gene expression. Epstein-Barr virus (EBV) is a human gammaherpesvirus that is present in Ͼ90% the world's population. EBV is associated with infectious mononucleosis and neoplasms such as Burkitt lymphoma, Hodgkin's lymphoma, posttransplant lymphoproliferative disease, nasopharyngeal carcinoma, and gastric carcinoma (1, 2).The EBV BDLF4 gene is an open reading frame (ORF), located between BGLF1 and BDLF3.5. Sequence similarities suggest that it is conserved among the members of the betaherpesvirus and gammaherpesvirus subfamilies (UL92 of human cytomegalovirus [HCMV] and ORF31 of murine gammaherpesvirus 68 and Kaposi's sarcoma-associated herpesvirus [KSHV]) but that it is not present in the alphaherpesviruses. It has been reported that KSHV ORF31, MHV-68 ORF31, HCMV UL92, and MCMV M92 are required for the efficient transcription of viral true late genes (3-7), but the EBV BDLF4 has not been characterized well.BDLF4 expression in B95-8 cells with early kinetics. We first prepared antiserum against EBV BDLF4 by immunizing rabbits with a glutathione S-transferase (GST)-fused BDLF4 protein expressed in bacteria. This anti-BDLF4 antiserum reacted with a protein of about ϳ22 kDa in the lysate from B95-8 cells, treated with tetradecanoyl phorbol acetate (TPA), A23187, and sodium butyrate (T/A/B) for 48 h (Fig. 1A). The BDLF4 protein could be detected readily by 24 h, and expression was increased at 48 h (Fig. 1B). While BALF4 mRNA expression was clearly blocked by phosphonoacetic acid (PAA), the amount of BDLF4 was decreased only slightly by the inhibitor (Fig. 1C), suggesting that the BDLF4 gene is expressed with E kinetics.Generation of BDLF4 knockout virus by frameshifting. To study the biological role of the BDLF4 gene in EBV lytic replication, we constructed a BDLF4-deficient recombinant virus and its revertant, as depicted in Fig. 2A. The integrity of the viral genomes was examined by restriction enzyme analysis, followed by agarose electrophoresis (Fig. 2B), and sequencing confirmed the intended one-nucleotide (nt) deletion.Knockout of BDLF4 decreased viral L gene expression and progeny production. The recombinant EBV-bacterial artificial chromosome (BAC) DNAs were transfected into HEK293 cells, and cells stably and latently containing EBV DNAs were cloned by hygromycin selection. We found that viral DNA synthesis levels of the wild-type, frameshift mutant, and revertant viruses were comparable (Fig. 3A). Disruption of BDLF4 decreased virus yield to approximately 10% to 20% compared with levels seen with the wild-type and revertant viruses (Fig. 3B)...