Lytic Cycle Gene Regulation of Epstein-Barr Virus

In this study, we report that murine cytomegalovirus (MCMV) protein pM92 regulates viral late gene expression during virus infection. Previously, we have shown that MCMV protein pM79 and its human cytomegalovirus (HCMV) homologue pUL79 are required for late viral gene transcription. Identification of additional factors involved is critical to dissecting the mechanism of this regulation. We show here that pM92 accumulated abundantly at late times of infection in a DNA synthesis-dependent manner and localized to nuclear viral replication compartments. To investigate the role of pM92, we constructed a recombinant virus SMin92, in which pM92 expression was disrupted by an insertional/frameshift mutation. During infection, SMin92 accumulated representative viral immediate-early gene products, early gene products, and viral DNA sufficiently but had severe reduction in the accumulation of late gene products and was thus unable to produce infectious progeny. Coimmunoprecipitation and mass spectrometry analysis revealed an interaction between pM92 and pM79, as well as between their HCMV homologues pUL92 and pUL79. Importantly, we showed that the growth defect of pUL92-deficient HCMV could be rescued in trans by pM92. This study indicates that pM92 is an additional viral regulator of late gene expression, that these regulators (represented by pM92 and pM79) may need to complex with each other for their activity, and that pM92 and pUL92 share a conserved function in CMV infection. pM92 represents a potential new target for therapeutic intervention in CMV disease, and a gateway into studying a largely uncharted viral process that is critical to the viral life cycle.

Section: Discussionmentioning

confidence: 99%

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

Chapa

Perng

French

et al. 2014

“…Promoter regions (600 to 1,000 bp upstream of the translation initiation codon) of genes ORF57 (early or ␤), M3 (early-late or ␥ 1 ), ORF26 (true-late or ␥ 2 ), and ORF65 (true-late or ␥ 2 ) were cloned upstream of the firefly luciferase coding gene. It was reported that the late promoters of herpesviruses require a functional viral origin of lytic replication (oriLyt) in cis besides viral transfactors for activity (3,24). Hence, the late promoters were cloned into a vector containing the MHV-68 minimum oriLyt (13).…”

Section: Vol 80 2006 Orf18 Controls Gammaherpesvirus Late Gene Exprmentioning

confidence: 99%

“…Although the regulation of gammaherpesvirus IE and early gene expression has been studied extensively in tumor cell lines (8,22,49,56), studies on the regulation of late gene expression have been limited by the lack of a robust lytic system. EBV late lytic gene promoter sequences essential for the regulation of late gene expression have been mapped using a plasmid containing a late promoter-reporter and origin of lytic replication (3,43). Nevertheless, the viral factor(s) specifically controlling the transcriptional regulation of gammaherpesvirus late genes is unknown.…”

Section: Human Gammaherpesviruses Epstein-barr Virus (Ebv) Andmentioning

confidence: 99%

ORF18 Is a Transfactor That Is Essential for Late Gene Transcription of a Gammaherpesvirus

Arumugaswami

Wu²,

Martinez-Guzman³

et al. 2006

Lytic replication of the tumor-associated human gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus has important implications in pathogenesis and tumorigenesis. Herpesvirus lytic genes have been temporally classified as exhibiting immediate-early (IE), early, and late expression kinetics. Though the regulation of IE and early gene expression has been studied extensively, very little is known regarding the regulation of late gene expression. Late genes, which primarily encode virion structural proteins, require viral DNA replication for their expression. We have identified a murine gammaherpesvirus 68 (MHV-68) early lytic gene, ORF18, essential for viral replication. ORF18 is conserved in both beta-and gammaherpesviruses. By generating an MHV-68 ORF18-null virus, we characterized the stage of the virus lytic cascade that requires the function of ORF18. Gene expression profiling and quantitation of viral DNA synthesis of the ORF18-null virus revealed that the expression of early genes and viral DNA replication were not affected; however, the transcription of late genes was abolished. Hence, we have identified a gammaherpesvirus-encoded factor essential for the expression of late genes independently of viral DNA synthesis. Human gammaherpesviruses Epstein-Barr virus (EBV) andKaposi's sarcoma-associated herpesvirus (KSHV) contribute to the development of epithelial, hematopoietic, and endothelial cell cancers. EBV is associated with a number of malignancies, including Burkitt's lymphoma, nasopharyngeal carcinoma, gastric carcinoma, and Hodgkin's disease (40). KSHV has been shown to be the causative agent of Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma (9, 10, 51). A murine gammaherpesvirus, MHV-68, is a model to study gammaherpesvirus biology due to its conservation with EBV and KSHV in both genomic content and gene expression program. In addition, MHV-68 retains the ability to lytically infect various cell lines, including those of human origin, and provides a small-animal model for experimental gammaherpesvirus infection in vivo (36,46,52,55).Herpesviruses undergo both lytic and latent phases of infection. Following primary lytic infection in epithelial cells and lymphocytes, gammaherpesviruses establish a life-long latent infection, with intermittent bursts of lytic reactivation (33,35). This sporadic reactivation allows the virus to maintain a dynamic infectious reserve for transmission and secondary infection. During latent infection, the virus expresses a limited number of genes which promote the survival and proliferation of infected cells, resulting in transformation of a small percentage of cells (32). In KSHV, a subset of the transformed cell population supports spontaneous reactivation, leading to the expression of virally encoded cellular cytokine and chemokine homologues, including viral macrophage inflammatory proteins I, II,34,37). These viral cytokines have a paracrine growth-promoting effect on neighboring infected cells; thus, lyti...

“…A hierarchy of viral gene expression is recognized in which the lytic transactivator ZTA (also called ZEBRA, Z, BZLF1, and EB1) is the first viral protein expressed, followed by the transcriptional and posttranscriptional regulatory proteins RTA (R) and MTA (SM) and subsequently the complement of EBV early and late genes (2,3,20). The key role of ZTA in the lytic reactivation process has resulted in an extensive examination of the regulation of the promoter that drives the BZLF1 open reading frame encoding ZTA.…”

mentioning

confidence: 99%

Contribution of C/EBP Proteins to Epstein-Barr Virus Lytic Gene Expression and Replication in Epithelial Cells

Huang

Liao

Chen

et al. 2006

Induction of the Epstein-Barr virus (EBV) lytic cycle has been examined predominantly in B cells. A hierarchy of viral gene expression is recognized in which the lytic transactivator ZTA (also called ZEBRA, Z, BZLF1, and EB1) is the first viral protein expressed, followed by the transcriptional and posttranscriptional regulatory proteins RTA (R) and MTA (SM) and subsequently the complement of EBV early and late genes (2,3,20). The key role of ZTA in the lytic reactivation process has resulted in an extensive examination of the regulation of the promoter that drives the BZLF1 open reading frame encoding ZTA. Induction of the lytic cycle in Akata cells through cross-linking of the B-cell receptor has defined a pathway in which primary cell-mediated responses are followed by a ZTA-induced autoamplification of ZTA protein expression (5, 9, 56, 71) mediated through two ZTA binding sites in the BZLF1 promoter (Zp) (21,38,62). Induction of the Zp following B-cell-receptor coupled signaling is dependent on the activity of the receptor-associated Syk and Btk protein tyrosine kinases (36) and on activation of the phosphatidylinositol 3-kinase signaling pathway (30). Expression of ZTA is also induced by the cytokine transforming growth factor beta (TGF-␤) (17,18,71), and three binding sites for SMAD proteins, the effectors of TGF-␤ signaling, have been identified in the Zp (37). Other EBV lytic cycle-inducing agents are phorbol esters, histone deacetylase inhibitors, and calcium ionophores. Phorbol ester treatment activates protein kinase C and the responsive cis-acting signals were mapped to the ZI and ZII (CRE/AP-1) Zp elements (7,22). The CRE/AP-1 sites bind activating transcription factor 1 (ATF-1) and 45,68), and both ZTA and RTA induce ATF-2 phosphorylation and activation (1). The Zp response to calcium-dependent signaling also mapped to the ZI and ZII sites. The ZIA, ZIB, and ZID sites bind myocyte enhancer factor 2D (MEF-2D) (9, 43), which recruits histone deacetylases to repress the uninduced Zp (25). Calcium activation of MEF-2 response elements is mediated by two calcium calmodulin-dependent enzymes, the phosphatase calcineurin and the kinase type IV/Gr (6, 11). Other negative regulatory elements in the Zp are the promoter proximal ZV element, which binds the zinc-finger E-box binding factor ZEB (32, 33), and promoter distal elements that bind the transcription factor YY1 (49) and the E-box binding protein E2-2 (59).The ZII and ZIIIB elements in the Zp have recently been shown to contain binding sites also for cellular CCAAT/enhancer binding protein ␣ (C/EBP␣) (70). Cross-linking the B-cell receptor of Akata cells, a process that induces the EBV lytic cycle, also induces C/EBP␣ expression (70). In a series of reinforcing interactions, C/EBP␣ activates the Zp, ZTA inter-* Corresponding author. Mailing address: