Genome-Wide Analyses of Zta Binding to the Epstein-Barr Virus Genome Reveals Interactions in both Early and Late Lytic Cycles and an Epigenetic Switch Leading to an Altered Binding Profile

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: Discussionmentioning

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

“…The master regulator of the latent-lytic cycle switch, BZLF1, binds directly to Zta response elements throughout the viral genome and activates both early and late lytic cycle genes (45). BZLF1 does not rely on DNA binding proteins, such as RBP-Jκ, to activate the promoters of the lytic cycle genes, so it is not regulated by Notch at this level.…”

Section: Discussionmentioning

confidence: 99%

Counteracting Effects of Cellular Notch and Epstein-Barr Virus EBNA2: Implications for Stromal Effects on Virus-Host Interactions

Rowe

Raithatha

Shannon-Lowe

2014

A number of diverse environmental cues have been linked to B lymphocyte differentiation and activation. One such cue, Notch-2, may be particularly relevant to the biology of infection with Epstein-Barr virus (EBV), which colonizes the B cell compartment. Activated Notch and EBV nuclear antigen 2 (EBNA2) both function as transcriptional activators by virtue of their interactions with the transcription factor RBP-Jκ. Although EBNA2 and activated Notch appear to have partially overlapping functions, we now report that activated Notch counteracts a crucial EBNA2 function both in newly infected primary B cells and in lymphoblastoid cell lines (LCLs). EBNA2 is directly responsible for the initiation of transcription of the majority of EBV proteins associated with type III latency, leading to the outgrowth of LCLs. One of the key proteins driving this outgrowth is latent membrane protein 1 (LMP1), which is regulated by an EBNA2-responsive element within its ED-L1 promoter. Activation of Notch-2 via Delta-like ligand 1 inhibits EBNA2-mediated initiation of LMP1 transcription. Furthermore, ligated Notch-2 also efficiently turns off LMP1 expression from the ED-L1 promoter in LCLs already expressing LMP1. Modulation of EBV gene expression by Notch was not confined to EBNA2-dependent events. Activated Notch-2 also inhibited EBV entry into the lytic cycle in a B cell non-Hodgkin's lymphoma line by upregulating the cellular transcription factor Zeb2, which represses the transcription of BZLF1. These results support the concept that in vivo, cumulative signals from the microenvironment downregulate EBV gene expression in B cells to the latency 0 gene expression profile observed in B cells entering the peripheral blood.IMPORTANCE Experimental infection of resting B cells by Epstein-Barr virus leads to the growth transformation program of virus gene expression and the outgrowth of lymphoblastoid cell lines. Previous studies at the single-cell level revealed complex cellular and viral signaling networks regulating transcription of the viral genome. This study demonstrates that viral gene expression can also be radically altered by molecules expressed on stromal cells in the microenvironment of lymphoid tissue, specifically, Delta-like ligand 1 on stromal cells ligating Notch-2 on infected B cells. Activation of Notch interferes with the transactivation function of EBNA2, downregulates the expression of LMP1 and LMP2a, and inhibits the activation of lytic virus replication in a B cell non-Hodgkin's lymphoma line by preventing expression of BZLF1. The significance of these observations is that they indicate new mechanisms whereby the microenvironment in normal lymphoid tissue may facilitate the repression of viral gene expression, enabling establishment of true latency in memory B cells.

“…These promoters contain CpG-containing Zta response elements (CpG-ZREs). Highly methylated ZREs were associated with high Zta occupancies (73). Our data suggest that Rta expression provides methylation marks in the viral promoters for Zta binding during latent-lytic cycle transition.…”

Section: Discussionmentioning

confidence: 63%

“…It has been demonstrated that a methylated EBV genome is essential for viral lytic reproduction (51,53,72), as the immediate-early protein Zta preferentially binds to methylated viral promoters (54,(73)(74)(75). In 293T cells stably infected with an EBV genome, high occupancies of Zta were observed in methylated promoters of BALF2, BMRF1, Rta, and BHLF1, whereas these bindings were disrupted once the EBV genome is hypomethylated (54).…”

Section: Discussionmentioning

confidence: 99%

Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes

Chen

Chang

et al. 2017

Rta, an Epstein-Barr virus (EBV) immediate-early protein, reactivates viral lytic replication that is closely associated with tumorigenesis. In previous studies, we demonstrated that in epithelial cells Rta efficiently induced cellular senescence, which is an irreversible G 1 arrest likely to provide a favorable environment for productive replications of EBV and Kaposi's sarcoma-associated herpesvirus (KSHV). To restrict progression of the cell cycle, Rta simultaneously upregulates CDK inhibitors and downregulates MYC, CCND1, and JUN, among others. Rta has long been known as a potent transcriptional activator, thus its role in gene repression is unexpected. In silico analysis revealed that the promoter regions of MYC, CCND1, and JUN are common in (i) the presence of CpG islands, (ii) strong chromatin immunoprecipitation (ChIP) signals of CCCTC-binding factor (CTCF), and (iii) having at least one Rta binding site. By combining ChIP assays and DNA methylation analysis, here we provide evidence showing that Rta binding accumulated CpG methylation and decreased CTCF occupancy in the regulatory regions of MYC, CCND1, and JUN, which were associated with downregulated gene expression. Stable residence of CTCF in the viral latency and reactivation control regions is a hallmark of viral latency. Here, we observed that Rta-mediated decreased binding of CTCF in the viral genome is concurrent with virus reactivation. Via interfering with CTCF binding, in the host genome Rta can function as a transcriptional repressor for gene silencing, while in the viral genome Rta acts as an activator for lytic gene loci by removing a topological constraint established by CTCF.IMPORTANCE CTCF is a multifunctional protein that variously participates in gene expression and higher-order chromatin structure of the cellular and viral genomes. In certain loci of the genome, CTCF occupancy and DNA methylation are mutually exclusive. Here, we demonstrate that the Epstein-Barr virus (EBV) immediate-early protein, Rta, known to be a transcriptional activator, can also function as a transcriptional repressor. Via enriching CpG methylation and decreasing CTCF reloading, Rta binding efficiently shut down the expression of MYC, CCND1, and JUN, thus impeding cell cycle progression. Rta-mediated disruption of CTCF binding was also detected in the latency/reactivation control regions of the EBV genome, and this in turn led to viral lytic cycle progression. As emerging evidence indicates that a methylated EBV genome is a preferable substrate for EBV Zta, the other immediate-early protein, our results suggest a mechanistic link in understanding the molecular processes of viral latent-lytic switch.KEYWORDS Epstein-Barr virus, immediate-early protein Rta, lytic cycle reactivation, CTCF, DNA methylation, cell cycle arrest, genome topology, immediate-early protein Rta