Epstein-Barr virus miR-BHRF1-3 targets the BZLF1 3’UTR and regulates the lytic cycle

2021 ◽  
Author(s):  
Devin N. Fachko ◽  
Yan Chen ◽  
Rebecca L. Skalsky
Keyword(s):  
Gene Expression ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Luciferase Reporter ◽  
Viral Gene ◽  
Epstein Barr Virus Infection ◽  
Barr Virus ◽  
Epstein Barr ◽  
The Impact

Suppression of lytic viral gene expression is a key aspect of the Epstein-Barr virus (EBV) life cycle to facilitate the establishment of latent infection. Molecular mechanisms regulating transitions between EBV lytic replication and latency are not fully understood. Here, we investigated the impact of viral microRNAs on the EBV lytic cycle. Through functional assays, we found that miR-BHRF1-3 attenuates EBV lytic gene expression following reactivation. To understand the miRNA targets contributing to this activity, we performed Ago PAR-CLIP analysis on EBV-positive, reactivated Burkitt’s lymphoma cells and identified multiple miR-BHRF1-3 interactions with viral transcripts. Using luciferase reporter assays, we confirmed a miRNA interaction site within the 3’UTR of BZLF1 which encodes the essential immediate early (IE) transactivator Zta. Comparison of >850 published EBV genomes identified sequence polymorphisms within the miR-BHRF1-3 locus that deleteriously affect miRNA expression and function. Molecular interactions between the homologous viral miRNA, miR-rL1-17, and IE transcripts encoded by rhesus lymphocryptovirus were further identified. Our data demonstrate that regulation of IE gene expression by a BHRF1 miRNA is conserved amongst lymphocryptoviruses, and further reveal virally-encoded genetic elements that orchestrate viral antigen expression during the lytic cycle. Importance Epstein-Barr virus infection is predominantly latent in healthy individuals, while periodic cycles of reactivation are thought to facilitate persistent lifelong infection. Lytic infection has been linked to development of certain EBV-associated diseases. Here, we demonstrate that EBV miR-BHRF1-3 can suppress lytic replication by directly inhibiting Zta expression. Moreover, we identify nucleotide variants that impact the function of miR-BHRF1-3, which may contribute to specific EBV pathologies.

scholarly journals The Impact of Epstein-Barr Virus Infection on Epigenetic Regulation of Host Cell Gene Expression in Epithelial and Lymphocytic Malignancies

2021 ◽  
Vol 11 ◽  
Author(s):  
Merrin Man Long Leong ◽  
Maria Li Lung
Keyword(s):  
Gene Expression ◽  
Epigenetic Regulation ◽  
Epstein Barr Virus ◽  
Regulation Of Gene Expression ◽  
Host Cells ◽  
Viral Gene ◽  
Epstein Barr Virus Infection ◽  
Barr Virus ◽  
Epstein Barr ◽  
The Impact

Epstein-Barr virus (EBV) infection is associated with a variety of malignancies including Burkitt’s lymphoma (BL), Hodgkin’s disease, T cell lymphoma, nasopharyngeal carcinoma (NPC), and ∼10% of cases of gastric cancer (EBVaGC). Disruption of epigenetic regulation in the expression of tumor suppressor genes or oncogenes has been considered as one of the important mechanisms for carcinogenesis. Global hypermethylation is a distinct feature in NPC and EBVaGC, whereas global reduction of H3K27me3 is more prevalent in EBVaGC and EBV-transformed lymphoblastoid cells. In BL, EBV may even usurp the host factors to epigenetically regulate its own viral gene expression to restrict latency and lytic switch, resulting in evasion of immunosurveillance. Furthermore, in BL and EBVaGC, the interaction between the EBV episome and the host genome is evident with respectively unique epigenetic features. While the interaction is associated with suppression of gene expression in BL, the corresponding activity in EBVaGC is linked to activation of gene expression. As EBV establishes a unique latency program in these cancer types, it is possible that EBV utilizes different latency proteins to hijack the epigenetic modulators in the host cells for pathogenesis. Since epigenetic regulation of gene expression is reversible, understanding the precise mechanisms about how EBV dysregulates the epigenetic mechanisms enables us to identify the potential targets for epigenetic therapies. This review summarizes the currently available epigenetic profiles of several well-studied EBV-associated cancers and the relevant distinct mechanisms leading to aberrant epigenetic signatures due to EBV.

scholarly journals Epstein–Barr virus origin of lytic replication mediates association of replicating episomes with promyelocytic leukaemia protein nuclear bodies and replication compartments

2006 ◽  
Vol 87 (5) ◽  
pp. 1133-1137 ◽  
Author(s):  
Wolfgang Amon ◽  
Robert E. White ◽  
Paul J. Farrell
Keyword(s):  
Gene Expression ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Nuclear Bodies ◽  
Lytic Cycle ◽  
Promyelocytic Leukaemia ◽  
Late Gene ◽  
Barr Virus ◽  
Pml Nuclear Bodies ◽  
Epstein Barr

Epstein–Barr virus (EBV) establishes a latent persistence from which it can be reactivated to undergo lytic replication. Late lytic-cycle gene expression is linked to lytic DNA replication, as it is sensitive to the same inhibitors that block lytic replication, and it has recently been shown that the viral origin of lytic replication (ori lyt) is required in cis for late-gene expression. During the lytic cycle, the viral genome forms replication compartments, which are usually adjacent to promyelocytic leukaemia protein (PML) nuclear bodies. A tetracycline repressor DNA-binding domain–enhanced green fluorescent protein fusion was used to visualize replicating plasmids carrying a tetracycline operator sequence array. ori lyt mediated the production of plasmid replication compartments that were associated with PML nuclear bodies. Plasmids carrying ori lyt and EBV itself were visualized in the same cells and replicated in similar regions of the nucleus, further supporting the validity of the plasmids for studying late-gene regulation.

scholarly journals Inhibition of the Epstein–Barr virus lytic cycle by Zta-targeted RNA interference

2004 ◽  
Vol 85 (6) ◽  
pp. 1371-1379 ◽  
Author(s):  
Yao Chang ◽  
Shih-Shin Chang ◽  
Heng-Huan Lee ◽  
Shin-Lian Doong ◽  
Kenzo Takada ◽  
...  
Keyword(s):  
Rna Interference ◽  
Epstein Barr Virus ◽  
Disease Risk ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Viral Gene ◽  
Barr Virus ◽  
Ebv Reactivation ◽  
Epstein Barr

Epstein–Barr virus (EBV) reactivation into the lytic cycle plays certain roles in the development of EBV-associated diseases, so an effective strategy to block the viral lytic cycle may be of value to reduce the disease risk or to improve the clinical outcome. This study examined whether the EBV lytic cycle could be inhibited using RNA interference (RNAi) directed against the essential viral gene Zta. In cases of EBV reactivation triggered by chemicals or by exogenous Rta, Zta-targeted RNAi prevented the induction of Zta and its downstream genes and further blocked the lytic replication of viral genomes. This antiviral effect of RNAi was not likely to be mediated by activation of the interferon pathway, as phosphorylation of STAT1 was not induced. In addition, novel EBV-infected epithelial cells showing constitutive activation of the lytic cycle were cloned; such established lytic infection was also suppressed by Zta-targeted RNAi. These results indicate that RNAi can be used to inhibit the EBV lytic cycle effectively in vitro and could also be of potential use to develop anti-EBV treatments.

scholarly journals The SWI/SNF Chromatin Regulator BRG1 Modulates the Transcriptional Regulatory Activity of the Epstein-Barr Virus DNA Polymerase Processivity Factor BMRF1

2017 ◽  
Vol 91 (9) ◽  
Author(s):  
Mei-Tzu Su ◽  
Ya-Ting Wang ◽  
Yen-Ju Chen ◽  
Su-Fang Lin ◽  
Ching-Hwa Tsai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Viral Gene ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT During the lytic phase of Epstein-Barr virus (EBV), binding of the transactivator Zta to the origin of lytic replication (oriLyt) and the BHLF1 transcript, forming a stable RNA-DNA hybrid, is required to initiate viral DNA replication. EBV-encoded viral DNA replication proteins form complexes to amplify viral DNA. BMRF1, the viral DNA polymerase accessory factor, is essential for lytic DNA replication and also known as a transcriptional regulator of the expression of BHLF1 and BALF2 (single-stranded DNA [ssDNA]-binding protein). In order to determine systematically how BMRF1 regulates viral transcription, a BMRF1 knockout bacmid was generated to analyze viral gene expression using a viral DNA microarray. We found that a subset of Rta-responsive late genes, including BcLF1, BLLF1, BLLF2, and BDLF3, were downregulated in cells harboring a BMRF1 knockout EBV bacmid (p2089ΔBMRF1). In reporter assays, BMRF1 appears to transactivate a subset of viral late promoters through distinct pathways. BMRF1 activates the BDLF3 promoter in an SP1-dependent manner. Notably, BMRF1 associates with the transcriptional regulator BRG1 in EBV-reactivated cells. BMRF1-mediated transactivation activities on the BcLF1 and BLLF1 promoters were attenuated by knockdown of BRG1. In BRG1-depleted EBV-reactivated cells, BcLF1 and BLLF1 transcripts were reduced in number, resulting in reduced virion secretion. BMRF1 and BRG1 bound to the adjacent upstream regions of the BcLF1 and BLLF1 promoters, and depletion of BRG1 attenuated the recruitment of BMRF1 onto both promoters, suggesting that BRG1 is involved in BMRF1-mediated regulation of these two genes. Overall, we reveal a novel pathway by which BMRF1 can regulate viral promoters through interaction with BRG1. IMPORTANCE The cascade of viral gene expression during Epstein-Barr virus (EBV) replication is exquisitely regulated by the coordination of the viral DNA replication machinery and cellular factors. Upon lytic replication, the EBV immediate early proteins Zta and Rta turn on the expression of early proteins that assemble into viral DNA replication complexes. The DNA polymerase accessory factor, BMRF1, also is known to transactivate early gene expression through its interaction with SP1 or Zta on specific promoters. Through a global analysis, we demonstrate that BMRF1 also turns on a subset of Rta-regulated, late structural gene promoters. Searching for BMRF1-interacting cellular partners revealed that the SWI/SNF chromatin modifier BRG1 contributes to BMRF1-mediated transactivation of a subset of late promoters through protein-protein interaction and viral chromatin binding. Our findings indicate that BMRF1 regulates the expression of more viral genes than thought previously through distinct viral DNA replication-independent mechanisms.

scholarly journals The Epstein–Barr virus lytic cycle activator Zta interacts with methylated ZRE in the promoter of host target gene egr1

2009 ◽  
Vol 90 (6) ◽  
pp. 1450-1454 ◽  
Author(s):  
James Heather ◽  
Kirsty Flower ◽  
Samine Isaac ◽  
Alison J. Sinclair
Keyword(s):  
Epstein Barr Virus ◽  
Target Gene ◽  
Distal Part ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Viral Gene ◽  
Reporter Construct ◽  
Barr Virus ◽  
Epstein Barr ◽  
Host Target

Activation of the host gene egr1 is essential for the lytic replication of Epstein–Barr virus (EBV). egr1 is activated by Zta (BZLF1, ZEBRA). Zta interacts directly with DNA through a series of closely related Zta-response elements (ZREs). Here we dissect the mechanism used by Zta to interact with the egr1 promoter and identify a weak interaction with egr1ZRE that is dependent on the distal part of egr1ZRE. Furthermore, we demonstrate that the ability of Zta to interact with egr1ZRE is enhanced at least tenfold by methylation. The ability of Zta to transactivate a reporter construct driven by the egr1 promoter can be enhanced by methylation. As the ability of Zta to interact with a methylated ZRE in the EBV genome correlates with its ability to activate the expression of the endogenous viral gene BRLF1, this suggests that Zta may also have the capability to overturn epigenetic control of egr1.

scholarly journals Functional Analysis of Epstein-Barr Virus SM Protein: Identification of Amino Acids Essential for Structure, Transactivation, Splicing Inhibition, and Virion Production

2004 ◽  
Vol 78 (1) ◽  
pp. 340-352 ◽  
Author(s):  
Vivian Ruvolo ◽  
Liang Sun ◽  
Karilynn Howard ◽  
Seung Sung ◽  
Henri-Jacques Delecluse ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Epstein Barr Virus ◽  
Protein Identification ◽  
Lytic Replication ◽  
Viral Gene ◽  
Barr Virus ◽  
Virion Production ◽  
Epstein Barr ◽  
Sm Protein

ABSTRACT The Epstein-Barr virus (EBV) SM protein is a posttranscriptional regulator of cellular and viral gene expression that binds and stabilizes target mRNAs and shuttles from nucleus to cytoplasm. SM enhances expression of several EBV genes required for lytic replication and is essential for virion production. SM increases accumulation of specific mRNAs but also inhibits expression of several intron-containing transcripts. The mechanism by which SM inhibits gene expression is poorly understood. The experiments described here had several aims: to determine whether specific domains of SM were responsible for activation or inhibition function; whether these functions could be separated; and whether one or more of these functions were essential for virion production. A mutational analysis of SM was performed, focusing on amino acids in SM that are evolutionarily conserved among SM homologs in other herpesviruses. Mutation of the carboxy-terminal region of SM revealed a region that is likely to be structurally important for SM protein conformation. In addition, several amino acids were identified that are critical for activation and inhibition function. A specific mutation of a highly conserved cysteine residue revealed that it was essential for gene inhibition but not for transactivation, indicating that these two functions operate through independent mechanisms. Furthermore, the ability of wild-type SM and the inability of the mutant to inhibit gene expression were shown to correlate with the ability to inhibit splicing of a human target gene and thereby prevent accumulation of its processed mRNA. Surprisingly, some mutations which preserved both activation and inhibition functions in vitro nevertheless abolished virion production, suggesting that other SM functions or protein-protein interactions are also required for lytic replication.

scholarly journals Interaction sites of the Epstein–Barr virus Zta transcription factor with the host genome in epithelial cells

2021 ◽  
Vol 3 (11) ◽  
Author(s):  
Anja Godfrey ◽  
Kay Osborn ◽  
Alison J. Sinclair
Keyword(s):  
Transcription Factor ◽  
B Cells ◽  
Epithelial Cells ◽  
Binding Sites ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Barr Virus ◽  
Epstein Barr ◽  
The Impact

Epstein–Barr virus (EBV) is present in a state of latency in infected memory B-cells and EBV-associated lymphoid and epithelial cancers. Cell stimulation or differentiation of infected B-cells and epithelial cells induces reactivation to the lytic replication cycle. In each cell type, the EBV transcription and replication factor Zta (BZLF1, EB1) plays a role in mediating the lytic cycle of EBV. Zta is a transcription factor that interacts directly with Zta response elements (ZREs) within viral and cellular genomes. Here we undertake chromatin-precipitation coupled to DNA-sequencing (ChIP-Seq) of Zta-associated DNA from cancer-derived epithelial cells. The analysis identified over 14 000 Zta-binding sites in the cellular genome. We assessed the impact of lytic cycle reactivation on changes in gene expression for a panel of Zta-associated cellular genes. Finally, we compared the Zta-binding sites identified in this study with those previously identified in B-cells and reveal substantial conservation in genes associated with Zta-binding sites.

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