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Species-specific functions of Epstein-Barr virus nuclear antigen 2 (EBNA2) reveal dual roles for initiation and maintenance of B cell immortalization.

ABSTRACT: Epstein-Barr virus (EBV) and related lymphocryptoviruses (LCV) from non-human primates infect B cells, transform their growth to facilitate life-long viral persistence in the host, and contribute to B cell oncogenesis. Co-evolution of LCV with their primate hosts has led to species-specificity so that LCVs preferentially immortalize B cells from their natural host in vitro. We investigated whether the master regulator of transcription, EBV nuclear antigen 2 (EBNA2), is involved in LCV species-specificity. Using recombinant EBVs, we show that EBNA2 orthologues of LCV isolated from chimpanzees, baboons, cynomolgus or rhesus macaques cannot replace EBV EBNA2 for the immortalization of human B cells. Thus, LCV species-specificity is functionally linked to viral proteins expressed during latent, growth-transforming infection. In addition, we identified three independent domains within EBNA2 that act through species-specific mechanisms. Importantly, the EBNA2 orthologues and species-specific EBNA2 domains separate unique roles for EBNA2 in the initiation of B cell immortalization from those responsible for maintaining the immortalized state. Investigating LCV species-specificity provides a novel approach to identify critical steps underlying EBV-induced B cell growth transformation, persistent infection, and oncogenesis.

SUBMITTER: Muhe J

PROVIDER: S-EPMC5754137 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Species-specific functions of Epstein-Barr virus nuclear antigen 2 (EBNA2) reveal dual roles for initiation and maintenance of B cell immortalization.

Mühe Janine J Wang Fred F

PLoS pathogens 20171220 12

Epstein-Barr virus (EBV) and related lymphocryptoviruses (LCV) from non-human primates infect B cells, transform their growth to facilitate life-long viral persistence in the host, and contribute to B cell oncogenesis. Co-evolution of LCV with their primate hosts has led to species-specificity so that LCVs preferentially immortalize B cells from their natural host in vitro. We investigated whether the master regulator of transcription, EBV nuclear antigen 2 (EBNA2), is involved in LCV species-sp ...[more]

PMID: 29261800

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Project description:Latent infection of B lymphocytes by Epstein-Barr virus (EBV) in vitro results in their immortalization into lymphoblastoid cell lines (LCLs); this latency program is controlled by the EBNA2 viral transcriptional activator, which targets promoters via RBPJ, a DNA binding protein in the Notch signaling pathway. Three other EBNA3 proteins (EBNA3A, EBNA3B, and EBNA3C) interact with RBPJ to regulate cell gene expression. The mechanism by which EBNAs regulate different genes via RBPJ remains unclear. Our chromatin immunoprecipitation with deep sequencing (ChIP-seq) analysis of the EBNA3 proteins analyzed in concert with prior EBNA2 and RBPJ data demonstrated that EBNA3A, EBNA3B, and EBNA3C bind to distinct, partially overlapping genomic locations. Although RBPJ interaction is critical for EBNA3A and EBNA3C growth effects, only 30 to 40% of EBNA3-bound sites colocalize with RBPJ. Using LCLs conditional for EBNA3A or EBNA3C activity, we demonstrate that EBNA2 binding at sites near EBNA3A- or EBNA3C-regulated genes is specifically regulated by the respective EBNA3. To investigate EBNA3 binding specificity, we identified sequences and transcription factors enriched at EBNA3A-, EBNA3B-, and EBNA3C-bound sites. This confirmed the prior observation that IRF4 is enriched at EBNA3A- and EBNA3C-bound sites and revealed IRF4 enrichment at EBNA3B-bound sites. Using IRF4-negative BJAB cells, we demonstrate that IRF4 is essential for EBNA3C, but not EBNA3A or EBNA3B, binding to specific sites. These results support a model in which EBNA2 and EBNA3s compete for distinct subsets of RBPJ sites to regulate cell genes and where EBNA3 subset specificity is determined by interactions with other cell transcription factors.Epstein-Barr virus (EBV) latent gene products cause human cancers and transform B lymphocytes into immortalized lymphoblastoid cell lines in vitro. EBV nuclear antigens (EBNAs) and membrane proteins constitutively activate pathways important for lymphocyte growth and survival. An important unresolved question is how four different EBNAs (EBNA2, -3A, -3B, and -3C) exert unique effects via a single transcription factor, RBPJ. Here, we report that each EBNA binds to distinct but partially overlapping sets of genomic sites. EBNA3A and EBNA3C specifically regulate EBNA2's access to different RBPJ sites, providing a mechanism by which each EBNA can regulate distinct cell genes. We show that IRF4, an essential regulator of B cell differentiation, is critical for EBNA3C binding specificity; EBNA3A and EBNA3B specificities are likely due to interactions with other cell transcription factors. EBNA3 titration of EBNA2 transcriptional function at distinct sites likely limits cell defenses that would be triggered by unchecked EBNA2 prooncogenic activity.

Project description:Biological macromolecule condensates formed by liquid-liquid phase separation (LLPS) have been discovered in recent years to be prevalent in biology. These condensates are involved in diverse processes, including the regulation of gene expression. LLPS of proteins have been found in animal, plant, and bacterial species but have scarcely been identified in viral proteins. Here, we discovered that Epstein-Barr virus (EBV) EBNA2 and EBNALP form nuclear puncta that exhibit properties of liquid-like condensates (or droplets), which are enriched in superenhancers of MYC and Runx3. EBNA2 and EBNALP are transcription factors, and the expression of their target genes is suppressed by chemicals that perturb LLPS. Intrinsically disordered regions (IDRs) of EBNA2 and EBNALP can form phase-separated droplets, and specific proline residues of EBNA2 and EBNALP contribute to droplet formation. These findings offer a foundation for understanding the mechanism by which LLPS, previously determined to be related to the organization of P bodies, membraneless organelles, nucleolus homeostasis, and cell signaling, plays a key role in EBV-host interactions and is involved in regulating host gene expression. This work suggests a novel anti-EBV strategy where developing appropriate drugs of interfering LLPS can be used to destroy the function of the EBV's transcription factors.IMPORTANCE Protein condensates can be assembled via liquid-liquid phase separation (LLPS), a process involving the concentration of molecules in a confined liquid-like compartment. LLPS allows for the compartmentalization and sequestration of materials and can be harnessed as a sensitive strategy for responding to small changes in the environment. This study identified the Epstein-Barr virus (EBV) proteins EBNA2 and EBNALP, which mediate virus and cellular gene transcription, as transcription factors that can form liquid-like condensates at superenhancer sites of MYC and Runx3. This study discovered the first identified LLPS of EBV proteins and emphasized the importance of LLPS in controlling host gene expression.

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Species-specific functions of Epstein-Barr virus nuclear antigen 2 (EBNA2) reveal dual roles for initiation and maintenance of B cell immortalization.

Publications

Species-specific functions of Epstein-Barr virus nuclear antigen 2 (EBNA2) reveal dual roles for initiation and maintenance of B cell immortalization.

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