Epstein-Barr virus tegument protein BGLF2 in exosomes released from virus-producing cells facilitates de novo infection.

BACKGROUND: Viruses must adapt to the environment of their host cells to establish infection and persist. Diverse mammalian cells, including virus-infected cells, release extracellular vesicles such as exosomes containing proteins and miRNAs, and use these vesicles to mediate intercellular communication. However, the roles of exosomes in viral infection remain unclear. RESULTS: We screened viral proteins to identify those responsible for the exosome-mediated enhancement of Epstein-Barr virus (EBV) infection. We identified BGLF2 protein encapsulated in exosomes, which were released by EBV-infected cells. BGLF2 protein is a tegument protein that exists in the space between the envelope and nucleocapsid, and it is released into the cytoplasm shortly after infection. BGLF2 protein-containing exosomes enhanced viral gene expression and repressed innate immunity, thereby supporting the EBV infection. CONCLUSIONS: The EBV tegument protein BGLF2 is encapsulated in exosomes and released by infected cells to facilitate the establishment of EBV infection. These findings suggest that tegument proteins support viral infection not only between the envelope and nucleocapsid, as well as in extraviral particles such as exosomes. Video abstract.

Suppression of JAK-STAT Signaling by Epstein-Barr Virus Tegument Protein BGLF2 through Recruitment of SHP1 Phosphatase and Promotion of STAT2 Degradation.

Some lytic proteins encoded by Epstein-Barr virus (EBV) suppress host interferon (IFN) signaling to facilitate viral replication. In this study, we sought to identify and characterize EBV proteins antagonizing IFN signaling. The induction of IFN-stimulated genes (ISGs) by IFN-ß was effectively suppressed by EBV. A functional screen was therefore performed to identify IFN-antagonizing proteins encoded by EBV. EBV tegument protein BGLF2 was identified as a potent suppressor of JAK-STAT signaling. This activity was found to be independent of its stimulatory effect on p38 and JNK pathways. Association of BGLF2 with STAT2 resulted in more pronounced K48-linked polyubiquitination and proteasomal degradation of the latter. Mechanistically, BGLF2 promoted the recruitment of SHP1 phosphatase to STAT1 to inhibit its tyrosine phosphorylation. In addition, BGLF2 associated with cullin 1 E3 ubiquitin ligase to facilitate its recruitment to STAT2. Consequently, BGLF2 suppressed ISG induction by IFN-ß. Furthermore, BGLF2 also suppressed type II and type III IFN signaling, although the suppressive effect on type II IFN response was milder. When pretreated with IFN-ß, host cells became less susceptible to primary infection of EBV. This phenotype was reversed when expression of BGLF2 was enforced. Finally, genetic disruption of BGLF2 in EBV led to more pronounced induction of ISGs. Our study unveils the roles of BGLF2 not only in the subversion of innate IFN response but also in lytic infection and reactivation of EBV. IMPORTANCE Epstein-Barr virus (EBV) is an oncogenic virus associated with the development of lymphoid and epithelial malignancies. EBV has to subvert interferon-mediated host antiviral response to replicate and cause diseases. It is therefore of great interest to identify and characterize interferon-antagonizing proteins produced by EBV. In this study, we perform a screen to search for EBV proteins that suppress the action of interferons. We further show that BGLF2 protein of EBV is particularly strong in this suppression. This is achieved by inhibiting two key proteins STAT1 and STAT2 that mediate the antiviral activity of interferons. BGLF2 recruits a host enzyme to remove the phosphate group from STAT1 thereby inactivating its activity. BGLF2 also redirects STAT2 for degradation. A recombinant virus in which BGLF2 gene has been disrupted can activate host interferon response more robustly. Our findings reveal a novel mechanism by which EBV BGLF2 protein suppresses interferon signaling.

Epstein-Barr Virus (EBV) Tegument Protein BGLF2 Suppresses Type I Interferon Signaling To Promote EBV Reactivation.

Interferon alpha (IFN-α) and IFN-ß are type I IFNs that are induced by virus infection and are important in the host's innate antiviral response. EBV infection activates multiple cell signaling pathways, resulting in the production of type I IFN which inhibits EBV infection and virus-induced B-cell transformation. We reported previously that EBV tegument protein BGLF2 activates p38 and enhances EBV reactivation. To further understand the role of BGLF2 in EBV infection, we used mass spectrometry to identify cellular proteins that interact with BGLF2. We found that BGLF2 binds to Tyk2 and confirmed this interaction by coimmunoprecipitation. BGLF2 blocked type I IFN-induced Tyk2, STAT1, and STAT3 phosphorylation and the expression of IFN-stimulated genes (ISGs) IRF1, IRF7, and MxA. In contrast, BGLF2 did not inhibit STAT1 phosphorylation induced by IFN-γ. Deletion of the carboxyl-terminal 66 amino acids of BGLF2 reduced the ability of the protein to repress type I IFN signaling. Treatment of gastric carcinoma and Raji cells with IFN-α blocked BZLF1 expression and EBV reactivation; however, expression of BGLF2 reduced the ability of IFN-α to inhibit BZLF1 expression and enhanced EBV reactivation. In summary, EBV BGLF2 interacts with Tyk2, inhibiting Tyk2, STAT1, and STAT3 phosphorylation and impairs type I IFN signaling; BGLF2 also counteracts the ability of IFN-α to suppress EBV reactivation.IMPORTANCE Type I interferons are important for controlling virus infection. We have found that the Epstein-Barr virus (EBV) BGLF2 tegument protein binds to a protein in the type I interferon signaling pathway Tyk2 and inhibits the expression of genes induced by type I interferons. Treatment of EBV-infected cells with type I interferon inhibits reactivation of the virus, while expression of EBV BGLF2 reduces the ability of type I interferon to inhibit virus reactivation. Thus, a tegument protein delivered to cells during virus infection inhibits the host's antiviral response and promotes virus reactivation of latently infected cells. Therefore, EBV BGLF2 might protect virus-infected cells from the type I interferon response in cells undergoing lytic virus replication.

Protein inhibitor of activated STAT1 (PIAS1) inhibits IRF8 activation of Epstein-Barr virus lytic gene expression.

Epstein-Barr virus (EBV), a major human oncogenic pathogen, establishes life-long persistent infections. In latently infected B lymphocytes, the virus persists as an episome in the nucleus. Periodic reactivation of latent virus is controlled by both viral and cellular factors. Our recent studies showed that interferon regulatory factor 8 (IRF8) is required for EBV lytic reactivation while protein inhibitor of activated STAT1 (PIAS1) functions as an EBV restriction factor to block viral reactivation. Here, we show that IRF8 directly binds to the EBV genome and regulates EBV lytic gene expression together with PU.1 and EBV transactivator RTA. Furthermore, our study reveals that PIAS1 antagonizes IRF8/PU.1-mediated lytic gene activation through binding to and inhibiting IRF8. Together, our study establishes IRF8 as a transcriptional activator in promoting EBV reactivation and defines PIAS1 as an inhibitor of IRF8 to limit lytic gene expression.

Interaction Between BGLF2 and BBLF1 Is Required for the Efficient Production of Infectious Epstein-Barr Virus Particles.

BGLF2 is a tegument protein of the Epstein-Barr virus (EBV). This study finds that BGLF2 is expressed in the late stage of the EBV lytic cycle. Microscopic investigations reveal that BGLF2 is present in both the nucleus and the cytoplasm and colocalized with BBLF1 and gp350 at juxtanuclear regions in the cytoplasm. This study also finds that the basic KKK69 motif of BGLF2 and acidic DYEE31 motif of BBLF1 are crucial for the interaction between BGLF2 and BBLF1, which is required for the recruitment of BGLF2 to the BBLF1 that is anchored on the trans-Golgi-network (TGN). In addition, BGLF2 in a density gradient is co-sedimented with un-enveloped capsids, revealing that BGLF2 associates with the EBV capsid before the final envelopment. The knockout of BGLF2 expression is demonstrated to reduce the numbers of infectious virions that are released into the culture medium, but they do not affect the expression of lytic proteins and viral DNA replication. The production of infectious viral particles by a BGLF2-knockout mutant can be rescued by exogenously expressed BGLF2 but only partially rescued by BGLF2-3KA, which is a mutant with reduced ability to interact with BBLF1 but does not affect its ability to activate the MAPK pathway and the expression of the EBV lytic proteins, suggesting that the interaction of BGLF2 with BBLF1 is important to the efficient production of infectious viral particles during the maturation. The results of this study improve our understanding of how BGLF2 promotes EBV viral production.

BGLF2 Increases Infectivity of Epstein-Barr Virus by Activating AP-1 upon De Novo Infection.

Epstein-Barr virus (EBV) is a human gammaherpesvirus that causes infectious mononucleosis and several malignancies, such as endemic Burkitt lymphoma and nasopharyngeal carcinoma. Herpesviruses carry genes that can modify cell functions, including transcription and ubiquitination, thereby facilitating viral growth and survival in infected cells. Using a reporter screening system, we revealed the involvement of several EBV gene products in such processes. Of these, BGLF2 activated the AP-1 signaling pathway through phosphorylation of p38 and c-Jun N-terminal kinase (JNK). Knockout of the BGLF2 gene did not affect viral gene expression and viral genome DNA replication, but resulted in marked reduction of progeny titer. We also found that the BGLF2 disruption resulted in significant loss of infectivity upon de novo infection. Interestingly, expression of a binding partner, BKRF4, repressed the activation of AP-1 by BGLF2. These results shed light on the physiological role of the tegument protein BGLF2.IMPORTANCE Epstein-Barr virus (EBV), an oncogenic gammaherpesvirus, carries ~80 genes. While several genes have been investigated extensively, most lytic genes remain largely unexplored. Therefore, we cloned 71 EBV lytic genes into an expression vector and used reporter assays to screen for factors that activate signal transduction pathways, viral and cellular promoters. BGLF2 activated the AP-1 signaling pathway, likely by interacting with p38 and c-Jun N-terminal kinase (JNK), and increased infectivity of the virus. We also revealed that BKRF4 can negatively regulate AP-1 activity. Therefore, it is suggested that EBV exploits and modifies the AP-1 signaling pathway for its replication and survival.

Molecular Characterization of the Epstein-Barr Virus BGLF2 Gene, its Expression, and Subcellular Localization.

BACKGROUND: Epstein-Barr virus (EBV) is a universal herpes virus which can cause a life-long and largely asymptomatic infection in the human population. However, the exact pathogenesis of the EBV infection is not well known. OBJECTIVE: A comprehensive bioinformatics prediction was carried out for investigating the molecular properties of the BGLF2 and to afford a foundation for future research of the role and instrument of BGLF2 in the course of EBV infection. MATERIALS AND METHODS: A 1011-base-pair sequence of BGLF2 gene from the Epstein-Barr virus (EBV) Akata strain genome was amplified using polymerase chain reaction and was further characterized by cloning, sequencing, and subcellular localization in the COS-7 cells. RESULTS: The bioinformatics analysis demonstrated that EBV BGLF2 gene encodes a putative BGLF2 polypeptide which contains a conservative Herpes_UL16 domain. It was established that the polypeptide shows a close relationship with the Herpes UL16 tegument protein family and is extremely conserved among its homologues proteins encoded by UL16 genes. Multiple sequence alignments of the nucleic acid and amino acid sequence showed that the gene product of EBV BGLF2 contains a comparatively higher homology with the BGLF2-like proteins of the subfamily Gammaherpesvirinae than that of other subfamilies of the herpes virus. Moreover, the phylogenetic analyses suggested that EBV BGLF2 has a close genetic relationship with the member of Gammaherpesvirinae; in particular with the members of Cercopithecine herpesvirus 15 and Callitrichine herpesvirus 3. An antigen epitope analysis indicated that BGLF2 contains several potential B-cell epitopes. In addition, the secondary structure, as well as the three dimensional structure prediction suggests that BGLF2 consists of the both α-helix and ß-strand. Besides, the subcellular localization prediction revealed that BGLF2 localizes in both nucleus and cytoplasm. CONCLUSIONS: Illustrating the relevance of the molecular properties and genetic evolution of EBV, BGLF2 will offer the perspectives for further study on the role and mechanism of the BGLF2 in course of EBV infection. These works will also conduct our understanding of the EBV at the molecular level as well as enriching the herpesvirus database.