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Epstein-Barr virus large tegument protein BPLF1 contributes to innate immune evasion through interference with toll-like receptor signaling.

ABSTRACT: Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-?B at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-?B activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts.

SUBMITTER: van Gent M

PROVIDER: S-EPMC3930590 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Epstein-Barr virus large tegument protein BPLF1 contributes to innate immune evasion through interference with toll-like receptor signaling.

van Gent Michiel M Braem Steven G E SG de Jong Annemieke A Delagic Nezira N Peeters Janneke G C JG Boer Ingrid G J IG Moynagh Paul N PN Kremmer Elisabeth E Wiertz Emmanuel J EJ Ovaa Huib H Griffin Bryan D BD Ressing Maaike E ME

PLoS pathogens 20140220 2

Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV ...[more]

PMID: 24586164

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Project description:Macroautophagy/autophagy plays an important role in the control of viral infections and viruses have evolved multiple strategies to interfere with autophagy to avoid destruction and promote their own replication and spread. Here we report that the deubiquitinase encoded in the N-terminal domain of the Epstein-Barr virus (EBV) large tegument protein, BPLF1, regulates selective autophagy. Mass spectrometry analysis identified several vesicular traffic and autophagy related proteins as BPLF1 interactors and potential substrates, suggesting that the viral protein targets this cellular defense during productive infection. Direct binding of BPLF1 to the autophagy receptor SQSTM1/p62 (sequestosome 1) was confirmed by co-immunoprecipitation of transfected BPLF1 and by in vitro affinity isolation of bacterially expressed proteins. Expression of the catalytically active BPLF1 was associated with decreased SQSTM1/p62 ubiquitination and failure to recruit LC3 to SQSTM1/p62-positive aggregates. Selective autophagy was inhibited as illustrated by the accumulation of large protein aggregates in BPLF1-positive cells co-transfected with an aggregate-prone HTT (huntingtin)-Q109 construct, and by a slower autophagy-dependent clearance of protein aggregates upon transfection of BPLF1 in cells expressing a tetracycline-regulated HTT-Q103. The inhibition of aggregate clearance was restored by overexpression of a SQSTM1/p62[E409A,K420R] mutant that does not require ubiquitination of Lys420 for cargo loading. These findings highlight a previously unrecognized role of the viral deubiquitinase in the regulation of selective autophagy, which may promote infection and the production of infectious virus.Abbreviations: BPLF1, BamH1 fragment left open reading frame-1; EBV, Epstein-Barr virus; GFP, green fluorescent protein; HTT, huntingtin; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; PB1, Phox and Bem1 domain; PE, phosphatidylethanolamine; SQSTM1/p62, sequestosome 1; UBA, ubiquitin-associated domain.

Project description:BackgroundMonocytes/macrophages are activated in several autoimmune diseases, including systemic sclerosis (scleroderma; SSc), with increased expression of interferon (IFN)-regulatory genes and inflammatory cytokines, suggesting dysregulation of the innate immune response in autoimmunity. In this study, we investigated whether the lytic form of Epstein-Barr virus (EBV) infection (infectious EBV) is present in scleroderma monocytes and contributes to their activation in SSc.MethodsMonocytes were isolated from peripheral blood mononuclear cells (PBMCs) depleted of the CD19+ cell fraction, using CD14/CD16 negative-depletion. Circulating monocytes from SSc and healthy donors (HDs) were infected with EBV. Gene expression of innate immune mediators were evaluated in EBV-infected monocytes from SSc and HDs. Involvement of Toll-like receptor (TLR)8 in viral-mediated TLR8 response was investigated by comparing the TLR8 expression induced by infectious EBV to the expression stimulated by CL075/TLR8/agonist-ligand in the presence of TLR8 inhibitor in THP-1 cells.ResultsInfectious EBV strongly induced TLR8 expression in infected SSc and HD monocytes in vitro. Markers of activated monocytes, such as IFN-regulated genes and chemokines, were upregulated in SSc- and HD-EBV-infected monocytes. Inhibiting TLR8 expression reduced virally induced TLR8 in THP-1 infected cells, demonstrating that innate immune activation by infectious EBV is partially dependent on TLR8. Viral mRNA and proteins were detected in freshly isolated SSc monocytes. Microarray analysis substantiated the evidence of an increased IFN signature and altered level of TLR8 expression in SSc monocytes carrying infectious EBV compared to HD monocytes.ConclusionThis study provides the first evidence of infectious EBV in monocytes from patients with SSc and links EBV to the activation of TLR8 and IFN innate immune response in freshly isolated SSc monocytes. This study provides the first evidence of EBV replication activating the TLR8 molecular pathway in primary monocytes. Immunogenicity of infectious EBV suggests a novel mechanism mediating monocyte inflammation in SSc, by which EBV triggers the innate immune response in infected cells.

Project description:Interferon alpha (IFN-?) and IFN-? 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-?. 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-? blocked BZLF1 expression and EBV reactivation; however, expression of BGLF2 reduced the ability of IFN-? 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-? 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.

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Epstein-Barr virus large tegument protein BPLF1 contributes to innate immune evasion through interference with toll-like receptor signaling.

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Epstein-Barr virus large tegument protein BPLF1 contributes to innate immune evasion through interference with toll-like receptor signaling.

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