Molecular Basis for Ebolavirus VP35 Suppression of Human Dendritic Cell Maturation

Ebola virus (EBOV) causes Filoviruses cause a severe hemorrhagic fever in humans and nonhuman primates with a mortality in humans of up to 90% (1). Filoviruses include five species, Zaire ebolavirus, Bundibugyo ebolavirus, Sudan ebolavirus, Taï Forest ebolavirus, and Reston ebolavirus, belonging to the genus Ebolavirus, and a single species, Marburg marburgvirus, belonging to the genus Marburgvirus (2). Filovirus outbreaks occur in Central Africa regularly; in 2012, four filovirus outbreaks occurred in Uganda and the Democratic Republic of the Congo (3), and a new outbreak with a previously unknown clade of Ebola virus (EBOV), which belongs to the species Zaire ebolavirus, started in Guinea in the winter of 2013-2014 (4) and spread to Liberia, Sierra Leone, Nigeria, Senegal, Mali, Spain, the United States, and the United Kingdom (5). Because of the extremely high mortality of the disease caused by filoviruses and the lack of approved vaccine and treatments, work with these viruses is performed under the biosafety level 4 (BSL-4) biocontainment.EBOV causes a severe immunosuppression in both humans and nonhuman primate models, characterized by a deficient T cell response, lymphopenia, and T cell apoptosis, despite the lack of infection of T cells (6)(7)(8)(9)(10)(11)(12). On the other hand, dendritic cells (DC), which are the most effective antigen-presenting cells and are infected by EBOV, do not undergo normal maturation despite the infection (13-15). Because DC play a key role in initiation of the adaptive immune response by processing viral antigens and presenting them to naive lymphocytes, the deficient T cell response may be linked to the deficient and/or aberrant stimulation of T cells by the infected DC.EBOV has two proteins, VP35 and VP24, which antagonize the

Section: Discussioncontrasting

confidence: 51%

Different Temporal Effects of Ebola Virus VP35 and VP24 Proteins on Global Gene Expression in Human Dendritic Cells

Ilinykh

Lubaki

Widen

et al. 2015

“…Replication-defective lentiviruses were generated as previously described (31,35). Expression plasmids used were pHCMV-G encoding VSV glycoprotein, packaging plasmid pNL4.3-gagpol and derivatives of pHR-SIN-CSIGW.…”

Section: Isolation Of Human Mddcs and Naive T Cells Human Mddcs Werementioning

confidence: 99%

“…These observations are likely relevant to in vivo infection since DCs have been demonstrated to be targets of infection in animal models and in infected humans. EBOV IFN-antagonist proteins have been implicated as potentially mediating this suppression (28)(29)(30)(31). Expression of eVP35 in monocyte-derived DCs (MDDCs) is sufficient to disrupt not only IFN-␣/␤ production but also production of proinflammatory cytokines, upregulation of cell surface expression of DC maturation markers, and activation of T cells following infection of the cells with RIG-I activator Sendai virus (SeV) or MDA5 activator encephalomyocarditis virus (EMCV) (31).…”

mentioning

confidence: 99%

“…EBOV IFN-antagonist proteins have been implicated as potentially mediating this suppression (28)(29)(30)(31). Expression of eVP35 in monocyte-derived DCs (MDDCs) is sufficient to disrupt not only IFN-␣/␤ production but also production of proinflammatory cytokines, upregulation of cell surface expression of DC maturation markers, and activation of T cells following infection of the cells with RIG-I activator Sendai virus (SeV) or MDA5 activator encephalomyocarditis virus (EMCV) (31). Infection of DCs with recombinant EBOVs bearing mutations that disrupt VP35 dsRNA-binding and RLR inhibition results in significant DC maturation, definitively linking eVP35 to DC suppression in the context of EBOV infection (28).…”

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confidence: 99%

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Effects of Filovirus Interferon Antagonists on Responses of Human Monocyte-Derived Dendritic Cells to RNA Virus Infection

Yen

Basler

2016

Self Cite

Dendritic cells (DCs) are major targets of filovirus infection in vivo. Previous studies have shown that the filoviruses Ebola virus (EBOV) and Marburg virus (MARV) suppress DC maturation in vitro.Both viruses also encode innate immune evasion functions. The EBOV VP35 (eVP35) and the MARV VP35 (mVP35) proteins each can block RIG-I-like receptor signaling and alpha/ beta interferon (IFN-␣/␤) production. The EBOV VP24 (eVP24) and MARV VP40 (mVP40) proteins each inhibit the production of IFN-stimulated genes (ISGs) by blocking Jak-STAT signaling; however, this occurs by different mechanisms, with eVP24 blocking nuclear import of tyrosine-phosphorylated STAT1 and mVP40 blocking Jak1 function. MARV VP24 (mVP24) has been demonstrated to modulate host cell antioxidant responses. Previous studies demonstrated that eVP35 is sufficient to strongly impair primary human monocyte-derived DC (MDDC) responses upon stimulation induced through the RIG-I-like receptor pathways. We demonstrate that mVP35, like eVP35, suppresses not only IFN-␣/␤ production but also proinflammatory responses after stimulation of MDDCs with RIG-I activators. In contrast, eVP24 and mVP40, despite suppressing ISG production upon RIG-I activation, failed to block upregulation of maturation markers or T cell activation. mVP24, although able to stimulate expression of antioxidant response genes, had no measurable impact of DC function. These data are consistent with a model where filoviral VP35 proteins are the major suppressors of DC maturation during filovirus infection, whereas the filoviral VP24 proteins and mVP40 are insufficient to prevent DC maturation. Zaire ebolavirus (EBOV) and Marburg marburgvirus (MARV) belong to the Filovirus family of emerging, zoonotic negativesense RNA viruses. Both EBOV and MARV cause severe, frequently fatal disease in humans (1). The West African Ebola epidemic, which was first recognized in early 2014, is the largest filovirus outbreak on record with over 28,000 cases worldwide and a reported case fatality rate of approximately 40% (2). The severity of EBOV and MARV infections reflects, at least in part, robust systemic virus replication that is not effectively restrained by host immune responses (3).Both EBOV and MARV encode multiple proteins that impair innate antiviral immune responses and likely contribute to excessive virus replication (3, 4). Both viruses encode VP35, a doublestranded RNA (dsRNA)-binding protein that carries out multiple functions critical for virus propagation. One such function is the inhibition of signaling by RIG-I and MDA5 through (RLR) signaling, which otherwise triggers alpha/beta interferon (IFN-␣/␤) gene expression (5-13). For both EBOV VP35 (eVP35) and MARV VP35 (mVP35), point mutations that disrupt VP35 dsR-NA-binding activity also greatly impair RLR inhibition activity (5,9,14,15). In vitro studies suggest that dsRNA-binding allows eVP35 to sequester RIG-I stimulatory dsRNAs (8, 9). eVP35 also interacts with the cellular protein PACT, which binds to and facilitates activatio...

“…In addition to VP35's critical role in virus replication as a cofactor of the viral polymerase, it has also been extensively studied for its function in inhibition of innate signaling pathways and expression of antiviral type I interferons (IFN-I) (10,11,(14)(15)(16)(17)(18)(19)(20). VP35 also inhibits the maturation of dendritic cells (DCs) and prevents efficient adaptive immune responses (21)(22)(23)(24)(25).…”

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confidence: 99%

The Host E3-Ubiquitin Ligase TRIM6 Ubiquitinates the Ebola Virus VP35 Protein and Promotes Virus Replication

Bharaj

Atkins

Luthra

et al. 2017

Self Cite

Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection.IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35 protein contributes to pathogenesis, because it serves as an essential cofactor of the viral polymerase as well as a potent antagonist of innate immunity. However, how VP35 function is regulated by host cellular factors is poorly understood. Here, we report that the host E3-ubiquitin ligase TRIM6 promotes VP35 ubiquitination and is important for efficient virus replication. Therefore, our study identifies a new host factor, TRIM6, as a potential target in the development of antiviral drugs against EBOV.KEYWORDS TRIM6, tripartite motif (TRIM) protein, VP35, viral RNA polymerase, Ebola virus, innate immunity, ubiquitination, unanchored ubiquitin, virus-host interactions