Chia-Yi Yu

Dengue Virus Targets the Adaptor Protein MITA to Subvert Host Innate Immunity

Dengue is one of the most important arboviral diseases caused by infection of four serotypes of dengue virus (DEN). We found that activation of interferon regulatory factor 3 (IRF3) triggered by viral infection and by foreign DNA and RNA stimulation was blocked by DEN-encoded NS2B3 through a protease-dependent mechanism. The key adaptor protein in type I interferon pathway, human mediator of IRF3 activation (MITA) but not the murine homologue MPYS, was cleaved in cells infected with DEN-1 or DEN-2 and with expression of the enzymatically active protease NS2B3. The cleavage site of MITA was mapped to LRR↓96G and the function of MITA was suppressed by dengue protease. DEN replication was reduced with overexpression of MPYS but not with MITA, while DEN replication was enhanced by MPYS knockdown, indicating an antiviral role of MITA/MPYS against DEN infection. The involvement of MITA in DEN-triggered innate immune response was evidenced by reduction of IRF3 activation and IFN induction in cells with MITA knockdown upon DEN-2 infection. NS2B3 physically interacted with MITA, and the interaction and cleavage of MITA could be further enhanced by poly(dA:dT) stimulation. Thus, we identified MITA as a novel host target of DEN protease and provide the molecular mechanism of how DEN subverts the host innate immunity.

The Interferon Stimulator Mitochondrial Antiviral Signaling Protein Facilitates Cell Death by Disrupting the Mitochondrial Membrane Potential and by Activating Caspases

ABSTRACT Interferon (IFN) signaling is initiated by the recognition of viral components by host pattern recognition receptors. Dengue virus (DEN) triggers IFN-β induction through a molecular mechanism involving the cellular RIG-I/MAVS signaling pathway. Here we report that the MAVS protein level is reduced in DEN-infected cells and that caspase-1 and caspase-3 cleave MAVS at residue D429. In addition to its well-known function in IFN induction, MAVS is also a proapoptotic molecule that triggers disruption of the mitochondrial membrane potential and activation of caspases. Although different domains are required for the induction of cytotoxicity and IFN, caspase cleavage at residue 429 abolished both functions of MAVS. The apoptotic role of MAVS in viral infection and double-stranded RNA (dsRNA) stimulation was demonstrated in cells with reduced endogenous MAVS expression induced by RNA interference. Even though IFN-β promoter activation was largely suppressed, DEN production was not affected greatly in MAVS knockdown cells. Instead, DEN- and dsRNA-induced cell death and caspase activation were delayed and attenuated in the cells with reduced levels of MAVS. These results reveal a new role of MAVS in the regulation of cell death beyond its well-known function of IFN induction in antiviral innate immunity.

Blocking Double-Stranded RNA-Activated Protein Kinase PKR by Japanese Encephalitis Virus Nonstructural Protein 2A

ABSTRACT Japanese encephalitis virus (JEV) is an enveloped flavivirus with a single-stranded, positive-sense RNA genome encoding three structural and seven nonstructural proteins. To date, the role of JEV nonstructural protein 2A (NS2A) in the viral life cycle is largely unknown. The interferon (IFN)-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) phosphorylates the eukaryotic translation initiation factor 2α subunit (eIF2α) after sensing viral RNA and results in global translation arrest as an important host antiviral defense response. In this study, we found that JEV NS2A could antagonize PKR-mediated growth inhibition in a galactose-inducible PKR-expressing yeast system. In human cells, PKR activation, eIF2α phosphorylation, and the subsequent translational inhibition and cell death triggered by dsRNA and IFN-α were also repressed by JEV NS2A. Moreover, among the four eIF2α kinases, NS2A specifically blocked the eIF2α phosphorylation mediated by PKR and attenuated the PKR-promoted cell death induced by the chemotherapeutic drug doxorubicin. A single point mutation of NS2A residue 33 from Thr to Ile (T33I) abolished the anti-PKR potential of JEV NS2A. The recombinant JEV mutant carrying the NS2A-T33I mutation showed reduced in vitro growth and in vivo virulence phenotypes. Thus, JEV NS2A has a novel function in blocking the host antiviral response of PKR during JEV infection.

Vimentin binding is critical for infection by the virulent strain of Japanese encephalitis virus

Japanese encephalitis virus (JEV), a mosquito‐borne flavivirus, causes acute encephalitis with high mortality in humans. We used a pair of virulent (RP‐9) and attenuated (RP‐2ms) variants of JEV to pull down the cell surface molecules bound with JEV particle; their identities were revealed by LC‐MS/MS analysis. One major protein bound with RP‐9 and weakly with RP‐2ms was identified as the intermediate filament protein vimentin. Infection of RP‐9 but not that of RP‐2ms was blocked by anti‐vimentin antibodies and by recombinant‐expressed vimentin proteins. Knockdown of vimentin expression reduced the levels of viral binding and viral production of RP‐9, but not that of RP‐2ms. The different vimentin dependency for JEV infection could be attributed to the major structural envelope protein, as the recombinant RP‐9 with an E‐E138K mutation became resistant to anti‐vimentin blockage. Furthermore, RP‐2ms mainly depended on cell surface glycosaminoglycans for viral binding and it became vimentin‐dependent only when binding to glycosaminoglycans was blocked. Thus, we suggest that vimentin contributes to virulent JEV infection and might be a new target to intervene in this deadly infection.

Dengue Virus Impairs Mitochondrial Fusion by Cleaving Mitofusins

Mitochondria are highly dynamic subcellular organelles participating in many signaling pathways such as antiviral innate immunity and cell death cascades. Here we found that mitochondrial fusion was impaired in dengue virus (DENV) infected cells. Two mitofusins (MFN1 and MFN2), which mediate mitochondrial fusion and participate in the proper function of mitochondria, were cleaved by DENV protease NS2B3. By knockdown and overexpression approaches, these two MFNs showed diverse functions in DENV infection. MFN1 was required for efficient antiviral retinoic acid-inducible gene I–like receptor signaling to suppress DENV replication, while MFN2 participated in maintaining mitochondrial membrane potential (MMP) to attenuate DENV-induced cell death. Cleaving MFN1 and MFN2 by DENV protease suppressed mitochondrial fusion and deteriorated DENV-induced cytopathic effects through subverting interferon production and facilitating MMP disruption. Thus, MFNs participate in host defense against DENV infection by promoting the antiviral response and cell survival, and DENV regulates mitochondrial morphology by cleaving MFNs to manipulate the outcome of infection.

Dengue Virus Serotype 2 Blocks Extracellular Signal-Regulated Kinase and Nuclear Factor-κB Activation to Downregulate Cytokine Production

Background Dengue virus (DENV) infection is the most common mosquito-borne viral disease threatening human health around the world. Type I interferon (IFN) and cytokine production are crucial in the innate immune system. We previously reported that DENV serotype 2 (DENV-2) induced low levels of interferon regulatory factor 3 and NF-κB activation, thus leading to reduced production of IFN-β in the early phase of infection. Here, we determined whether DENV infection not only hampers type I IFN activation but also cytokine production triggered by Toll-like receptor (TLR) signaling. Methodology/Principal Findings We used quantitative RT-PCR and found that only low levels of IFN-β and inflammatory cytokines such as interleukin 10 (IL-10), IL-12 and tumor necrosis factor α (TNFα) mRNA were detected in DENV-2–infected bone-marrow–derived dendritic cells. Furthermore, DENV-2 infection repressed cytokine production triggered by TLR signaling. To elucidate the molecular mechanisms underlying this suppression event, we measured NF-κB activation by p65 nuclear translocation and luciferase reporter assay and found that NF-κB activation triggered by TLR ligands was blocked by DENV-2 infection. As well, extracellular signal-regulated kinase (ERK) activity was suppressed by DENV-2 infection. Conclusions/Significance To downregulate the host innate immunity, DENV-2 by itself is a weak inducer of type I IFN and cytokines, furthermore DENV-2 can also block the TLR-triggered ERK–NF-κB activation and cytokine production.

Ubiquitin-Specific Protease 13 Regulates IFN Signaling by Stabilizing STAT1

The IFN immune system comprises type I, II, and III IFNs, signals through the JAK-STAT pathway, and plays central roles in host defense against viral infection. Posttranslational modifications such as ubiquitination regulate diverse molecules in the IFN pathway. To search for the deubiquitinating enzymes (DUBs) involved in the antiviral activity of IFN, we used RNA interference screening to identify a human DUB, ubiquitin-specific protease (USP) 13, whose expression modulates the antiviral activity of IFN-α against dengue virus serotype 2 (DEN-2). The signaling events and anti–DEN-2 activities of IFN-α and IFN-γ were reduced in cells with USP13 knockdown but enhanced with USP13 overexpression. USP13 may regulate STAT1 protein because the protein level and stability of STAT1 were increased with USP13 overexpression. Furthermore, STAT1 ubiquitination was reduced in cells with USP13 overexpression and increased with USP13 knockdown regardless of with or without IFN-α treatment. Thus, USP13 positively regulates type I and type II IFN signaling by deubiquitinating and stabilizing STAT1 protein. Overall, to our knowledge, USP13 is the first DUB identified to modulate STAT1 and play a role in the antiviral activity of IFN against DEN-2 replication.

SUMO Modification Stabilizes Dengue Virus Nonstructural Protein 5 To Support Virus Replication

ABSTRACT Small ubiquitin-like modifier (SUMO) participates in a reversible posttranslational modification process (SUMOylation) that regulates a wide variety of cellular processes and plays important roles for numerous viruses during infection. However, the roles of viral protein SUMOylation in dengue virus (DENV) infection have not been elucidated. In this study, we found that the SUMOylation pathway was involved in the DENV life cycle, since DENV replication was reduced by silencing the cellular gene Ubc9, which encodes the sole E2-conjugating enzyme required for SUMOylation. By in vivo and in vitro SUMOylation assays, the DENV NS5 protein was identified as an authentic SUMO-targeted protein. By expressing various NS5 mutants, we found that the SUMO acceptor sites are located in the N-terminal domain of NS5 and that a putative SUMO-interacting motif (SIM) of this domain is crucial for its SUMOylation. A DENV replicon harboring the SUMOylation-defective SIM mutant showed a severe defect in viral RNA replication, supporting the notion that NS5 SUMOylation is required for DENV replication. SUMOylation-defective mutants also failed to suppress the induction of STAT2-mediated host antiviral interferon signaling. Furthermore, the SUMOylation of NS5 significantly increased the stability of NS5 protein, which could account for most of the biological functions of SUMOylated NS5. Collectively, these findings suggest that the SUMOylation of DENV NS5 is one of the mechanisms regulating DENV replication. IMPORTANCE SUMOylation is a common posttranslational modification that regulates cellular protein functions but has not been reported in the proteins of dengue virus. Here, we found that the replicase of DENV, nonstructural protein 5 (NS5), can be SUMOylated. It is well known that providing RNA-dependent RNA polymerase activity and antagonizing host antiviral IFN signaling are a “double indemnity” of NS5 to support DENV replication. Without SUMOylation, NS5 fails to maintain its protein stability, which consequently disrupts its function in viral RNA replication and innate immunity antagonism. DENV threatens billions of people worldwide, but no licensed vaccine or specific therapeutics are currently available. Thus, our findings suggest that rather than specifically targeting NS5 enzyme activity, NS5 protein stability is a novel drug target on the growing list of anti-DENV strategies.