[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Hydroxychloroquine (HCQ) is an antimalarial drug also used in treating autoimmune diseases. Its antiviral activity was demonstrated in restricting HIV infection in vitro; however, the clinical implications remain controversial. Infection with dengue virus (DENV) is a global public health problem, and we lack an antiviral drug for DENV. Here, we evaluated the anti-DENV potential of treatment with HCQ. Immunofluorescence assays demonstrated that HCQ could inhibit DENV serotype 1-4 infection in vitro. RT-qPCR analysis of HCQ-treated cells showed induced expression of interferon (IFN)-related antiviral proteins and certain inflammatory cytokines. Mechanistic study suggested that HCQ activated the innate immune signaling pathways of IFN-β, AP-1, and NFκB. Knocking down mitochondrial antiviral signaling protein (MAVS), inhibiting TANK binding kinase 1 (TBK1)/inhibitor-κB kinase ɛ (IKKɛ), and blocking type I IFN receptor reduced the efficiency of HCQ against DENV-2 infection. Furthermore, HCQ significantly induced cellular production of reactive oxygen species (ROS), which was involved in the host defense system. Suppression of ROS production attenuated the innate immune activation and anti-DENV-2 effect of HCQ. In summary, HCQ triggers the host defense machinery by inducing ROS- and MAVS-mediated innate immune activation against DENV infection and may be a candidate drug for DENV infection.
Full-text · Article · Oct 2014 · Journal of Interferon & Cytokine Research
[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · Aug 2013 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] 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.
Preview · Article · Jul 2012 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: 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↓(96)G 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · Jun 2011 · Cellular Microbiology
[Show abstract][Hide abstract] 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.
Full-text · Article · Mar 2010 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: The unfolded protein response (UPR) is a coordinated change in gene expression triggered by perturbations in functions of
the endoplasmic reticulum (ER). XBP1, a key transcription factor of the UPR, is activated by an IRE1-mediated splicing event,
which results in a frameshift and encodes a protein with transcriptional activity. Here, we report that XBP1 was activated
during flaviviral infection, as evidenced by XBP1 mRNA splicing and protein expression, as well as induction of the downstream genes ERdj4, EDEM1, and p58(IPK) in Japanese encephalitis virus (JEV)- and dengue virus serotype 2 (DEN-2)-infected cells. Reporter systems based on IRE1-mediated
XBP1 splicing were established, and several flaviviral proteins associated with the ER, including glycoproteins and small hydrophobic
membrane-anchored proteins, were found to trigger the splicing event. Notably, nonstructural protein NS2B-3 of DEN-2, but
not of JEV, was a potent inducer of XBP1 splicing through an unclear mechanism(s). Reduction of XBP1 by a small interfering RNA had no effect on cells' susceptibility
to the two viruses but exacerbated the flavivirus-induced cytopathic effects. Overall, flaviviruses trigger the XBP1 signaling
pathway and take advantage of this cellular response to alleviate virus-induced cytotoxicity.
Preview · Article · Jan 2007 · Journal of Virology