DEN replication is reduced by MPYS but not MITA.

<p>(A) A549 cells infected with DEN-2 (MOI 5) for various times were harvested for western blot analysis. Immunoblotting was done with antibodies against pIRF3, IRF3, DEN NS3, RIG-I, MAVS, MITA, and actin as indicated. The band density was quantified with ImageJ and the relative ratios of the indicated proteins are shown. (B) A549 cells infected with DEN-2 for 30 h with the indicated MOI were harvested for western blot analysis. (C) Immunoblotting of A549 stable cell lines expressing HA-MITA-V5, HA-MPYS-V5, or HA-GFP control infected with DEN-2 (MOI 10) for various times. The relative ratios of pIRF3/IRF3 and DEN-2 NS3/actin were analyzed as described in panel A. The positions of full-length MITA and the cleaved MITA are indicated by black arrows and open arrows, respectively. (D) IFNβ mRNA expression levels in A549 cells with GFP, MITA or MPYS overexpression were quantified by RT-qPCR after DEN-2 infection for 24 h. (E) The conditioned medium collected from DEN-2-infected cell lines expressing GFP, MITA or MPYS was analyzed for antiviral activity against IFN-sensitive dSinF-Luc/2A as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002780#s4" target="_blank">Materials and Methods</a>. (F) DEN-2 virus production from A549 cells with GFP, MITA or MPYS overexpression was determined by plaque forming assays at 24, 36, and 48 h post infection. The data in panels D, E, and F are mean and SD (<i>n = 3</i> per group), and were compared by two-tailed Student's <i>t</i> test.</p

Mapping of the dengue protease cleavage site of MITA.

<p>(A) Schematic diagram and summarized properties of MITA constructs. Constructs were N-terminal HA- and C-terminal V5-tagged and are numbered according to the amino acid residues. The potential cleavage site LRRG in human MITA and the corresponding sequence IHCM in murine MPYS are indicated. (B) A549 cells were transfected with the full-length or deletion constructs of MITA with or without the Flag-tagged dengue NS2B3. Transfectants were harvested for immunoblotting with antibodies indicated at the right. The positions for full-length MITA are indicated by black arrows and the cleaved MITA by open arrows. (C) A549 cells were cotransfected with Vip-Luc (0.2 µg), IRF3/pCR3.1 (0.3 µg), pRL-TK (0.1 µg), plus GFP control or the indicated MITA constructs (0.4 µg) for 24 h. The cells were harvested and analyzed by dual-luciferase assay. The relative normalized luciferase activities are expressed as mean and SD (<i>n = 3</i> per group), and were compared by two-tailed Student's <i>t</i> test. (D) Immunoblotting of A549 cells cotransfected with DEN-2 NS2B3 plus the indicated constructs of MITA or MPYS for 24 h. (E) Dual-luciferase assay of A549 cells cotransfected with Vip-Luc (0.15 µg), IRF3/pCR3.1 (0.15 µg), pRL-TK (0.05 µg), plus the wild-type or S135A-mutated dengue NS2B3 (0.35 µg) with MITA or MPYS (0.3 µg) for 24 h. GFP was used as the negative control. The cells were harvested and analyzed by dual-luciferase assay. Data are expressed as mean and SD (<i>n = 3</i> per group), and were compared by two-tailed Student's <i>t</i> test.</p

Dengue protease NS2B3 interacts with MITA.

<p>(A) Immunoprecipitation and western blot analysis (IP-WB) of 293T/17 cells cotransfected with dengue NS2B3 (WT or S135A-mutated) plus MITA or MPYS for 18 h with the indicated antibodies. (B) IP-WB analysis of 293T/17 cells cotransfected with S135A-mutated NS2B3 and MITA for 48 h, then the cells were treated with different doses of poly(dA:dT) or poly(I:C) as indicated for 18 h. (C) Western blot analysis of A549 stable cell lines overexpressing dengue NS2B3(WT) plus MITA or MPYS treated with poly(dA:dT) or poly(I:C) (0, 0.5, and 1 µg/ml) for 24 h with antibody against V5-tag. Black arrow, full-length MITA; open arrow, cleaved MITA. A549 stable cell lines overexpressing dengue NS2B3 (WT or S135A) were stimulated with poly(dA:dT) (0.5 µg/ml) for 24 h and then analyzed by western blotting (D) and by IP-WB (E) analysis with the indicated antibodies. Black arrow indicates the full-length endogenous MITA.</p

Silencing MITA/MPYS attenuates host antiviral signaling.

<p>(A) Human A549 cells stably expressing shRNA targeting control <i>LacZ</i> or <i>MITA</i> were infected with DEN-2 (MOI 0.1 and 10) for various times. Immunoblotting was performed with antibodies against DEN NS3, pIRF3, IRF3, RIG-I, MAVS, MITA, and actin. (B) The conditioned medium collected from mock or DEN-2-infected (MOI 10, 24 h p.i.) iLacZ or iMITA cells was analyzed for antiviral activity against IFN-sensitive dSinF-Luc/2A as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002780#s4" target="_blank">Materials and Methods</a>. Data are expressed as mean and SD (<i>n = 3</i> per group), and were compared by two-tailed Student's <i>t</i> test. (C and D) Murine Hepa 1–6 cells stably expressing shRNA targeting control <i>LacZ</i> or <i>MPYS</i> were infected with DEN-2 (MOI 5) for 24 h. Cell lysates were analyzed by western blotting with indicated antibodies (C) and culture supernatants were harvested for DEN-2 virus titration by plaque forming assays (<i>n</i> = 3) (D).</p

MITA is targeted by dengue protease.

<p>(A) N-terminal Flag-tagged RIG-I, MITA, or MAVS was cotransfected with dengue NS2B3 (DNS2B3) in 293T/17 (lanes 1 and 2) or A549 (lanes 3–6) cells. Cells were harvested for western blotting at 18 h post transfection with the indicated antibodies. Molecule weight (kDa) markers are shown on the sides. The position for full-length MITA is indicated by a black arrow and for the cleaved MITA by an open arrow. (B) A549 cells were cotransfected with HA-MITA-V5 plus the viral protease encoded by JEV or DEN-2 (WT and S135A) and analyzed by immunoblotting with antibodies against HA-tag and NS3. (C) A549 cells stably expressing HA-MITA-V5 were infected with the indicated virus (MOI 5) for 24 h and then analyzed by immunoblotting with antibodies against HA-tag, NS3, and actin. (D) A549 cells were cotransfected with HA-MITA-V5 (0.3 µg), Vip-Luc (0.15 µg), IRF3/pCR3.1 (0.15 µg), pRL-TK (0.05 µg), and different doses of WT or S135A-mutated dengue NS2B3 (0.3, 0.45, or 0.6 µg) for 24 h. GFP plasmid was used as transfection plasmid control. The cell lysates were harvested and analyzed by dual-luciferase assay. <i>Firefly</i> luciferase activity was normalized to that of <i>Renilla</i> luciferase. The relative luciferase activity to that of cotransfection of GFP plus MITA was calculated. Data are expressed as mean and SD (<i>n = 3</i> per group), and were compared by two-tailed Student's <i>t</i> test. (E) Quantification of endogenous IFNβ mRNA levels by RT-qPCR. The WT and S135A NS2B3-expressing A549 cells were transfected with MITA or GFP for 24 h and harvested for RT-qPCR of IFNβ and actin. Data are expressed as mean and SD (<i>n = 3</i> per group), and were compared by two-tailed Student's <i>t</i> test. (F) Vero cells were pretreated with culture medium derived from NS2B3-expressing A549 cells transfected with MITA or GFP as indicated. The conditioned Vero cells were infected with dSinF-Luc/2A (500 pfu/well) for 24 h and then harvested for luciferase assay. Data are expressed as mean and SD (<i>n = 3</i> per group), and were compared by two-tailed Student's <i>t</i> test.</p

DEN antagonizes MITA-mediated antiviral signaling.

<p>Activated MITA translocates from ER to associate with Sec5 translocon complex, and then reaches the cytoplasmic punctate structures to assemble with TBK1. This activation process leads to phosphorylation and translocation of IRF3, and then induces antiviral IFN production. DEN-encoded protease NS2B3 targets human MITA at LRR↓⁹⁶G but not the murine homologue MPYS for cleavage, thus subverts the MITA-triggered antiviral signaling.</p

Japanese Encephalitis Virus Nonstructural Protein NS5 Interacts with Mitochondrial Trifunctional Protein and Impairs Fatty Acid β-Oxidation

<div><p>Infection with Japanese encephalitis virus (JEV) can induce the expression of pro-inflammatory cytokines and cause acute encephalitis in humans. β-oxidation breaks down fatty acids for ATP production in mitochondria, and impaired β-oxidation can induce pro-inflammatory cytokine expression. To address the role of fatty-acid β-oxidation in JEV infection, we measured the oxygen consumption rate of mock- and JEV-infected cells cultured with or without long chain fatty acid (LCFA) palmitate. Cells with JEV infection showed impaired LCFA β-oxidation and increased interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) expression. JEV nonstructural protein 5 (NS5) interacted with hydroxyacyl-CoA dehydrogenase α and β subunits, two components of the mitochondrial trifunctional protein (MTP) involved in LCFA β-oxidation, and NS5 proteins were detected in mitochondria and co-localized with MTP. LCFA β-oxidation was impaired and higher cytokines were induced in cells overexpressing NS5 protein as compared with control cells. Deletion and mutation studies showed that the N-terminus of NS5 was involved in the MTP association, and a single point mutation of NS5 residue 19 from methionine to alanine (NS5-M19A) reduced its binding ability with MTP. The recombinant JEV with NS5-M19A mutation (JEV-NS5-M19A) was less able to block LCFA β-oxidation and induced lower levels of IL-6 and TNF-α than wild-type JEV. Moreover, mice challenged with JEV-NS5-M19A showed less neurovirulence and neuroinvasiveness. We identified a novel function of JEV NS5 in viral pathogenesis by impairing LCFA β-oxidation and inducing cytokine expression by association with MTP.</p></div

Reduced neurovirulence of NS5-M19A—mutated JEV in challenged mice.

<p>(A) Survival in C57BL/6 mice infected with 0.2, 2 or 20 plaque-forming units (PFU) of JEV-WT or JEV-NS5-M19A by an intracerebral (i.c.) injection. The animal number (<i>n</i>) and survival rate for each group are shown. (B-D) RT-qPCR of relative JEV RNA (B), IL-6 (C), and TNF-α (D) mRNA levels in brain tissues of mice inoculated with JEV-WT or JEV-NS5-M19A (20 PFU) (n = 3). Data are mean±SD.*P < 0.05.</p

Subcellular localization of JEV NS5.

<p>(A-C) Cellular lysates of HEK293T cells infected with JEV (MOI = 5) or transfected with NS5-Flag for 24 h underwent Qproteome Mitochondria Isolation (A) or biochemical fractionation as outlined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004750#ppat.1004750.s005" target="_blank">S5B and S5C Fig</a>, respectively (B and C). Western blot analysis of indicated proteins in cytosolic and crude mitochondrial fractions. C, cytosolic fraction; H, heavy membrane fraction/crude mitochondrial fraction; L, light microsomal membrane fraction. (D) The crude mitochondrial fraction isolated from HEK293T cells infected with JEV (MOI = 3) or transfected with NS5-Flag for 24 h was treated with Proteinase K (100 μg/ml) for 30 min on ice. The reactants were developed by Western blot analysis with antibodies against NS5 or the indicated mitochondrial proteins. (E) Confocal microscopy of pEYFP-Mito-NS5-A549 cells stained with anti-Flag plus Alexa Fluor 568 goat anti-rabbit and anti-HADHα plus Alexa Fluor 647 goat anti-mouse antibody. (F) Confocal microscopy of pEYFP-Mito-NS5-A549 cells transfected with HADHβ-HA for 24 h and stained with anti-Flag plus Alexa Fluor 568 goat anti-rabbit and anti-HA plus Alexa Fluor 647 goat anti-mouse antibody.</p

N-terminus of NS5 is essential for its interaction with MTP.

<p>(A) Schematic diagram and properties of full-length and truncated NS5 constructs. (B and C) IP—Western and Western blot analysis with anti-Flag affinity gel and the indicated antibodies for Flag-tag, V5-tag and HA-tag in HEK293T cells co-transfected with full-length and truncated NS5-Flag plus HADHα-V5-His (B) or HADHβ-HA (C) for 24 h.</p