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

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

<div><h3>Background</h3><p>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.</p> <h3>Methodology/Principal Findings</h3><p>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.</p> <h3>Conclusions/Significance</h3><p>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.</p> </div

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 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

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

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

Genome and Infection Characteristics of Human Parechovirus Type 1: The Interplay between Viral Infection and Type I Interferon Antiviral System

<div><p>Human parechoviruses (HPeVs), members of the family <i>Picornaviridae</i>, are associated with severe human clinical conditions such as gastrointestinal disease, encephalitis, meningitis, respiratory disease and neonatal sepsis. A new contemporary strain of HPeV1, KVP6 (accession no. KC769584), was isolated from a clinical specimen. Full-genome alignment revealed that HPeV1 KVP6 shares high genome homology with the German strain of HPeV1, 7555312 (accession no. FM178558) and could be classified in the clade 1B group. An intertypic recombination was shown within the P2-P3 genome regions of HPeV1. Cell-type tropism test showed that T84 cells (colon carcinoma cells), A549 cells (lung carcinoma cells) and DBTRG-5MG cells (glioblastoma cells) were susceptible to HPeV1 infection, which might be relevant clinically. A facilitated cytopathic effect and increased viral titers were reached after serial viral passages in Vero cells, with viral genome mutation found in later passages. HPeV1 is sensitive to elevated temperature because 39C incubation impaired virion production. HPeV1 induced innate immunity with phosphorylation of interferon (IFN) regulatory transcription factor 3 and production of type I IFN in A549 but not T84 cells. Furthermore, type I IFN inhibited HPeV1 production in A549 cells but not T84 cells; T84 cells may be less responsive to type I IFN stimulation. Moreover, HPeV1-infected cells showed downregulated type I IFN activation, which indicated a type I IFN evasion mechanism. The characterization of the complete genome and infection features of HPeV1 provide comprehensive information about this newly isolated HPeV1 for further diagnosis, prevention or treatment strategies.</p></div

NF-κB activation triggered by LPS and polyI:C is suppressed by DENV-2 infection.

<p>(<b>A</b>) Dual luciferase assay of reporter activity. A549 cells (1×10⁵) transfected with NF-κB-Luc reporter (0.7 µg) and pRL-TK (0.02 µg), were infected with DENV-2 for 24 h and then were stimulated with LPS or polyI:C (both 1 µg/ml) for various times. Data are mean±SD from 3 determinations. *, <i>P</i>≤0.001 (<b>B</b>) The cells lysates collected from cells as described in panel A were subjected for immunoblotting assay to detect the expression of DENV-2 NS3 and actin.</p

DENV-2 infection blocks TLR-triggered IFN-β and cytokine production.

<p>J774A.1 macrophages were mock-infected or infected with DENV-2 (MOI 5) for 24 h before TLR ligand stimulation. qPCR analysis of mRNA expression of IFN-β and IL-10 on cells coincubated with lipopolysaccharide (LPS; 100 ng/ml) (<b>A</b>) or polyI:C (100 µg/ml) (<b>B</b>) for the indicated times. (<b>C</b>) BMDCs were mock-infected or infected with DENV-2 (MOI 5) for 24 h before polyI:C (100 µg/ml) stimulation. qPCR analysis of mRNA expression of IFN-β and IL-10 was analyzed at the indicated times. Normalization was done with the expression level of internal control HPRT. Data are mean±SD of 3 determinations. <b>*</b>, <i>p</i><0.01; **, <i>p</i><0.005.</p