IFN signaling and mechanisms of evasion by WNV.

<p>(A) Infection by WNV produces dsRNA intermediates within the cytoplasm that display motifs recognized by the RIG-I and MDA5 helicases. Binding of viral RNA promotes an interaction with IPS-1 that results in recruitment of signaling proteins (e.g., NEMO and TRAF3) that lead to activation of IRF-3 and NF-κB. These factors translocate to the nucleus and bind to the promoter region of the IFN-β gene, leading to transcription and translation. In some cells, TLR3 and TLR7 in endosomes recognize dsRNA and ssRNA motifs, leading to recruitment of cytoplasmic adaptor molecules (MyD88 and TRIF), which initiate signaling cascades that activate IRF-3, IRF-7, and NF-κB, resulting in IFN-β gene transcription. Mechanisms of evasion by WNV include the following: (a) reduction in IFN-β gene transcription by the viral NS2A protein; (b) impairment of RIP-1 signaling by high mannose carbohydrates on the structural E protein and attenuation of TLR3 signaling by NS1; and (c) a delay in recognition of WNV RNA by RIG-I. (B) Secretion of IFN by WNV-infected cells results in autocrine and paracrine signaling through the heterodimeric receptor for IFN-α and β (IFNAR). Binding by IFN results in activation and tyrosine phosphorylation of JAK family members (JAK1 and Tyk2) and the cytoplasmic tail of the IFN-αβR. This promotes recruitment of the STAT1 and STAT2, which themselves become phosphorylated by the JAKs. Phosphorylated STAT1 and STAT2 proteins heterodimerize, associate with IRF-9, and translocate to the nucleus, where they bind IFN-stimulated response element (ISRE) sequences to induce expression of hundreds of ISGs. Mechanisms of evasion by WNV include (d) blockade of phosphorylation of Tyk2 and JAK1 by NS5; (e) down-regulation of the IFNAR through virus-induced redistribution of cellular cholesterol; and (f) attenuation of STAT signaling by NS4B.</p

The IFN-β response in mDC and MEF is IPS-1-dependent but MyD88-independent.

<p>mDC and MEF generated from wild type, IPS-1^−/− and MyD88^−/−, mice were infected at an MOI of 0.1 and levels of (A, C, and E) IFN-α and (B, D, and F) IFN-β mRNA were quantified by qRT-PCR. Values are an average of duplicate samples generated from three independent experiments. Asterisks indicate values that are statistically significant (***, P<0.0001).</p

Survival and viral burden analysis in mice.

<p>A. Eight to 12 week-old C57BL/6 mice were inoculated with 10² PFU of WNV by footpad injection and followed for mortality for 21 days. Survival differences were statistically significant between immunodeficient and wild type mice (n = 11, IFN-αβR^−/−; n = 20, DKO; and n = 20, wild type mice, P<0.0001). Average survival time between IFN-αβR^−/− (3.5 days) and DKO (6 days) mice was also statistically different (P<0.001). B–G. Viral burden in peripheral and CNS tissues after WNV infection. WNV RNA in (B) serum and (C) draining lymph node, and infectious virus in (D) spleen, (E) kidney, (F) brain and (G) spinal cord were determined from samples harvested on the indicated days using qRT-PCR (B and C) or viral plaque assay (D–G). Data is shown as viral RNA equivalents or PFU per gram of tissue for 10 to 12 mice per time point. For all viral load data, the solid line represents the median PFU per gram at the indicated time point, and the dotted line represents the limit of sensitivity of the assay.</p

IRF-3 and IRF-7 restrict WNV infection by regulating the IFN-α/β response and ISG expression in primary cortical neurons.

<p>A. Primary cortical neurons generated from wild type or DKO mice were infected at an MOI of 0.001 and virus production was evaluated at the indicated times by plaque assay. Values are an average of triplicate samples generated from three independent experiments. Asterisks indicate values that are statistically significant (***, P<0.0001). B and C. Levels of (B) IFN-α and (C) IFN-β mRNA in WNV-infected cortical neurons were measured by qRT-PCR as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000607#ppat-1000607-g003" target="_blank">Figure 3</a>. D. Whole cell lysates were generated at the indicated times from wild type or DKO MEF that were uninfected (Un) or infected with WNV (W). Protein levels of ISG54, RIG-I, and MDA5 were examined by immunoblot analysis. The data is the average of at least three independent experiments performed in quadruplicate (***, P<0.0001).</p

Levels of type I IFN levels in serum of wild type and DKO mice infected with WNV.

<p>Mice were inoculated with 10² PFU of WNV by footpad injection and sacrificed at the indicated times. Type I IFN levels were determined from serum collected on days 1 to 4 after WNV infection by an EMCV bioassay in L929 cells. Data reflect averages of serum samples from 5 to 10 mice per time point and the data are expressed as international units (IU) of IFN-α per ml. The specificity of the assay was confirmed with an anti-IFN-αβR neutralizing antibody (data not shown). Asterisks indicate values that are statistically significant (**, P<0.005, *, P<0.05).</p

The WNV-induced IFN-β response and ISG expression is primarily IRF-3 and IRF-7-independent in mDC.

<p>A. mDC generated from wild type, IFN-αβR^−/− and DKO mice were infected at an MOI of 0.001 and virus production was evaluated at the indicated times post infection by plaque assay. Values are an average of quadruplicate samples generated from at least three independent experiments (***, P<0.0001). B–E. Levels of (B) IFN-α and (D) IFN-β mRNA as well as (C) IFN-α and (E) IFN-β protein in WNV-infected mDC were measured by qRT-PCR or ELISA as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000607#ppat-1000607-g003" target="_blank">Figure 3</a>. F. Whole cell lysates were generated at the indicated times from wild type and DKO mDC that were uninfected (Un) or infected with WNV (W). Protein levels of ISG49, ISG54, PKR, STAT1, RIG-I, MDA5 and tubulin were examined by immunoblot analysis.</p

The early phase of the type I IFN regulation in MEF partially involves TRAF3 and TBK1 whereas the late phase requires TRAF6.

<p>TRAF3^−/−, TBK1^−/− and TRAF6^−/− MEF were infected at an MOI of 0.1 and levels of (A and C) IFN-α and (B and D) IFN-β mRNA and secreted protein were measured by qRT-PCR and ELISA. Since basal mRNA expression of the IFN-β gene in uninfected TBK1^−/− and TRAF6^−/− MEF was lower than that observed in congenic wild type cells, for these cells only, we compared IFN-β mRNA levels to the wild type MEF. Values are an average of duplicate samples generated from three independent experiments. Asterisks indicate values that are statistically significant (***, P<0.0001, **, P<0.005, *, P<0.05); n.s. indicates differences that were not statistically significant.</p

Pharmacological inhibition of NF-κB and p38 in wild type and DKO mDC and MEF.

<p>A–B. mDC (A) and MEF (B) generated from wild type and DKO mice were infected at an MOI of 0.1 in the presence of 1% DMSO or increasing concentrations of BAY 11-7082 (in 1% DMSO) and/or increasing concentrations of SB 202190 (in 1% DMSO). Levels of IFN-β mRNA were evaluated at the indicated times post-infection by qRT-PCR. Cell viability was analyzed using a cytotoxicity assay as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000607#s4" target="_blank">Materials and Methods</a>. C. Efficiency of BAY 11-7082 in inhibiting NF-κB transcriptional activity. Wild type and DKO mDC were treated with 1% DMSO or 5 µM and 10 µM of BAY 11-7082 and stimulated with LPS (2 µg/ml) for 24 hours. Levels of secreted TNF-α were measured by ELISA. Values are an average of duplicate samples generated from three independent experiments. Asterisks indicate values that are statistically significant (***, P<0.0001, **, P<0.005, *, P<0.05); n.s. indicates differences that were not statistically significant.</p

IRF-3 and IRF-7 control the IFN-α/β gene induction and ISG expression in MEF.

<p>A–D. MEF generated from wild type or DKO mice were infected at an MOI of 0.1 and analyzed for IFN-α/β gene induction. Total RNA from uninfected and WNV-infected MEF was harvested at the indicated times after infection and levels of (A) IFN-α and (C) IFN-β mRNA were measured by qRT-PCR. Data are normalized to 18S rRNA and are expressed as the relative fold increase over normalized RNA from uninfected controls. Accumulation of (B) IFN-α and (D) IFN-β protein in supernatants was evaluated by ELISA. E. Whole cell lysates were generated at the indicated times from wild type or DKO MEF that were uninfected (Un) or infected with WNV (W). Protein levels of ISG49, ISG54, RIG-I, WNV and tubulin were examined by immunoblot analysis. F. MEF generated from wild type, IFN-αβR^−/− and DKO mice were infected at an MOI of 0.001 and virus production was evaluated by plaque assay. The data is the average of at least three independent experiments performed in quadruplicate, (***, P<0.0001).</p

IRF-3 and IRF-7 partially modulate the IFN-β response and ISG expression in primary Mφ.

<p>A. Mφ generated from wild type, IFN-αβR^−/− and DKO mice were infected at an MOI of 0.01 and virus production was evaluated at the indicated times post infection by plaque assay. Values are an average of quadruplicate samples generated from at least three independent experiments. B. Whole cell lysates were generated at the indicated times from wild type and DKO Mφ that were uninfected (Un) or infected with WNV (W). Protein levels of ISG49, ISG54, PKR, STAT1, RIG-I, MDA5 and tubulin were examined by immunoblot analysis. C and D. The induction of (C) IFN-α and (D) IFN-β mRNA in WNV-infected Mφ was analyzed by qRT-PCR as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000607#ppat-1000607-g003" target="_blank">Figure 3</a>. Asterisks indicate values that are statistically significant (***, P<0.0001, **, P<0.005, *, P<0.05).</p