Figure 1.
Survival and viral burden analysis in mice.
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.
Figure 2.
Levels of type I IFN levels in serum of wild type and DKO mice infected with WNV.
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).
Figure 3.
IRF-3 and IRF-7 control the IFN-α/β gene induction and ISG expression in MEF.
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).
Figure 4.
IRF-3 and IRF-7 restrict WNV infection by regulating the IFN-α/β response and ISG expression in primary cortical neurons.
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 Figure 3. 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).
Figure 5.
IRF-3 and IRF-7 partially modulate the IFN-β response and ISG expression in primary Mφ.
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 Figure 3. Asterisks indicate values that are statistically significant (***, P<0.0001, **, P<0.005, *, P<0.05).
Figure 6.
The WNV-induced IFN-β response and ISG expression is primarily IRF-3 and IRF-7-independent in mDC.
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 Figure 3. 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.
Figure 7.
The IFN-β response in mDC after TLR stimulation and viral infection.
mDC generated from wild type and DKO mice were (A) stimulated with 50 µg/ml of poly(I∶C) or 2 µg/ml LPS for 24 h or (B) infected with EMCV (MOI 0.1), WNV (MOI 0.1) and CHIK (MOI 1) for 24 h. Levels of IFN-β were measured either by ELISA or qRT-PCR. Values are an average of triplicate samples generated from three independent experiments. Asterisks indicate values that are statistically significant (***, P<0.0001, ** P<0.0005) from wild type cells; n.s. indicates differences that were not statistically significant.
Figure 8.
The IFN-β response in mDC is IRF-1, IRF-8, IFN-α/β-independent but partially IRF-5-dependent.
mDC generated from wild type, (A and B) IRF-1−/−, IRF-8−/−, (C and D) IFN-α/βR−/−, or (E and F) IRF-5−/− mice were infected at an MOI of 0.1 and levels of IFN-α (A, C, and E) and IFN-β (B, D, and F) mRNA were measured by qRT-PCR. Values are an average of triplicate samples generated from three independent experiments. Asterisks indicate values that are statistically significant (***, P<0.0001, **, P<0.005, *, P<0.05) compared to wild type.
Figure 9.
Pharmacological inhibition of NF-κB and p38 in wild type and DKO mDC and MEF.
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 Materials and Methods. 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.
Figure 10.
The IFN-β response in mDC and MEF is IPS-1-dependent but MyD88-independent.
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).
Figure 11.
The early phase of the type I IFN regulation in MEF partially involves TRAF3 and TBK1 whereas the late phase requires TRAF6.
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.
Figure 12.
A model for detection of WNV and IFN-α/β gene activation in MEF.
(1). The host through recognition of an as yet undefined viral RNA PAMP in the cytoplasm detects WNV. RIG-I acts as the primary PRR sensor for WNV during the early stages of infection. RIG-I activation promotes association with IPS-1, which leads to recruitment of TRAF3 and TBK1, and phosphorylation of IRF-3. NF-κB and ATF-2/c-Jun and the small amounts of constitutively expressed IRF-7 may also be activated via this IPS-1-dependent pathway. IRF-3, IRF-7 NF-κB, ATF-2/c-Jun translocate to the nucleus, bind the IFN-β gene promoter and promote transcription. Secretion of IFN-β by infected cells during this early phase results in autocrine and paracrine type I IFN signaling through binding of the IFN-αβR. (2). Activation of IFN-αβR results in phosphorylation of JAK1 and Tyk2, which activate STAT1 and STAT2 leading to formation of the heterotrimer ISGF3 (STAT1, STAT2 and IRF-9). Nuclear translocation and promoter binding of ISGF3 upregulates hundreds of different ISG, including IRF-7. (3). During a later phase of infection, detection of WNV in MEF also relies on MDA5 and PKR. Recruitment of TRAF3 and TRAF6 activates IRF-3 and IRF-7. NF-κB and ATF-2/c-Jun are also activated via an as yet undefined mechanism. Subsequently, IRF-3, IRF-7, NF-κB, and ATF-2/c-Jun translocate to the nucleus, bind the IFN-β gene promoter and induce optimal transcription. Induction of IFN-α genes occurs through TRAF6 and the transcriptional activation of IRF-7.