Inhibitor of κB Kinase ϵ (IKKϵ), STAT1, and IFIT2 Proteins Define Novel Innate Immune Effector Pathway against West Nile Virus Infection

Abstract

Background: IFN activates JAK-STAT signaling, where STAT1 phosphorylation is crucial for ISG induction and expression of IFIT2 to limit West Nile virus infection. Results: IKK⑀ mediates STAT1 serine 708 phosphorylation exclusive of tyrosine phosphorylation but dependent on nuclear export and ISG synthesis. Conclusion: IKK⑀-mediated STAT1 S708 phosphorylation is crucial for IFIT2 expression to control WNV. Significance: We define a novel anti-WNV innate immune effector pathway.

Infectious Clones of Novel Lineage 1 and Lineage 2 West Nile Virus Strains WNV-TX02 and WNV-Madagascar

“…Lineage 1 WNV-TX, but not lineage 2 WNV-MAD, has been shown to inhibit type I interferon (IFN)-induced phosphorylation of STAT1 and STAT2 by blocking the activity of the IFN receptor-associated kinase Tyk2 (9). We found that WNV-TX also blocks an inhibitor of B kinase ε (IKKε)-dependent phosphorylation event on STAT1, resulting in a temporal regulation of STAT1 phosphorylation and subsequent expression of IFN-stimulated genes that are essential for the control of WNV infection (18). However, studies to identify specific viral determinants that regulate WNV inhibition of IFN-mediated signaling have been hindered by the lack of appropriate reagents.…”

Fucose-based PAMPs prime dendritic cells for follicular T helper cell polarization via DC-SIGN-dependent IL-27 production

“…However, although IKKe-mediated serine phosphorylation of STAT1 precedes TLR4-induced IFNR signalling, IFNR signalling is required for STAT1 phosphorylation at Tyr701 to induce heterodimerization between STAT1 and STAT2 (for ISGF3 formation) and STAT1 homodimerization 30,31 . A previous report suggested that phosphorylation of Ser708 and Tyr701 are mutually exclusive when Tyr701 phosphorylation preceded Ser708 phosphorylation 47 ; however, during costimulatory TLR4-DC-SIGN triggering, Ser708 phosphorylation exhibits faster kinetics than Tyr701 phosphorylation. Thus, de novo type I IFN synthesis induced by TLR signalling intersects with IKKe signalling to generate ISGF3 complexes.…”

The Essential, Nonredundant Roles of RIG-I and MDA5 in Detecting and Controlling West Nile Virus Infection

“…Moreover, our data indicate that MDA5 contributes to innate immune induction at later times after initial WNV infection. This response leads to an amplification of innate immune signaling to IFN-␤, as well as response diversification due to the induced expression (and activation) of IRF-7 and other innate immune signal transducers to drive the expression of IFN-␣2a and increased ISG and cytokine expression (17,50). Consistent with this, collaborative studies reveal that MDA5 is essential for the optimal priming of effector T cells and that this process occurs in a T cell-nonautonomous manner, with MDA5 function required for the priming environment (48).…”

“…Immunoblot analysis demonstrated that WNV proteins accumulated to higher levels in MDA5 Ϫ/Ϫ M and DCs than in WT cells (especially at 36 h), whereas DCs exhibited a concomitant reduction in the virus-induced expression of IFIT2, an IFN-stimulated gene (ISG) that is downstream of RLR signaling and restricts WNV infection ( Fig. 3c and d) (50,55). We also found that MDA5 Ϫ/Ϫ M and DCs produced lower levels of IFN-␤ postinfection than WT cells (Fig.…”

“…Protein extracts were prepared as previously described (50), and 20 g of protein lysate was analyzed by SDS-poly-acrylamide gel electrophoresis followed by immunoblotting. The following primary antibodies were used: anti-murine IFIT2 (gift of G. Sen, Cleveland Clinic), goat anti-WNV NS3 (R&D Systems), mouse antitubulin (Sigma), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; Santa Cruz).…”