IPS-1 Is Essential for the Control of West Nile Virus Infection and Immunity

Suthar, Mehul S.; Ma, Daphne Y.; Thomas, Sunil; Lund, Jennifer M.; Zhang, Nu; Daffis, Stephane; Rudensky, Alexander Y.; Bevan, Michael J.; Clark, Edward A.; Kaja, Murali-Krishna; Diamond, Michael S.; Gale, Michael

The RLR signaling pathway triggers the innate immune response to WNV infection in primary cortical neurons Neurons represent the target cell of WNV infection in the CNS and their death after infection is a key factor in pathogenesis and neurological sequelae [32, 33] . To define the role of RLR signaling in restricting virus replication in neurons, primary cortical neurons were generated from wild type and IPS-1 2/2 mice. Cells were infected at an MOI of 1.0 with WN-TX and virus yield, IFN-b induction, and ISG expression were evaluated. In the absence of IPS-1, WNV replicated faster and to higher levels resulting in a 2.2 and 4.2-fold (p,0.05) increase in viral production at 24 hrs and 48 pi, respectively as compared to infected wild type neuronal cells ( Fig 3A) . This relatively modest virologic effect in neurons compared to that observed in IPS-1 2/2 DC and Mw was expected, as IFN-a or -b pre-treatment only inhibits WNV infection in cortical neurons to a maximum of 5 to 8-fold [12] , suggesting that the IFN response is comparably less potent in neurons. IFN-b expression was induced to lower levels in IPS-1 2/2 neurons compared to wild type infected neurons at 24 (10-fold, p,0.05) and 36 hours pi (5-fold, p,0.05) despite the higher levels of virus replication (Fig 3A and 3B) . Expression of ISGs, (including RIG-I and MDA5) and IRF-3 target genes (including ISG56 and ISG49) followed this pattern and were dependent on IPS-1 for rapid and high level expression ( Fig 3C) . The presence of IFN-b and ISG transcripts in IPS-1 2/2 cells at 48 hrs pi is consistent with the finding that TLR3 has an independent and subordinate role in triggering innate immune responses in cortical neurons at later time points after WNV infection [23] . These results demonstrate that the RLR signaling pathway controls virus replication and induces innate immune responses against WNV infection in cortical neurons.