FIG 8.

NS6 hinders the combination of dsRNA with RIG-I/MDA5. (A and B) HEK-293T cells were individually transfected with plasmids encoding Flag-tagged RIG-I (A), MDA5 (B), and increasing quantities of NS6 expression plasmids for 28 h. Lysates from the cells overexpressing NS6 were incubated with an equal volume of lysates from cells overexpressing RIG-I or MDA5, followed by treatment with poly(I·C)-coated agarose beads for 4 h at 4°C. The beads were then washed three times with lysis buffer by centrifugation and subjected to Western blotting as described in the legend to Fig. 7. The numbers below the images represent the relative level of RIG-I/MDA5 compared to that of the control group via ImageJ software analysis. (C) A schematic diagram, where RIG-I acts as a representative protein, of the mechanism for NS6 protein inhibition of the RLR signaling pathway. In the inactivated state of RIG-I, the CARDs are bound to Hel-2i, which is unavailable for downstream signaling in this autoinhibited state. The CTD, which is tethered to the red bridging helix by a flexible linker, is able to sense RNA PAMPs. Upon virus infection, the CTD-bound dsRNA is preoriented to form a network of interactions with the helicase domains Hel-1 and Hel-2i but not Hel-2, leading to the segregation of interaction of CARDs with Hel-2i and the subsequent availability for interaction with downstream signaling molecules (64).