NS3/4A cleaves MAVS at Cys-508. (A) HEK293 (lanes 1–6) or Huh7 (lanes 7–12) cells were transfected with expression vectors for the WT (lanes 3,4, 9, and 10) or S139A mutant (lanes 5, 6, 11, and 12) of NS3/4A or vector (lanes 1, 2, 7, and 8). Cell lysates were separated into membrane pellet (P) or cytosolic supernatant (S) and then immunoblotted with an Ab against MAVS or Flag. The cleavage product of MAVS was indicated as MAVS*. (B) HEK293 cells were transfected with the expression vectors for NS3/4A together with a MAVS mutant containing only the CARD and TM domains (miniMAVS; Top). The activation of IFN-β by the CARD-TM fragment of MAVS in the presence of WT or S139A mutant of NS3/4A was determined by measuring the expression of the IFN-β-Luc reporter (Middle). Cell lysates were analyzed by immunoblotting with the Flag Ab that detects both NS3/4A and MAVS proteins (Bottom). (C) Alignment of the junction sequences of NS proteins of HCV and the putative cleavage site at the C terminus of MAVS from different species. (D) HEK293 cells were transfected with expression vectors encoding the WT or C508R mutant of MAVS together with those encoding the WT or S139A mutant of NS3/4A. Cell lysates were separated by differential centrifugation into the membrane pellet and cytosolic supernatant, which were then resolved by 7% (Top and Bottom) or 4–20% (Middle) SDS/PAGE, followed by immunoblotting with the indicated Ab that detects the N terminus (Flag; Top) or C terminus (HA; Middle) of MAVS, or NS3/4A (Myc; Bottom). (E) HEK293 cells were transfected with MAVS and NS3/4A expression vectors as in D, except that IFN-β-Luc was also cotransfected to measure the induction of IFN-β by Luc assay.

MAVS is cleaved at Cys-508 in a HCV replicon cell line. (A) Cell lysates from Huh7 (lanes 1 and 2) or Replicon cells (K2040; lanes 3 and 4) were separated by centrifugation into the membrane pellet (P) and cytosolic supernatant (S), which then were analyzed by immunoblotting with an Ab against MAVS or NS3. The cleavage products of MAVS are indicated as MAVS*. MAVS-ΔN, N-terminal truncated fragment of MAVS (see ref. 10); MAVS-ΔN*, N-terminal truncated fragment of MAVS in which the TM domain is also cleaved by NS3/4A. (B and C) Expression vectors for MAVS, C508 mutant of MAVS, or RIG-I(N) were cotransfected with the IFN-β-Luc reporter into Huh7 (B) or Replicon (C) cell lines. Twenty-four hours after transfection, cells were infected with SeV or mock infected for another 24 h before harvesting for Luc assay.

NS3/4A blocks IFN induction by MAVS. (A) HEK293 cells were transfected with IFN-β-Luc and the expression vector for either the WT or S139A mutant of NS3/4A. The next day, cells were transfected with expression vectors encoding MAVS, TBK1, or RIG-I(N), or infected with SeV. The Luc activity was measured 24 h later and normalized for transfection efficiency. The error bar represents standard deviation from the mean value of duplicated experiments. (B) Similar to A, except that NF-κB Luc reporter was used in lieu of IFN-β-Luc. (C) Similar to A, except that cells were transfected with the UAS-Luc reporter together with the Gal4-IRF3 expression vector. (D) HEK293 cells were transfected with the WT or S139A mutant of NS3/4A together with the expression vectors for MAVS (lanes 5–7) or TBK1 (lanes 8–10). In lanes 2–4, cells were infected with SeV for 24 h. Cell lysates were resolved by electrophoresis under native (Top) or denaturing condition (Middle) and then immunoblotted with an Ab against IRF3. The expression of NS3/4A and TBK1 also was analyzed by immunoblotting with a Flag Ab (Bottom).

NS3/4A binds to and colocalizes with MAVS in the mitochondria, and it cleaves MAVS in vitro.(A) Flag-tagged WT or S139A mutant of NS3/4A was expressed in HEK293 cells and then purified by using Flag-Sepharose. The purified NS3/4A proteins (lanes 4, 5, 9, and 10) or lysates containing NS3/4A (lanes 1–3 and 6–8) were incubated with ³⁵S-labeled MAVS or C508R mutant of MAVS, which was synthesized by in vitro translation and purified by using Flag-Sepharose. After incubation at 30°C for 2 h, proteins were separated by SDS/PAGE and analyzed by PhosphorImaging (Upper) or immunoblotting (Lower). The cleavage product of MAVS is indicated as MAVS*. (B) HEK293 cells were transfected with expression vectors for Flag-NS3/4A and HA-MAVS as indicated. Cell lysates were immunoprecipitated with the MAVS Ab (lanes 3, 6, 9, and 12) or control IgG (lanes 2, 5, 8, and 11). The precipitated proteins were analyzed by immunoblotting with an Ab against HA (Upper) or Flag (Lower). (C) Expression vectors for Flag-NS3/4A and HA-MAVS or C508R mutant were transfected into HeLa cells, and the localization of these proteins was stained by the indicated Abs and visualized by confocal microscopy.