Purified DENV NS1 and WNV NS1 promote C4 cleavage. (A and B) Western blot analysis. (A) Purified DENV NS1, WNV NS1, BSA, or lysozyme (50 µg/ml) was preincubated with C4 (6.25 µg/ml) for 1 h on ice. Subsequently, the NS1-C4, BSA-C4, and lysozyme-C4 solutions were incubated at 30°C for 15 min. (B) Purified C4 was incubated with C1s (12 µg/ml) for 1 h at 30°C. Subunits and cleavage fragments of C4 and C4b are labeled to the right of the gel; 93 kD α chain of C4; 84 kD α′ chain of C4b. (C and D) Inhibition of C4 cleavage in the presence of NS1 by C1 inhibitor (C1-INH). (C) C4a ELISA. C4 was incubated with purified DENV NS1 or lysozyme (30 µg/ml) in the presence or absence of C1-INH (1.4 µg/ml) on ice for 1 h. (D) Single-step hemolysis assay. EA were incubated with a 160-fold dilution of C4-deficient GPS supplemented with human C4 (0.1 µg/ml) that was preincubated with DENV or WNV NS1 (30 µg/ml) in the presence or absence of C1-INH (40 µg/ml). In some experiments, C1 inhibitor (C1-INH; 40 µg/ml) was added to before the incubation with EA- and C4-deficient GPS. Data are the mean ± SD for four independent experiments. Asterisks denote C4a liberation (C) as measured by ELISA that is statistically different (**, P < 0.005) or hemolysis conditions (D) that are statistically different from untreated cells (***, P < 0.0005).