FIG. 7.

HAstV-1 CP inhibits iC3b formation, terminal complement cascade activation, and C4d formation. Reaction mixtures containing NHS were preincubated alone, with 5.4 μg CP, or with 1 μg CVF at 37°C for 3 h and then aliquoted. CVF is a positive control for complement activation. (A) Reaction products were resolved by SDS-PAGE and subsequently analyzed by immunoblotting using antisera to C3. CVF is a positive control for iC3b formation and corresponding C3 alpha chain depletion. Purified standards for C3 (alpha [114 kDa] and beta [75 kDa] chains) and iC3b (α′₁ [68 kDa] and α′₂ [42 kDa]) products were included in the gel, as indicated to the left and right of the gel, respectively. Under these gel conditions, the 68-kDa iC3b band comigrated with the C3 beta chain product. Aliquots of the above reaction mixtures were analyzed by ELISA (ng/ml) for iC3b (B) and SC5b-9 (C) formation. (D) NHS was incubated for 1 h in the presence of heat-aggregated IgG (agg-IgG; a classical pathway activator), CP, or both and measured for C4 cleavage by C4d ELISA. Standard curves were generated using purified iC3b, SC5b-9, and C4d. Data are the means for four independent experiments for each ELISA. Error bars denote SEM.