FIG 7.

Effects of mutations in nsP1 of CHIKV for infectious virus rescue and trans-replicase activities in mammalian and mosquito cells. (A) BHK-21 cells were transfected with one of the following plasmids: CMV-ICRES1, CMV-ICRES1^DA, CMV-ICRES1^YA, CMV-ICRES1^RE, or CMV-ICRES1^3C3A. (Left) Results of ICA. (Right) Western blot of lysates from transfected cells collected at 24 h posttransfection. The CHIKV capsid protein was revealed by the corresponding rabbit polyclonal antiserum; β-actin was used as a loading control. Data from one reproducible experiment out of two independent experiments are shown. (B) U2OS cells were all cotransfected with HSPolI-Fluc-Gluc and one of CMV-P1234, CMV-P1^DA234, CMV-P1^YA234, CMV-P1^RE234, CMV-P1^WA234, CMV-P1^3C3A234, or CMV-P1234^GAA. Samples were collected at 18 h posttransfection. Production of positive-strand RNAs was estimated by measuring the activities of Fluc (left) and Gluc (right) as described in the legend to Fig. 3A. Each column represents an average from three independent experiments; error bars represent standard deviations. Viral protein expression was verified by Western blotting using anti-nsP1 antiserum. (C) AF319 cells were all cotransfected with AegPolI-Fluc-Gluc and one of Ubi-P1234, Ubi-P1^DA234, Ubi-P1^YA234, Ubi-P1^RE234, Ubi-P1^WA234, Ubi-P1^3C3A234, or Ubi-P1234^GAA. Samples were collected at 48 h posttransfection. Production of positive-strand RNAs was estimated by measuring the activities of Fluc (left) and Gluc (right) as described in the legend to Fig. 3A. Each column represents an average from three independent experiments; error bars represent standard deviations. Viral protein expression was verified by Western blotting as described in the legend to panel B.