FIG. 3.

The designed CHIKV nsP2 mutants demonstrate an efficient synthesis of virus-specific RNAs and viral structural proteins. (A) 5x10^s cells in the 6-well Costar plate were infected with parental CHIKV 181/25 and designed mutants at an MOI of 20 PFU/cell. At 7 h PI, they were washed with PBS, and proteins were metabolically labeled for 30 min with [³⁵S]methionine (see Materials and Methods for details). Equal amounts of lysates were analyzed by gel electrophoresis in 10% NuPAGE gels (Invitrogen), followed by autoradiography. (B) 5x10⁵ cells in the 6-well Costar plate were infected with the indicated viruses at an MOI of 20 PFU/cell. Virus-specific RNAs were metabolically labeled between 4 and 8 h PI in complete media, supplemented with [³H]uridine (20 μCi/ml) and Actinomycin D (1 μg/ml), then RNAs were isolated and analyzed by agarose gel electrophoresis as described in Materials and Methods.