FIG 4.

Defined amino acid sequences of VLoop in CHIKV nsP2 make CHIKrep/Pac replicon noncytopathic in BHK-21 cells. (A) Schematic presentations of CHIKV replicon with a cloned library of VLoops in nsP2 protein and the scheme of selection of noncytopathic replicons. Cells were electroporated with the in vitro-synthesized replicon RNA and then treated with puromycin to select colonies of Pur^r cells. (B) Amino acid sequences identified in nsP2 of the noncytopathic replicons in randomly selected clones of Pur^r cells. (C) Amino acid sequences of VLoop and detected additional mutations in CHIKV nsP2 identified in the noncytopathic replicons after 3-week-long passaging of the pool of Pur^r cells (see the text for details). In panels B and C, amino acid sequences used in further experiments are indicated in red.