FIG 5.
The replacement of VLoop in CHIKrep/GFP/Pac by selected sequences makes replicons noncytopathic and capable of persistent replication. (A) Schematic presentation of the designed replicons containing indicated mutations in VLoop and additional mutation in nsP2 (in CHIKrep/RLH(A730V)/GFP/Pac) and their efficiencies in the formation of colonies of Purr, GFP-positive cells upon electroporation of the in vitro-synthesized RNAs. (B) BHK-21 cells were electroporated with equal amounts of the in vitro-synthesized replicon RNAs and harvested either at 8 h posttransfection (CHIKV/GFP RNA-transfected cells) or 3 to 7 days after transfection and puromycin selection of cells with mutant replicons. Equal numbers of cells were used for analysis. Levels of indicated nsPs and GFP in the cell lysates were evaluated by Western blotting using specific Abs. Membranes were scanned on an Odyssey imager (LI-COR).