FIG. 3.

Effect of multiple CS mutations on RNA replication and viral genome packaging. (A) Schematic representation of WNR RNA structure. The nucleotide sequences of CS are given next to their names. Boxes indicate the eight nucleotides that are 100% conserved among all mosquito-borne flaviviruses. Nucleotide substitutions in the 5′ CS and 3′ CS are in lower case. (B) RNA replication of WT and mutant replicon in BHK cells. Averages of triplicate relative luciferase activities at 2 days posttransfection were normalized to that at 5 h, and the WT control value was set to 100%; error bars show standard deviations. The experiment was repeated at least twice, producing similar results. (C) Photograph of monolayers of BHK packaging cells infected with WT or m4/7 mutant VLP, overlaid with semisolid medium, fixed, and immunostained with anti-NS1 antibody 48 h after incubation at 37°C.