FIG. 1.

Effects of nucleotide sequence of CS on RNA replication. (A) Schematic representation of WNV replicon RNA structure (WNR-CNS1-5Rluc) and position of the Rluc-encoding cistron. The nucleotide sequences of CS are indicated next to their names (see the text). 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. Underlined nucleotides were exceptions to the rules used to swap codons (see the text) that were implemented to avoid generation of stop codons in 5′ CS that reside in the C coding region. ΔNS5 contains WT CS and a frameshift mutation upstream of the active-site GDD motif of the RNA-dependent RNA polymerase of NS5. (B) RNA replication of WT and mutant replicons in BHK cells. BHK cells were transfected with identical amounts of in vitro-transcribed Rluc-expressing WNR RNAs. Rluc activities were measured at 5 h and 2 days posttransfection. The Rluc activity obtained at 2 days is shown normalized to activity obtained at 5 h to account for slight differences in transfection efficiency (see the text). Data for each condition are averages of triplicate values with error bars showing standard deviations; the WT control value was set to 100%. The experiment was repeated at least twice, with consistent results.