FIG 3.
P4 Arg-to-His substitution rescues CHIKV trans-replicase activity in the absence of the nsP3-G3BP interaction. (A) Schematic representation of amino acid substitutions in CHIKV R532H (RH), CHIKV F3ANC, and CHIKV F3ANC-RH. (B) U2OS WT or ΔΔ cells were cotransfected with WT CHIKV (WT) or CHIKV R532H (RH) replicase expression plasmids and corresponding templates. Fluc (replication) and Gluc (transcription) activities were measured 20 h posttransfection and normalized to signals obtained from control cells transfected with plasmids encoding catalytic-inactive replicases and templates and plotted as fold change. Data are means of the results from three independent experiments. Error bars indicate the standard deviation (SD). Statistical differences were as follows: ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (C) U2OS WT or ΔΔ cells were infected with indicated CHIKV variants at an MOI of 0.1, and viral titers at 24 hpi, 48 hpi, and 72 hpi were quantified by plaque assay. Data are means of the results from three independent experiments. Error bars indicate the SEM. Statistical differences were as follows: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (D and E) U2OS WT (D) or Aedes albopictus C6/36 (E) cells were cotransfected with indicated trans-replicases and templates and 18 h or 48 h posttransfection, respectively, analyzed as described for panel B. *, P ≤ 0.05, **, P ≤ 0.01, ***, P ≤ 0.001, ****, P ≤ 0.0001.