Fig 7.
Effect of amino acid substitutions in NS4B on the viral RNA replication efficiency. (A) A GPE−-derived bicistronic replicon consisting of the 5′ and 3′ UTRs flanking a chimeric Npro-firefly luciferase gene separated from the NS3 to NS5B genes by the IRES of EMCV was constructed. Single amino acid substitutions at positions 2475 and 2563 were introduced by site-directed mutagenesis as indicated. (B) In order to analyze the effects of the amino acid substitutions on the viral RNA replication efficiency, 106.8 CPK cells were transfected with 1 μg of each replicon RNA and with 100 ng of pRL-TK (Int−) reporter plasmid for constitutive Renilla luciferase expression. After 6 h, 12 h, and 24 h of incubation at 37°C in the presence of 5% CO2, the firefly and Renilla luciferase activities expressed in relative light units were assayed in cell lysates. The firefly luciferase activity was normalized with the corresponding Renilla luciferase activity from the same lysate. Each bar represents the mean value from 3 independent experiments, with error bars showing the standard deviations. The values obtained with each construct were compared pairwise using the t test. *, P < 0.05.