Fig. 3: D1467G ablates RLR signalling and reduces the abundance of dsRNA.

A Whole cell lysates of Huh7-Lunet naïve and neoJFH1 wt/D1467G were analysed regarding NS5A, MAVS and Calnexin expression by WB. Representative of three independent experiments. B-C) Huh7 MAVS KO MAVScr cells, expressing the cleavage-resistant MAVS-mutant C508R, were electroporated with ivt RNA of JFH1 wt/D1467G subgenomic reporter replicons, compared to a replication-deficient control (∆GDD). B Luciferase activity at the indicated time point was monitored as a correlate for RNA replication (n = 3 independent biological replicates). C IFIT1 mRNA expression fold respective ∆GDD control was quantified by RT-qPCR (n = 4 independent biological replicates). D HCV RNA levels in Huh7-Lunet neoJFH1 wt/D1467G relative to wt cells were quantified by RT-qPCR (n = 4 independent biological replicates). E Huh7-Lunet naïve and neoJFH1 wt/D1467G were stained for dsRNA and NS3. Representative of n = 3 independent experiments. Scale bar indicates 30 µm. F At least 364 imaged cells per condition and independent replicate (n = 3 independent biological replicates) were segmented using cellpose, and dsRNA and NS3 per cell intensity were quantified using FIJI. Mean of all cells of each replicate is graphed. G dsRNA levels in total RNA isolated from neoJFH1 wt/D1467G cells (n = 3 independent biological replicates) were analysed using a commercially available dsRNA bioassay. Statistical analysis was performed by an unpaired two-sided t-test using GraphPad prism. P-values are indicated. B–D, F, and G Mean value is indicated by the bar graph and standard deviation is indicated by error bars.