FIG 11.

NS2A and NS3 mutations did not alter the proximity and interaction between NS2A and NS3. (A) Duo-link analysis. A Duo-link assay was performed to examine the effects of NS2A and NS3 mutations on the intracellular proximity of NS2A and NS3 proteins. BHK-21 cells were cotransfected with two plasmids: one plasmid encoding C-terminally myc-tagged WT or mutant NS3 (A373V or E477D) and another plasmid encoding C-terminally HA-tagged WT or K188-del NS2A. Positive fluorescence signals are shown in red. (B) Co-IP of NS2A and NS3 proteins. HEK 293T cells were cotransfected with two plasmids: one encoding GST or GST-tagged NS2A (WT or K188-del) and the other encoding HA-tagged NS3 (WT, A373V, or E477D). (C) Densitometry analysis. The intensity of each band from the immunoblot in panel B was quantified with ImageJ software. The relative pulldown efficiencies were calculated as follows: [(a/b)/(c/d)] × 100%, where a is the intensity of HA-NS3 (WT or mutant), b is the intensity of GST or NS2A-GST (WT or K188-del), c is the intensity of the WT HA-NS3 in lane 2, and d is the intensity of the WT NS2A-GST in lane 2. The means and standard deviations from the results of three independent experiments are shown. Statistical analysis was performed using an unpaired Student t test. ***, P < 0.001 (extremely significant); ns, nonsignificant (P > 0.05).