Figure 5. Complex formation between drug susceptible and drug-resistant NS5A in the absence of RNA replication.

(A) HCV NS3-5B constructs driven by T7 polymerase and encoding tandem tagged copies of NS5A (Berger et al., 2014) were created to co-express HA- and GFP-tagged NS5A alleles. Huh7-Lunet-T7 cells were transfected with pTM-Dual-NS5A constructs that contained two drug-resistant (R), two drug susceptible (S), or mixed alleles of NS5A. Proteins from transfected cell extracts that were incubated in the absence (B) or presence (C) of SR2486 were subjected to SDS-PAGE without further fractionation (Input) or after immunoprecipitation with anti-HA antibodies (IP αHA). The gel was subjected to immunoblotting with GFP or HA antibodies as indicated. (D) Cells were transfected with dual-NS5A constructs in the absence or presence of SR2486 for 24 hr. Cells were then fixed, stained with anti-HA antibodies and visualized by confocal microscopy. Representative images from over 25 cells are presented. (E) Cells transfected with dual-NS5A constructs that expressed drug-susceptible (S) and drug-resistant (R) alleles as shown were prepared for electron microscopy by high-pressure freezing and freeze-substitution and visualized by transmission electron microscopy. (F) Numbers of cytoplasmic lipid droplets per cell formed in the absence (left) or presence (right) of SR2486; at least 25 images per sample such as those shown in (E) were quantified.