Extended Data Figure 6: SEC14L2 does not interact with HCV nonstructural proteins under the tested conditions.

a, Yeast two-hybrid assay was performed to examine direct interaction between SEC14L2 and HCV nonstructural proteins. SEC14L2-AD (GAL4 activation domain) fusion or control AD vector was co-expressed with DBD (DNA binding domain) fusion of the individual HCV non-structural proteins or control DBD vector and tested for positive yeast two-hybrid interactions under selective nutritional conditions (lacking leucine, tryptophan, histidine, and adenosine). The strong signal obtained for NS5A most likely reflects the intrinsic trans-activating activity of NS5A⁴⁵. b, Co-immunoprecipitation did not reveal binding of SEC14L2 with HCVNS5Aprotein. As two-hybrid assay (a) did not yield unambiguous results on interaction between SEC14L2 and NS5A, we employed co-immunoprecipitation assay to probe binding between these proteins. Huh-7.5 cells stably expressing SEC14L2-EGFP and harbouring wild-type Con1/SGneo replicon were lysed and subjected to immunoprecipitation with anti-NS5A antibody, anti-EGFP antibody or control immunoglobulins (IgG). The bound proteins were analysed by immunoblotting. Endogenous MOBKL1B, a previously described binding partner of the NS5A protein⁴¹, served as a positive control. c, Subcellular fractionation of SEC14L2-EGFP/Huh-7.5 cells harbouring wild-type Con1/SG-neo replicon did not reveal obvious co-fractionation of SEC14L2 and HCV NS5A protein