Plasmid Construction and Protein Expression

HJ3-5, JFH1-QL, and HJ3-5/^HAp7 were described previously [18 (link),67 (link),68 (link)]. JFH1/^HAp7 was generated by inserting the HA-epitope sequence between the E2 and p7 coding region of JFH1/QL. The pFN11A (BIND) Flexi Vector (Promega) was modified by deleting the GAL4 fusion protein-coding region and used to generate plasmids expressing E1-E2, E1-E2-p7, E1-E2(AR)-p7, and NS2 from two different genotypes, H77 and JFH1. In brief, E1-E2, E1-E2-p7, E1-E2(AR)-p7, NS2 sequences were PCR amplified from H77 and JFH1 with the primer sets introducing SgfI and PmeI restriction enzyme sites at their N- and C-terminus, respectively, and then cloned into the modified vector digested with SgfI and PmeI enzymes (Promega, WI, USA). The E2 (AR) mutations were introduced by using the QuikChange II XL site-directed mutagenesis kit (Agilent Technology, Santa Clara, CA). The sequences of regions manipulated within each plasmid were verified by DNA sequencing. The SPCS1 sequence was cloned from cellular RNA isolated from Huh-7 cells and inserted into the modified pFN11A vector described above, without or with Flag- or HA-tags.