Generating HCV-specific Ramos B cells

HCV-specific Ramos B cells stably expressing gl-AR3C, AR3C, gl-HEPC74, or HEPC74 BCRs were generated as described elsewhere^{52 (link)}. In short, the 2-1261gl gene of the pRRL EuB29 2-1261gl IgG TM.BCR.GFP.WPRE plasmid^{109 (link)} was exchanged for the heavy and light chain genes of either gl-AR3C, AR3C, gl-HEPC74, or HEPC74 using Gibson assembly (Integrated DNA Technologies). Lentiviruses were produced by co-transfecting the generated expression plasmid with pVSV-g, pMDL, and pRSV-Rev into HEK293T cells using lipofectamine 2000 (Invitrogen). Two days post-transfection, HEK293T supernatant was used to transduce IgM-negative Ramos B. Seven days post-transduction, BCR-expressing B cells were FACS sorted on GFP and IgG double-positivity (i.e., BCR-expressing cells) using a FACS Aria-II SORP (BD Biosciences). B cells were expanded and cultured indefinitely. Prior to B cell binding assays and calcium flux assays, cells were selected for GFP expression to ensure similar levels if IgG (as BCR) across the cell lines (Supplemental Fig. 3b).