HCV Pseudoparticle Generation and Analysis

HCVpp was generated by cotransfection of 293T cells with pNL4-3.lucR−E− and the corresponding expression plasmid encoding wildtype or mutant E1E2 genes as described previously [35 (link)]. Cell lysates and culture supernatants were harvested 72 hours post-transfection for immunoblotting, infectivity assay and purification of virions. HCVpp was pelleted by centrifugation of culture supernatant at 16,000 rpm for 1 hour, resuspended, and purified over a 20% (wt/vol) sucrose cushion [35 (link), 76 (link)]. Envelope glycoproteins E1 and E2 were detected by immunoblotting using biotinylated mouse mAb A4 [28 (link)] and mAb HCV1 [24 (link)] and the IRDye680RD Streptavidin (1:2,000) and IRDye800CW goat anti-human IgG secondary antibodies (1:10,000) (LI-COR Biosciences), respectively. HIV-1-p24 was detected using biotinylated mouse monoclonal antibody (diluted to 1:1,000; Aalto Bio Reagents). The immunoblots were analyzed with the Odyssey Infrared Imaging System and Image Studio software (LI-COR Biosciences).