HCV Pseudoparticle Production and Infection

Human Immunodeficiency virus (HIV)-based particles bearing HCV envelope proteins were produced by HEK293T cells. 1.2x10⁶ cells were seeded in a 6 cm-diameter-dish and transfected with 2.16 μg envelope protein expression construct pcDNAΔcE1E2-Con1 [72 (link)], pcDNAΔcE1E2-GLT1 or pcDNAΔcE1E2-GLT1cc, 6.42 μg HIV gag-pol expression construct pCMVΔ8.74 [73 (link)] and 6.42 μg firefly luciferase transducing retroviral vector [78 (link)] using polyethylenimine (PEI). Medium was replaced after 6 h. After 48 h, supernatant containing the pseudoparticles was passed through a 0.45 μm filter and used to infect 4x10⁴ naïve Huh7-Lunet CD81 cells seeded in a 12-well plate the day before. After 72 h, luciferase assay was performed. SYBR Green based Product Enhanced Reverse Transcriptase assay (SG-PERT) was performed using the Takyon SYBR green kit (Eurogentec) to quantify HCVpp titers used for infection [79 (link)].