Production and characterization of HCV particles from serum-free culture

Abstract

Hepatitis C virus (HCV) is a major cause of liver cancer, and it is therefore important to develop a prophylactic strategy for HCV infection. In recent years, a system for cell culture of the infectious HCV particle has been established, and the inactivated particle has potential as an antigen for vaccine development. In this study, we aimed to establish highly efficient HCV particle purification procedures using the following serum-free culture of HCV particles. First, naïve human hepatoma Huh7 cells were grown in serum-free medium that was supplemented with human-derived insulin, transferrin and sodium selenite. Then, in vitro transcribed JFH-1 or J6/JFH-1 chimeric HCV-RNA was transfected into the serum-free conditioned Huh7 cells. Infectious HCV was secreted into the culture supernatant with the same efficiency as that from cells cultured in FBS-containing medium. The HCV-core protein and RNA continued to be detected in the culture supernatant when the infected cells were subcultured in serum-free medium. Sucrose gradient centrifugation analyses indicated that the profiles of HCV-core, HCV-RNA and the infectivity of HCV particles were almost identical between HCV from FBS-supplemented and serum-free cultures. We further determined that anti-CD81, anti-SR-BI and anti-E2 antibodies inhibited infection by serum-free cultured HCV to a greater extent than infection by HCV from FBS-supplemented cultures. These HCV particles also differed in the level of associated apoplipoproteins: the ApoE level was lower in serum-free cultured HCV. ApoB and ApoE antibody-depletion assays suggested that infection of serum-free cultured HCV was independent of ApoB and ApoE proteins. These data suggest that lipids conjugated with HCV affect infection and neutralization.