建立 C 型肝炎病毒感染之報導系統

Abstract

Hepatitis C virus (HCV) contains a single-stranded positive-sense RNA genome that encodes 3 structural and 7 non-structural proteins. HCV infection may lead to chronic liver disease, including hepatitis, steatosis, cirrhosis, and hepatocellular carcinoma. Recently, HCV replicons or chimeric viruses that contained reporter genes (luciferase or green fluorescence protein) have been useful to study HCV replication in cells. However, the reporter genes inserted into HCV genome may attenuate HCV replication and subsequent virus production. To avoid the drawbacks of chimeric replicons, we established a HCV-producing cell line, JFH1-SEAP cells, with separatedly expressed reporter gene and HCV genome. The JFH1-SEAP cells expressed enhancement green fluorescence protein-NS3/4 protease-cleavable △4AB peptide-secreted alkaline phosphatase fusion protein that was transduced by lentivirus and replicating HCV genotype 2a full-length sequences to produce infectious HCVcc. The functional HCV NS3/4 protease could cleave the △4AB peptide to release SEAP for quantitative measurement of virus replication. The SEAP activity was increased linearly as JFH1-SEAP replicated. Moreover, we cultured JFH1-SEAP cells and HuH7.5-SEAP cells simultaneously to allow HCV infection to the naive cells. Agreed with the previous reports, our results showed that interferon-α prohibited the anti-HCV activity. Vitamin E enhanced HCV replication. Furthermore, folic acid, EGF, LiCl and LPL decreased the SEAP activity. Our results demonstrated that a novel HCV-producing cell line with a SEAP reporter, JFH1-SEAP cell, was established. The system is a powerful platform to monitor HCV infection or replication and holds a potential for high-throughput investigation of the effects of nutrients and biochemical compounds on HCV replication.