Baculovirus Mediated Production Of Infectious Hepatitis C Virus In Human Hepatoma Cells Stably Expressing T7 RNA Polymerase

Hepatitis C virus(HCV) is a major causative agent of posttransfusion of non-A non-Bhepatitis. Over 170 million people are persistently infected with HCV world wide. It hasproven to be a difficlult public health problem. For many years, the main limitation of HCVresearch is the lack of efficient and reliable cell culture system to amplify the virus. Untilrecently, this problem was partly solved after identifying a unique HCV genotype 2astrain,which was shown to replicate and produce infeetiours viral particles efficiently incell culture. The baculovirus is a diverse family of arthropod-specific virus with adouble-stranded genomic DNA of which most members infect Lepidoptera.With thedevelopment of biology, baeulovirus became a new gene transfering vetor. The aims of thisthesis was to establish an efficient and reliable cell culture system for HCV via baculovirusvector. We transducted the recombinant baulovirus containing the cDNA of HCV JFH1into Huh7-lunetT7 cells. The HCV genome transcription was driven by T7 promoter withthe help of T7 RNA polymerase expressed in this cell and the efficency of this cell culturesystem was subsequently evaluate.In chapter one, an overview briefly outlines the character of HCV, major in thedevelopment of cell cultures system for HCV. Then reviewed the character of baculovirusand the application of baculovirus vector in HCV reseach.In chapter two, we characterized biochemical characteristics for the HCV produced inthe cell culture system via the baculovirus vector. First of all, we constructed recombinantbaculovirus vector containing the cDNA of HCV JFH1genome named vAcJFH1 and thecontrol recombinant bacuclovirus vAcJFH1 (GND).Then screen the Huh7-lunet cell linestably expressed T7 RNA polymerase, called Huh7-lunetT7 cell line. Finally wetransducted the recombinant baeuloviurs vAcJFH1 and the control vAcJFH1 (GND) intoHuh7-lunetT7 cell line. Synthesis of positive-strand and negative HCV strand could be detected in the Huh7-1unetT7 cells transducted by RT-PCR and strand specific RT-PCR.Western blot and immunofluorescene analysis revealed that HCV structural and non-structural proteins were correctly processed and were localized in the cytoplasm of thetarget cell. Sucrose density gradient centrifugation of the culture medium revealedcolocalization of HCV RNA and structural proteins in the fraction with the density of1.08-1.10g/mL. Electron microscopy showed viral particles of≈55nm in diameter, whichcould be recognized by anti-HCV E2 antibodies. Real-time RT-PCR detected that the levelof HCV RNA in the supematant was 107 copies/ml at 72h post-transduction.In chapter 3, we analysis whether the HCV particles produced via baculovirus vectorsis infectious. The supematant from Huh7-1unetCDgl cell was filtrated and infected theHCV permissive cell line Huh7-1unetCDS1. RT-PCR and strand specific RT-PCRdemonstrate the presense of positive and negative-strand HCV in the Huh7-1unetCDS1infected. Immunofluorescene detected the expression of HCV E2 protein at 72h posttransduction. This indicate that HCV particles produced from baculovirus vetor wereinfectious, which could replicate efficiently in Huh7-1unetT7 cell and express HCVproteins.In chapter 4 we research the regulation of HBV promoters by HCV proteins duiringHCV/HBV coinfection. The result showed that HCV NS2 protein can activate HBV Xpromoter.In summary, we use baculovirus vector to establish an efficient cell culture system forHCV. HCV JFH1 genome could replicate efficiently and produce infectious HCV particlesin this system. In addition, we use T7 promoter but not CMV promoter to initiate thetranscription of HCV genome, so the transcripted HCV genome neither containing capstructure in 5'UTR nor enter the nucleous of the host cell, which consistent with the naturelife cycle of HCV. This cell culture system could be useful in the searching for the antiviralagents, the molecular mechanism of HCV infection etc.