Baculovirus mediated production of infectious hepatitis C virus in human hepatoma cells stably expressing T7 RNA polymerase.
Although much has been learned about Hepatitis C virus (HCV), research progress has been hindered by the lack of a suitable cell culture system supporting its replication. Recently, a unique HCV strain JFH1 has been found to replicate efficiently in cell culture with production of infectious HCV (HCVcc). Baculovirus vectors were found to be efficient delivery vehicles and a HBV recombinant baculovirus/HepG2 system efficiently delivered the HBV genome into HepG2 resulting in HBV replication. In this study, we developed a recombinant baculovirus expression system to generate infectious HCV particles in hepatoma cell line Huh7-lunetT7 by using cDNA from the HCV JFH1 genotype. Results show that HCV positive, negative RNA strands and proteins were produced in this system. Furthermore, HCV particles were produced and secreted into the culture medium. Sucrose density gradient centrifugation of the culture medium revealed co-localization of HCV RNA and structural proteins in the fraction with a density of 1.08-1.13 g/ml. Electron microscopy (EM) showed viral particles approximately 55 nm in diameter, which could be recognized by anti-HCV E2 antibodies. Real-time RT-PCR detected that the level of HCV vRNA in the supernatant was 10(7) copies/ml at 72 h post-transduction (hpt). In addition, the JFH1 virus produced by the recombinant baculovirus was confirmed to be infectious in vitro. In summary, this system provides a novel tool not only for the analysis of the replication and pathogenesis of HCV but also to screen for potential therapeutic targets.
Yao
Xiangjie
X
State Key Lab of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, People's Republic of China.
Han
Qingxia
Q
Song
Jianhua
J
Liang
Changyong
C
Wakita
Takaji
T
Yang
Rongge
R
Chen
Xinwen
X
D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D016679:Genome, Viral / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008854:Microscopy, Electron; D019076:Transgenes / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*; D014771:Virion / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases