Robust Production of Infectious Hepatitis C Virus (HCV) from Stably HCV cDNA-Transfected Human Hepatoma Cells

Abstract

Hepatitis C virus (HCV) chronically infects approximately 170 million people worldwide, with an increased risk of developing cirrhosis and hepatocellular carcinoma. The study of HCV replication and pathogenesis has been hampered by the lack of an efficient stable cell culture system and small-animal models of HCV infection and propagation. In an effort to develop a robust HCV infection system, we constructed stable human hepatoma cell lines that contain a chromosomally integrated genotype 2a HCV cDNA and constitutively produce infectious virus. Transcriptional expression of the full-length HCV RNA genome is under the control of a cellular Pol II polymerase promoter at the 5 end and a hepatitis delta virus ribozyme at the 3 end. The resulting HCV RNA was expressed and replicated efficiently, as shown by the presence of high levels of HCV proteins as well as both positive-and negative-strand RNAs in the stable Huh7 cell lines. Stable cell lines robustly produce HCV virions with up to 10 8 copies of HCV viral RNA per milliliter (ml) of the culture medium. Subsequent infection of naïve Huh7.5 cells with HCV released from the stable cell lines resulted in high levels of HCV proteins and RNAs. Additionally, HCV infection was inhibited by monoclonal antibodies specific to CD81 and the HCV envelope glycoproteins E1 and E2, and HCV replication was suppressed by alpha interferon. Collectively, these results demonstrate the establishment of a stable HCV culture system that robustly produces infectious virus, which will allow the study of each aspect of the entire HCV life cycle.Discovered in 1989 by molecular cloning (10), hepatitis C virus (HCV) has been recognized as a major cause of viral hepatitis in humans. HCV infection is characterized by the establishment of chronic infection in the majority (up to 85%) of individuals exposed to HCV. It is estimated that approximately 4 million people in the United States and 170 million people worldwide are persistently infected (9, 38). The chronic HCV infection carries an increased risk of developing fatal liver diseases such as cirrhosis, liver failure, and hepatocellular carcinoma. HCV is a single-stranded positive-sense RNA virus belonging to the Hepacivirus genus of the family Flaviviridae (30). The 9.6-kb RNA genome encodes a single polyprotein that is cleaved by cellular and viral proteases into at least 10 structural (C, E1, E2, and probably p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins that play important roles in virus entry, replication, assembly, and pathogenesis (24, 29). The sequence and structures of the untranslated regions (UTR) at both the 5Ј and 3Ј ends of the HCV RNA genome, which contain cis-acting RNA elements required for HCV RNA translation and replication, are highly conserved (7,13,20,24,25,35,40,41).A great deal of progress has been made with respect to the HCV genome organization, properties and roles of viral proteins and conserved RNA sequence/structures, virus-host interactions, and mechanisms of HCV replication since the dis...

Hepatitis C virus production by human hepatocytes dependent on assembly and secretion of very low-density lipoproteins

“…We then examined whether VLDL secretion is required for the release of infectious HCV particles from Huh7 cells. Huh7-GL cells, a line of Huh7 cells that contain a chromosomally integrated genotype 2a HCV cDNA and constitutively produce infectious virus (27), were incubated in the absence or presence of the MTP inhibitor. After incubation in serum-free medium, culture medium was harvested, and the amount of HCV RNA, HCV titer, and apoB in the medium was measured by real-time PCR, foci formation, and immunoblot analysis, respectively.…”

“…They were maintained in medium A supplemented with 200 g/ml G418. Huh7-GL cells, a line of Huh7 cells that contain a chromosomally integrated genotype 2a HCV cDNA and constitutively produce infectious virus (27), were maintained in medium A supplemented with 5 g/ml blasticidine. All cells were maintained in monolayer culture at 37°C in 5% CO 2 .…”

Centrifugal enhancement of hepatitis C virus infection of human hepatocytes

“…Although HCV replication in vivo is extremely robust (Neumann et al, 1998), the growth of HCV in cell cultures in vitro is very difficult. Several groups recently reported the establishment of efficient in vitro cell systems for complete and infectious HCV replication based on the hepatic cell lines and HCV genotype 2a JFH1 clone (Cai et al, 2005;Lindenbach et al, 2006;Wakita et al, 2005;Zhong et al, 2005).…”

New developments in small molecular compounds for anti-hepatitis C virus (HCV) therapy

“…The discovery of HCV's autonomous subgenomic replication system in 1999 (Lohmann et al, 1999) boosted knowledge of the HCV replication cycle, and a robust cell culture infection system JFH-1/HCVcc was established in 2005. This system has allowed researchers to study the HCV life cycle (Cai et al, 2005;Lindenbach et al, 2005;Zhong et al, 2005) and has accelerated the development of new HCV therapies.…”