In this study, we establish that cholesterol and sphingolipid associated with hepatitis C virus (HCV) particles are important for virion maturation and infectivity. In a recently developed culture system enabling study of the complete life cycle of HCV, mature virions were enriched with cholesterol as assessed by the molar ratio of cholesterol to phospholipid in virion and cell membranes. Depletion of cholesterol from the virus or hydrolysis of virion-associated sphingomyelin almost completely abolished HCV infectivity. Supplementation of cholesterol-depleted virus with exogenous cholesterol enhanced infectivity to a level equivalent to that of the untreated control. Cholesterol-depleted or sphingomyelin-hydrolyzed virus had markedly defective internalization, but no influence on cell attachment was observed. Significant portions of HCV structural proteins partitioned into cellular detergent-resistant, lipid-raft-like membranes. Combined with the observation that inhibitors of the sphingolipid biosynthetic pathway block virion production, but not RNA accumulation, in a JFH-1 isolate, our findings suggest that alteration of the lipid composition of HCV particles might be a useful approach in the design of anti-HCV therapy.Hepatitis C virus (HCV) is recognized as a major cause of chronic liver disease, including chronic hepatitis, hepatic steatosis, cirrhosis, and hepatocellular carcinoma. It presently affects approximately 200 million people worldwide (26). HCV is an enveloped positive-strand RNA virus belonging to the Hepacivirus genus of the family Flaviviridae. Its genome of ϳ9.6 kb encodes a polyprotein precursor of ϳ3,000 residues, and the structural proteins (core, E1, and E2) reside in its N-terminal region.Little is known about the assembly of HCV and its virion structure, because efficient production of authentic HCV particles has only recently been achieved. Nucleocapsid assembly generally involves oligomerization of the capsid protein and encapsidation of genomic RNA. This process is thought to occur upon interaction of the core protein with viral RNA, and this core-RNA interaction may induce a change from RNA replication to packaging. As with related viruses, the mature HCV virion likely consists of a nucleocapsid and an outer envelope composed of a lipid membrane and envelope proteins. Expression of the structural proteins in mammalian cells has been observed to generate virus-like particles with ultrastructural properties similar to those of HCV virions (5, 29). Packaging of these HCV-like particles into intracellular vesicles as a result of budding from the endoplasmic reticulum (ER) has also been observed (8,34). However, HCV structural proteins are observed both in the ER and in the Golgi apparatus (45). Moreover, complex N-linked glycans have been detected on the surfaces of HCV particles isolated from patient sera, suggesting that the glycans transit through the Golgi apparatus (44). Interactions between the core and E1/E2 proteins are thought to determine viral morphology and are mediated thro...

Hepatitis C virus (HCV) infection causes chronic liver disease and is a worldwide health problem. Despite ever-increasing demand for knowledge on viral replication and pathogenesis, detailed analysis has been hampered by a lack of efficient viral culture systems. We isolated HCV genotype 2a strain JFH-1 from a patient with fulminant hepatitis. This strain replicates efficiently in Huh7 cells. Efficient replication and secretion of recombinant viral particles can be obtained in cell culture by transfection of in vitro-transcribed full-length JFH-1 RNA into Huh7 cells. JFH-1 virus generated in cell culture is infectious for both naive Huh7 cells and chimpanzees. The efficiency of viral production and infectivity of generated virus is substantially improved with permissive cell lines. This protocol describes how to use this system, which provides a powerful tool for studying viral life cycle and for the construction of antiviral strategies and the development of effective vaccines. Viral particles can be obtained in 12 days with this protocol.

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Hepatitis C virus (HCV) nonstructural protein 3-4A (NS3-4A) is a complex composed of NS3 and its cofactor NS4A. It harbours serine protease as well as NTPase/RNA helicase activities and is essential for viral polyprotein processing, RNA replication and virion formation. Specific inhibitors of the NS3-4A protease significantly improve sustained virological response rates in patients with chronic hepatitis C when combined with pegylated interferon-α and ribavirin. The NS3-4A protease can also target selected cellular proteins, thereby blocking innate immune pathways and modulating growth factor signalling. Hence, NS3-4A is not only an essential component of the viral replication complex and prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. This review provides a concise update on the biochemical and structural aspects of NS3-4A, its role in the pathogenesis of chronic hepatitis C and the clinical development of NS3-4A protease inhibitors.