Hepatitis C virus (HCV) infection is a global health problem affecting an estimated 170 million individuals worldwide. We report the identification of multiple independent adaptive mutations that cluster in the HCV nonstructural protein NS5A and confer increased replicative ability in vitro. Among these adaptive mutations were a single amino acid substitution that allowed HCV RNA replication in 10% of transfected hepatoma cells and a deletion of 47 amino acids encompassing the interferon (IFN) sensitivity determining region (ISDR). Independent of the ISDR, IFN-alpha rapidly inhibited HCV RNA replication in vitro. This work establishes a robust, cell-based system for genetic and functional analyses of HCV replication.

Hepatitis C virus (HCV) replication appears to be restricted to the human hepatoma cell line Huh-7, indicating that a favorable cellular environment exists within these cells. Although adaptive mutations in the HCV nonstructural proteins typically enhance the replicative capacity of subgenomic replicons in Huh-7 cells, replication can only be detected in a subpopulation of these cells. Here we show that self-replicating subgenomic RNA could be eliminated from Huh-7 clones by prolonged treatment with alpha interferon (IFN-␣) and that a higher frequency of cured cells could support both subgenomic and full-length HCV replication. The increased permissiveness of one of the cured cell lines allowed us to readily detect HCV RNA and antigens early after RNA transfection, eliminating the need for selection of replication-positive cells. We also demonstrate that a single amino acid substitution in NS5A is sufficient for establishing HCV replication in a majority of cured cells and that the major phosphate acceptor site of subtype 1b NS5A is not essential for HCV replication.An estimated 3% of the world's population is seropositive for hepatitis C virus (HCV) (32). The acute phase of infection is often subclinical; however, approximately 70% of seropositive individuals develop a chronic infection, predisposing the infected patient to the development of progressive liver pathology, including fibrosis, cirrhosis, and hepatocellular carcinoma (1, 28). The current treatments for HCV infection are alpha interferon (IFN-␣) in combination with ribavirin or, more recently, a polyethylene glycol-modified form of IFN-␣; however, sustained responses are only observed in ϳ50% of treated patients, and effectiveness varies depending on the infecting HCV genotype (19).HCV has been classified within its own genus, Hepacivirus, within the family Flaviviridae, which comprises three genera of small enveloped positive-strand RNA viruses (27). The 9.6-kb genome consists of a single open reading frame (ORF) flanked by 5Ј and 3Ј nontranslated regions (NTRs) (reviewed in references 3, 4, and 16). The 5Ј NTR contains an internal ribosome entry site (IRES), mediating cap-independent translation of the ORF of ϳ3,011 amino acids. The resulting polyprotein is processed co-and posttranslationally into at least 10 individual proteins. Host signal peptidase cleavages within the N-terminal portion of the polyprotein generate the structural proteins core (C), E1, and E2. Two HCV-encoded proteases mediate downstream cleavages, liberating the nonstructural (NS) proteins involved in viral replication. The NS2-3 protease spanning the C-terminal half of NS2 and the N-terminal one-third of NS3 catalyzes autocatalytic cleavage between NS2 and NS3. The N-terminal one-third of NS3 also encodes a serine protease that functions in concert with NS4A to cleave downstream sites, while the C-terminal two-thirds harbors RNA helicase and RNA-stimulated nucleoside triphosphatase activities. The NS5B protein exhibits an RNA-dependent RNA polymerase activity. Although the N...

More than 1% of the world's population is chronically infected with hepatitis C virus (HCV). HCV infection can result in acute hepatitis, chronic hepatitis, and cirrhosis, which is strongly associated with development of hepatocellular carcinoma. Genetic studies of HCV replication have been hampered by lack of a bona fide infectious molecular clone. Full-length functional clones of HCV complementary DNA were constructed. RNA transcripts from the clones were found to be infectious and to cause disease in chimpanzees after direct intrahepatic inoculation. This work defines the structure of a functional HCV genome RNA and proves that HCV alone is sufficient to cause disease.

Hepatitis C virus (HCV) genotype 1 (subtypes 1a and 1b) is responsible for the majority of treatmentresistant liver disease worldwide. Thus far, efficient HCV RNA replication has been observed only for subgenomic and full-length RNAs derived from genotype 1b isolates. Here, we report the establishment of efficient RNA replication systems for genotype 1a strain H77. Replication of subgenomic and full-length H77 1a RNAs required the highly permissive Huh-7.5 hepatoma subline and adaptive amino acid substitutions in both NS3 and NS5A. Replication could be detected by RNA quantification, fluorescence-activated cell sorting, and metabolic labeling of HCV-specific proteins. Replication efficiencies were similar for subgenomic and fulllength RNAs and were most efficient for HCV RNAs lacking heterologous RNA elements. Interestingly, both subtype 1a and 1b NS3 adaptive mutations are surface exposed and present on only one face of the NS3 structure. The cell culture-adapted subtype 1a replicons should be useful for basic replication studies and for antiviral development. These results are also encouraging for the development of adapted replicons for the remaining HCV genotypes.Persistent infection with hepatitis C virus (HCV) is one of the primary causes of chronic liver disease. Progression to chronic active hepatitis with cirrhosis occurs in ϳ20 to 30% of infected individuals, and HCV-associated liver disease is now the leading cause of liver transplantation in the United States (7). Genotypes 1a and 1b, the most prevalent worldwide, have the poorest rates of response to the present treatment regimen, a combination of pegylated alfa interferon 2b with ribavirin (4, 5, 18).HCV, a member of the family Flaviviridae, is a small enveloped virus whose genome is a 9.6-kb single-stranded RNA with positive polarity consisting of a 5Ј nontranslated region (NTR), a large open reading frame encoding the virus-specific proteins, and a 3Ј NTR (reviewed in references 1, 15, and 21). The 5Ј NTR contains an internal ribosome entry site (IRES) mediating translation of a single polyprotein of ϳ3,000 amino acids with the structural proteins (C, E1, and E2) located in the N terminus and the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) encoded in the remainder. The NS3-5B coding region is sufficient for RNA replication in cell culture (17), and these proteins are presumed to function as components of the HCV replicase. The NS3 protein possesses serine protease and nucleoside triphosphatase-helicase activities, NS4A is a cofactor for the NS3 serine protease, and NS5B is the RNA-dependent RNA polymerase. The functions of NS4B and NS5A remain elusive, although NS5A, a phosphorylated protein, has been a target for adaptive mutation, allowing efficient initiation of HCV replication in vitro (2, 9, 13). Amino acid substitutions in the NS3, NS4B, and NS5B proteins can also enhance replication to various degrees (9, 13, 16).Initially, only the genotype 1b Con1 RNA sequence was replication competent in the human hepatoma cell line Hu...