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...

The E2 glycoprotein of hepatitis C virus (HCV) mediates viral attachment and entry into target hepatocytes and elicits neutralizing antibodies in infected patients. To characterize the structural and functional basis of HCV neutralization, we generated a novel panel of 78 monoclonal antibodies (MAbs) against E2 proteins from genotype 1a and 2a HCV strains. Using high-throughput focus-forming reduction or luciferase-based neutralization assays with chimeric infectious HCV containing structural proteins from both genotypes, we defined eight MAbs that significantly inhibited infection of the homologous HCV strain in cell culture. Two of these bound E2 proteins from strains representative of HCV genotypes 1 to 6, and one of these MAbs, H77. Hepatitis C virus (HCV) is a blood-borne hepatotropic virus that infects ϳ170 million people worldwide. Approximately 70% of infected individuals progress to chronic liver disease, which carries an increased risk of cirrhosis and hepatocellular carcinoma (7). In general, treatment of chronic HCV infection is complicated by resistance due to extensive genetic diversity. HCV has been classified into seven major genotypes, which differ by ϳ30% at the nucleotide level (4), and this positivesense, single-stranded RNA virus has a capacity for rapid evolution of variant viruses during persistent infection. The current treatment, pegylated ␣ 2a interferon (IFN-␣ 2a ) and ribavirin, has variable side effects and response rates depending on the virus and host genotype (16). No vaccine is currently available, and preclinical development has been hampered by a lack of understanding of which conserved epitopes on the HCV structural proteins should be targeted.HCV contains an ϳ9.6-kb RNA genome that is translated as a single polyprotein and then cleaved by viral and host proteases into structural proteins (core, E1, and E2), p7, and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (39). Viral attachment and entry are mediated by the envelope glycoproteins, E1 and E2. Four attachment or entry receptors that are required for infection of hepatocytes have been identified, including CD81 (53), scavenger receptor B1 (SR-B1) (56), and the tight-junction proteins claudin 1 (CLDN1) (14) and occludin (OCLN) (54). The importance of E2 binding to the large extracellular loop of CD81 has been established in vitro (13,18,28,50,53), and interactions between E2 hypervariable region 1 (HVR1) and SR-B1

The hepatitis C virus (HCV) glycoproteins (E1 and E2) interact to form a heterodimeric complex, which has been proposed as a functional subunit of the HCV virion envelope. As examined in cell culture transient-expression assays, the formation of properly folded, noncovalently associated E1E2 complexes is a slow and inefficient process. Due to lack of appropriate immunological reagents, it has been difficult to distinguish between glycoprotein molecules that undergo productive folding and assembly from those which follow a nonproductive pathway leading to misfolding and aggregation. Here we report the isolation and characterization of a conformation-sensitive E2-reactive monoclonal antibody (H2). The H2 monoclonal antibody selectively recognizes slowly maturing E1E2 heterodimers which are noncovalently linked, protease resistant, and no longer associated with the endoplasmic reticulum chaperone calnexin. This complex probably represents the native prebudding form of the HCV glycoprotein heterodimer. Besides providing a novel reagent for basic studies on HCV virion assembly and entry, this monoclonal antibody should be useful for optimizing production and isolation of native HCV glycoprotein complexes for serodiagnostic and vaccine applications.

The RNA genome of hepatitis C virus (HCV) terminates with a highly conserved 98-base sequence. Enzymatic and chemical approaches were used to define the secondary structure of this 3-terminal element in RNA transcribed in vitro from cloned cDNA. Both approaches yielded data consistent with a stable stem-loop structure within the 3-terminal 46 bases. In contrast, the 5 52 nucleotides of this 98-base element appear to be less ordered and may exist in multiple conformations. Under the experimental conditions tested, interaction between the 3 98 bases and upstream HCV sequences was not detected. These data provide valuable information for future experiments aimed at identifying host and/or viral proteins which interact with this highly conserved RNA element. MATERIALS AND METHODS Construction of plasmids for RNA synthesis. The full-length HCV consensus sequence (genotype 1a) was cloned downstream of the T7 RNA polymerase promoter in a pBR322 derivative, pTET, where the sequences between DraI-EcoRI were deleted and replaced with a polylinker (20) (Fig. 1). The sequence between the ApaI sites in the ORF were deleted, leaving the entire 5Ј and 3Ј NTRs (⌬Apa [Fig. 1]). A deletion between XcmI and MseI in the ⌬Apa plasmid created a construct containing 9 nt of the 5Ј NTR and 4 nt of the poly(U/UC) tract upstream of the 98-base element (⌬Xcm [Fig. 1]). The fourth construct contained only 52 nt of the 98-base element (⌬SL I [Fig. 1]). Plasmid DNAs were linearized with either BsmI or EcoRV such that transcribed RNA would presumably have the same 3Ј terminus as HCV RNA or additional nonviral sequences to serve as a primer binding site during cDNA synthesis, respectively (Fig. 1). RNA was transcribed according to standard procedures, using T7 RNA polymerase (Epicentre). Radioactive end labeling. Dephosphorylated in vitro-transcribed RNA and gel-purified oligonucleotides were 5Ј end labeled by using T4 polynucleotide kinase (Gibco-BRL) and [␥-32 P]ATP (6,000 Ci/mmol; Amersham). Dephosphorylated RNA and oligonucleotides were incubated in a 20-l reaction mixture containing 70 mM Tris-HCl (pH 7.6), 10 mM MgCl 2 , 100 mM KCl, 1 mM 2-mercaptoethanol, 100 Ci of [␥-32 P]ATP, and 10 U of T4 polynucleotide kinase for 30 min at 37°C. The reaction mixture was phenol-chloroform extracted and ethanol precipitated. Labeling at the 3Ј end of RNA was performed by ligation of [␣-32 P]pCp (3,000 Ci/mmol; Amersham) with T4 RNA ligase (Gibco-BRL) as described by England and Uhlenbeck (10). All radiolabeled RNA was purified by denaturing polyacrylamide gel electrophoresis, eluted in 100 mM Tris-HCl (pH 8.0)-1 mM EDTA, phenol-chloroform extracted, and ethanol precipitated. Structure-specific enzymatic probing. Enzymatic probing of in vitro-transcribed RNAs was performed at 0°C for 30 min with the following RNases: cobra venom RNase V 1 (0.01 and 0.1 U; Pharmacia), RNase CL3 (0.1 U; U.S. Biochemical [USB]), and RNases T 1 (USB), T 2 (Gibco-BRL), Phy M (Gibco-BRL), and B. cereus (USB) (each at 1 U). RNase digestion of 10 5 cpm of 32 P-labeled RNA was ...

We generated recombinant vesicular stomatitis viruses (VSV) expressing genes encoding hybrid proteins consisting of the extracellular domains of hepatitis C virus (HCV) glycoproteins fused at different positions to the transmembrane and cytoplasmic domains of the VSV G glycoprotein (E1G and E2G). We show that these chimeric proteins are transported to the cell surface and incorporated into VSV virions efficiently. We also generated VSV recombinants in which the gene encoding the VSV G protein was deleted and replaced by one or both of the E1G and E2G genes, together with a green fluorescent protein gene. These ⌬G viruses incorporated E1G and E2G proteins at levels approximately equivalent to the normal level of VSV G itself, or about 1,200 molecules of each protein per virion. Given the potency of VSV recombinants as vaccines in other studies, this high-level expression and incorporation of HCV proteins into virions could be very important for development of an HCV vaccine. Despite the presence of E1G and E2G proteins at high levels in the virions, these virions did not infect cell lines that have been reported to support at least a low level of HCV infection and replication.Hepatitis C virus (HCV), the major cause of non-A, non-B viral hepatitis, was first identified in 1989 (3) and has infected approximately 170 million people worldwide (34), including about 4 million in the United States (1). HCV infection leads to chronic hepatitis, cirrhosis, and hepatocellular carcinoma and is now the leading cause of liver transplantation in the United States (7). Current treatment for HCV infection leads to sustained viral clearance in only about 50% of patients (24), and no vaccine is available to prevent new infections.HCV is a positive-stranded RNA virus with a genome of 9.6 kb encoding a polyprotein precursor of 3,000 amino acids (19). The polyprotein is proteolytically cleaved into 10 distinct products: the structural proteins (C, E1, E2, and P7) and several nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). E1 and E2 are type I transmembrane proteins which are highly glycosylated and associate to form a noncovalently linked heterodimer (6). The C-terminal hydrophobic regions of both proteins contain signals that are responsible for retaining these proteins in the endoplasmic reticulum (4,5,8). Deletions in these C-terminal regions and replacement with foreign transmembrane and cytoplasmic domains result in the expression of both E1 and E2 at the cell surface. E2 binds with high affinity to CD81, a tetraspanin protein expressed on various cell types including hepatocytes and B lymphocytes (23). However, definitive evidence that CD81 is an HCV receptor has not yet been obtained. Vesicular stomatitis virus (VSV) is a nonsegmented negative-strand RNA virus and the prototype of the rhabdovirus family. VSV infection in animals induces strong cellular and humoral immunity to its own proteins and to additional proteins encoded by recombinant viruses (12,20,26,35). VSV has an 11-kb RNA genome of negative (noncod...