We identified an N-terminal amphipathic helix (AH) in one of hepatitis C virus (HCV)'s nonstructural proteins, NS5A. This AH is necessary and sufficient for membrane localization and is conserved across isolates. Genetically disrupting the AH impairs HCV replication. Moreover, an AH peptide-mimic inhibits the membrane association of NS5A in a dose-dependent manner. These results have exciting implications for the HCV life cycle and novel antiviral strategies.Hepatitis C virus (HCV) is a significant cause of morbidity and mortality, infecting over 100 million people worldwide (1, 9). Despite recent progress, current therapies remain inadequate for the majority of patients (27,29,45). HCV is a positive, single-stranded RNA virus. Its 9.6-kb genome encodes a single ϳ3,000-amino-acid polyprotein which is proteolytically processed by cellular and viral proteinases into structural (components of the mature virus) and nonstructural (NS) (elements proposed to help replicate new virions) proteins (2,6,33). Like other plus-strand RNA viruses, HCV is thought to replicate its RNA in association with cytoplasmic membranes (4,8,12,25,34), although how the RNA replication complex is assembled and maintained remains unknown. That NS5A, one of the NS proteins of HCV, may play a key role in membrane-associated RNA replication is suggested by its apparent association with host cell membranes (17,30,37). NS5A has also been reported to interact with a variety of host cell proteins (13,41,44) and to determine the response to interferon therapy in some patients (11), although to date its precise role in HCV replication is not clear.The study of HCV replication has been hampered by the lack of a convenient cell culture system. The recent advent of high-efficiency HCV subgenomic replicons (5), however, now opens the prospect of performing detailed molecular genetic studies. Such replicons, based on the original report by Lohmann et al. (28), contain all the cis and trans elements required for HCV RNA replication and should allow an analysis of structure-function relationships of engineered HCV mutants.In the hope of further characterizing the role of NS5A in the HCV life cycle and identifying potential novel targets for antiviral therapy, we have been studying the cell biology of this NS protein. We were particularly interested in its mechanism of membrane association since it has no obvious transmembrane or endoplasmic reticulum (ER)-targeting domains (32).Here we report the identification of a key mechanism of the membrane association of NS5A and show that disrupting this mechanism abolishes HCV RNA replication. Disruption of the N-terminal AH of NS5A abolishes membrane localization. To study the determinants of NS5A intracellular localization, we first expressed NS5A in Huh-7 cells (a liver-derived cell line) by using the vaccinia virus expression system and a vector that encodes most of the HCV NS proteins (16). The cells were fixed with 4% formaldehyde and stained with a monoclonal antibody against NS5A (Virostat, Portland, Maine) ...

More effective therapies are urgently needed against hepatitis C virus (HCV), a major cause of viral hepatitis. We used in vitro protein expression and microfluidic affinity analysis to study RNA binding by the HCV transmembrane protein NS4B, which plays an essential role in HCV RNA replication. We show that HCV NS4B binds RNA and that this binding is specific for the 3′ terminus of the negative strand of the viral genome with a dissociation constant (Kd) of ~3.4 nM. A high-throughput microfluidic screen of a compound library identified 18 compounds that substantially inhibited binding of RNA by NS4B. One of these compounds, clemizole hydrochloride, was found to inhibit HCV RNA replication in cell culture that was mediated by its suppression of NS4B’s RNA binding, with little toxicity for the host cell. These results yield new insight into the HCV life cycle and provide a candidate compound for pharmaceutical development.

Like other positive-strand RNA viruses, hepatitis C virus (HCV) is believed to replicate its RNA in association with host cell cytoplasmic membranes. Because of its association with such membranes, NS4B, one of the virus's nonstructural proteins, may play an important role in this process, although the mechanistic details are not well understood. We identified a putative N-terminal amphipathic helix (AH) in NS4B that mediates membrane association. Introduction of site-directed mutations designed to disrupt the hydrophobic face of the AH abolishes the AH's ability to mediate membrane association. An AH in NS4B is conserved across HCV isolates. Completely disrupting the amphipathic nature of NS4B's N-terminal helix abolished HCV RNA replication, whereas partial disruption resulted in an intermediate level of replication. Finally, immunofluorescence studies revealed that HCV replication complex components were mislocalized in the AH-disrupted mutant. These results identify a key membrane-targeting domain which can form the basis for developing novel antiviral strategies.Hepatitis C virus (HCV) is a positive-strand RNA virus that belongs to the family Flaviviridae and was classified into a separate genus, Hepacivirus (39). HCV infects over 100 million people worldwide and causes chronic hepatitis that can progress to liver cirrhosis and hepatocellular carcinoma (1, 11). Despite recent progress, current therapies remain inadequate for the majority of patients (30,33,50).HCV's genome is composed of a 9.6-kb positive, singlestranded RNA molecule that encodes an ϳ3,000-amino-acid polyprotein, which is proteolytically processed by cellular and viral proteinases into structural proteins (components of the mature virus) and nonstructural proteins (proteins proposed to be involved in the replication of the virus) (2,8,38). Like that of other positive-strand RNA viruses (3,10,18,28,39), HCV's RNA replication is believed to take place on cytoplasmic membranes (15, 21), although the details of the replication complex assembly and maintenance are largely unknown. A better understanding of these mechanistic details should help elucidate a fundamental stage in the viral life cycle and may reveal potential new targets for antiviral therapy.The introduction of the high-efficiency HCV subgenomic replicon (4, 31) and, recently, of the full-length replicon (5, 6) enables the design and implementation of detailed molecular genetic studies of the HCV replication process. Such replicons contain all the cis and trans elements required for HCV RNA replication and allow studies of engineered HCV mutants (4, 16).The function of NS4B, one of HCV's nonstructural proteins, is incompletely understood. NS4B is a membrane-associated protein that colocalizes predominantly with endoplasmic reticulum (ER) markers, suggesting an ER or ER-derived membrane localization (24,26,32,42). This membrane association was shown to occur cotranslationally, and NS4B behaves biochemically as an integral membrane protein (24). The precise topology of NS4B with respe...

Hepatitis C virus (HCV) is a major cause of viral hepatitis. There is no effective therapy for most patients. We have identified a nucleotide binding motif (NBM) in one of the virus's nonstructural proteins, NS4B. This structural motif binds and hydrolyzes GTP and is conserved across HCV isolates. Genetically disrupting the NBM impairs GTP binding and hydrolysis and dramatically inhibits HCV RNA replication. These results have exciting implications for the HCV life cycle and novel antiviral strategies.Over 150 million people are infected with hepatitis C virus (HCV) worldwide (1). Current therapies are inadequate for most of these individuals (18). HCV is a positive singlestranded RNA virus. Its 9.6-kb genome encodes a single ϳ3,000-amino-acid polyprotein, which is proteolytically processed into structural proteins, which are components of the mature virus, and nonstructural proteins, which are involved in replicating the viral genome (26). A characteristic feature of positive-strand RNA viruses is their use of cytoplasmic membranes as platforms for replication (27). These membranes can either be preexisting host cell compartments or novel structures induced by the virus (2,4,8,17,27). HCV is also believed to replicate in association with intracellular membranes, although how the RNA replication complex is assembled and maintained remains unknown. Recently the HCV NS4B protein has been shown to induce the formation of a distinct membranous structure designated the membranous web (5), which represents the candidate site for HCV RNA replication (12). The mechanism whereby NS4B mediates its function(s) in membrane-associated RNA replication, however, remains to be elucidated and may offer insights for the development of novel antiviral strategies. Here we report the identification of a nucleotide binding motif (NBM) within NS4B and show that this motif mediates both binding and hydrolysis of GTP and HCV RNA replication. MATERIALS AND METHODSCell cultures. Cell monolayers of the human hepatoma cell line Huh-7 were routinely grown in complete medium consisting of equal volumes of Dulbecco's modified minimal essential medium (Gibco) and RPMI 1640 (Gibco), supplemented with 1% L-glutamine (Gibco), 1% penicillin, 1% streptomycin, and 10% fetal bovine serum. Cell lines were passaged twice weekly after treatment with 0.05% trypsin-0.02% EDTA and seeding at a dilution of 1:10. Antibodies.A rabbit polyclonal antibody against green fluorescent protein (GFP) and an anti-rabbit secondary antibody were purchased from Molecular Probes. A monoclonal antibody against glutathione S-transferase (GST) was purchased from Cell Signaling Technology.Plasmids. Standard recombinant DNA technology was used to construct and purify all plasmids. All regions that were amplified by PCR were analyzed by automated DNA sequencing. Plasmid DNAs were prepared from large-scale bacterial cultures and purified by a Maxiprep kit (Marligen Biosciences). Restriction enzymes were purchased from New England Biolabs. The Bart79I plasmid was described previou...

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