The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is a multifunctional enzyme with serine protease and DEXH/D-box helicase domains. A crystal structure of the NS3 helicase domain (NS3h) was generated in the presence of a single-stranded oligonucleotide long enough to accommodate binding of two molecules of enzyme. Several amino acid residues at the interface of the two NS3h molecules were identified that appear to mediate a proteinprotein interaction between domains 2 and 3 of adjacent molecules. Mutations were introduced into domain 3 to disrupt the putative interface and subsequently examined using an HCV subgenomic replicon, resulting in significant reduction in replication capacity. The mutations in domain 3 were then examined using recombinant NS3h in biochemical assays. The mutant enzyme showed RNA binding and RNA-stimulated ATPase activity that mirrored wild type NS3h. In DNA unwinding assays under single turnover conditions, the mutant NS3h exhibited a similar unwinding rate and only ϳ2-fold lower processivity than wild type NS3h. Overall biochemical activities of the mutant NS3h were similar to the wild type enzyme, which was not reflective of the large reduction in HCV replicative capacity observed in the biological experiment. Hence, the biological results suggest that the known biochemical properties associated with the helicase activity of NS3h do not reveal all of the likely biological roles of NS3 during HCV replication. Domain 3 of NS3 is implicated in protein-protein interactions that are necessary for HCV replication. Hepatitis C virus (HCV),5 a member of the family Flaviviridae, is a leading cause of liver cirrhosis and hepatocellular carcinoma and has infected more than 170 million people worldwide (1-3). Standard treatment with interferon-␣ and ribavirin is frequently ineffective or toxic, and no vaccine for HCV is currently available (4). Identification of a novel viral target for therapeutic intervention could lead to the development of a more effective treatment.The HCV genome is a 9.6-kb positive, single-stranded RNA that supports translation of a 3000-amino acid polyprotein, which is subsequently cleaved to produce both structural (C, E1, and E2) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) viral proteins (5). Two viral proteases are encoded by the NS2 and NS3 genes (6, 7). In addition to its protease domain, the NS3 protein also contains a DEXH/ D-box helicase domain (8). NS4A encodes a peptide co-factor for NS3 protease (9), and NS5B encodes an RNA-dependent RNA polymerase (10). The functions of NS4B and NS5A have not been as well characterized, but NS5A is involved in viral interferon sensitivity (11) and in adaptation of subgenomic replicons for growth in cultured hepatoma cells (12).One obstacle to studying viral protein interactions is that HCV cannot be maintained efficiently in cell culture, although recent progress may overcome this difficulty (13). In order to circumvent this problem, selectable replicons carrying the neomycin phosphotransferase gene in add...

HCV NS3 helicase exhibits activity toward DNA and RNA substrates. The DNA helicase activity of NS3 has been proposed to be optimal when multiple NS3 molecules are bound to the same substrate molecule. NS3 catalyzes little or no measurable DNA unwinding under single cycle conditions in which the concentration of substrate exceeds the concentration of enzyme by 5-fold. However, when NS3 (100 nM) is equimolar with the substrate, a small burst amplitude of ϳ8 nM is observed. The burst amplitude increases as the enzyme concentration increases, consistent with the idea that multiple molecules are needed for optimal unwinding. Protein-protein interactions may facilitate optimal activity, so the oligomeric properties of the enzyme were investigated. Chemical cross-linking indicates that fulllength NS3 forms higher order oligomers much more readily than the NS3 helicase domain. Dynamic light scattering indicates that full-length NS3 exists as an oligomer, whereas NS3 helicase domain exists in a monomeric form in solution. Size exclusion chromatography also indicates that full-length NS3 behaves as an oligomer in solution, whereas the NS3 helicase domain behaves as a monomer. When NS3 was passed through a small pore filter capable of removing protein aggregates, greater than 95% of the protein and the DNA unwinding activity was removed from solution. In contrast, only ϳ10% of NS3 helicase domain and ϳ20% of the associated DNA unwinding activity was removed from solution after passage through the small pore filter. The results indicate that the optimally active form of fulllength NS3 is part of an oligomeric species in vitro.Helicases are molecular motors that catalyze unwinding of double-stranded DNA or RNA by converting chemical energy from ATP hydrolysis into mechanical energy for nucleic acid strand separation. Helicases are required for virtually all cellular processes involving nucleic acids, including replication, transcription, translation, repair, and recombination (1-4). Several diseases characterized by premature aging and increased incidence of cancer, including Bloom and Werner syndromes, have been linked to mutations in helicase genes (5).The quaternary structure of the active forms of helicases varies considerably, with some requiring dimer or oligomer formation for strand separation activity and others functioning efficiently as monomers. Bacteriophage T4 gp41 helicase, for example, forms a hexameric structure that sequesters singlestranded DNA by encircling it (6). Numerous other helicases function as hexamers (7). Bacteriophage T4 Dda helicase is active as a monomer (8) but unwinds DNA substrates more efficiently under conditions that allow binding of multiple helicase molecules per substrate molecule (9). Escherichia coli helicase TraI, also called helicase 1, can function as a robust, highly processive helicase as a monomer (10). In order to understand the mechanism by which helicases unwind duplex DNA, the quaternary structure of the active species must be known.More than 170 million people worldwide a...

Hepatitis C virus (HCV) infects over 170 million persons worldwide. It is the leading cause of liver disease in the U.S. and is responsible for most liver transplants. Current treatments for this infectious disease are inadequate; therefore, new therapies must be developed. Several labs have obtained evidence for a protein complex that involves many of the nonstructural (NS) proteins encoded by the virus. NS3, NS4A, NS4B, NS5A, and NS5B appear to interact structurally and functionally. In this study, we investigated the interaction between the helicase, NS3, and the RNA polymerase, NS5B. Pull-down experiments and surface plasmon resonance data indicate a direct interaction between NS3 and NS5B that is primarily mediated through the protease domain of NS3. This interaction reduces the basal ATPase activity of NS3. However, NS5B stimulates product formation in RNA unwinding experiments under conditions of excess nucleic acid substrate. When the concentrations of NS3 and NS5B are in excess of nucleic acid substrate, NS5B reduces the rate of NS3-catalyzed unwinding. Under pre-steady-state conditions, in which NS3 and substrate concentrations are similar, product formation increased in the presence of NS5B. The increase was consistent with 1:1 complex formed between the two proteins. A fluorescently labeled form of NS3 was used to investigate this interaction through fluorescence polarization binding assays. Results from this assay support interactions that include a 1:1 complex formed between NS3 and NS5B. The modulation of NS3 by NS5B suggests that these proteins may function together during replication of the HCV genome.

Hepatitis C virus NS3 helicase can unwind double-stranded DNA and RNA and has been proposed to form oligomeric structures. Here we examine the DNA unwinding activity of monomeric NS3. Oligomerization was measured by preparing a fluorescently labeled form of NS3, which was titrated with unlabeled NS3, resulting in a hyperbolic increase in fluorescence anisotropy and providing an apparent equilibrium dissociation constant of 236 nM. To evaluate the DNA binding activity of individual subunits within NS3 oligomers, two oligonucleotides were labeled with fluorescent donor or acceptor molecules and then titrated with NS3. Upon the addition of increasing concentrations of NS3, fluorescence energy transfer was observed, which reached a plateau at a 1:1 ratio of NS3 to oligonucleotides, indicating that each subunit within the oligomeric form of NS3 binds to DNA. DNA unwinding was measured under multiple turnover conditions with increasing concentrations of NS3; however, no increase in specific activity was observed, even at enzyme concentrations greater than the apparent dissociation constant for oligomerization. An ATPase-deficient form of NS3, NS3(D290A), was prepared to explore the functional consequences of oligomerization. Under single turnover conditions in the presence of excess concentration of NS3 relative to DNA, NS3(D290A) exhibited a dominant negative effect. However, under multiple turnover conditions in which DNA concentration was in excess to enzyme concentration, NS3(D290A) did not exhibit a dominant negative effect. Taken together, these data support a model in which monomeric forms of NS3 are active. Oligomerization of NS3 occurs, but subunits can function independently or cooperatively, dependent upon the relative concentration of the DNA.Helicases are ubiquitous enzymes required for virtually all cellular processes involving nucleic acids, including replication, transcription, translation, repair, and recombination (1-5). These enzymes catalyze unwinding of double-stranded DNA or RNA by converting chemical energy from ATP hydrolysis into mechanical energy for nucleic acid strand separation. However, there is considerable variability in the quaternary structure of the active forms of helicases. Some helicases function effectively in unwinding activities as monomers, whereas others are active as dimers or oligomers. For example, bacteriophage T4 gp41 helicase and Escherichia coli DnaB helicase form hexameric structures that encircle and sequester single-stranded DNA (6, 7). Indeed, a large number of helicases form and function as hexameric structures (3). PcrA, a Gram-positive bacterial helicase, translocates on single-stranded DNA as a monomer (8) and has been proposed to unwind double-stranded DNA as a monomer (9). Bacteriophage T4 Dda helicase has activity in the monomeric form (10) but demonstrates more efficient unwinding activity under conditions where multiple helicase molecules bind a given substrate molecule (11). It has been shown that E. coli Rep helicase unwinds DNA as a dimer (12). UvrD has...