Structure-Based Mutagenesis Study of Hepatitis C Virus NS3 Helicase

The NS3 protein of hepatitis C virus (HCV) is a bifunctional protein containing a serine protease in the N-terminal one-third, which is stimulated upon binding of the NS4A cofactor, and an RNA helicase in the C-terminal two-thirds. In this study, a C-terminal hexahistidine-tagged helicase domain of the HCV NS3 protein was expressed in Escherichia coli and purified to homogeneity by conventional chromatography. The purified HCV helicase domain has a basal ATPase activity, a polynucleotide-stimulated ATPase activity, and a nucleic acid unwinding activity and binds efficiently to single-stranded polynucleotide. Detailed characterization of the purified HCV helicase domain with regard to all four activities is presented. Recently, we published an X-ray crystallographic structure of a binary complex of the HCV helicase with a (dU)(8) oligonucleotide, in which several conserved residues of the HCV helicase were shown to be involved in interactions between the HCV helicase and oligonucleotide. Here, site-directed mutagenesis was used to elucidate the roles of these residues in helicase function. Four individual mutations, Thr to Ala at position 269, Thr to Ala at position 411, Trp to Leu at position 501, and Trp to Ala at position 501, produced a severe reduction of RNA binding and completely abolished unwinding activity and stimulation of ATPase activity by poly(U), although the basal ATPase activity (activity in the absence of polynucleotide) of these mutants remained intact. Alanine substitution at Ser-231 or Ser-370 resulted in enzymes that were indistinguishable from wild-type HCV helicase with regard to all four activities. A mutant bearing Phe at Trp-501 showed wild-type levels of basal ATPase, unwinding activity, and single-stranded RNA binding activity. Interestingly, ATPase activity of this mutant became less responsive to stimulation by poly(U) but not to stimulation by other polynucleotides, such as poly(C). Given the conservation of some of these residues in other DNA and RNA helicases, their role in the mechanism of unwinding of double-stranded nucleic acid is discussed.     

ATP binding modulates the nucleic acid affinity of hepatitis C virus helicase

The helicase of hepatitis C virus (HCV) unwinds nucleic acid using the energy of ATP hydrolysis. The ATPase cycle is believed to induce protein conformational changes to drive helicase translocation along the length of the nucleic acid. We have investigated the energetics of nucleic acid binding by HCV helicase to understand how the nucleotide ligation state of the helicase dictates the conformation of its nucleic acid binding site. Because most of the nucleotide ligation states of the helicase are transient due to rapid ATP hydrolysis, several compounds were analyzed to find an efficient unhydrolyzable ATP analog. We found that the beta-gamma methylene/amine analogs of ATP, ATPgammaS, or [AlF4]ADP were not effective in inhibiting the ATPase activity of HCV helicase. On the other hand, [BeF3]ADP was found to be a potent inhibitor of the ATPase activity, and it binds tightly to HCV helicase with a 1:1 stoichiometry. Equilibrium binding studies showed that HCV helicase binds single-stranded nucleic acid with a high affinity in the absence of ATP or in the presence of ADP. Upon binding to the ATP analog, a 100-fold reduction in affinity for ssDNA was observed. The reduction in affinity was also observed in duplex DNA with 3' single-stranded tail and in RNA but not in duplex DNA. The results of this study indicate that the nucleic acid binding site of HCV helicase is allosterically modulated by the ATPase reaction. The binding energy of ATP is used to bring HCV helicase out of a tightly bound state to facilitate translocation, whereas ATP hydrolysis and product release steps promote tight rebinding of the helicase to the nucleic acid. On the basis of these results we propose a Brownian motor model for unidirectional translocation of HCV helicase along the nucleic acid length.     

Two novel conserved motifs in the hepatitis C virus NS3 protein critical for helicase action

The hepatitis C virus (HCV) NS3 helicase shares several conserved motifs with other superfamily 2 (SF2) helicases. Besides these sequences, several additional helicase motifs are conserved among the various HCV genotypes and quasispecies. The roles of two such motifs are examined here. The first motif (YRGXDV) forms a loop that connects SF2 helicase motifs 4 and 5, at the tip of which is Arg393. When Arg393 is changed to Ala, the resulting protein (R393A) retains a nucleic acid stimulated ATPase but cannot unwind RNA. R393A also unwinds DNA more slowly than wild type and translocates poorly on single-stranded DNA (ssDNA). DNA and RNA stimulate ATP hydrolysis catalyzed by R393A like the wild type, but the mutant protein binds ssDNA more weakly both in the presence and absence of the non-hydrolyzable ATP analog ADP(BeF3). The second motif (DFSLDPTF) forms a loop that connects two anti-parallel sheets between SF2 motifs 5 and 6. When Phe444 in this Phe-loop is changed to Ala, the resulting protein (F444A) is devoid of all activities. When Phe438 is changed to Ala, the protein (F438A) retains nucleic acid-stimulated ATPase, but does not unwind RNA. F438A unwinds DNA and translocates on ssDNA at about half the rate of the wild type. Equilibrium binding data reveal that this uncoupling of ATP hydrolysis and unwinding is due to the fact that the F438A mutant does not release ssDNA upon ATP binding like the wild type. A model is presented explaining the roles of the Arg-clamp and the Phe-loop in the unwinding reaction.     

Fuel Specificity of the Hepatitis C Virus NS3 Helicase

The hepatitis C virus (HCV) NS3 protein is a helicase capable of unwinding duplex RNA or DNA. This study uses a newly developed molecular-beacon-based helicase assay (MBHA) to investigate how nucleoside triphosphates (NTPs) fuel HCV helicase-catalyzed DNA unwinding. The MBHA monitors the irreversible helicase-catalyzed displacement of an oligonucleotide-bound molecular beacon so that rates of helicase translocation can be directly measured in real time. The MBHA reveals that HCV helicase unwinds DNA at different rates depending on the nature and concentration of NTPs in solution, such that the fastest reactions are observed in the presence of CTP followed by ATP, UTP, and GTP. 3′-Deoxy-NTPs generally support faster DNA unwinding, with dTTP supporting faster rates than any other canonical (d)NTP. The presence of an intact NS3 protease domain makes HCV helicase somewhat less specific than truncated NS3 bearing only its helicase region (NS3h). Various NTPs bind NS3h with similar affinities, but each NTP supports a different unwinding rate and processivity. Studies with NTP analogs reveal that specificity is determined by the nature of the Watson-Crick base-pairing region of the NTP base and the nature of the functional groups attached to the 2′ and 3′ carbons of the NTP sugar. The divalent metal bridging the NTP to NS3h also influences observed unwinding rates, with Mn²⁺ supporting about 10 times faster unwinding than Mg²⁺. Unlike Mg²⁺, Mn²⁺ does not support HCV helicase-catalyzed ATP hydrolysis in the absence of stimulating nucleic acids. Results are discussed in relation to models for how ATP might fuel the unwinding reaction.     

The hepatitis C virus NS3 protein: a model RNA helicase and potential drug target

The C-terminal portion of hepatitis C virus (HCV) nonstructural protein 3 (NS3) forms a three domain polypeptide that possesses the ability to travel along RNA or single-stranded DNA (ssDNA) in a 3' to 5' direction. Fueled byATP hydrolysis, this movement allows the protein to displace complementary strands of DNA or RNA and proteins bound to the nucleic acid. HCV helicase shares two domains common to other motor proteins, one of which appears to rotate upon ATP binding. Several models have been proposed to explain how this conformational change leads to protein movement and RNA unwinding, but no model presently explains all existing experimental data. Compounds recently reported to inhibit HCV helicase, which include numerous small molecules, RNA aptamers and antibodies, will be useful for elucidating the role of a helicase in positive-sense single-stranded RNA virus replication and might serve as templates for the design of novel antiviral drugs.     

Interactions of Escherichia coli replicative helicase PriA protein with single-stranded DNA

Quantitative analyses of the interactions of the Escherichia coli replicative helicase PriA protein with a single-stranded DNA have been performed, using the thermodynamically rigorous fluorescence titration technique. The analysis of the PriA helicase interactions with nonfluorescent, unmodified nucleic acids has been performed, using the macromolecular competition titration (MCT) method. Thermodynamic studies of the PriA helicase binding to ssDNA oligomers, as well as competition studies, show that independently of the type of nucleic acid base, as well as the salt concentration, the type of salt in solution, and nucleotide cofactors, the PriA helicase binds the ssDNA as a monomer. The enzyme binds the ssDNA with significant affinity in the absence of any nucleotide cofactors. Moreover, the presence of AMP-PNP diminishes the intrinsic affinity of the PriA protein for the ssDNA by a factor approximately 4, while ADP has no detectable effect. Analyses of the PriA interactions with different ssDNA oligomers, over a large range of nucleic acid concentrations, indicates that the enzyme has a single, strong ssDNA-binding site. The intrinsic affinities are salt-dependent. The formation of the helicase-ssDNA complexes is accompanied by a net release of 3-4 ions. The experiments have been performed with ssDNA oligomers encompassing the total site size of the helicase-ssDNA complex and with oligomers long enough to encompass only the ssDNA-binding site of the enzyme. The obtained results indicate that salt dependence of the intrinsic affinity results predominantly, if not exclusively, from the interactions of the ssDNA-binding site of the helicase with the nucleic acid. There is an anion effect on the studied interactions, which suggests that released ions originate from both the protein and the nucleic acid. Contrary to the intrinsic affinities, cooperative interactions between bound PriA molecules are accompanied by a net uptake of approximately 3 ions. The PriA protein shows preferential intrinsic affinity for pyrimidine ssDNA oligomers. In our standard conditions (pH 7.0, 10 degrees C, 100 mM NaCl), the intrinsic binding constant for the pyrimidine oligomers is approximately 1 order of magnitude higher than the intrinsic binding constant for the purine oligomers. The significance of these results for the mechanism of action of the PriA helicase is discussed.     

Role of divalent metal cations in ATP hydrolysis catalyzed by the hepatitis C virus NS3 helicase: magnesium provides a bridge for ATP to fuel unwinding

This study investigates the role of magnesium ions in coupling ATP hydrolysis to the nucleic acid unwinding catalyzed by the NS3 protein encoded by the hepatitis C virus (HCV). Analyses of steady-state ATP hydrolysis rates at various RNA and magnesium concentrations were used to determine values for the 15 dissociation constants describing the formation of a productive enzyme-metal-ATP-RNA complex and the four rate constants describing hydrolysis of ATP by the possible enzyme-ATP complexes. These values coupled with direct binding studies, specificity studies and analyses of site-directed mutants reveal only one ATP binding site on HCV helicase centered on the catalytic base Glu291. An adjacent residue, Asp290, binds a magnesium ion that forms a bridge to ATP, reorienting the nucleotide in the active site. RNA stimulates hydrolysis while decreasing the affinity of the enzyme for ATP, magnesium, and MgATP. The binding scheme described here explains the unusual regulation of the enzyme by ATP that has been reported previously. Binding of either free magnesium or free ATP to HCV helicase competes with MgATP, the true fuel for helicase movements, and leads to slower hydrolysis and nucleic acid unwinding.     

Escherichia coli replicative helicase PriA protein-single-stranded DNA complex. Stoichiometries, free energy of binding, and cooperativities

Analyses of interactions of the Escherichia coli replicative helicase, PriA protein, with a single-stranded (ss) DNA have been performed, using the quantitative fluorescence titration technique. The stoichiometry of the PriA helicase.ssDNA complex has been examined in binding experiments with a series of ssDNA oligomers. The total site-size of the PriA.ssDNA complex, i.e. the maximum number of nucleotide residues occluded by the PriA helicase in the complex, is 20 +/- 3 residues per protein monomer. However, the protein can efficiently form a complex with a minimum of 8 nucleotides. Thus, the enzyme has a strong ssDNA-binding site that engages in direct interactions with a significantly smaller number of nucleotides than the total site-size. The ssDNA-binding site is located in the center of the enzyme molecule, with the protein matrix protruding over a distance of approximately 6 nucleotides on both sides of the binding site. The analysis of the binding of two PriA molecules to long oligomers was performed using statistical thermodynamic models that take into account the overlap of potential binding sites, cooperative interactions, and the protein.ssDNA complexes with different stoichiometries. The intrinsic affinity depends little upon the length of the ssDNA. Moreover, the binding is accompanied by weak cooperative interactions.     

Structurally conserved amino Acid w501 is required for RNA helicase activity but is not essential for DNA helicase activity of hepatitis C virus NS3 protein

Hepatitis C virus (HCV) is a positive-strand RNA virus that encodes a helicase required for viral replication. Although HCV does not replicate through a DNA intermediate, HCV helicase unwinds both RNA and DNA duplexes. An X-ray crystal structure of the HCV helicase complexed with (dU)(8) has been solved, and the substrate-amino acids interactions within the catalytic pocket were shown. Among these, residues W501 and V432 were reported to have base stacking interactions and to be important for the unwinding function of HCV helicase. It has been hypothesized that specific interactions between the enzyme and substrate in the catalytic pocket are responsible for the substrate specificity phenotype. We therefore mutagenized W501 and V432 to investigate their role in substrate specificity in HCV helicase. Replacement of W501, but not V432, with nonaromatic residues resulted in complete loss of RNA unwinding activity, whereas DNA unwinding activity was largely unaffected. The loss of unwinding activity was fully restored in the W501F mutant, indicating that the aromatic ring is crucial for RNA helicase function. Analysis of ATPase and nucleic acid binding activities in the W501 mutant enzymes revealed that these activities are not directly responsible for the substrate specificity phenotype. Molecular modeling of the enzyme-substrate interaction at W501 revealed a putative pi-facial hydrogen bond between the 2'-OH of ribose and the aromatic tryptophan ring. This evidence correlates with biochemical results suggesting that the pi-facial bond may play an important role in the RNA unwinding activity of the HCV NS3 protein.     

The Escherichia coli PriA Helicase Specifically Recognizes Gapped DNA Substrates: EFFECT OF THE TWO NUCLEOTIDE-BINDING SITES OF THE ENZYME ON THE RECOGNITION PROCESS*

Energetics and specificity of interactions between the Escherichia coli PriA helicase and the gapped DNAs have been studied, using the quantitative fluorescence titration and analytical ultracentrifugation methods. The gap complex has a surprisingly low minimum total site size, corresponding to ∼7 nucleotides of the single-stranded DNA (ssDNA), as compared with the site size of ∼20 nucleotides of the enzyme-ssDNA complex. The dramatic difference in stoichiometries indicates that the enzyme predominantly engages the strong DNA-binding subsite in interactions with the gap and assumes a very different orientation in the gap complex, as compared with the complex with the ssDNA. The helicase binds the ssDNA gaps with 4–5 nucleotides with the highest affinity, which is ∼3 and ∼2 orders of magnitude larger than the affinities for the ssDNA and double-stranded DNA, respectively. In the gap complex, the protein does not engage in cooperative interactions with the enzyme predominantly associated with the surrounding dsDNA. Binding of nucleoside triphosphate to the strong and weak nucleotide-binding sites of the helicase eliminates the selectivity of the enzyme for the size of the gap, whereas saturation of both sites with ADP leads to amplified affinity for the ssDNA gap containing 5 nucleotides and engagement of an additional protein area in interactions with the nucleic acid.     

The helicase from hepatitis C virus is active as an oligomer.

The helicase from hepatitis C virus (HCV NS3h) residing on the C-terminal domain of nonstructural protein 3 was considered to be monomeric by several researchers. Here we demonstrate, based on biochemical kinetic data, that the HCV helicase acts as an oligomer. The increase in the ATPase k(cat) of the NS3h protein with increasing protein concentration provided evidence for oligomerization. A sharp decrease in the unwinding rate was observed when the wild type NS3h was mixed with the ATPase deficient mutants of NS3h protein. This provided strong support for both mixed oligomer formation and subunit interactions for the HCV helicase. Chemical cross-linking of NS3h protein was an inefficient process, but yielded cross-linked protein oligomers of various sizes. The information currently available for HCV helicase is consistent with the hypothesis that oligomers of NS3h are not stable and the helicase subunits exchange during unwinding. Nevertheless, oligomerization of HCV helicase stimulates the ATPase activity, and it is required for the helicase activity.     

Enhanced nucleic acid binding to ATP-bound hepatitis C virus NS3 helicase at low pH activates RNA unwinding

The molecular basis of the low-pH activation of the helicase encoded by the hepatitis C virus (HCV) was examined using either a full-length NS3 protein/NS4A cofactor complex or truncated NS3 proteins lacking the protease domain, which were isolated from three different viral genotypes. All proteins unwound RNA and DNA best at pH 6.5, which demonstrate that conserved NS3 helicase domain amino acids are responsible for low-pH enzyme activation. DNA unwinding was less sensitive to pH changes than RNA unwinding. Both the turnover rate of ATP hydrolysis and the K_m of ATP were similar between pH 6 and 10, but the concentration of nucleic acid needed to stimulate ATP hydrolysis decreased almost 50-fold when the pH was lowered from 7.5 to 6.5. In direct-binding experiments, HCV helicase bound DNA weakly at high pH only in the presence of the non-hydrolyzable ATP analog, ADP(BeF₃). These data suggest that a low-pH environment might be required for efficient HCV RNA translation or replication, and support a model in which an acidic residue rotates toward the RNA backbone upon ATP binding repelling nucleic acid from the binding cleft.     

The functional interaction of the hepatitis C virus helicase molecules is responsible for unwinding processivity

Although helicases participate in virtually every cellular process involving nucleic acids, the details of their mechanism including the role of interaction between the subunits remains unclear. Here we study the unwinding kinetics of the helicase from hepatitis C virus using DNA substrates with a range of tail and duplex lengths. The binding of the helicase to the substrates was characterized by electron microscopy and fluorimetric titrations. Depending on the length of the ssDNA tail, one or more helicase molecules can be loaded on the DNA. Unwinding was measured under single-turnover conditions, and the results show that a monomer is active on short duplexes yet multiple molecules are needed to unwind long duplexes. Thus, increasing the ssDNA tail length increases the unwinding efficiency. The unwinding kinetics was modeled as a stepwise process performed by single or multiple helicase molecules. The model programmed in MATLAB was used for global fitting of the kinetics, yielding values for the rate of unwinding, processivity, cooperativity, step size, and occlusion site. The results indicate that a single hepatitis C virus helicase molecule unwinds DNA with a low processivity. The multiple helicase molecules present on the DNA substrate show functional cooperativity and unwind with greater efficiency, although they bind and release the substrate non-cooperatively, and the ATPase cycle of the helicase molecules is not coordinated. The functional interaction model explains the efficient unwinding by multiple helicases and is generally applicable.     

Analysis of the DNA substrate specificity of the human BACH1 helicase associated with breast cancer

We have investigated the DNA substrate specificity of BACH1 (BRCA1-associated C-terminal helicase). The importance of various DNA structural elements for efficient unwinding by purified recombinant BACH1 helicase was examined. The results indicated that BACH1 preferentially binds and unwinds a forked duplex substrate compared with a duplex flanked by only one single-stranded DNA (ssDNA) tail. In support of its DNA substrate preference, helicase sequestration studies revealed that BACH1 can be preferentially trapped by forked duplex molecules. BACH1 helicase requires a minimal 5 ' ssDNA tail of 15 nucleotides for unwinding of conventional duplex DNA substrates; however, the enzyme is able to catalytically release the third strand of the homologous recombination intermediate D-loop structure irrespective of DNA tail status. In contrast, BACH1 completely fails to unwind a synthetic Holliday junction structure. Moreover, BACH1 requires nucleic acid continuity in the 5 ' ssDNA tail of the forked duplex substrate within six nucleotides of the ssDNA-dsDNA junction to initiate efficiently DNA unwinding. These studies provide the first detailed information on the DNA substrate specificity of BACH1 helicase and provide insight to the types of DNA structures the enzyme is likely to act upon to perform its functions in DNA repair or recombination.     

Parvovirus initiator protein NS1 and RPA coordinate replication fork progression in a reconstituted DNA replication system

We show here that the DNA helicase activity of the parvoviral initiator protein NS1 is highly directional, binding to the single strand at a recessed 5' end and displacing the other strand while progressing in a 3'-to-5' direction on the bound strand. NS1 and a cellular site-specific DNA binding factor, PIF, also known as glucocorticoid modulating element binding protein, bind to the left-end minimal replication origin of minute virus of mice, forming a ternary complex. In this complex, NS1 is activated to nick one DNA strand, becoming covalently attached to the 5' end of the nick in the process and providing a 3' OH for priming DNA synthesis. In this situation, the helicase activity of NS1 did not displace the nicked strand, but the origin duplex was distorted by the NS1-PIF complex, as assayed by its sensitivity to KMnO(4) oxidation, and a stretch of about 14 nucleotides on both strands of the nicked origin underwent limited unwinding. Addition of Escherichia coli single-stranded DNA binding protein (SSB) did not lead to further unwinding. However, addition of recombinant human single-stranded DNA binding protein (RPA) to the initiation reaction catalyzed extensive unwinding of the nicked origin, suggesting that RPA may be required to form a functional replication fork. Accordingly, the unwinding mediated by NS1 and RPA promoted processive leading-strand synthesis catalyzed by recombinant human DNA polymerase delta, PCNA, and RFC, using the minimal left-end origin cloned in a plasmid as a template. The requirement for RPA, rather than SSB, in the unwinding reaction indicated that specific NS1-RPA protein interactions were formed. NS1 was tested by enzyme-linked immunosorbent assay for binding to two- or three-subunit RPA complexes expressed from recombinant baculoviruses. NS1 efficiently bound each of the baculovirus-expressed complexes, indicating that the small subunit of RPA is not involved in specific NS1 binding. No NS1 interactions were observed with E. coli SSB or other proteins included as controls.     

Substrate evokes translocation of both domains in the mitochondrial processing peptidase alpha-subunit during which the C-terminus acts as a stabilizing element

To determine whether the two domains of hepatitis C virus (HCV) NS3 and the NS4A interact with each other to regulate the RNA unwinding activity, this study compares the RNA unwinding, ATPase and RNA binding activities of three forms of NS3 proteins--the NS3H protein, containing only the helicase domain, the full-length NS3 protein, and the NS3-NS4A complex. The results revealed that NS3 displayed the weakest RNA helicase activity, not because it had lower ATPase or RNA binding activity than did NS3H or NS3-NS4A, but because it had the lowest RNA unwinding processivity. A mutant protein, R1487Q, which contained a mutation in the helicase domain, displayed a reduced protease activity as compared to the wild-type NS3-NS4A. Together, these results suggest the existence of interactions between the two domains of NS3 and the NS4A, which regulates the HCV NS3 protease and RNA helicase activities.     

Double-stranded DNA-induced localized unfolding of HCV NS3 helicase subdomain 2

The NS3 helicase of the hepatitis C virus (HCV) unwinds double-stranded (ds) nucleic acid (NA) in an NTP-dependent fashion. Mechanistic details of this process are, however, largely unknown for the HCV helicase. We have studied the binding of dsDNA to an engineered version of subdomain 2 of the HCV helicase (d(2Delta)NS3h) by NMR and circular dichroism. Binding of dsDNA to d(2Delta)NS3h induces a local unfolding of helix (alpha(3)), which includes residues of conserved helicase motif VI (Q(460)RxxRxxR(467)), and strands (beta(1) and beta(8)) from the central beta-sheet. This also occurs upon lowering the pH (4.4) and introducing an R461A point mutation, which disrupt salt bridges with Asp 412 and Asp 427 in the protein structure. NMR studies on d(2Delta)NS3h in the partially unfolded state at low pH map the dsDNA binding site to residues previously shown to be involved in single-stranded DNA binding. Sequence alignment and structural comparison suggest that these Arg-Asp interactions are highly conserved in SF2 DEx(D/H) proteins. Thus, modulation of these interactions by dsNA may allow SF2 helicases to switch between conformations required for helicase function.     

N-Naphthoyl-substituted indole thio-barbituric acid analogs inhibit the helicase activity of the hepatitis C virus NS3

The hepatitis C virus (HCV) represents a substantial threat to human health worldwide. The virus expresses a dual-function protein, NS3 having both protease and RNA helicase activities that are essential for productive viral replication and sustained infections. While viral protease and polymerase inhibitors have shown great successes in treating chronic HCV infections, drugs that specifically target the helicase activity have not advanced. A robust and quantitative 96-well plate-based fluorescent DNA unwinding assay was used to screen a class of indole thio-barbituric acid (ITBA) analogs using the full-length, recombinant HCV NS3, and identified three naphthoyl-containing analogs that efficiently inhibited NS3 helicase activity in a dose-dependent manner, with observed IC₅₀ values of 21–24?µM. Standard gel electrophoresis helicase assays using radiolabeled duplex DNA and RNA NS3 substrates confirmed the inhibition of NS3 unwinding activity. Subsequent anisotropy measurements demonstrated that the candidate compounds did not disrupt NS3 binding to nucleic acids. Additionally, the rate of ATP hydrolysis and the protease activity were also not affected by the inhibitors. Thus, these results indicate that the three ITBA analogs containing N-naphthoyl moieties are the foundation of a potential series of small molecules capable of inhibiting NS3 activity via a novel interaction with the helicase domain that prevents the productive unwinding of nucleic acid substrates, and may represent the basis for a new class of therapeutic agents with the potential to aid in the treatment and eradication of hepatitis C virus.     

Hepatitis C virus subgenomic replicon requires an active NS3 RNA helicase

Mutations were introduced into the NS3 helicase region of a hepatitis C virus (HCV) Con1 subgenomic replicon to ascertain the role of the helicase in viral replication. One new replicon lacked two-thirds of the NS3 helicase (Deltahel), and six others contained one of the following six amino acid substitutions in NS3: R393A, F438A, T450I, E493K, W501A, and W501F. It has been previously reported that purified R393A, F438A, and W501A HCV helicase proteins do not unwind RNA but unwind DNA, bind RNA, and hydrolyze ATP. On the other hand, previous data suggest that a W501F protein retains most of its unwinding abilities and that purified T450I and E493K HCV helicase proteins have enhanced unwinding abilities. In a hepatoma cell line that has been cured of HCV replicons using interferon, the T450I and W501F replicons synthesized both negative-sense and positive-sense viral RNA and formed colonies after selection with similar efficiencies as the parent replicon. However, the Deltahel, R393A, F438A, and W501A replicons encoded and processed an HCV polyprotein but did not synthesize additional viral RNA or form colonies. Surprisingly the same phenotype was seen for the E493K replicon. The inability of the E493K replicon to replicate might point to a role of pH in viral replication because a previous analysis has shown that, unlike the wild-type NS3 protein, the helicase activity of an E493K protein is not sensitive to pH changes. These results demonstrate that the RNA-unwinding activity of the HCV NS3 helicase is needed for RNA replication.     

Interactions of the RepA helicase hexamer of plasmid RSF1010 with the ssDNA. Quantitative analysis of stoichiometries, intrinsic affinities, cooperativities, and heterogeneity of the total ssDNA-binding site

Interactions between the replicative RepA helicase hexamer of plasmid RSF1010 with the single-stranded DNA (ssDNA) have been studied, using the quantitative fluorescence titration, analytical sedimentation velocity, and sedimentation equilibrium techniques. Experiments were performed with fluorescein-labeled ssDNA oligomers. Studies with unmodified ssDNA oligomers were accomplished using the macromolecular competition titration method. Analyses of RepA helicase interactions with a series of the ssDNA provide direct evidence that the total site-size of the RepA hexamer-ssDNA complex is 19 +/- 1 nucleotide residues. The total ssDNA-binding site of the hexamer has a heterogeneous structure. Part of the total binding site constitutes the proper ssDNA-binding site of the enzyme, an area that possesses strong ssDNA-binding capability and encompasses only 8 +/- 1 residues of the ssDNA. The statistical effect on the macroscopic binding constant for the proper ssDNA-binding site indicates that it is structurally separated from the remaining part of the total ssDNA-binding site. Engagement in interactions with the ssDNA is accompanied by net ion release. Moreover, the proper ssDNA-binding site shows little base specificity. On the other hand, with long ssDNA oligomers, the entire total ssDNA-binding site of the RepA hexamer engages in interactions with the ssDNA resulting in a dramatic change in the nature of interactions with the nucleic acid. The association includes an uptake of ions by the protein. Moreover, unlike the proper-ssDNA-binding site, the total binding site shows a significant preference for pyrimidine oligomers. In this aspect, the RepA helicase is different from the Escherichia coli DnaB hexamer that shows large preference for purine homo-oligomers. In similar solution conditions, the ssDNA intrinsic affinity of the RepA hexamer is similar to the intrinsic affinity of the DnaB helicase. The RepA helicase binds to ssDNA oligomers that can accept more than one RepA hexamer with significant positive cooperative interactions.