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.     

The Macroscopic Rate of Nucleic Acid Translocation by Hepatitis C Virus Helicase NS3h Is Dependent on Both Sugar and Base Moieties

The nonstructural protein 3 helicase (NS3h) of hepatitis C virus is a 3′-to-5′ superfamily 2 RNA and DNA helicase that is essential for the replication of hepatitis C virus. We have examined the kinetic mechanism of the translocation of NS3h along single-stranded nucleic acid with bases uridylate (rU), deoxyuridylate (dU), and deoxythymidylate (dT), and have found that the macroscopic rate of translocation is dependent on both the base moiety and the sugar moiety of the nucleic acid, with approximate macroscopic translocation rates of 3 nt s^− 1 (oligo(dT)), 35 nt s^− 1 (oligo(dU)), and 42 nt s^− 1 (oligo(rU)), respectively. We found a strong correlation between the macroscopic translocation rates and the binding affinity of the translocating NS3h protein for the respective substrates such that weaker affinity corresponded to faster translocation. The values of K_0.5 for NS3h translocation at a saturating ATP concentration are as follows: 3.3 ± 0.4 μM nucleotide (poly(dT)), 27 ± 2 μM nucleotide (poly(dU)), and 36 ± 2 μM nucleotide (poly(rU)). Furthermore, results of the isothermal titration of NS3h with these oligonucleotides suggest that differences in TΔS⁰ are the principal source of differences in the affinity of NS3h binding to these substrates. Interestingly, despite the differences in macroscopic translocation rates and binding affinities, the ATP coupling stoichiometries for NS3h translocation were identical for all three substrates (∼ 0.5 ATP molecule consumed per nucleotide translocated). This similar periodicity of ATP consumption implies a similar mechanism for NS3h translocation along RNA and DNA substrates.     

Hyperpolization-activated Ca channels in guard cell plasma membrane are involved in extracellular ATP-promoted stomatal opening in Vicia faba

Extracellular ATP (eATP) plays essential roles in plant growth, development, and stress tolerance. Extracellular ATP-regulated stomatal movement of Arabidopsis thaliana has been reported. Here, ATP was found to promote stomatal opening of Vicia faba in a dose-dependent manner. Three weakly hydrolysable ATP analogs (adenosine 5′-O-(3-thio) triphosphate (ATPγS), 3′-O-(4-benzoyl) benzoyl adenosine 5′-triphosphate (Bz-ATP) and 2-methylthio-adenosine 5′-triphosphate (2meATP)) showed similar effects, indicating that ATP acts as a signal molecule rather than an energy charger. ADP promoted stomatal opening, while AMP and adenosine did not affect stomatal movement. An ATP-promoted stomatal opening was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI), the reductant dithiothreitol (DTT) or the Ca²⁺ channel blockers GdCl₃ and LaCl₃. A hyperpolarization-activated Ca²⁺ channel was detected in plasma membrane of guard cell protoplast. Extracellular ATP and weakly hydrolyzable ATP analogs activated this Ca²⁺ channel significantly. Extracellular ATP-promoted Ca²⁺ channel activation was markedly inhibited by DPI or DTT. These results indicated that eATP may promote stomatal opening via reactive oxygen species that regulate guard cell plasma membrane Ca²⁺ channels.     

Analysis of the Enzymatic Activity of an NS3 Helicase Genotype 3a Variant Sequence Obtained from a Relapse Patient

The hepatitis C virus (HCV) is a species of diverse genotypes that infect over 170 million people worldwide, causing chronic inflammation, cirrhosis and hepatocellular carcinoma. HCV genotype 3a is common in Brazil, and it is associated with a relatively poor response to current direct-acting antiviral therapies. The HCV NS3 protein cleaves part of the HCV polyprotein, and cellular antiviral proteins. It is therefore the target of several HCV drugs. In addition to its protease activity, NS3 is also an RNA helicase. Previously, HCV present in a relapse patient was found to harbor a mutation known to be lethal to HCV genotype 1b. The point mutation encodes the amino acid substitution W501R in the helicase RNA binding site. To examine how the W501R substitution affects NS3 helicase activity in a genotype 3a background, wild type and W501R genotype 3a NS3 alleles were sub-cloned, expressed in E. coli, and the recombinant proteins were purified and characterized. The impact of the W501R allele on genotype 2a and 3a subgenomic replicons was also analyzed. Assays monitoring helicase-catalyzed DNA and RNA unwinding revealed that the catalytic efficiency of wild type genotype 3a NS3 helicase was more than 600 times greater than the W501R protein. Other assays revealed that the W501R protein bound DNA less than 2 times weaker than wild type, and both proteins hydrolyzed ATP at similar rates. In Huh7.5 cells, both genotype 2a and 3a subgenomic HCV replicons harboring the W501R allele showed a severe defect in replication. Since the W501R allele is carried as a minor variant, its replication would therefore need to be attributed to the trans-complementation by other wild type quasispecies.     

Molecular Basis for Recognition of Nucleoside Triphosphate by Gene 4 Helicase of Bacteriophage T7

The translocation of DNA helicases on single-stranded DNA and the unwinding of double-stranded DNA are fueled by the hydrolysis of nucleoside triphosphates (NTP). Although most helicases use ATP in these processes, the DNA helicase encoded by gene 4 of bacteriophage T7 uses dTTP most efficiently. To identify the structural requirements of the NTP, we determined the efficiency of DNA unwinding by T7 helicase using a variety of NTPs and their analogs. The 5-methyl group of thymine was critical for the efficient unwinding of DNA, although the presence of a 3′-ribosyl hydroxyl group partially overcame this requirement. The NTP-binding pocket of the protein was examined by randomly substituting amino acids for several amino acid residues (Thr-320, Arg-504, Tyr-535, and Leu-542) that the crystal structure suggests interact with the nucleotide. Although positions 320 and 542 required aliphatic residues of the appropriate size, an aromatic side chain was necessary at position 535 to stabilize NTP for efficient unwinding. A basic side chain of residue 504 was essential to interact with the 4-carbonyl of the thymine base of dTTP. Replacement of this residue with a small aliphatic residue allowed the accommodation of other NTPs, resulting in the preferential use of dATP and the use of dCTP, a nucleotide not normally used. Results from this study suggest that the NTP must be stabilized by specific interactions within the NTP-binding site of the protein to achieve efficient hydrolysis. These interactions dictate NTP specificity.     

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.     

ATP Binding and Hydrolysis by Mcm2 Regulate DNA Binding by Mcm Complexes

The essential minichromosome maintenance (Mcm) proteins Mcm2 through Mcm7 likely comprise the replicative helicase in eukaryotes. In addition to Mcm2-7, other subcomplexes, including one comprising Mcm4, Mcm6, and Mcm7, unwind DNA. Using Mcm4/6/7 as a tool, we reveal a role for nucleotide binding by Saccharomyces cerevisiae Mcm2 in modulating DNA binding by Mcm complexes. Previous studies have shown that Mcm2 inhibits DNA unwinding by Mcm4/6/7. Here, we show that interaction of Mcm2 and Mcm4/6/7 is not sufficient for inhibition; rather, Mcm2 requires nucleotides for its regulatory role. An Mcm2 mutant that is defective for ATP hydrolysis (K549A), as well as ATP analogues, was used to show that ADP binding by Mcm2 is required to inhibit DNA binding and unwinding by Mcm4/6/7. This Mcm2-mediated regulation of Mcm4/6/7 is independent of Mcm3/5. Furthermore, the importance of ATP hydrolysis by Mcm2 to the regulation of the native complex was apparent from the altered DNA binding properties of Mcm2^KA-7. Moreover, together with the finding that Mcm2_K549A does not support yeast viability, these results indicate that the nucleotide-bound state of Mcm2 is critical in regulating the activities of Mcm4/6/7 and Mcm2-7 complexes.     

HCV NS3 protein helicase domain assists RNA structure conversion

NS3H, the helicase domain of HCV NS3, possesses RNA-stimulated ATPase and ATP hydrolysis-dependent dsRNA unwinding activities. Here, the ability of NS3H to facilitate RNA structural rearrangement is studied using relatively long RNA strands as the model substrates. NS3H promotes intermolecular annealing, resolves three-stranded RNA duplexes, and assists dsRNA and ssRNA inter-conversions to establish a steady state among RNA structures. NS3H facilitates RNA structure conversions in a mode distinct from an ATP-independent RNA chaperone. These findings expand the known function of HCV NS3 helicase and reveal a role for viral helicase in assisting RNA structure conversions during virus life cycle.     

Hepatitis C virus helicase/NTPase: an efficient expression system and new inhibitors

A method has been developed for obtaining a full-length protein NS3 of hepatitis C virus with the yield of 6.5 mg/liter of cell culture, and conditions for measuring its NTPase and helicase activities have been optimized. The helicase reaction can proceed in two modes depending on the enzyme and substrate concentration ratio: it can be non-catalytic in the case of enzyme excess and catalytic in the case of tenfold substrate excess. In the latter case, helicase activity is coupled with NTPase and is stimulated by ATP. A number of NTP and inorganic pyrophosphate analogs were studied as substrates and/or inhibitors of NS3 NTPase activity, and it was found that the structure of nucleic base and ribose fragment of NTP molecule has a slight effect on its inhibitory (substrate) properties. Among the nucleotide derivatives, the most efficient inhibitor of NTPase activity is 2 -deoxythymidine 5 -phosphoryl-beta,gamma-hypophosphate, and among pyrophosphate analogs imidodiphosphate exhibited maximal inhibitory activity. These compounds were studied as inhibitors of the helicase reaction, and it was shown that imidodiphosphate efficiently inhibited the ATP-dependent helicase reaction and had almost no effect on the ATP-independent duplex unwinding. However, the inhibitory effect of 2 -deoxythymidine 5 -phosphoryl-beta,gamma-hypophosphate was insignificant in both cases, which is due to the possibility of helicase activation by this ATP analog.     

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.     

Primuline Derivatives That Mimic RNA to Stimulate Hepatitis C Virus NS3 Helicase-catalyzed ATP Hydrolysis

ATP hydrolysis fuels the ability of helicases and related proteins to translocate on nucleic acids and separate base pairs. As a consequence, nucleic acid binding stimulates the rate at which a helicase catalyzes ATP hydrolysis. In this study, we searched a library of small molecule helicase inhibitors for compounds that stimulate ATP hydrolysis catalyzed by the hepatitis C virus (HCV) NS3 helicase, which is an important antiviral drug target. Two compounds were found that stimulate HCV helicase-catalyzed ATP hydrolysis, both of which are amide derivatives synthesized from the main component of the yellow dye primuline. Both compounds possess a terminal pyridine moiety, which was critical for stimulation. Analogs lacking a terminal pyridine inhibited HCV helicase catalyzed ATP hydrolysis. Unlike other HCV helicase inhibitors, the stimulatory compounds differentiate between helicases isolated from various HCV genotypes and related viruses. The compounds only stimulated ATP hydrolysis catalyzed by NS3 purified from HCV genotype 1b. They inhibited helicases from other HCV genotypes (e.g. 1a and 2a) or related flaviviruses (e.g. Dengue virus). The stimulatory compounds interacted with HCV helicase in the absence of ATP with dissociation constants of about 2 μm. Molecular modeling and site-directed mutagenesis studies suggest that the stimulatory compounds bind in the HCV helicase RNA-binding cleft near key residues Arg-393, Glu-493, and Ser-231.     

The interdomain interface in bifunctional enzyme protein 3/4A (NS3/4A) regulates protease and helicase activities

Hepatitis C (HCV) protein 3/4A (NS3/4A) is a bifunctional enzyme comprising two separate domains with protease and helicase activities, which are essential for viral propagation. Both domains are stable and have enzymatic activity separately, and the relevance and implications of having protease and helicase together as a single protein remains to be explored. Altered in vitro activities of isolated domains compared with the full‐length NS3/4A protein suggest the existence of interdomain communication. The molecular mechanism and extent of this communication was investigated by probing the domain–domain interface observed in HCV NS3/4A crystal structures. We found in molecular dynamics simulations that the two domains of NS3/4A are dynamically coupled through the interface. Interestingly, mutations designed to disrupt this interface did not hinder the catalytic activities of either domain. In contrast, substrate cleavage and DNA unwinding by these mutants were mostly enhanced compared with the wild‐type protein. Disrupting the interface did not significantly alter RNA unwinding activity; however, the full‐length protein was more efficient in RNA unwinding than the isolated protease domain, suggesting a more direct role in RNA processing independent of the interface. Our findings suggest that HCV NS3/4A adopts an “extended” catalytically active conformation, and interface formation acts as a switch to regulate activity. We propose a unifying model connecting HCV NS3/4A conformational states and protease and helicase function, where interface formation and the dynamic interplay between the two enzymatic domains of HCV NS3/4A potentially modulate the protease and helicase activities in vivo.     

High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon

We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5′-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3′-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the screening for NS3 helicase inhibitors from cell extracts of microorganisms, and found several cell extracts containing potential inhibitors.     

Solution structure and backbone dynamics of an engineered arginine-rich subdomain 2 of the hepatitis C virus NS3 RNA helicase

The NS3 protein of the hepatitis C virus (HCV) is a 631 amino acid residue bifunctional enzyme with a serine protease localized to the N-terminal 181 residues and an RNA helicase located in the C-terminal 450 residues. The HCV NS3 RNA helicase consists of three well-defined subdomains which all contribute to its helicase activity. The second subdomain of the HCV helicase is flexibly linked to the remainder of the NS3 protein and could undergo rigid-body movements during the unwinding of double-stranded RNA. It also contains several motifs that are implicated in RNA binding and in coupling NTP hydrolysis to nucleic acid unwinding and translocation. As part of our efforts to use NMR techniques to assist in deciphering the enzyme's structure-function relationships and developing specific small molecule inhibitors, we have determined the solution structure of an engineered subdomain 2 of the NS3 RNA helicase of HCV, d(2Delta)-HCVh, and studied the backbone dynamics of this protein by (15)N-relaxation experiments using a model-free approach. The NMR studies on this 142-residue construct reveal that overall subdomain 2 of the HCV helicase is globular and well structured in solution even in the absence of the remaining parts of the NS3 protein. Its solution structure is very similar to the corresponding parts in the X-ray structures of the HCV NS3 helicase domain and intact bifunctional HCV NS3 protein. Slow hydrogen-deuterium exchange rates map to a well-structured, stable hydrophobic core region away from the subdomain interfaces. In contrast, the regions facing the subdomain interfaces in the HCV NS3 helicase domain are less well structured in d(2Delta)-HCVh, show fast hydrogen-deuterium exchange rates, and the analysis of the dynamic properties of d(2Delta)-HCVh reveals that these regions of the protein show distinct dynamical features. In particular, residues in motif V, which may be involved in transducing allosteric effects of nucleotide binding and hydrolysis on RNA binding, exhibit slow conformational exchange on the milli- to microsecond time-scale. The intrinsic conformational flexibility of this loop region may facilitate conformational changes required for helicase function.     

Kinetic control of Mg2+-dependent melting of duplex DNA ends by Escherichia coli RecBC

Escherichia coli RecBCD is a highly processive DNA helicase involved in double-strand break repair and recombination that possesses two helicase/translocase subunits with opposite translocation directionality (RecB (3′ to 5′) and RecD (5′ to 3′)). RecBCD has been shown to melt out ∼ 5-6 bp upon binding to a blunt-ended duplex DNA in a Mg²⁺-dependent, but ATP-independent reaction. Here, we examine the binding of E. coli RecBC helicase (minus RecD), also a processive helicase, to duplex DNA ends in the presence and in the absence of Mg²⁺ in order to determine if RecBC can also melt a duplex DNA end in the absence of ATP. Equilibrium binding of RecBC to DNA substrates with ends possessing pre-formed 3′ and/or 5′ single-stranded (ss)-(dT)_n flanking regions (tails) (n ranging from zero to 20 nt) was examined by competition with a fluorescently labeled reference DNA and by isothermal titration calorimetry. The presence of Mg²⁺ enhances the affinity of RecBC for DNA ends possessing 3′ or 5′-(dT)_n ssDNA tails with n < 6 nt, with the relative enhancement decreasing as n increases from zero to six nt. No effect of Mg²⁺ was observed for either the binding constant or the enthalpy of binding (ΔH_obs) for RecBC binding to DNA with ssDNA tail lengths, n ≥ 6 nucleotides. Upon RecBC binding to a blunt duplex DNA end in the presence of Mg²⁺, at least 4 bp at the duplex end become accessible to KMnO₄ attack, consistent with melting of the duplex end. Since Mg²⁺ has no effect on the affinity or binding enthalpy of RecBC for a DNA end that is fully pre-melted, this suggests that the role of Mg²⁺ is to overcome a kinetic barrier to melting of the DNA by RecBC and presumably also by RecBCD. These data also provide an accurate estimate (ΔH_obs = 8 ± 1 kcal/mol) for the average enthalpy change associated with the melting of a DNA base-pair by RecBC.     

A novel recombinant single-chain hepatitis C virus NS3-NS4A protein with improved helicase activity.

Hepatitis C virus (HCV) nonstructural protein 3 (NS3) has been shown to possess protease and helicase activities and has also been demonstrated to spontaneously associate with nonstructural protein NS4A (NS4A) to form a stable complex. Previous attempts to produce the NS3/NS4A complex in recombinant baculovirus resulted in a protein complex that aggregated and precipitated in the absence of nonionic detergent and high salt. A single-chain form of the NS3/NS4A complex (His-NS4A21-32-GSGS-NS3-631) was constructed in which the NS4A core peptide is fused to the N-terminus of the NS3 protease domain as previously described (Taremi et al., 1998). This protein contains a histidine tagged NS4A peptide (a.a. 21-32) fused to the full-length NS3 (a.a. 3-631) through a flexible tetra amino acid linker. The recombinant protein was expressed to high levels in Escherichia coli, purified to homogeneity, and examined for NTPase, nucleic acid unwinding, and proteolytic activities. The single-chain recombinant NS3-NS4A protein possesses physiological properties equivalent to those of the NS3/NS4A complex except that this novel construct is stable, soluble and sixfold to sevenfold more active in unwinding duplex RNA. Comparison of the helicase activity of the single-chain recombinant NS3-NS4A with that of the full-length NS3 (without NS4A) and that of the helicase domain alone suggested that the presence of the protease domain and at least the NS4A core peptide are required for optimal unwinding activity.     

Helicase from hepatitis C virus,energetics of DNA binding

The ability of a helicase to bind single-stranded nucleic acid is critical for nucleic acid unwinding. The helicase from the hepatitis C virus, NS3 protein, binds to the 3'-DNA or the RNA strand during unwinding. As a step to understand the mechanism of unwinding, DNA binding properties of the helicase domain of NS3 (NS3h) were investigated by fluorimetric binding equilibrium titrations. The global analysis of the binding data by a combinatorial approach was done using MATLAB. NS3h interactions with single-stranded DNA (ssDNA) are 300-1000-fold tighter relative to duplex DNA. The NS3h protein binds to ssDNA less than 15 nt in length with a stoichiometry of one protein per DNA. The minimal ssDNA binding site of NS3h helicase was determined to be 8 nucleotides with the microscopic K(d) of 2-4 nm or an observed free energy of -50 kJ/mol. These NS3h-DNA interactions are highly sensitive to salt, and the K(d) increases 4 times when the NaCl concentration is doubled. Multiple HCV helicase proteins bind to ssDNA >15 nucleotides in length, with an apparent occluded site of 8-11 nucleotides. The DNA binding data indicate that the interactions of multiple NS3h protein molecules with long ssDNA are both noncooperative and sequence-independent. We discuss the DNA binding properties of HCV helicase in relation to other superfamily 1 and 2 helicases. These studies provide the basis to investigate the DNA binding interactions with the unwinding substrate and their modulation by the ATPase activity of HCV helicase.     

An RNA ligand inhibits hepatitis C virus NS3 protease and helicase activities

The hepatitis C virus non-structural protein 3 (HCV NS3) possesses both protease and helicase activities that are essential for viral replication. In a previous study, we obtained RNA aptamers that specifically and efficiently inhibited NS3 protease activity (G9 aptamers). In order to add helicase-inhibition capability, we attached (U)14 to the 3'-terminal end of a minimized G9 aptamer, DeltaNEO-III. NEO-III-14U was shown to inhibit the NS3 protease activity more efficiently than the original aptamer and, furthermore, to efficiently inhibit the unwinding reaction by NS3 helicase. In addition, NEO-III-14U has the potential to diminish specific interactions between NS3 and the 3'-UTR of HCV-positive and -negative strands. NEO-III-14U showed effective inhibition against NS3 protease in living cells.     

Crystal structure and activity of Kunjin virus NS3 helicase; protease and helicase domain assembly in the full length NS3 protein

Flaviviral NS3 is a multifunctional protein displaying N-terminal protease activity in addition to C-terminal helicase, nucleoside 5'-triphosphatase (NTPase), and 5'-terminal RNA triphosphatase (RTPase) activities. NS3 is held to support the separation of RNA daughter and template strands during viral replication. In addition, NS3 assists the initiation of replication by unwinding the RNA secondary structure in the 3' non-translated region (NTR). We report here the three-dimensional structure (at 3.1 A resolution) of the NS3 helicase domain (residues 186-619; NS3:186-619) from Kunjin virus, an Australian variant of the West Nile virus. As for homologous helicases, NS3:186-619 is composed of three domains, two of which are structurally related and held to host the NTPase and RTPase active sites. The third domain (C-terminal) is involved in RNA binding/recognition. The NS3:186-619 construct occurs as a dimer in solution and in the crystals. We show that NS3:186-619 displays both ATPase and RTPase activities, that it can unwind a double-stranded RNA substrate, being however inactive on a double-stranded DNA substrate. Analysis of different constructs shows that full length NS3 displays increased helicase activity, suggesting that the protease domain plays an assisting role in the RNA unwinding process. The structural interaction between the helicase and protease domain has been assessed using small angle X-ray scattering on full length NS3, disclosing that the protease and helicase domains build a rather elongated molecular assembly differing from that observed in the NS3 protein from hepatitis C virus.     

The ATPase Cycle of PcrA Helicase and Its Coupling to Translocation on DNA

The superfamily 1 bacterial helicase PcrA has a role in the replication of certain plasmids, acting with the initiator protein (RepD) that binds to and nicks the double-stranded origin of replication. PcrA also translocates single-stranded DNA with discrete steps of one base per ATP hydrolyzed. Individual rate constants have been determined for the DNA helicase PcrA ATPase cycle when bound to either single-stranded DNA or a double-stranded DNA junction that also has RepD bound. The fluorescent ATP analogue 2′(3′)-O-(N-methylanthraniloyl)ATP was used throughout all experiments to provide a complete ATPase cycle for a single nucleotide species. Fluorescence intensity and anisotropy stopped-flow measurements were used to determine rate constants for binding and release. Quenched-flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor MDCC-PBP was used to measure phosphate release kinetics. The chemical cleavage step is the rate-limiting step in the cycle and is essentially irreversible and would result in the bound ATP complex being a major component at steady state. This cleavage step is greatly accelerated by bound DNA, producing the high activation of this protein compared to the protein alone. The data suggest the possibility that ADP is released in two steps, which would result in bound ADP also being a major intermediate, with bound ADP·P_i being a very small component. It therefore seems likely that the major transition in structure occurs during the cleavage step, rather than P_i release. ATP rebinding could then cause reversal of this structural transition. The kinetic mechanism of the PcrA ATPase cycle is very little changed by potential binding to RepD, supporting the idea that RepD increases the processivity of PcrA by increasing affinity to DNA rather than affecting the enzymatic properties per se.