INTRODUCTION

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Hepatitis C virus (HCV) is the major causative agent of parenterally transmitted non-A, non-B hepatitis (6, 8, 25). The infection easily becomes persistent and causes chronic hepatitis (17), which may lead to liver cirrhosis (38, 42) and hepatocellular carcinoma (9, 36). Alpha interferon and ribavirin combination therapy is the only effective therapy for chronic hepatitis C; however, less than 50% of patients respond to the treatment (2, 3, 5, 28). Therefore, new, more effective treatments for hepatitis C urgently need to be developed.

The HCV genome encodes a large polyprotein comprised of 3,008 to 3,037 amino acids (aa) (4). Nonstructural protein 3 (NS3) of HCV, ranging from aa 1027 to 1657 of the polyprotein, is a multifunctional protein. The N terminus of NS3 expresses a serine protease (27), while two-thirds of the C- terminal region of the protein expresses an RNA helicase (18, 20, 40, 41). The homologous NS3s in all flaviviruses and pestiviruses sequenced to date contain conserved sequence motifs (31), suggesting that the enzyme may be important in viral replication and may be a potential target for developing antiviral drugs.

Helicases are the enzymes that unwind duplex DNA or RNA at the expense of energy derived from nucleoside triphosphate (NTP) hydrolysis (13). A large number of putative RNA and DNA helicases from different organisms have been identified (14, 23). Sequence comparisons have classified the helicases into three superfamilies (SF): SFI, SFII, and SFIII (14, 19, 37). The HCV NS3 helicase belongs to SFII, which contains eight conserved motifs (14, 24). Motifs I (AxxGxGKS/T) and II (DExH), known as Walker motifs A and B, respectively (44), are responsible for binding the NTP-Mg²⁺ complex (46), while motif VI (QRxGRxGR) is thought to bind nucleic acids (11, 19) because it possesses many basic residues (notably Arg).

The crystal structure of HCV NS3 helicase has recently been uncovered (7, 22, 48). The reported structures shared similar global conformation, which consisted of three domains forming a Y-shaped configuration. The structure reported by Kim et al. is the only one based on a cocrystallization of the helicase with a sulfate ion and a (dU)₈ oligonucleotide (22). Notably, Kim et al. predicted that conserved motif VI (QRRGRTGR) would not interact with the single-stranded DNA oligonucleotide as originally expected but would be involved in ATP hydrolysis. On the other hand, hydrophobic residue Val-432 and aromatic residue Trp-501 are associated with the (dU)₈ oligonucleotide. They also noted that His-293 and Gln-460 from motifs II and VI, respectively, lie on opposite sides of the interdomain cleft and referred to them as "gatekeepers." However, the importance of gatekeepers has not been biochemically determined yet.

To decipher the link between the biochemical role and three-dimensional structure of the important amino acid residues, this study investigated the activities of 17 point mutation clones of NS3.

	MATERIALS AND METHODS

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Site-directed mutagenesis. The plasmid 5'-1175/pET21a (41), which contains the RNA helicase domain encompassing aa 1175 (nucleotide [nt] 3864) to 1657 (nt 5312) cloned at the BamHI and HindIII sites of the pET21a plasmid, was used as the target plasmid for site-directed mutagenesis. Mutants A204V, K210N, D290N, E291Q, C292A, H293A, C292A/H293D, and R461Q were constructed using the Transformer site-directed mutagenesis kit (Clontech) according to the manufacturer's instructions. Meanwhile, mutations were confirmed by DNA sequencing. Table 1 lists the sequences of the mutagenic primers. The remaining mutants E291A, H293K, H293Q, T322A, T324A, V432A, C292A/H293D/Q460H, W501A, and V432A/W501A were constructed using recombinant PCR, with the desired mutations introduced in the internal mutagenic primers (Table 1). The mutated forms of the BamHI (nt 3864)-EcoRI (nt 4578) DNA fragments (for mutants E291A, H293K, H293Q, T322A, and T324A) or the EcoRI (nt 4578)-StuI (nt 5034) DNA fragments (for mutants V432A and W501A) were then used in place of the corresponding regions of the parental 5'-1175/pET21a plasmid. Mutant Q460H/C292A/H293D was generated by substituting the mutated EcoRI-StuI fragment harboring the Q460H mutation for the corresponding region of the C292A/H293D plasmid, while mutant V432A/W501A was constructed by substituting the EcoRI-StuI DNA fragment harboring the W501A mutation for the same region of the V432A plasmid. To ensure that only the desired mutation was introduced, the PCR portions were sequenced with the dideoxy DNA sequencing method.

Expression and purification of mutant proteins. The mutant plasmids were transformed into Escherichia coli BL21(DE3). Expression of the plasmids was induced by adding 0.2 mM isopropyl--D-thiogalactopyranoside (IPTG) at 30°C for 3 h. Purification of the proteins was performed by using an Ni-affinity column as previously described (41). The final eluted proteins were dialyzed in TNE buffer (20 mM Tris-HCl, pH 7.9, 50 mM NaCl, and 1 mM EDTA) and were concentrated using Centriprep-10 (Amicon). The purified proteins were quantified using a bicinchoninic acid protein assay reagent (Pierce).

ATPase activity assays. ATPase activity was assessed by measuring the extent of [-³²P]ATP hydrolysis as previously described (41). The reaction mixture (10 µl) contained 20 mM HEPES-KOH (pH 7.0), 2 mM dithiothreitol, 1.5 mM MgCl₂, 5 µCi of [-³²P]ATP (3,000 Ci/mmol; Amersham), 100 µg of poly(U)/ml, 0.05 µg of wild-type or mutant helicases, and various concentrations of cold ATP ranging from 0.067 mM to 0.8 mM. For kinetic analysis, the reaction was carried out at room temperature for just 5 min to constrain the reaction rate within the linear phase and was then halted by adding EDTA to 20 mM. Reaction products were analyzed by thin-layer chromatography. One-half microliter of the reaction mixture was spotted onto plastic-backed polyethyleneimine cellulose F sheets (Merck) and was developed by ascending chromatography in 0.375 M potassium phosphate (pH 3.5) buffer. The sheets were dried and exposed to a FUJIX imaging plate, and the conversion rate was quantified by phosphorimager analysis (FUJIX BAS1000).

ATP binding assays. UV cross-linking (29, 49) was used to assess the ATP binding ability of those mutants whose ATPase activity was undetectable. However, this is a nonequilibrium method of measuring ATP binding. One microgram of wild-type or mutant helicase protein and 100 µg of poly(U)/ml were premixed in 10 µl of a buffer containing 20 mM HEPES-KOH, pH 7.5, and 5 mM Mg(CH₃CO₂)₂. Then 6 µCi (2 pmol) of [-³²P]ATP (3,000 Ci/mmol; Amersham) and 181 pmol of cold ATP were added. The reaction mixture was incubated on ice and was then irradiated using a UV cross-linker (Stratagene) (254 nm) situated at a distance of 4 cm for 25 min. Samples were boiled in sample buffer (100 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate [SDS], 20% -mercaptoethanol, 20% glycerol, 4 mM EDTA, and 0.01% bromophenol blue) for 5 min and were then separated by SDS-12.5% polyacrylamide gel electrophoresis (PAGE). The gels were stained with Coomassie brilliant blue, dried, and processed for autoradiography.

Preparation of partial dsRNA substrates. The partial double-stranded RNA (dsRNA) substrates were prepared by transcribing in vitro a portion of the multiple cloning sequences of a pGEM vector (Promega) in both orientations and then annealing these two RNA strands (41). After in vitro transcription, the transcripts were combined at a molar ratio of released strand (labeled) to template strand (unlabeled) of approximately 1:10 in a solution containing 20 mM HEPES-KOH (pH 7.6), 0.5 M NaCl, 1 mM EDTA, and 0.1% SDS. The mixture was boiled for 10 min, transferred to 65°C for 30 min, and then incubated at 25°C overnight. The hybridized products were mixed with 5× RNA loading dye (0.1 M Tris-HCl [pH 7.4], 20 mM EDTA, 0.5% SDS, 0.1% bromophenol blue, 0.1% xylene cyanol, and 50% glycerol) and then subjected to electrophoresis on an 8% native polyacrylamide (acrylamide/bisacrylamide ratio, 30:1)-1× Tris-borate-EDTA gel. The duplex RNA band was localized by autoradiography. The gel slice was excised, pulverized, and extracted with 0.5 M ammonium acetate (pH 7.0)-0.1% SDS-10 mM EDTA for 2 h at 25°C. The eluted substrate was then extracted with phenol-chloroform, ethanol precipitated, and resuspended in storage buffer (20 mM HEPES [pH 7.6] and 0.1 mM EDTA). The annealed product contains both 5' and 3' overhang regions and an internal double-stranded region.

RNA binding assays. The binding of the partial dsRNA substrate to the HCV NS3 helicase or to the mutant proteins was analyzed by gel mobility shift assay. One-half microgram of the wild-type or mutant helicase protein was incubated with 1.3 pmol of radiolabeled partial dsRNA substrate and 15 pmol of cold single release strand in 20 µl of a helicase reaction buffer containing nonhydrolyzable ATP-S. The binding reaction was incubated at 37°C for 15 min and was then terminated by adding 5× RNA loading dye containing 0.5% Nonidet P-40 instead of SDS and was electrophoresed on a 4% polyacrylamide (acrylamide/bisacrylamide ratio, 80:1)-1/3× Tris-borate-EDTA gel containing 5% glycerol. The bound complexes were visualized by autoradiography (41).

RNA helicase assays. Helicase assays were conducted by measuring the unwinding of the radiolabeled dsRNA substrate as previously described (41) except that, for kinetic analysis, the reaction was performed for just 5 min to constrain the reaction rate within the linear phase. The reaction mixture (20 µl) contained 20 mM HEPES-KOH (pH 7.0), 2 mM dithiothreitol, 1.5 mM MnCl₂, 2.5 mM ATP, 0.1 mg of bovine serum albumin per ml, 2 U of RNasin, 0.1 µg of wild-type or mutant helicases, and various amounts of dsRNA substrate ranging from 0.66 pmol to 10.56 pmol. The reaction was carried out at 37°C for 5 min and was then terminated by adding 5 µl of 5× RNA loading dye. An aliquot (12.5 µl) of each reaction was loaded onto an 8% native polyacrylamide (acrylamide/bisacrylamide ratio, 30:1)-1× Tris-borate-EDTA gel and electrophoresed. The gel was dried and autoradiographed. The ratio of single-stranded products to double-stranded substrates was quantified by phosphorimager analysis (FUJIX BAS1000).

	RESULTS

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Mutation, expression, and purification of HCV helicase proteins. Like many other SFII helicases, the HCV NS3 helicase contains several conserved motifs. This work investigates the roles of conserved motifs I, II, III, and VI and the two possible RNA-interacting residues, Val-432 and Trp-501, in ATP hydrolysis, RNA binding, and RNA helicase activities. A total of 17 mutants were constructed using site-directed mutagenesis. Figure 1A illustrates the mutated positions and the amino acids replaced. The wild type and the mutant clones were expressed in an E. coli pET expression system, and the recombinant proteins were purified using the Ni-agarose affinity column. Figure 1B illustrates the SDS-PAGE results for 2 µg of each of the purified proteins. The estimated molecular mass of the wild-type NS3 helicase or its mutant proteins is approximately 56 kDa. The data revealed that all these proteins had similar purities (ranging from 80 to 88%).

View larger version (28K): [in this window] [in a new window]   FIG. 1.   Site-directed mutagenesis of the HCV NS3 helicase domain. (A) Summary of all mutant clones. The top line sequences represent conserved motifs I, II, III, and VI, respectively, of HCV NS3 helicase. The amino acid residue replacements are shown below the arrows. (B) Purities of mutant proteins. The wild-type and mutant proteins were expressed in E. coli and purified as described in Materials and Methods. Two micrograms of each of the purified proteins was resolved by SDS-12.5% PAGE and stained with Coomassie brilliant blue. WT denotes the wild-type helicase protein.

ATPase activity of helicase mutants. ATPase activity was measured in the presence of poly(U) to stimulate the ATP hydrolysis activity of HCV NS3 (40, 41). Enzymatic activity was measured kinetically and expressed as k_cat/K_m, determined by varying the concentration of ATP substrate. All mutant proteins displayed a degree of reduced ATPase activity when compared to the wild-type protein (Table 2).

View larger version (50K): [in this window] [in a new window]   FIG. 2.   ATP binding activity of the wild type and some mutant helicase proteins. One microgram of the wild-type or mutant helicase proteins was incubated with [-32P]ATP in the presence of poly(U) and was UV cross-linked [WT(-UV)] as described in Materials and Methods. Samples were separated by SDS-12.5% PAGE. Following electrophoresis, the gels were stained with Coomassie brilliant blue, dried, and processed for autoradiography. WT, wild-type helicase protein; BSA, bovine serum albumin.

RNA binding activity of helicase mutants. RNA binding activity was measured by gel mobility shift assay. As illustrated in Fig. 3, all mutant proteins but R461Q, W501A, and V432A/W501A bound RNA as efficiently as the wild-type protein did. Mutants A204V, K210N, H293K, H293Q, and T322A exhibited even slightly higher RNA binding activity than the wild-type protein. Notably, the V432A mutant exhibited virtually normal RNA binding activity. The W501A mutant retained only residual RNA binding activity, but the double mutation clone V432A/W501A almost completely lost RNA binding activity (Fig. 3). These results suggested that Trp-501 was crucial to RNA binding, whereas Val-432 might play an auxiliary role.

View larger version (58K): [in this window] [in a new window]   FIG. 3.   RNA binding activity of wild-type and mutant helicase proteins. The wild-type or mutant helicase proteins, cold release strand RNA, and labeled dsRNA substrate were incubated as described in Materials and Methods. The reaction mixtures were electrophoresed by native PAGE and were processed for autoradiography. WT denotes the wild-type helicase protein.

RNA helicase activity of helicase mutants. The helicase activity of all mutant proteins was measured and expressed as k_cat/K_m, determined by varying the concentration of dsRNA substrate. Results given in Table 2 indicate that the helicase activity was completely abolished in two types of mutants: namely, those whose ATPase activity was completely lost (for example, K210N, D290N, E291Q, C292A/H293D, and C292A/H293D/Q460H) and those whose RNA binding activity was dramatically reduced (for example, R461Q, W501A, and V432A/W501A). The helicase activity of other mutants was only moderately affected, including mutants whose ATPase activity was severely reduced but not completely abolished (for example, H293A and T324A) (see below).

Relationship of ATPase activity and RNA helicase activity. To correlate the ATPase activity and RNA helicase activity of these mutant proteins, both activities were translated into their ratios relative to wild-type protein activity (Table 2). The relationship between RNA helicase activity and ATPase activity is presented in Fig. 4. The unwinding of dsRNA is an ATP-dependent process, and ATP hydrolysis is a prerequisite for RNA helicase activity. Therefore, as anticipated, mutants with abolished ATPase activity (such as K210N, D290N, E291Q, C292A/H293D, and C292A/H293D/Q460H) would impede RNA helicase activity. Unexpectedly, some mutants (for example, H293A and T324A) which exhibited ATPase activity as low as 6 to 16% of that exhibited by the wild-type protein still displayed sufficient RNA helicase activity (75 and 62%, respectively) (Fig. 4). These analytical results suggest that a low ATPase activity threshold (e.g., 6%) is sufficient for the present RNA helicase assay. Accordingly, the loss of RNA helicase activity in mutants R461Q, W501A, and V432A/W501A is probably caused by reduced RNA binding ability or is a synergistic effect of both reduced ATPase and reduced RNA binding activities.

View larger version (21K): [in this window] [in a new window]   FIG. 4.   Relationship of ATPase activity and RNA helicase activity. Both ATPase activity and RNA helicase activity of all mutant proteins were translated into ratios relative to activity of the wild-type (WT) helicase protein (data shown in Table 2). The relationship was then plotted as the RNA helicase activity versus ATPase activity.

	DISCUSSION

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The structure of domains I and II of the HCV NS3 helicase displays a fold similar to that of domains 1A and 2A, respectively, of the PcrA DNA helicase, whose structure was recently solved by cocrystallizing the protein with a nonhydrolyzable ATP analog and a magnesium ion (43). In comparing the PcrA DNA helicase structure with those reported structures for the HCV NS3 helicase (7, 22, 48), some different aspects among these models are noted. The results of this study may thus provide clues to elucidating the relationship between the function and the three-dimensional structure of each critical amino acid residue.

Residues that influence ATP hydrolysis. The amino acids in the conserved Walker A motif (Lys-210) and Walker B motif (Asp-290, Glu-291, and His-293) are crucial to ATP hydrolysis, as predicted. This investigation additionally identified other residues not contained in Walker A and B motifs but also vital to ATPase activity, for example, Gln-460, Thr-322, Thr-324, and Arg-461. To function in ATP hydrolysis, all of these residues except Arg-461 are conformationally proximal to NTP (Fig. 5).

View larger version (54K): [in this window] [in a new window]   FIG. 5.   Locations of the important amino acid residues in the three-dimensional conformation. The structure of HCV NS3 helicase was based on that determined by Kim et al. (22) using Protein Data Bank (PDB) accession code 1A1V. The important residues identified herein and the Arg-464/Arg-467 previously demonstrated (21, 22, 43) are emphasized by being represented in stick format, while the sulfate ion is represented in Corey-Pauling-Koltun (CPK) format. Residues His-293 and Gln-460 are located in domains I and II, respectively. It is hypothesized that the amine group of the histidine ring of His-293 formed a hydrogen bond with the carbonyl oxygen of the side chain of Gln-460. Residue Arg-461 is located in the interior of domain II. The inset indicates that the Arg-461 can form hydrogen bonds with Asp-412 and Asp-427, which are located at conserved motif V. These figures were drawn using InsightII.

Residues that influence RNA binding. The crystal structures determined by Cho et al. and Yao et al. located the single-stranded RNA (ssRNA) in a channel formed by the interdomain cleft between domains I and II (7, 48), whereas that determined by Kim et al. located the oligonucleotide in a groove between the first two domains and the third (22). The latter thus resembles that of PcrA DNA helicase (43). The mutagenesis data described herein demonstrated that Trp-501 was crucial for RNA binding, thus supporting the model proposed by Kim et al. (22). But our data demonstrated that Val-432, one of the "bookends" for the ssRNA binding proposed by the model, might play only a minor role. Notably, however, simultaneous mutation at Val-432 and Trp-501 synergistically degraded RNA binding activity. The role of Val-432 determined in this study is also different from that reported by other groups using full-length NS3 (33, 35). The discrepancy is not clear but may be due to different proteins (helicase domain versus full-length NS3) and assay systems used in different laboratories.

Factors that affect RNA helicase activity and the implications. Unwinding of RNA is an ATP-dependent process which requires ATP binding, ATP hydrolysis, and RNA binding. It is reasonable to hypothesize that the mutants whose ATPase activity is abolished will also impede RNA helicase activity. Mutants K210N, D290N, E291Q, C292A/H293D, and C292A/H293D/Q460H indeed followed this rule (Table 2). Surprisingly, however, some mutants with fairly low ATPase activity (6 to 16% of the wild-type helicase) still exhibited sufficiently high RNA helicase activity (for example, H293A and T324A). Similar phenomena were observed in previous mutational studies (21, 30). These results thus suggest that the ATPase activity required for RNA unwinding may be minimal. On the contrary, reduction of RNA binding activity markedly influenced RNA helicase activity, e.g., R461Q and W501A (Table 2). Based on these findings, we suggest that interfering with RNA binding activity rather than interfering with ATPase activity should be considered more in designing antiviral therapeutics for hepatitis C.