Modulation of the nucleoside triphosphatase/RNA helicase and 5'-RNA triphosphatase activities of Dengue virus type 2 nonstructural protein 3 (NS3) by interaction with NS5, the RNA-dependent RNA polymerase

Dengue virus type 2 (DEN2), a member of the Flaviviridae family, is a re-emerging human pathogen of global significance. DEN2 nonstructural protein 3 (NS3) has a serine protease domain (NS3-pro) and requires the hydrophilic domain of NS2B (NS2BH) for activation. NS3 is also an RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicase and a 5'-RNA triphosphatase (RTPase). In this study the first biochemical and kinetic properties of full-length NS3 (NS3FL)-associated NTPase, RTPase, and RNA helicase are presented. The NS3FL showed an enhanced RNA helicase activity compared with the NS3-pro-minus NS3, which was further enhanced by the presence of the NS2BH (NS2BH-NS3FL). An active protease catalytic triad is not required for the stimulatory effect, suggesting that the overall folding of the N-terminal protease domain contributes to this enhancement. In DEN2-infected mammalian cells, NS3 and NS5, the viral 5'-RNA methyltransferase/polymerase, exist as a complex. Therefore, the effect of NS5 on the NS3 NTPase activity was examined. The results show that NS5 stimulated the NS3 NTPase and RTPase activities. The NS5 stimulation of NS3 NTPase was dose-dependent until an equimolar ratio was reached. Moreover, the conserved motif, 184RKRK, of NS3 played a crucial role in binding to RNA substrate and modulating the NTPase/RNA helicase and RTPase activities of NS3.     

In vitro processing of dengue virus type 2 nonstructural proteins NS2A, NS2B, and NS3.

We have tested the hypothesis that the flavivirus nonstructural protein NS3 is a viral proteinase that generates the termini of several nonstructural proteins by using an efficient in vitro expression system and monospecific antisera directed against the nonstructural proteins NS2B and NS3. A series of cDNA constructs was transcribed by using T7 RNA polymerase, and the RNA was translated in reticulocyte lysates. The resulting protein patterns indicated that proteolytic processing occurred in vitro to generate NS2B and NS3. The amino termini of NS2B and NS3 produced in vitro were found to be the same as the termini of NS2B and NS3 isolated from infected cells. Deletion analysis of cDNA constructs localized the protease domain within NS3 to the first 184 amino acids but did not eliminate the possibility that sequences within NS2B were also required for proper cleavage. Kinetic analysis of processing events in vitro and experiments to examine the sensitivity of processing to dilution suggested that an intramolecular cleavage between NS2A and NS2B preceded an intramolecular cleavage between NS2B and NS3. The data from these expression experiments confirm that NS3 is the viral proteinase responsible for cleavage events generating the amino termini of NS2B and NS3 and presumably for cleavages generating the termini of NS4A and NS5 as well.     

Functional characterization of cis and trans activity of the Flavivirus NS2B-NS3 protease

Flaviviruses are serious human pathogens for which treatments are generally lacking. The proteolytic maturation of the 375-kDa viral polyprotein is one target for antiviral development. The flavivirus serine protease consists of the N-terminal domain of the multifunctional nonstructural protein 3 (NS3) and an essential 40-residue cofactor (NS2B(40)) within viral protein NS2B. The NS2B-NS3 protease is responsible for all cytoplasmic cleavage events in viral polyprotein maturation. This study describes the first biochemical characterization of flavivirus protease activity using full-length NS3. Recombinant proteases were created by fusion of West Nile virus (WNV) NS2B(40) to full-length WNV NS3. The protease catalyzed two autolytic cleavages. The NS2B/NS3 junction was cleaved before protein purification. A second site at Arg(459) decreasing Gly(460) within the C-terminal helicase region of NS3 was cleaved more slowly. Autolytic cleavage reactions also occurred in NS2B-NS3 recombinant proteins from yellow fever virus, dengue virus types 2 and 4, and Japanese encephalitis virus. Cis and trans cleavages were distinguished using a noncleavable WNV protease variant and two types of substrates as follows: an inactive variant of recombinant WNV NS2B-NS3, and cyan and yellow fluorescent proteins fused by a dodecamer peptide encompassing a natural cleavage site. With these materials, the autolytic cleavages were found to be intramolecular only. Autolytic cleavage of the helicase site was insensitive to protein dilution, confirming that autolysis is intramolecular. Formation of an active protease was found to require neither cleavage of NS2B from NS3 nor a free NS3 N terminus. Evidence was also obtained for product inhibition of the protease by the cleaved C terminus of NS2B.     

Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing, NS2B-NS3 complex formation, and viral replication.

To study the role of specific regions of the yellow fever virus NS2B protein in proteolytic processing and association with the NS3 proteinase domain, a series of mutations were created in the hydrophobic regions and in a central conserved hydrophilic region proposed as a domain important for NS2B function. The effects of these mutations on cis cleavage at the 2B/3 cleavage site and on processing at other consensus cleavage sites for the NS3 proteinase in the nonstructural region were then characterized by cell-free translation and transient expression in BHK cells. Association between NS2B and the NS3 proteinase domain and the effects of mutations on complex formation were investigated by nondenaturing immunoprecipitation of these proteins expressed in infected cells, by cell-free translation, or by recombinant vaccinia viruses. Mutations within the hydrophobic regions had subtle effects on proteolytic processing, whereas mutations within the conserved domain dramatically reduced cleavage efficiency or abolished all cleavages. The conserved domain of NS2B is also implicated in formation of an NS2B-NS3 complex on the basis of the ability of mutations in this region to eliminate both association of these two proteins and trans-cleavage activity. In addition, mutations which either eliminated proteolytic processing or had no apparent effect on processing were found to abolish recovery of infectious virus following RNA transfection. These results suggest that the conserved region of NS2B is a domain essential for the function of the NS3 proteinase. Hydrophobic regions of NS2B whose structural integrity may not be essential for proteolytic processing may have additional functions during viral replication.     

Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins.

The cleavages at the junctions of the flavivirus nonstructural (NS) proteins NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 share an amino acid sequence motif and are presumably catalyzed by a virus-encoded protease. We constructed recombinant vaccinia viruses expressing various portions of the NS region of the dengue virus type 4 polyprotein. By analyzing immune precipitates of 35S-labeled lysates of recombinant virus-infected cells, we could monitor the NS2A/NS2B, NS2B/NS3, and NS3/NS4A cleavages. A polyprotein composed of NS2A, NS2B, and the N-terminal 184 amino acids of NS3 was cleaved at the NS2A/NS2B and NS2B/NS3 junctions, whereas a similar polyprotein containing only the first 77 amino acids of NS3 was not cleaved. This finding is consistent with the proposal that the N-terminal 180 amino acids of NS3 constitute a protease domain. Polyproteins containing NS2A and NS3 with large in-frame deletions of NS2B were not cleaved at the NS2A/NS2B or NS2B/NS3 junctions. Coinfection with a recombinant expressing NS2B complemented these NS2B deletions for NS2B/NS3 cleavage and probably also for NS2A/NS2B cleavage. Thus, NS2B is also required for the NS2A/NS2B and NS2B/NS3 cleavages and can act in trans. Other experiments showed that NS2B was needed, apparently in cis, for NS3/NS4A cleavage and for a series of internal cleavages in NS3. Indirect evidence that NS3 can also act in trans was obtained. Models are discussed for a two-component protease activity requiring both NS2B and NS3.     

Crystal structure of the NS3 protease-helicase from dengue virus

Several flaviviruses are important human pathogens, including dengue virus, a disease against which neither a vaccine nor specific antiviral therapies currently exist. During infection, the flavivirus RNA genome is translated into a polyprotein, which is cleaved into several components. Nonstructural protein 3 (NS3) carries out enzymatic reactions essential for viral replication, including proteolysis of the polyprotein through its serine protease N-terminal domain, with a segment of 40 residues from the NS2B protein acting as a cofactor. The ATPase/helicase domain is located at the C terminus of NS3. Atomic structures are available for these domains separately, but a molecular view of the full-length flavivirus NS3 polypeptide is still lacking. We report a crystallographic structure of a complete NS3 molecule fused to 18 residues of the NS2B cofactor at a resolution of 3.15 Å. The relative orientation between the protease and helicase domains is drastically different than the single-chain NS3-NS4A molecule from hepatitis C virus, which was caught in the act of cis cleavage at the NS3-NS4A junction. Here, the protease domain sits beneath the ATP binding site, giving the molecule an elongated shape. The domain arrangement found in the crystal structure fits nicely into an envelope determined ab initio using small-angle X-ray scattering experiments in solution, suggesting a stable molecular conformation. We propose that a basic patch located at the surface of the protease domain increases the affinity for nucleotides and could also participate in RNA binding, explaining the higher unwinding activity of the full-length enzyme compared to that of the isolated helicase domain.     

The two-component NS2B-NS3 proteinase represses DNA unwinding activity of the West Nile virus NS3 helicase

Similar to many flavivirus types including Dengue and yellow fever viruses, the nonstructural NS3 multifunctional protein of West Nile virus (WNV) with an N-terminal serine proteinase domain and an RNA triphosphatase, an NTPase domain, and an RNA helicase in the C-terminal domain is implicated in both polyprotein processing and RNA replication and is therefore a promising drug target. To exhibit its proteolytic activity, NS3 proteinase requires the presence of the cofactor encoded by the upstream NS2B sequence. During our detailed investigation of the biology of the WNV helicase, we characterized the ATPase and RNA/DNA unwinding activities of the full-length NS2B-NS3 proteinase-helicase protein as well as the individual NS3 helicase domain lacking both the NS2B cofactor and the NS3 proteinase sequence and the individual NS3 proteinase-helicase lacking only the NS2B cofactor. We determined that both the NS3 helicase and NS3 proteinase-helicase constructs are capable of unwinding both the DNA and the RNA templates. In contrast, the full-length NS2B-NS3 proteinase-helicase unwinds only the RNA templates, whereas its DNA unwinding activity is severely repressed. Our data suggest that the productive, catalytically competent fold of the NS2B-NS3 proteinase moiety represents an essential component of the RNA-DNA substrate selectivity mechanism in WNV and, possibly, in other flaviviruses. Based on our data, we hypothesize that the mechanism we have identified plays a role yet to be determined in WNV replication occurring both within the virus-induced membrane-bound replication complexes in the host cytoplasm and in the nuclei of infected cells.     

An amino-terminal domain of the hepatitis C virus NS3 protease is essential for interaction with NS4A.

Hepatitis C virus (HCV) genomic RNA is translated into a large polyprotein that is processed into structural and nonstructural proteins. Processing at the N termini of several nonstructural proteins requires sequences contained in both NS3 and NS4A. NS3 contains a serine protease, whereas the function of NS4A in proteolysis is yet to be determined. By using the vaccinia virus-T7 hybrid expression system to transiently express HCV polypeptides in HeLa cells, we studied the effect of several N-terminal and C-terminal deletions of HCV NS3 on the processing activity at all the downstream cleavage sites. In this way, we have delineated the minimal domain of NS3 required for the serine protease activity associated with this protein. In addition, we demonstrate the formation of a stable complex between NS3 and NS4A: analysis of the deletion mutants reveals a region at the N terminus of NS3 that is necessary for both complex formation and modulation of the proteolytic activity by NS4A but not for the NS4A-independent serine protease activity of NS3.     

Both NS3 and NS4A are required for proteolytic processing of hepatitis C virus nonstructural proteins.

The proteolytic cleavages at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions of hepatitis C virus (HCV) polyprotein are effected by the virus-encoded serine protease contained within NS3. Using transient expression in HeLa cells of cDNA fragments that code for regions of the HCV polyprotein, we studied whether viral functions other than NS3 are required for proteolytic processing at these sites. We found that, in addition to NS3, a C-terminal 33-amino-acid sequence of the NS4A protein is required for cleavage at the NS3-NS4A and NS4B-NS5A sites and that it accelerates the rate of cleavage at the NS5A-NS5B junction. In addition, we show that NS4A can activate the NS3 protease when supplied in trans. Our data suggest that HCV NS4A may be the functional analog of flavivirus NS2B and pestivirus p10 proteins.     

Multiple Enzymatic Activities Associated with Recombinant NS3 Protein of Hepatitis C Virus

The hepatitis C virus (HCV) nonstructural 3 protein (NS3) contains at least two domains associated with multiple enzymatic activities; a serine protease activity resides in the N-terminal one-third of the protein, whereas RNA helicase activity and RNA-stimulated nucleoside triphosphatase activity are associated with the C-terminal portion. To study the possible mutual influence of these enzymatic activities, a full-length NS3 polypeptide of 67 kDa was expressed as a nonfusion protein in Escherichia coli, purified to homogeneity, and shown to retain all three enzymatic activities. The protease activity of the full-length NS3 was strongly dependent on the activation by a synthetic peptide spanning the central hydrophobic core of the NS4A cofactor. Once complexed with the NS4A-derived peptide, the full-length NS3 protein and the isolated N-terminal protease domain cleaved synthetic peptide substrates with comparable efficiency. We show that, as in the case of the isolated protease domain, the protease activity of full-length NS3 undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B and NS5A-NS5B. We have also characterized and quantified the NS3 ATPase, RNA helicase, and RNA-binding activities under optimized reaction conditions. Compared with the isolated N-terminal and C-terminal domains, recombinant full-length NS3 did not show significant differences in the three enzymatic activities analyzed in independent in vitro assays. We have further explored the possible interdependence of the NS3 N-terminal and C-terminal domains by analyzing the effect of polynucleotides on the modulation of all NS3 enzymatic functions. Our results demonstrated that the observed inhibition of the NS3 proteolytic activity by single-stranded RNA is mediated by direct interaction with the protease domain rather than with the helicase RNA-binding domain.     

NS3 protease from flavivirus as a target for designing antiviral inhibitors against dengue virus

The development of novel therapeutic agents is essential for combating the increasing number of cases of dengue fever in endemic countries and among a large number of travelers from non-endemic countries. The dengue virus has three structural proteins and seven non-structural (NS) proteins. NS3 is a multifunctional protein with an N-terminal protease domain (NS3pro) that is responsible for proteolytic processing of the viral polyprotein, and a C-terminal region that contains an RNA triphosphatase, RNA helicase and RNA-stimulated NTPase domain that are essential for RNA replication. The serine protease domain of NS3 plays a central role in the replicative cycle of dengue virus. This review discusses the recent structural and biological studies on the NS2B-NS3 protease-helicase and considers the prospects for the development of small molecules as antiviral drugs to target this fascinating, multifunctional protein.     

Proper processing of dengue virus nonstructural glycoprotein NS1 requires the N-terminal hydrophobic signal sequence and the downstream nonstructural protein NS2a.

Expression of dengue virus gene products involves specific proteolytic cleavages of a precursor polyprotein. To study the flanking sequences required for expression of the dengue virus nonstructural glycoprotein NS1, we constructed a series of recombinant vaccinia viruses that contain the coding sequence for NS1 in combination with various lengths of upstream and downstream sequences. The NS1 products expressed by these viruses in infected CV-1 cells were immune precipitated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data show that the 24-residue hydrophobic sequence preceding NS1 was necessary and sufficient for the production of glycosylated NS1 and that this sequence was cleaved from NS1 in the absence of most dengue virus proteins. This finding is consistent with previous proposals that this hydrophobic sequence serves as an N-terminal signal sequence that is cleaved by signal peptidase. The cleavage between the C terminus of NS1 and the downstream protein NS2a occurred when the complete NS2a was present. Recombinant viruses containing NS1 plus 15 or 49% of NS2a produced proteins larger than authentic NS1, indicating that the cleavage between NS1 and NS2a had not occurred. Failure of cleavage was not corrected by coinfection with a recombinant virus capable of cleavage. These results suggest that NS2a may be a cis-acting protease that cleaves itself from NS1, or NS2a may provide sequences for recognition by a specific cellular protease that cleaves at the NS1-NS2a junction.     

Stimulation of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase activity by the NS3 protease domain and by HCV RNA-dependent RNA polymerase

Hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses multiple enzyme activities. The N-terminal one-third of NS3 primarily functions as a serine protease, while the remaining two-thirds of NS3 serve as a helicase and nucleoside triphosphatase. Whether the multiple enzyme activities of NS3 are functionally interdependent and/or modulated by other viral NS proteins remains unclear. We performed biochemical studies to examine the functional interdependence of the NS3 protease and helicase domains and the modulation of NS3 helicase by NS5B, an RNA-dependent RNA polymerase (RdRp). We found that the NS3 protease domain of the full-length NS3 (NS3FL) enhances the NS3 helicase activity. Additionally, HCV RdRp stimulates the NS3FL helicase activity by more than sevenfold. However, the helicase activity of the NS3 helicase domain was unaffected by HCV RdRp. Glutathione S-transferase pull-down as well as fluorescence anisotropy results revealed that the NS3 protease domain is required for specific NS3 and NS5B interaction. These findings suggest that HCV RdRp regulates the functions of NS3 during HCV replication. In contrast, NS3FL does not increase NS5B RdRp activity in vitro, which is contrary to a previously published report that the HCV NS3 enhances NS5B RdRp activity.     

Functional interplay among the flavivirus NS3 protease,helicase, and cofactors

Flaviviruses are positive-sense RNA viruses, and many are important human pathogens. Nonstructural protein 2B and 3 of the flaviviruses (NS2BNS3) form an endoplasmic reticulum (ER) membrane-associated hetero-dimeric complex through the NS2B transmembrane region. The NS2BNS3 complex is multifunctional. The N-terminal region of NS3, and its cofactor NS2B fold into a protease that is responsible for viral polyprotein processing, and the C-terminal domain of NS3 possesses NTPase/RNA helicase activities and is involved in viral RNA replication and virus particle formation. In addition, NS2BNS3 complex has also been shown to modulate viral pathogenesis and the host immune response. Because of the essential functions that the NS2BNS3 complex plays in the flavivirus life cycle, it is an attractive target for antiviral development. This review focuses on the recent biochemical and structural advances of NS2BNS3 and provides a brief update on the current status of drug development targeting this viral protein complex.     

Functional interplay among the flavivirus NS3 protease,helicase, and cofactors

Flaviviruses are positive-sense RNA viruses, and many are important human pathogens. Nonstructural protein 2B and 3 of the flaviviruses(NS2BNS3) form an endoplasmic reticulum(ER) membrane-associated hetero-dimeric complex through the NS2B transmembrane region. The NS2BNS3 complex is multifunctional. The N-terminal region of NS3, and its cofactor NS2B fold into a protease that is responsible for viral polyprotein processing, and the C-terminal domain of NS3 possesses NTPase/RNA helicase activities and is involved in viral RNA replication and virus particle formation. In addition, NS2BNS3 complex has also been shown to modulate viral pathogenesis and the host immune response. Because of the essential functions that the NS2BNS3 complex plays in the flavivirus life cycle, it is an attractive target for antiviral development. This review focuses on the recent biochemical and structural advances of NS2BNS3 and provides a brief update on the current status of drug development targeting this viral protein complex.     

Mechanism of NS2B-mediated activation of NS3pro in dengue virus: molecular dynamics simulations and bioassays

The NS2B cofactor is critical for proteolytic activation of the flavivirus NS3 protease. To elucidate the mechanism involved in NS2B-mediated activation of NS3 protease, molecular dynamic simulation, principal component analysis, molecular docking, mutagenesis, and bioassay studies were carried out on both the dengue virus NS3pro and NS2B-NS3pro systems. The results revealed that the NS2B-NS3pro complex is more rigid than NS3pro alone due to its robust hydrogen bond and hydrophobic interaction networks within the complex. These potent networks lead to remodeling of the secondary and tertiary structures of the protease that facilitates cleavage sequence recognition and binding of substrates. The cofactor is also essential for proper domain motion that contributes to substrate binding. Hence, the NS2B cofactor plays a dual role in enzyme activation by facilitating the refolding of the NS3pro domain as well as being directly involved in substrate binding/interactions. Kinetic analyses indicated for the first time that Glu92 and Asp50 in NS2B and Gln27, Gln35, and Arg54 in NS3pro may provide secondary interaction points for substrate binding. These new insights on the mechanistic contributions of the NS2B cofactor to NS3 activation may be utilized to refine current computer-based search strategies to raise the quality of candidate molecules identified as potent inhibitors against flaviviruses.     

Regulated cleavages at the West Nile virus NS4A-2K-NS4B junctions play a major role in rearranging cytoplasmic membranes and Golgi trafficking of the NS4A protein

A common feature associated with the replication of most RNA viruses is the formation of a unique membrane environment encapsulating the viral replication complex. For their part, flaviviruses are no exception, whereupon infection causes a dramatic rearrangement and induction of unique membrane structures within the cytoplasm of infected cells. These virus-induced membranes, termed paracrystalline arrays, convoluted membranes, and vesicle packets, all appear to have specific functions during replication and are derived from different organelles within the host cell. The aim of this study was to identify which protein(s) specified by the Australian strain of West Nile virus, Kunjin virus (KUNV), are responsible for the dramatic membrane alterations observed during infection. Thus, we have shown using immunolabeling of ultrathin cryosections of transfected cells that expression of the KUNV polyprotein intermediates NS4A-4B and NS2B-3-4A, as well as that of individual NS4A proteins with and without the C-terminal transmembrane domain 2K, resulted in different degrees of rearrangement of cytoplasmic membranes. The formation of the membrane structures characteristic for virus infection required coexpression of an NS4A-NS4B cassette with the viral protease NS2B-3pro which was shown to be essential for the release of the individual NS4A and NS4B proteins. Individual expression of NS4A protein retaining the C-terminal transmembrane domain 2K resulted in the induction of membrane rearrangements most resembling virus-induced structures, while removal of the 2K domain led to a less profound membrane rearrangement but resulted in the redistribution of the NS4A protein to the Golgi apparatus. The results show that cleavage of the KUNV polyprotein NS4A-4B by the viral protease is the key initiation event in the induction of membrane rearrangement and that the NS4A protein intermediate containing the uncleaved C-terminal transmembrane domain plays an essential role in these membrane rearrangements.     

In vitro determination of dengue virus type 2 NS2B-NS3 protease activity with fluorescent peptide substrates

The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1 M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2BNS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.     

The serine protease and RNA-stimulated nucleoside triphosphatase and RNA helicase functional domains of dengue virus type 2 NS3 converge within a region of 20 amino acids

NS3 protein of dengue virus type 2 has a serine protease domain within the N-terminal 180 residues. NS2B is required for NS3 to form an active protease involved in processing of the viral polyprotein precursor. The region carboxy terminal to the protease domain has conserved motifs present in several viral RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicases. To define the functional domains of protease and NTPase/RNA helicase activities of NS3, full-length and amino-terminal deletion mutants of NS3 were expressed in Escherichia coli and purified. Deletion of 160 N-terminal residues of NS3 (as in NS3del.2) had no detrimental effect on the basal and RNA-stimulated NTPase as well as RNA helicase activities. However, mutagenesis of the conserved P-loop motif of the RNA helicase domain (K199E) resulted in loss of ATPase activity. The RNA-stimulated NTPase activity was significantly affected by deletion of 20 amino acid residues from the N terminus or by substitutions of the cluster of basic residues, 184RKRK-->QNGN, of NS3del.2, although both mutant proteins retained the conserved RNA helicase motifs. Furthermore, the minimal NS3 protease domain, required for cleavage of the 2B-3 site, was precisely defined to be 167 residues, using the in vitro processing of NS2B-NS3 precursors. Our results reveal that the functional domains required for serine protease and RNA-stimulated NTPase activities map within the region between amino acid residues 160 and 180 of NS3 protein and that a novel motif, the cluster of basic residues 184RKRK, plays an important role for the RNA-stimulated NTPase activity.