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.     

Hepatitis C virus-encoded NS2-3 protease: cleavage-site mutagenesis and requirements for bimolecular cleavage.

Cleavage at the 2/3 site of hepatitis C virus (HCV) is thought to be mediated by a virus-encoded protease composed of the region of the polyprotein encoding NS2 and the N-terminal one-third of NS3. This protease is distinct from the NS3 serine protease, which is responsible for downstream cleavages in the nonstructural region. Site-directed mutagenesis of residues surrounding the 2/3 cleavage site showed that cleavage is remarkably resistant to single-amino-acid substitutions from P5 to P3' (GWRLL decreases API). The only mutations which dramatically inhibited cleavage were the ones most likely to alter the conformation of the region, such as Pro substitutions at the P1 or P1' position, deletion of both amino acids at P1 and P1', or simultaneous substitution of multiple Ala residues. Cotransfection experiments were done to provide additional information on the polypeptide requirements for bimolecular cleavage. Polypeptides used in these experiments contained amino acid substitutions and/or deletions in NS2 and/or the N-terminal one-third of NS3. Polypeptides with defects in either NS2 or the N-terminal portion of NS3 but not both were cleaved when cotransfected with constructs expressing intact versions of the defective region. Cotransfection experiments also showed that certain defective NS2-3 constructs partially inhibited cleavage of wild-type polypeptides. Although these results show that inefficient cleavage can occur in a bimolecular reaction, they suggest that both molecules must contribute a functional subunit to allow formation of a protease which is capable of cleavage at the 2/3 site. This reaction may resemble the cis cleavage thought to occur at the 2/3 site during processing of the wild-type HCV polyprotein.     

Hyperphosphorylation of the hepatitis C virus NS5A protein requires an active NS3 protease, NS4A, NS4B, and NS5A encoded on the same polyprotein

The nonstructural protein NS5A of hepatitis c virus (HCV) has been demonstrated to be a phosphoprotein with an apparent molecular mass of 56 kDa. In the presence of other viral proteins, p56 is converted into a slower-migrating form of NS5A (p58) by additional phosphorylation events. In this report, we show that the presence of NS3, NS4A, and NS4B together with NS5A is necessary and sufficient for the generation of the hyperphosphorylated form of NS5A (p58) and that all proteins must be encoded on the same polyprotein (in cis). Kinetic studies of NS5A synthesis and pulse-chase experiments demonstrate that fully processed NS5A is the substrate for the formation of p58 and that p56 is converted to p58. To investigate the role of NS3 in NS5A hyperphosphorylation, point and deletion mutations were introduced into NS3 in the context of a polyprotein containing the proteins from NS3 to NS5A. Mutation of the catalytic serine residue into alanine abolished protease activity of NS3 and resulted in total inhibition of NS5A hyperphosphorylation, even if polyprotein processing was allowed by addition of NS3 and NS4A in trans. The same result was obtained by deletion of the first 10 or 28 N-terminal amino acids of NS3, which are known to be important for the formation of a stable complex between NS3 and its cofactor NS4A. These data suggest that the formation of p58 is closely connected to HCV polyprotein processing events. Additional data obtained with NS3 containing the 34 C-terminal residues of NS2 provide evidence that in addition to NS3 protease activity the authentic N-terminal sequence is required for NS5A hyperphosphorylation.     

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.     

Hepatitis C viral NS3-4A protease activity is enhanced by the NS3 helicase

Non-structural protein 3 (NS3) is a multifunctional enzyme possessing serine protease, NTPase, and RNA unwinding activities that are required for hepatitis C viral (HCV) replication. HCV non-structural protein 4A (NS4A) binds to the N-terminal NS3 protease domain to stimulate NS3 serine protease activity. In addition, the NS3 protease domain enhances the RNA binding, ATPase, and RNA unwinding activities of the C-terminal NS3 helicase domain (NS3hel). To determine whether NS3hel enhances the NS3 serine protease activity, we purified truncated and full-length NS3-4A complexes and examined their serine protease activities under a variety of salt and pH conditions. Our results indicate that the helicase domain enhances serine protease activity, just as the protease domain enhances helicase activity. Thus, the two enzymatic domains of NS3-4A are highly interdependent. This is the first time that such a complete interdependence has been demonstrated for a multifunctional, single chain enzyme. NS3-4A domain interdependence has important implications for function during the viral lifecycle as well as for the design of inhibitor screens that target the NS3-4A protease.     

In vitro characterization of a purified NS2/3 protease variant of hepatitis C virus

The cleavage of the hepatitis C virus polyprotein between the nonstructural proteins NS2 and NS3 is mediated by the NS2/3 protease, whereas the NS3 protease is responsible for the cleavage of the downstream proteins. Purification and in vitro characterization of the NS2/3 protease has been hampered by its hydrophobic nature. NS2/3 protease activity could only be detected in cells or in in vitro translation assays with the addition of microsomal membranes or detergent. To facilitate purification of this poorly characterized protease, we truncated the N-terminal hydrophobic domain, resulting in an active enzyme with improved biophysical properties. We define a minimal catalytic region of NS2/3 protease retaining autocleavage activity that spans residues 904-1206 and includes the C-terminal half of NS2 and the N-terminal NS3 protease domain. The NS2/3 (904-1206) variant was purified from Escherichia coli inclusion bodies and refolded by gel filtration chromatography. The purified inactive form of NS2/3 (904-1206) was activated by the addition of glycerol and detergent to induce autocleavage at the predicted site between Leu(1026) and Ala(1027). NS2/3 (904-1206) activity was dependent on zinc ions and could be inhibited by NS4A peptides, peptides that span the cleavage site, or an N-terminal peptidic cleavage product. This NS2/3 variant will facilitate the development of an assay suitable for identifying inhibitors of HCV replication.     

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.     

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.     

Mutagenesis of the NS3 Protease of Dengue Virus Type 2

The flavivirus protease is composed of two viral proteins, NS2B and NS3. The amino-terminal portion of NS3 contains sequence and structural motifs characteristic of bacterial and cellular trypsin-like proteases. We have undertaken a mutational analysis of the region of NS3 which contains the catalytic serine, five putative substrate binding residues, and several residues that are highly conserved among flavivirus proteases and among all serine proteases. In all, 46 single-amino-acid substitutions were created in a cloned NS2B-NS3 cDNA fragment of dengue virus type 2, and the effect of each mutation on the extent of self-cleavage of the NS2B-NS3 precursor at the NS2B-NS3 junction was assayed in vivo. Twelve mutations almost completely or completely inhibited protease activity, 9 significantly reduced it, 14 decreased cleavage, and 11 yielded wild-type levels of activity. Substitution of alanine at ultraconserved residues abolished NS3 protease activity. Cleavage was also inhibited by substituting some residues that are conserved among flavivirus NS3 proteins. Two (Y150 and G153) of the five putative substrate binding residues could not be replaced by alanine, and only Y150 and N152 could be replaced by a conservative change. The two other putative substrate binding residues, D129 and F130, were more freely substitutable. By analogy with the trypsin model, it was proposed that D129 is located at the bottom of the substrate binding pocket so as to directly interact with the basic amino acid at the substrate cleavage site. Interestingly, we found that significant cleavage activity was displayed by mutants in which D129 was replaced by E, S, or A and that low but detectable protease activity was exhibited by mutants in which D129 was replaced by K, R, or L. Contrary to the proposed model, these results indicate that D129 is not a major determinant of substrate binding and that its interaction with the substrate, if it occurs at all, is not essential. This mutagenesis study provided us with an array of mutations that alter the cleavage efficiency of the dengue virus protease. Mutations that decrease protease activity without abolishing it are candidates for introduction into the dengue virus infectious full-length cDNA clone with the aim of creating potentially attenuated virus stocks.     

Deletion analysis of dengue virus type 4 nonstructural protein NS2B: identification of a domain required for NS2B-NS3 protease activity.

Most proteolytic cleavages in the nonstructural protein (NS) region of the flavivirus polyprotein are effected by a virus-encoded protease composed of two viral proteins, NS2B and NS3. The N-terminal 180-amino-acid-region of NS3 includes sequences with homology to the active sites of serine proteases, and there is evidence that this portion of NS3 can mediate proteolytic cleavages. In contrast, nothing is known about required sequences in NS2B. We constructed a series of deletion mutations in the NS2B portion of plasmid pTM/NS2B-30% NS3, which expresses dengue virus type 4 (DEN4) cDNA encoding NS2B and the N-terminal 184 residues of NS3 from the T7 RNA polymerase promoter. Mutant or wild-type plasmids were transfected into cells that had been infected with a recombinant vaccinia virus expressing T7 RNA polymerase, and the protease activities of the expressed polyproteins were assayed by examining the extent of self-cleavage at the NS2B-NS3 junction. The results identify a 40-amino-acid segment of NS2B (DEN4 amino acids 1396 to 1435) essential for protease activity. A hydrophobicity profile of DEN4 NS2B predicts this segment constitutes a hydrophilic domain surrounded by hydrophobic regions. Hydrophobicity profiles of the NS2B proteins of other flaviviruses show similar patterns. Amino acid sequence alignment of this domain of DEN4 NS2B with comparable regions of other proteins of flaviviruses indicates significant sequence conservation, especially at the N-terminal end. These observations suggest that the central hydrophilic domain of NS2B of these other flaviviruses will also prove to be essential for protease activity.     

Identification of residues in the dengue virus type 2 NS2B cofactor that are critical for NS3 protease activation

Proteolytic processing of the dengue virus polyprotein is mediated by host cell proteases and the virus-encoded NS2B-NS3 two-component protease. The NS3 protease represents an attractive target for the development of antiviral inhibitors. The three-dimensional structure of the NS3 protease domain has been determined, but the structural determinants necessary for activation of the enzyme by the NS2B cofactor have been characterized only to a limited extent. To test a possible functional role of the recently proposed Phix(3)Phi motif in NS3 protease activation, we targeted six residues within the NS2B cofactor by site-specific mutagenesis. Residues Trp62, Ser71, Leu75, Ile77, Thr78, and Ile79 in NS2B were replaced with alanine, and in addition, an L75A/I79A double mutant was generated. The effects of these mutations on the activity of the NS2B(H)-NS3pro protease were analyzed in vitro by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of autoproteolytic cleavage at the NS2B/NS3 site and by assay of the enzyme with the fluorogenic peptide substrate GRR-AMC. Compared to the wild type, the L75A, I77A, and I79A mutants demonstrated inefficient autoproteolysis, whereas in the W62A and the L75A/I79A mutants self-cleavage appeared to be almost completely abolished. With exception of the S71A mutant, which had a k(cat)/K(m) value for the GRR-AMC peptide similar to that of the wild type, all other mutants exhibited drastically reduced k(cat) values. These results indicate a pivotal function of conserved residues Trp62, Leu75, and Ile79 in the NS2B cofactor in the structural activation of the dengue virus NS3 serine protease.     

Chimeric Sindbis viruses dependent on the NS3 protease of hepatitis C virus.

The hepatitis C virus (HCV) NS3 protease cleaves the viral polyprotein at specific sites to release the putative components of the HCV replication machinery. Selective inhibition of this enzyme is predicted to block virus replication, and NS3 is thus considered an attractive candidate for development of anti-HCV therapeutics. To set up a system for analysis of NS3 protease activity in cultured cells, we constructed a family of chimeric Sindbis viruses which carry sequences coding for NS3 and its activator, NS4A, in their genomes. HCV sequences were fused to the gene coding for the Sindbis virus structural polyprotein via an NS3-specific cleavage site, with the expectation that processing of the chimeric polyprotein, nucleocapsid assembly, and generation of viable viral particles would occur only upon NS3-dependent proteolysis. Indeed, the chimeric genomes encoding an active NS3 protease produced infectious viruses in mammalian cells, while those encoding NS3 inactivated by alanine substitution of the catalytic serine did not. However, in infected cells chimeric genomes recombined, splicing out HCV sequences and reverting to pseudo-wild-type Sindbis virus. To force retention of HCV sequences, we modified one of the initial chimeras by introducing a second NS3 cleavage site in the Sindbis virus portion of the recombinant polyprotein, anticipating that revertants not encoding an active NS3 protease would not be viable. The resulting chimera produced infectious viruses which replicated at a lower rate than the parental construct and displayed a marked temperature dependence in the formation of lysis plaques yet stably expressed NS3.     

Virus-specific cofactor requirement and chimeric hepatitis C virus/GB virus B nonstructural protein 3

GB virus B (GBV-B) is closely related to hepatitis C virus (HCV) and causes acute hepatitis in tamarins (Saguinus species), making it an attractive surrogate virus for in vivo testing of anti-HCV inhibitors in a small monkey model. It has been reported that the nonstructural protein 3 (NS3) serine protease of GBV-B shares similar substrate specificity with its counterpart in HCV. Authentic proteolytic processing of the HCV polyprotein junctions (NS4A/4B, NS4B/5A, and NS5A/5B) can be accomplished by the GBV-B NS3 protease in an HCV NS4A cofactor-independent fashion. We further characterized the protease activity of a full-length GBV-B NS3 protein and its cofactor requirement using in vitro-translated GBV-B substrates. Cleavages at the NS4A/4B and NS5A/5B junctions were readily detectable only in the presence of a cofactor peptide derived from the central region of GBV-B NS4A. Interestingly, the GBV-B substrates could also be cleaved by the HCV NS3 protease in an HCV NS4A cofactor-dependent manner, supporting the notion that HCV and GBV-B share similar NS3 protease specificity while retaining a virus-specific cofactor requirement. This finding of a strict virus-specific cofactor requirement is consistent with the lack of sequence homology in the NS4A cofactor regions of HCV and GBV-B. The minimum cofactor region that supported GBV-B protease activity was mapped to a central region of GBV-B NS4A (between amino acids Phe22 and Val36) which overlapped with the cofactor region of HCV. Alanine substitution analysis demonstrated that two amino acids, Val27 and Trp31, were essential for the cofactor activity, a finding reminiscent of the two critical residues in the HCV NS4A cofactor, Ile25 and Ile29. A model for the GBV-B NS3 protease domain and NS4A cofactor complex revealed that GBV-B might have developed a similar structural strategy in the activation and regulation of its NS3 protease activity. Finally, a chimeric HCV/GBV-B bifunctional NS3, consisting of an N-terminal HCV protease domain and a C-terminal GBV-B RNA helicase domain, was engineered. Both enzymatic activities were retained by the chimeric protein, which could lead to the development of a chimeric GBV-B virus that depends on HCV protease function.     

Hepatitis C NS3 protease: restoration of NS4A cofactor activity by N-biotinylation of mutated NS4A using synthetic peptides

The NS3 serine protase of Hepatitis C virus (HCV) requires NS4A protein as a cofactor for efficient cleavage at four sites in the nonstructural region. The cofactor activity has been mapped to the central hydrophobic region (aa 22-34) of this 54-amino-acid NS4A protein, and site-directed mutagenesis has identified alternating hydrophobic amino acids, particularly Ile25 and Ile29, as critically important. A double mutant of NS4A cofactor peptide, I25A/I29A, completely abolished the cofactor activity. We now report that the cofactor peptide activity in the I25A/I29A double mutant can be restored specifically by introducing a biotin-aminohexanoic acid fusion at the N-terminus. In addition, a similar N-terminal fusion of biotin-aminohexanoic acid with the wild-type 4A peptide significantly enhanced cofactor activity. Our data corroborate the crystal structure-based hypothesis of hydrophobic interaction between the N-terminus of NS4A and the N-terminal alpha(0) helix of NS3 protease.     

Hepatitis C Virus NS2 Protein Contributes to Virus Particle Assembly via Opposing Epistatic Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes

The hepatitis C virus NS2 protein has been recently implicated in virus particle assembly. To further understand the role of NS2 in this process, we conducted a reverse genetic analysis of NS2 in the context of a chimeric genotype 2a infectious cell culture system. Of 32 mutants tested, all were capable of RNA replication and 25 had moderate-to-severe defects in virus assembly. Through forward genetic selection for variants capable of virus spread, we identified second-site mutations in E1, E2, NS2, NS3, and NS4A that suppressed NS2 defects in assembly. Two suppressor mutations, E1 A78T and NS3 Q221L, were further characterized by additional genetic and biochemical experiments. Both mutations were shown to suppress other NS2 defects, often with mutual exclusivity. Thus, several NS2 mutants were enhanced by NS3 Q221L and inhibited by E1 A78T, while others were enhanced by E1 A78T and inhibited by NS3 Q221L. Furthermore, we show that the NS3 Q221L mutation lowers the affinity of native, full-length NS3-NS4A for functional RNA binding. These data reveal a complex network of interactions involving NS2 and other viral structural and nonstructural proteins during virus assembly.Hepatitis C virus (HCV) is a major cause of acute and chronic liver disease and contributes to the development of hepatocellular carcinoma. HCV is an enveloped, positive-strand RNA virus, the type member of the Hepacivirus genus in the family Flaviviridae (43). HCV exhibits high levels of sequence diversity that cluster into seven major genotypes and numerous subtypes (21).HCV genomes are 9.6 kb and encode a single long open reading frame of ∼3,011 codons (43). Translation of this genome produces a large polyprotein that is co- and posttranslationally processed by viral and host proteases into 10 distinct products. The N-terminal one-third of the polyprotein encodes the structural proteins, which are thought to compose the virus particle. These include an RNA-binding nucleocapsid protein, core (C), and two viral envelope glycoproteins, E1 and E2. E1 and E2 are type I membrane proteins that coordinately fold into a heterodimer complex (36). The remainder of the genome encodes the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B, which mediate the intracellular aspects of the viral life cycle. In addition, a small viroporin-like protein, p7, resides between the structural and NS genes.HCV encodes two proteases, the NS2-NS3 cysteine autoprotease and the NS3-NS4A serine protease. The only known substrate of the NS2-NS3 autoprotease is the NS2/3 junction. This enzyme is encoded by the C-terminal 121 amino acids (aa) of NS2, which forms a homodimer with twin composite active sites composed of two residues from one chain and one residue from the other (45). In addition, the serine protease domain of NS3 plays a noncatalytic role in stimulating NS2/3 cleavage (69). Upstream of the cysteine protease domain, the N-terminal hydrophobic region of NS2 mediates interaction with cellular membranes. While the membrane topology of NS2 is not yet fully known (67, 80), N-terminal cleavage by endoplasmic reticulum-resident signal peptidase and C-terminal cleavage by the cytosolic NS2-NS3 cysteine protease indicate that NS2 likely contains one or three transmembrane (TM) domains.The NS3-NS4A serine protease is encoded by the N-terminal domain of NS3 and is responsible for downstream cleavages at the NS3/4A, NS4A/B, NS4B/5A, and NS5A/B junctions. NS4A, a small (54-aa), membrane-anchored protein, acts as a cofactor for the serine protease activity by helping to complete the chymotrypsin-like fold of NS3 (14, 46). In addition to polyprotein processing, NS3-NS4A serine protease helps to dampen the innate antiviral response by cleaving cellular proteins involved in signal transduction (65).The C-terminal region of NS3 encodes an RNA helicase/NTPase activity that is essential for viral replication, although it is not yet clear which specific step(s) of the replication cycle requires this activity (29, 33). Interestingly, the NS3 serine protease and RNA helicase domains enhance each other''s activities, suggesting that proteolysis and RNA replication may be functionally coordinated (5, 6). In addition, NS4A helps to promote RNA-stimulated ATP hydrolysis by the NS3 helicase domain (4).In addition to their role in polyprotein processing, emerging evidence indicates that NS2 and NS3-NS4A participate in virus particle assembly (52). Prior work showed that NS2 is not essential for RNA replication of subgenomic replicons engineered to express NS3 through NS5B (44). The first evidence for an additional function of NS2 came from the construction of improved chimeric genotype 2a cDNA clones that replicated to high titers in cell culture (HCVcc). Pietschmann and colleagues showed that the Jc1 chimera containing a J6-JFH1 junction between the first and second putative TM domains of NS2 yielded higher-titer viruses than the original infectious J6/JFH chimera (41, 58). Furthermore, a number of adaptive mutations that improve virus production have been mapped to NS2 and NS3 (22, 23, 27, 53, 64, 68, 82). By using bicistronic constructs to express NS2 and NS3 independently of NS2/3 cleavage, two groups showed that full-length NS2, but not uncleaved NS2-NS3 or the NS2 cysteine protease active sites, was required for virus production (24, 25). Moreover, a limited number of mutations in NS2 were shown to inhibit virus assembly (24, 79, 83).Despite these observations, the role of NS2 in virus assembly remains unclear. We have therefore undertaken a genetic analysis to target conserved residues in NS2 for site-directed mutagenesis and identified a number of key residues that are important for virus assembly. Further analysis revealed that a network of genetic interactions among NS2, E1-E2, and NS3-NS4A helps to direct virus assembly. Finally, a suppressor mutation in NS3 was shown to influence functional RNA binding by the RNA helicase/ATPase.     

Specificity of the hepatitis C virus NS3 serine protease: effects of substitutions at the 3/4A, 4A/4B, 4B/5A, and 5A/5B cleavage sites on polyprotein processing.

Cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the hepatitis C virus polyprotein requires a viral serine protease activity residing in the N-terminal one-third of the NS3 protein. Sequence comparison of the residues flanking these cleavage sites reveals conserved features including an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position. In this study, we used site-directed mutagenesis to assess the importance of these and other residues for NS3 protease-dependent cleavages. Substitutions at the P7 to P2' positions of the 4A/4B site had varied effects on cleavage efficiency. Only Arg at the P1 position or Pro at P1' substantially blocked processing at this site. Leu was tolerated at the P1 position, whereas five other substitutions allowed various degrees of cleavage. Substitutions with positively charged or other hydrophilic residues at the P7, P3, P2, and P2' positions did not reduce cleavage efficiency. Five substitutions examined at the P6 position allowed complete cleavage, demonstrating that an acidic residue at this position is not essential. Parallel results were obtained with substrates containing an active NS3 protease domain in cis or when the protease domain was supplied in trans. Selected substitutions blocking or inhibiting cleavage at the 4A/4B site were also examined at the 3/4A, 4B/5A, and 5A/5B sites. For a given substitution, a site-dependent gradient in the degree of inhibition was observed, with a 3/4A site being least sensitive to mutagenesis, followed by the 4A/4B, 4B/5A, and 5A/5B sites. In most cases, mutations abolishing cleavage at one site did not affect processing at the other serine protease-dependent sites. However, mutations at the 3/4A site which inhibited cleavage also interfered with processing at the 4B/5A site. Finally, during the course of these studies an additional NS3 protease-dependent cleavage site has been identified in the NS4B region.     

GB virus B and hepatitis C virus NS3 serine proteases share substrate specificity.

GB virus B (GBV-B) is a recently discovered virus responsible for hepatitis in tamarins (Saguinus species). GBV-B belongs to the Flaviviridae family and is closely related to the human pathogen hepatitis C virus (HCV). Nonstructural protein 3 (NS3) of HCV has been shown to encompass a serine protease domain required for viral maturation. GBV-B and HCV share only about 30% of the amino acid sequence within the NS3 protease domain. The catalytic triad is conserved, and the residue Phe-154, presumed to be a crucial amino acid for determining the S1 specificity pocket of the HCV NS3 protease, is also conserved. We have expressed a synthetic gene encoding the GBV-B NS3 protease domain in Escherichia coli and have characterized the purified recombinant protein for its activity on HCV substrates. We have shown that the NS3 region of the GBV-B genome actually encodes a serine protease that, despite the low sequence homology, shares substrate specificity with the HCV NS3 protease.     

Synthesis of new acylsulfamoyl benzoxaboroles as potent inhibitors of HCV NS3 protease

HCV NS3/4A serine protease is essential for the replication of the HCV virus and has been a clinically validated target. A series of HCV NS3/4A protease inhibitors containing a novel acylsulfamoyl benzoxaborole moiety at the P1' region was synthesized and evaluated. The resulting P1-P3 and P2-P4 macrocyclic inhibitors exhibited sub-nanomolar potency in the enzymatic assay and low nanomolar activity in the cell-based replicon assay. The in vivo PK evaluations of selected compounds are also described.     

Modulation of hepatitis C virus NS3 protease and helicase activities through the interaction with NS4A

The hepatitis C virus nonstructural 3 protein (NS3) possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion. The serine protease activity is required for proteolytic processing at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B polyprotein cleavage sites. NS3 forms a complex with NS4A, a 54-residue polypeptide that was shown to act as an essential cofactor of the NS3 protease. We have expressed in Escherichia coli the NS3-NS4A precursor; cleavage at the junction between NS3 and NS4A occurs during expression in the bacteria cells, resulting in the formation of a soluble noncovalent complex with a sub-nanomolar dissociation constant. We have assessed the minimal ionic strength and detergent and glycerol concentrations required for maximal proteolytic activity and stability of the purified NS3-NS4A complex. Using a peptide substrate derived from the NS5A-NS5B junction, the catalytic efficiency (kcat/Km) of NS3-NS4A-associated protease under optimized conditions was 55 000 s-1 M-1, very similar to that measured with a recombinant complex purified from eukaryotic cells. Dissociation of the NS3-NS4A complex was found to be fully reversible. No helicase activity was exhibited by the purified NS3-NS4A complex, but NS3 was fully active as a helicase upon dissociation of NS4A. On the other hand, both basal and poly(U)-induced NTPase activity and ssRNA binding activity associated with the NS3-NS4A complex were very similar to those exhibited by NS3 alone. Therefore, NS4A appears to uncouple the ATPase/ssRNA binding and RNA unwinding activities associated with NS3.     

Mechanistic role of NS4A and substrate in the activation of HCV NS3 protease

Hepatitis C virus (HCV) NS3 protease is the key enzyme for its maturation. Three hypotheses have been advanced in the literature to demonstrate the mechanism of the activation of the HCV NS3 protease. A virus-encoded protein NS4A and substrate are proposed to be involved in the activation of the HCV NS3 protease. However, the three hypotheses are not completely consistent with one another. Multiple molecular dynamics simulations were performed on various NS3 protease systems: free NS3 protease, NS3/4A, NS3/inhibitor, and NS3/4A/inhibitor complexes, to further unravel the mechanism of the activation of the NS3 protease. Simulation results suggest that the binding of NS4A induces a classic serine protease conformation of the catalytic triad of the NS3 protease. NS4A rearranges the secondary structure of both the N-terminus and catalytic site of the NS3 protease, reduces the mobility of the global structure of the NS3 protease, especially the catalytic site, and provides a rigid and tight structure, except for the S1 pocket, for the binding and hydrolysis of substrates. The binding of substrate also contributes to the activation of the NS3 protease by an induced-fit of the classic serine protease catalytic triad. However, the global structure of the NS3 protease is still loose and highly flexible without stable secondary structural elements, such as helix α0 at the N-terminus and helix α1 and β-sheet E1-F1 at the catalytic site. The structure of the NS3 protease without NS4A is not suitable for the binding and hydrolysis of substrates.