Unmasking the active helicase conformation of nonstructural protein 3 from hepatitis C virus

The nonstructural protein 3 (NS3) helicase/protease is an important component of the hepatitis C virus (HCV) replication complex. We hypothesized that a specific β-strand tethers the C terminus of the helicase domain to the protease domain, thereby maintaining HCV NS3 in a compact conformation that differs from the extended conformations observed for other Flaviviridae NS3 enzymes. To test this hypothesis, we removed the β-strand and explored the structural and functional attributes of the truncated NS3 protein (NS3ΔC7). Limited proteolysis, hydrodynamic, and kinetic measurements indicate that NS3ΔC7 adopts an extended conformation that contrasts with the compact form of the wild-type (WT) protein. The extended conformation of NS3ΔC7 allows the protein to quickly form functional complexes with RNA unwinding substrates. We also show that the unwinding activity of NS3ΔC7 is independent of the substrate 3'-overhang length, implying that a monomeric form of the protein promotes efficient unwinding. Our findings indicate that an open, extended conformation of NS3 is required for helicase activity and represents the biologically relevant conformation of the protein during viral replication.     

The NTPase/helicase domain of hepatitis C virus nonstructural protein 3 inhibits protein kinase C independently of its NTPase activity

Helicase motif VI is a short arginine-rich motif within the NTPase/helicase domain of the non-structural protein 3 (NS3) of the hepatitis C virus (HCV). We previously demonstrated that it reduces the catalytic activity and intracellular shuttling of protein kinase C (PKC). Thus, NS3-mediated PKC inhibition may be involved in HCV-associated hepatocellular carcinoma (HCC). In this study, we expand on our earlier results, which were obtained in experiments with short fragments of NS3, to show for the first time that the catalytically active, longer C-terminal NTPase/helicase of NS3 acts as a potent PKC inhibitor in vitro. PKC inhibition assays with the NTPase-inactive mutant NS3h-D1316A revealed a mixed type kinetic inhibition pattern. A broad range of 11 PKC isotypes was tested and all of the PKC isotypes were inhibited with IC50-values in the low micromolar range. These findings were confirmed for the wild-type NTPase/helicase domain in a non-radiometric PKC inhibition assay with ATP regeneration to rule out any effect of ATP hydrolysis caused by its NTPase activity. PKCα was inhibited with a micromolar IC₅₀ in this assay, which compares well with our result for NS3h-D1316A (IC₅₀ = 0.7 μM). In summary, these results confirm that catalytically active NS3 NTPase/helicase can act in an analogous manner to shorter NS3 fragments as a pseudosubstrate inhibitor of PKC.     

Isolation and characterization of RNA aptamers specific for the hepatitis C virus nonstructural protein 3 protease.

Nonstructural protein 3 (NS3) from hepatitis C virus (HCV) is a serine protease that provides an essential function in maturation of the virus by cleaving the nonstructural regions of the viral polyprotein. The goal of this work was to isolate RNA aptamers that bind specifically to the NS3 protease active site in the truncated polypeptide DeltaNS3. RNA aptamers were selected in vitro by systematic evolution of ligands by exponential enrichment (SELEX). The RNA pool for SELEX had a 30-nucleotide randomized core region. After nine selection cycles, a pool of DeltaNS3-specific RNA aptamers were obtained. This RNA pool included 45 clones that divided into three main classes (G9-I, II and III). These classes include the conserved sequence GA(A/U)UGGGAC. These aptamers bind to DeltaNS3 with a binding constant of about 10 nM and inhibit approximately 90% of the protease activity of DeltaNS3 and MBP-NS3 (full-length of NS3 fused with maltose binding protein). In addition, these aptamers inhibited approximately 70% of the MBP-NS3 protease activity in the presence of the NS4A peptide P41. G9-I aptamer appeared to be a noncompetitive inhibitor for DeltaNS3 with a Ki approximately 100 nM in the presence of P41. These results suggest that the pool of selected aptamers have potential as anti-HCV compounds. Mutational analysis of the G9-I aptamer demonstrated that the sequences required for protease inhibition are in stem I, stem III and loop III of the aptamer. These regions include the conserved sequence GA(A/U)UGGGAC.     

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.     

Identification of active-site residues in protease 3C of hepatitis A virus by site-directed mutagenesis.

Picornavirus 3C proteases (3Cpro) are cysteine proteases related by amino acid sequence to trypsin-like serine proteases. Comparisons of 3Cpro of hepatitis A virus (HAV) to those of other picornaviruses have resulted in prediction of active-site residues: histidine at position 44 (H44), aspartic acid (D98), and cysteine (C172). To test whether these residues are key members of a putative catalytic triad, oligonucleotide-directed mutagenesis was targeted to 3Cpro in the context of natural polypeptide precursor P3. Autocatalytic processing of the polyprotein containing wild-type or variant 3Cpro was tested by in vivo expression of vaccinia virus-HAV chimeras in an animal cell-T7 hybrid system and by in vitro translation of corresponding RNAs. Comparison with proteins present in HAV-infected cells showed that both expression systems mimicked authentic polyprotein processing. Individual substitutions of H44 by tyrosine and of C172 by glycine or serine resulted in complete loss of the virus-specific proteolytic cascade. In contrast, a P3 polyprotein in which D98 was substituted by asparagine underwent only slightly delayed processing, while an additional substitution of valine (V47) by glycine within putative protein 3A caused a more pronounced loss of processing. Therefore, apparently H44 and C172 are active-site constituents whereas D98 is not. The results, furthermore, suggest that substitution of amino acid residues distant from polyprotein cleavage sites may reduce proteolytic activity, presumably by altering substrate conformation.     

RNA-Stimulated ATPase and RNA helicase activities and RNA binding domain of hepatitis G virus nonstructural protein 3

Hepatitis G virus (HGV) nonstructural protein 3 (NS3) contains amino acid sequence motifs typical of ATPase and RNA helicase proteins. In order to examine the RNA helicase activity of the HGV NS3 protein, the NS3 region (amino acids 904 to 1580) was fused with maltose-binding protein (MBP), and the fusion protein was expressed in Escherichia coli and purified with amylose resin and anion-exchange chromatography. The purified MBP-HGV/NS3 protein possessed RNA-stimulated ATPase and RNA helicase activities. Characterization of the ATPase and RNA helicase activities of MBP-HGV/NS3 showed that the optimal reaction conditions were similar to those of other Flaviviridae viral NS3 proteins. However, the kinetic analysis of NTPase activity showed that the MBP-HGV/NS3 protein had several unique properties compared to the other Flaviviridae NS3 proteins. The HGV NS3 helicase unwinds RNA-RNA duplexes in a 3'-to-5' direction and can unwind RNA-DNA heteroduplexes and DNA-DNA duplexes as well. In a gel retardation assay, the MBP-HGV/NS3 helicase bound to RNA, RNA/DNA, and DNA duplexes with 5' and 3' overhangs but not to blunt-ended RNA duplexes. We also found that the conserved motif VI was important for RNA binding. Further deletion mapping showed that the RNA binding domain was located between residues 1383 and 1395, QRRGRTGRGRSGR. Our data showed that the MBP-HCV/NS3 protein also contains the RNA binding domain in the similar domain.     

A time-resolved, internally quenched fluorescence assay to characterize inhibition of hepatitis C virus nonstructural protein 3-4A protease at low enzyme concentrations

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) with its cofactor NS4A is a pivotal enzyme for the replication of HCV. Inhibition of NS3-4A protease activity has been validated as an antiviral target in clinical studies of inhibitors of the enzyme. We have developed a sensitive time-resolved fluorescence (TRF) assay capable of detecting very low NS3-4A concentrations. A depsipeptide substrate is used that contains a europium-cryptate moiety and an efficient quenching group, QSY-7. The TRF assay is at least 30-fold more sensitive than a fluorescence energy transfer (FRET) assay and allows evaluation of NS3 protease inhibitors in reactions catalyzed by low enzyme concentrations (30 pM). Use of low enzyme concentrations allows for accurate measurement of inhibition by compounds with subnanomolar inhibition constants. The inhibitory potency of the potent protease inhibitor, BILN-2061, is significantly greater than previously reported. The ability to accurately determine inhibitory potency in reactions with low picomolar concentrations of NS3-4A is crucially important to allow valid comparisons between potent inhibitors. Studies of the interaction of NS3 with its NS4A cofactor at low enzyme concentration also reveal that the protease activity is salt dependent. This salt dependence of the enzyme activity is not present when high enzyme concentrations are used in the FRET assay.     

Serpin mechanism of hepatitis C virus nonstructural 3 (NS3) protease inhibition: induced fit as a mechanism for narrow specificity

Hepatitis C virus (HCV) nonstructural 3 (NS3) serine protease disrupts important cellular antiviral signaling pathways and plays a pivotal role in the proteolytic maturation of the HCV polyprotein precursor. This recent discovery has fostered the search for NS3 protease inhibitors. However, the enzyme's unusual induced fit behavior and peculiar molecular architecture have imposed considerable obstacles to the development of small molecule inhibitors. In this article, we demonstrate that such unique induced fit behavior and the chymotrypsin-like catalytic domain can provide the structural plasticity necessary to generate protein-based inhibitors of the NS3 protease. We took advantage of the macromolecular scaffold of a Drosophila serpin, SP6, which intrinsically supports chymotrypsin-like enzyme inhibition, to design a novel class of potent and selective inhibitors. We show that altering the SP6 reactive site loop (RSL) resulted in the development of the first effective (K(i) of 34 nm) and selective serpin, SP6(EVC/S), directed at the NS3 protease. SP6(EVC/S) operates as a suicide substrate inhibitor, and its partitioning between the complex-forming and proteolytic pathways for the NS3 protease is HCV NS4A cofactor-dependent and -specific. Once bound to the protease active site, SP6(EVC/S) partitions with equal probability to undergo proteolysis by NS3 at the C-terminal site of the engineered RSL, (P(6))Glu-Ile-(P(4))Val-Met-Thr-(P(1))Cys- downward arrow -(P(1)')Ser, or to form a covalent acyl-enzyme complex characteristic of cognate protease-serpin pairs. Our results also reveal a novel cofactor-induced serpin mechanism of enzyme inhibition that could be explored for developing effective and selective inhibitors of other important induced fit viral proteases of the Flaviviridae family such as the West Nile virus NS3 endoprotease.     

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.     

The nonstructural protein 3 protease/helicase requires an intact protease domain to unwind duplex RNA efficiently

The nonstructural 3 (NS3) protein encoded by the hepatitis C virus possesses both an N-terminal serine protease activity and a C-terminal 3'-5' helicase activity. This study examines the effects of the protease on the helicase by comparing the enzymatic properties of the full-length NS3 protein with truncated versions in which the protease is either deleted or replaced by a polyhistidine (His tag) or a glutathione S-transferase fusion protein (GST tag). When the NS3 protein lacks the protease domain it unwinds RNA more slowly and does not unwind RNA in the presence of excess nucleic acid that acts as an enzyme trap. Some but not all of the RNA helicase activity can be restored by adding a His tag or GST tag to the N terminus of the truncated helicase, suggesting that the effects of the protease are both specific and nonspecific. Similar but smaller effects are also seen in DNA helicase and translocation assays. While translocating on RNA (or DNA) the full-length protein hydrolyzes ATP more slowly than the truncated protein, suggesting that the protease allows for more efficient ATP usage. Binding assays reveal that the full-length protein assembles on single-stranded DNA as a higher order oligomer than the truncated fragment, and the binding appears to be more cooperative. The data suggest that hepatitis C virus RNA helicase, and therefore viral replication, could be influenced by the rotations of the protease domain which likely occur during polyprotein processing.     

Protein kinase C recognizes the protein kinase A-binding motif of nonstructural protein 3 of hepatitis C virus.

The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) inhibits the nuclear transport and the enzymatic activity of the catalytic subunit of protein kinase A. This inhibition is mediated by an arginine-rich domain localized between amino acids 1487-1500 of the HCV polyprotein. The data presented here indicate that the arginine-rich domain, when embedded in recombinant fragments of NS3, interacts with the catalytic site of protein kinase C (PKC) and inhibits the phosphorylation mediated by this enzyme in vitro and in vivo. Furthermore, a direct binding of PKC to the NS3 fragments leads to an inhibition of the free shuttling of the kinase between the cytoplasm and the particulate fraction. In contrast, a peptide corresponding to the arginine-rich domain (HCV (1487-1500)), despite also being a PKC inhibitor, did not influence the PKC shuttling process and was transported to the particulate fraction by the translocating kinase upon activation with tetradecanoylphorbol-13-acetate. Using the tetradecanoylphorbol-13-acetate -stimulated respiratory burst of NS3-introduced neutrophils as a model system, we could demonstrate that NS3 is able to block PKC-mediated functions within intact cells. Our data support the possibility that NS3 disrupts the PKC-mediated signal transduction.     

Partial nonstructural 3 region analysis of hepatitis C virus genotype 3a

Molecular Biology Reports - The hepatitis C virus (HCV) is a major cause of illness around the world. HCV genotype 3a is the most prevalent genotype in Thailand. Direct-acting antiviral (DAA) drugs...     

Conformational stability of hepatitis C virus NS3 protease

The hepatitis C virus NS3 protease is responsible for the processing of the nonstructural region of viral precursor polyprotein in infected hepatic cells. NS3 has been considered a target for drug discovery for a long time. NS3 is a zinc-dependent serine protease. However, the zinc ion is not involved in the catalytic mechanism, because it is bound far away from the active site. Thus, zinc is essential for the structural integrity of the protein and it is considered to have a structural role. The first thermodynamic study on the conformational equilibrium and stability of NS3 and the effect of zinc on such equilibrium is presented here. In agreement with a previous calorimetric study on the binding of zinc to NS3, the global unfolding heat capacity is dominated by the zinc dissociation step, suggesting that the binding of zinc induces a significant structural rearrangement of the protein. In addition, contrary to other homologous zinc-dependent proteases, the zinc-free NS3 protease is not completely unstructured. It is apparent that the conformational landscape of hepatitis C virus NS3 protease is fairly complex due to its intrinsic plasticity, and to the interactions with its different effectors (zinc and the accessory viral protein NS4A) and their modulation of the population of the different conformational states.     

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.     

Structure-based mutational analysis of the hepatitis C virus NS3 helicase

The carboxyl terminus of the hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses ATP-dependent RNA helicase activity. Based on the conserved sequence motifs and the crystal structures of the helicase domain, 17 mutants of the HCV NS3 helicase were generated. The ATP hydrolysis, RNA binding, and RNA unwinding activities of the mutant proteins were examined in vitro to determine the functional role of the mutated residues. The data revealed that Lys-210 in the Walker A motif and Asp-290, Glu-291, and His-293 in the Walker B motif were crucial to ATPase activity and that Thr-322 and Thr-324 in motif III and Arg-461 in motif VI significantly influenced ATPase activity. When the pairing between His-293 and Gln-460, referred to as gatekeepers, was replaced with the Asp-293/His-460 pair, which makes the NS3 helicase more like the DEAD helicase subgroup, ATPase activity was not restored. It thus indicated that the whole microenvironment surrounding the gatekeepers, rather than the residues per se, was important to the enzymatic activities. Arg-461 and Trp-501 are important residues for RNA binding, while Val-432 may only play a coadjutant role. The data demonstrated that RNA helicase activity was possibly abolished by the loss of ATPase activity or by reduced RNA binding activity. Nevertheless, a low threshold level of ATPase activity was found sufficient for helicase activity. Results in this study provide a valuable reference for efforts under way to develop anti-HCV therapeutic drugs targeting NS3.     

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

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

Immunodominant CD4+ T-cell epitope within nonstructural protein 3 in acute hepatitis C virus infection.

In acute hepatitis C virus infection, 50 to 70% of patients develop chronic disease. Considering the low rate of spontaneous viral clearance during chronic hepatitis C infection, the first few months of interaction between the patient's immune system and the viral population seem to be crucial in determining the outcome of infection. We previously reported the association between a strong and sustained CD4+ T-cell response to nonstructural protein 3 (NS3) of the hepatitis C virus and a self-limited course of acute hepatitis C infection. In this study, we identify an immunodominant CD4+ T-cell epitope (amino acids 1248 to 1261) that was recognized by the majority (14 of 23) of NS3-specific CD4+ T-cell clones from four of five patients with acute hepatitis C infection. This epitope can be presented to CD4+ T cells by HLA-DR4, -DR11, -DR12, -DR13, and -DR16. HLA-binding studies revealed a high binding affinity for 10 of 13 common HLA-DR alleles. Two additional CD4+ T-cell epitopes, amino acids 1388 to 1407 and amino acids 1450 to 1469, showed a very narrow pattern of binding to individual HLA-DR alleles. Our data suggest that the NS3-specific CD4+ T-cell response in acute hepatitis C infection is dominated by a single, promiscuous peptide epitope which could become a promising candidate for the development of a CD4+ T-cell vaccine.     

The hepatitis C virus NS2/3 protease

The hepatitis C virus NS2/3 protein is a highly hydrophobic protease responsible for the cleavage of the viral polypeptide between non-structural proteins NS2 and NS3. However, many aspects of the NS2/3 protease's role in the viral life cycle and mechanism of action remain unknown. Based on the recently elucidated crystal structure of NS2, NS2/3 has been proposed to function as a cysteine protease despite its lack of sequence homology to proteases of known function. In addition, although shown to be required for HCV genome replication and persistent infection in a chimpanzee, the role of NS2/3 cleavage in the viral life cycle has not yet been fully investigated. However, several recent studies are beginning to clarify possible roles of the cleaved NS2 protein in modulation of host cell gene expression and apoptosis.     

丙型肝炎病毒非结构蛋白NS2的研究进展

丙型肝炎病毒（hepatitis C virus,HCV）是一种严重危害人类健康的病原体,全球感染率约3％,中国普通人群抗HCV阳性率约3．2％。然而,到目前为止,HCV感染还没有有效的治疗方法。近年的研究发现,HCV非结构蛋白NS2在HCV感染中扮演着重要角色,具有许多重要功能。NS2可以在HCV病毒的包装过程中发挥其功能,还可调节宿主细胞的基因表达及凋亡过程。此外,NS2蛋自还可参与NS5A磷酸蛋白的高度磷酸化修饰过程及为感染性HCV病毒粒子产生所必需。本文综述近几年来关于NS2蛋白的研究进展。