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.     

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.     

Poliovirus-encoded 2C polypeptide specifically binds to the 3'-terminal sequences of viral negative-strand RNA.

The poliovirus-encoded, membrane-associated polypeptide 2C is believed to be required for initiation and elongation of RNA synthesis. We have expressed and purified recombinant, histidine-tagged 2C and examined its ability to bind to the first 100 nucleotides of the poliovirus 5' untranslated region of the positive strand and its complementary 3'-terminal negative-strand RNA sequences. Results presented here demonstrate that the 2C polypeptide specifically binds to the 3'-terminal sequences of poliovirus negative-strand RNA. Since this region is believed to form a stable cloverleaf structure, a number of mutations were constructed to examine which nucleotides and/or structures within the cloverleaf are essential for 2C binding. Binding of 2C to the 3'-terminal cloverleaf of the negative-strand RNA is greatly affected when the conserved sequence, UGUUUU, in stem a of the cloverleaf is altered. Mutational studies suggest that interaction of 2C with the 3'-terminal cloverleaf of negative-strand RNA is facilitated when the sequence UGUUUU is present in the context of a double-stranded structure. The implication of 2C binding to negative-strand RNA in viral replication is discussed.     

The serine protease domain of hepatitis C viral NS3 activates RNA helicase activity by promoting the binding of RNA substrate

Nonstructural (NS) protein 3 is a DEXH/D-box motor protein that is an essential component of the hepatitis C viral (HCV) replicative complex. The full-length NS3 protein contains two functional modules, both of which are essential in the life cycle of HCV: a serine protease domain at the N terminus and an ATPase/helicase domain (NS3hel) at the C terminus. Truncated NS3hel constructs have been studied extensively; the ATPase, nucleic acid binding, and helicase activities have been examined and NS3hel has been used as a target in the development of antivirals. However, a comprehensive comparison of NS3 and NS3hel activities has not been performed, so it remains unclear whether the protease domain plays a vital role in NS3 helicase function. Given that many DEXH/D-box proteins are activated upon interaction with cofactor proteins, it is important to establish if the protease domain acts as the cofactor for stimulating NS3 helicase function. Here we show that the protease domain greatly enhances both the direct and functional binding of RNA to NS3. Whereas electrostatics plays an important role in this process, there is a specific allosteric contribution from the interaction interface between NS3hel and the protease domain. Most importantly, we establish that the protease domain is required for RNA unwinding by NS3. Our results suggest that, in addition to its role in cleavage of host and viral proteins, the NS3 protease domain is essential for the process of viral RNA replication and, given its electrostatic contribution to RNA binding, it may also assist in packaging of the viral RNA.     

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.     

Structure/function analysis of an RNA aptamer for hepatitis C virus NS3 protease

RNA aptamers that bind specifically to hepatitis C virus (HCV) NS3 protease domain (DeltaNS3) were identified in previous studies. These aptamers, G9-I, -II, and -III, were isolated using an in vitro selection method and they share a common loop with the sequence 5'-GA(A/U)UGGGAC-3'. The aptamers are potent inhibitors of the NS3 protease in vitro and may have potential as anti-HCV compounds. G9-I has a 3-way stem-loop structure and was selected for further characterization using site-directed mutagenesis. Mutations or deletions in stem-loop II do not interfere with binding or inhibition of DeltaNS3, but mutations or deletions in stem I and stem-loop III destroy the G9-I active conformation and interfere with inhibition of NS3 protease. A 51 nt fragment of 74 nt G9-I was identified (DeltaNEO-III) as is the minimal fragment of G9-I that is an effective inhibitor of the NS3 protease. Tertiary interactions involving functionally important nucleotides were identified in the active structure of G9-I using nucleotide analog interference mapping (NAIM). Strong interferences were focused in the conserved loop involving stem-loop III and stem I. For example, analog-interference caused at A(+8) and C(+24)-G(-36) base pair implied an A-minor motif involving the intramolecular base triple A(+8).C(+24)-G(-36), which is further supported by mutagenesis. These results suggested the interaction of stem I and stem-loop III is essential for the function of G9-I aptamer.     

In trans interaction of hepatitis C virus helicase domains mediates protease activity critical for internal NS3 cleavage and cell transformation

Hepatitis C virus (HCV) internal non-structural protein 3 (NS3) cleavage can occur in trans in the presence of NS4A. In this study, we have further demonstrated a critical role of the helicase domain in the internal NS3 cleavage, different from HCV polyprotein processing which requires only the serine protease domain. The NTPase domain of NS3 helicase interacts with the RNA binding domain to facilitate internal NS3 cleavage. In addition, NS3 protease activity contributes to the transforming ability of the major internal cleavage product NS3(1-402). These findings imply important roles of the internal cleavage and protease activity of the NS3 protein in the pathogenesis of HCV.

Structured summary

MINT-7306465: NS3 (uniprotkb:P29846) physically interacts (MI:0915) with NS3 (uniprotkb:P29846) by anti tag coimmunoprecipitation (MI:0007).     

The hepatitis C viral NS3 protein is a processive DNA helicase with cofactor enhanced RNA unwinding

The RNA helicase/protease NS3 plays a central role in the RNA replication of hepatitis C virus (HCV), a cytoplasmic RNA virus that represents a major worldwide health problem. NS3 is, therefore, an important drug target in the effort to combat HCV. Most work has focused on the protease, rather than the helicase, activities of the enzyme. In order to further characterize NS3 helicase activity, we evaluated individual stages of duplex unwinding by NS3 alone and in complex with cofactor NS4A. Despite a putative replicative role in RNA unwinding, we found that NS3 alone is a surprisingly poor helicase on RNA, but that RNA activity is promoted by cofactor NS4A. In contrast, NS3 alone is a highly processive helicase on DNA. Phylogenetic analysis suggests that this robust DNA helicase activity is not vestigial and may have specifically evolved in HCV. Given that HCV has no replicative DNA intermediate, these findings suggest that NS3 may have the capacity to affect host DNA.     

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.     

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.     

Effect of interaction between hepatitis C virus NS5A and NS5B on hepatitis C virus RNA replication with the hepatitis C virus replicon

Hepatitis C virus (HCV) NS5A has been reported to be important for the establishment of replication by adaptive mutations or localization, although its role in viral replication remains unclear. It was previously reported that NS5A interacts with NS5B via two regions of NS5A in the isolate JK-1 and modulates the activity of NS5B RdRp (Y. Shirota et al., J. Biol. Chem., 277:11149-11155, 2002), but the biological significance of this interaction has not been determined. In this study, we addressed the effect of this interaction on HCV RNA replication with an HCV replicon system derived from the isolate M1LE (H. Kishine et al., Biochem. Biophys. Res. Commun., 293:993-999, 2002). We constructed three internal deletion mutants, M1LE/5Adel-1 and M1LE/5Adel-2, each encoding NS5A which cannot bind NS5B, and M1LE/5Adel-3, encoding NS5A that can bind NS5B. After transfection into Huh-7 cells, M1LE/5Adel-3 was replication competent, but both M1LE/5Adel-1 and M1LE/5Adel-2 were not. Next we prepared 20 alanine-substituted clustered mutants within both NS5B-binding regions and examined the effect of these mutants on HCV RNA replication. Only 5 of the 20 mutants were replication competent. Subsequently, we introduced a point mutation, S225P, a deletion of S229, or S232I into NS5A and prepared cured Huh-7 cells that were cured of RNA replication by alpha interferon. Finally, with these point mutations and cured cells, we established a highly improved replicon system. In this system, only the same five mutants were replication competent. These results strongly suggest that the interaction between NS5A and NS5B is critical for HCV RNA replication in the HCV replicon system.     

RNA unwinding activity of the hepatitis C virus NS3 helicase is modulated by the NS5B polymerase

Hepatitis C virus (HCV) infects over 170 million persons worldwide. It is the leading cause of liver disease in the U.S. and is responsible for most liver transplants. Current treatments for this infectious disease are inadequate; therefore, new therapies must be developed. Several labs have obtained evidence for a protein complex that involves many of the nonstructural (NS) proteins encoded by the virus. NS3, NS4A, NS4B, NS5A, and NS5B appear to interact structurally and functionally. In this study, we investigated the interaction between the helicase, NS3, and the RNA polymerase, NS5B. Pull-down experiments and surface plasmon resonance data indicate a direct interaction between NS3 and NS5B that is primarily mediated through the protease domain of NS3. This interaction reduces the basal ATPase activity of NS3. However, NS5B stimulates product formation in RNA unwinding experiments under conditions of excess nucleic acid substrate. When the concentrations of NS3 and NS5B are in excess of nucleic acid substrate, NS5B reduces the rate of NS3-catalyzed unwinding. Under pre-steady-state conditions, in which NS3 and substrate concentrations are similar, product formation increased in the presence of NS5B. The increase was consistent with 1:1 complex formed between the two proteins. A fluorescently labeled form of NS3 was used to investigate this interaction through fluorescence polarization binding assays. Results from this assay support interactions that include a 1:1 complex formed between NS3 and NS5B. The modulation of NS3 by NS5B suggests that these proteins may function together during replication of the HCV genome.     

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.     

Specific targeting of hepatitis C virus NS3 RNA helicase. Discovery of the potent and selective competitive nucleotide-mimicking inhibitor QU663

Hepatitis C virus (HCV) infection is an emerging global epidemic, and no effective cure is yet available. Interferon-alpha (INFalpha) and pegylated INFs, in combination or otherwise with ribavirin, have proven to be effective in no more than 50% of chronically infected patients. New and better therapeutic strategies are therefore needed. HCV nonstructural protein 3 (NS3) RNA helicase (h) is a promising target for developing new therapeutics. QU663 was discovered as a potent new selective inhibitor of the helicase reaction of HCV NS3 (K(i) = 0.75 microM), competing with the nucleic acid substrate without affecting ATPase function, even at high concentrations. QU663 is one of a new generation of small-molecule nucleotide-mimicking inhibitors which are potential anti-HCV agents. A thorough molecular modeling study was carried out to explain the molecular basis of NS3h inhibition by QU663. The resulting three-dimensional interaction model is discussed.     

Isolation of specific and high-affinity RNA aptamers against NS3 helicase domain of hepatitis C virus

Hepatitis C virus (HCV)-encoded nonstructural protein 3 (NS3) possesses protease, NTPase, and helicase activities, which are considered essential for viral proliferation. Thus, HCV NS3 is a good putative therapeutic target protein for the development of anti-HCV agents. In this study, we isolated specific RNA aptamers to the helicase domain of HCV NS3 from a combinatorial RNA library with 40-nucleotide random sequences using in vitro selection techniques. The isolated RNAs were observed to very avidly bind the HCV helicase with an apparent Kd of 990 pM in contrast to original pool RNAs with a Kd of >1 microM. These RNA ligands appear to impede binding of substrate RNA to the HCV helicase and can act as potent decoys to competitively inhibit helicase activity with high efficiency compared with poly(U) or tRNA. The minimal binding domain of the ligands was determined to evaluate the structural features of the isolated RNA molecules. Interestingly, part of binding motif of the RNA aptamers consists of similar secondary structure to the 3'-end of HCV negative-strand RNA. Moreover, intracellular NS3 protein can be specifically detected in situ with the RNA aptamers, indicating that the selected RNAs are very specific to the HCV NS3 helicase. Furthermore, the RNA aptamers partially inhibited RNA synthesis of HCV subgenomic replicon in Huh-7 hepatoma cell lines. These results suggest that the RNA aptamers selected in vitro could be useful not only as therapeutic and diagnostic agents of HCV infection but also as a powerful tool for the study of HCV helicase mechanism.     

The 5'-terminal region of the Aichi virus genome encodes cis-acting replication elements required for positive- and negative-strand RNA synthesis

Aichi virus is a member of the family Picornaviridae. It has already been shown that three stem-loop structures (SL-A, SL-B, and SL-C, from the 5' end) formed at the 5' end of the genome are critical elements for viral RNA replication. In this study, we further characterized the 5'-terminal cis-acting replication elements. We found that an additional structural element, a pseudoknot structure, is formed through base-pairing interaction between the loop segment of SL-B (nucleotides [nt] 57 to 60) and a sequence downstream of SL-C (nt 112 to 115) and showed that the formation of this pseudoknot is critical for viral RNA replication. Mapping of the 5'-terminal sequence of the Aichi virus genome required for RNA replication using a series of Aichi virus-encephalomyocarditis virus chimera replicons indicated that the 5'-end 115 nucleotides including the pseudoknot structure are the minimum requirement for RNA replication. Using the cell-free translation-replication system, we examined the abilities of viral RNAs with a lethal mutation in the 5'-terminal structural elements to synthesize negative- and positive-strand RNAs. The results showed that the formation of three stem-loops and the pseudoknot structure at the 5' end of the genome is required for negative-strand RNA synthesis. In addition, specific nucleotide sequences in the stem of SL-A or its complementary sequences at the 3' end of the negative-strand were shown to be critical for the initiation of positive-strand RNA synthesis but not for that of negative-strand synthesis. Thus, the 5' end of the Aichi virus genome encodes elements important for not only negative-strand synthesis but also positive-strand synthesis.     

An NS3 serine protease inhibitor abrogates replication of subgenomic hepatitis C virus RNA

The hepatitis C virus (HCV) NS3 protease is essential for polyprotein maturation and viral propagation, and it has been proposed as a suitable target for antiviral drug discovery. An N-terminal hexapeptide cleavage product of a dodecapeptide substrate identified as a weak competitive inhibitor of the NS3 protease activity was optimized to a potent and highly specific inhibitor of the enzyme. The effect of this potent NS3 protease inhibitor was evaluated on replication of subgenomic HCV RNA and compared with interferon-alpha (IFN-alpha), which is currently used in the treatment of HCV-infected patients. Treatment of replicon-containing cells with the NS3 protease inhibitor or IFN-alpha showed a dose-dependent decrease in subgenomic HCV RNA that reached undetectable levels following a 14-day treatment. Kinetic studies in the presence of either NS3 protease inhibitor or IFN-alpha also revealed similar profiles in HCV RNA decay with half-lives of 11 and 14 h, respectively. The finding that an antiviral specifically targeting the NS3 protease activity inhibits HCV RNA replication further validates the NS3 enzyme as a prime target for drug discovery and supports the development of NS3 protease inhibitors as a novel therapeutic approach for HCV infection.     

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.     

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.     

The hepatitis C virus NS3 protein: a model RNA helicase and potential drug target

The C-terminal portion of hepatitis C virus (HCV) nonstructural protein 3 (NS3) forms a three domain polypeptide that possesses the ability to travel along RNA or single-stranded DNA (ssDNA) in a 3' to 5' direction. Fueled byATP hydrolysis, this movement allows the protein to displace complementary strands of DNA or RNA and proteins bound to the nucleic acid. HCV helicase shares two domains common to other motor proteins, one of which appears to rotate upon ATP binding. Several models have been proposed to explain how this conformational change leads to protein movement and RNA unwinding, but no model presently explains all existing experimental data. Compounds recently reported to inhibit HCV helicase, which include numerous small molecules, RNA aptamers and antibodies, will be useful for elucidating the role of a helicase in positive-sense single-stranded RNA virus replication and might serve as templates for the design of novel antiviral drugs.