Full-length Nonstructural Protein 3 Research Articles

Hepatitis C virus nonstructural protein NS3 unfolds viral G-quadruplex RNA structures

Hepatitis C virus (HCV) is a major cause of liver-related diseases and hepatocellular carcinoma. The helicase domain of one of the nonstructural proteins of HCV, NS3 (nonstructural protein 3), is essential for viral replication; however, its specific biological role is still under investigation. Here, we set out to determine the interaction between a purified recombinant full length NS3 and synthetic guanine-rich substrates that represent the conserved G-quadruplex (G4)-forming sequences in the HCV-positive and HCV-negative strands. We performed fluorescence anisotropy binding, G4 reporter duplex unwinding, and G4RNA trapping assays to determine the binding and G4 unfolding activity of NS3. Our data suggest that NS3 can unfold the conserved G4 structures present within the genome and the negative strand of HCV. Additionally, we found the activity of NS3 on a G4RNA was reduced significantly in the presence of a G4 ligand. The ability of NS3 to unfold HCV G4RNA could imply a novel biological role of the viral helicase in replication.

Screening and identification of bioactive compounds from citrus against non-structural protein 3 protease of hepatitis C virus genotype 3a by fluorescence resonance energy transfer assay and mass spectrometry.

BACKGROUNDHepatitis C virus genotype 3a (HCV G3a) is highly prevalent in Pakistan. Due to the elevated cost of available Food and Drug Administration-approved drugs against HCV, medicinal natural products of potent antiviral activity should be screened for the cost-effective treatment of the disease. Furthermore, from natural products, active compounds against vital HCV proteins like non-structural protein 3 (NS3) protease could be identified to prevent viral proliferation in the host.AIMTo develop cost-effective HCV genotype 3a NS3 protease inhibitors from citrus fruit extracts.METHODSFull-length NS3 without co-factor non-structural protein 4A (NS4A) and codon optimized NS3 protease in fusion with NS4A were expressed in Escherichia coli. The expressed protein was purified by metal ion affinity chromatography and gel filtration. Citrus fruit extracts were screened using fluorescence resonance energy transfer (FRET) assay against the protease and polyphenols were identified as potential inhibitors using electrospray ionization-mass spectrometry (MS)/MS technique. Among different polyphenols, highly potent compounds were screened using molecular modeling approaches and consequently the most active compound was further evaluated against HCV NS4A-NS3 protease domain using FRET assay.RESULTSNS4A fused with NS3 protease domain gene was overexpressed and the purified protein yield was high in comparison to the lower yield of the full-length NS3 protein. Furthermore, in enzyme kinetic studies, NS4A fused with NS3 protease proved to be functionally active compared to full-length NS3. So it was concluded that co-factor NS4A fusion is essential for the purification of functionally active protease. FRET assay was developed and validated by the half maximal inhibitory concentration (IC50) values of commercially available inhibitors. Screening of citrus fruit extracts against the native purified fused NS4A-NS3 protease domain showed that the grapefruit mesocarp extract exhibits the highest percentage inhibition 91% of protease activity. Among the compounds identified by LCMS analysis, hesperidin showed strong binding affinity with the protease catalytic triad having S-score value of -10.98.CONCLUSIONFused NS4A-NS3 protease is functionally more active, which is effectively inhibited by hesperidin from the grapefruit mesocarp extract with an IC50 value of 23.32 µmol/L.

Crystal structures of full length DENV4 NS2B-NS3 reveal the dynamic interaction between NS2B and NS3

Flavivirus is a genus of the Flaviviridae family which includes significant emerging and re-emerging human disease-causing arboviruses such as dengue and Zika viruses. Flaviviral non-structural protein 3 (NS3) protease-helicase plays essential roles in viral replication and is an attractive antiviral target. A construct which connects the cytoplasmic cofactor region of NS2B and NS3 protease with an artificial glycine-rich flexible linker has been widely used for structural, biochemical and drug-screening studies. The effect of this linker on the dynamics and enzymatic activity of the protease has been studied by several biochemical and NMR methods but the findings remained inconclusive. Here, we designed and carried out a comparative study of constructs of NS2B cofactor joined to the full length DENV4 NS3 in three different ways, namely bNS2B47NS3 (bivalent), eNS2B47NS3(enzymatically cleavable) and gNS2B47NS3 (glycine-rich linker). We report the crystal structures of linked and unlinked NS2B47-NS3 constructs in their free state and in complex with bovine pancreatic trypsin inhibitor (BPTI). These structures demonstrate that the NS2B cofactor predominantly adopts a closed conformation in complex with full-length NS3. The glycine-rich linker between NS2B and NS3 may promote the open conformation which interferes with protease activity. This negative impact on the enzyme structure and function is restricted to the protease activity as the ATPase activity is not affected in vitro.

Selection of suitable detergents for obtaining an active dengue protease in its natural form from E. coli

Dengue protease is a two-component enzyme and is an important drug target against dengue virus. The protease activity and protein stability of dengue nonstructural protein 3 (NS3) require a co-factor region from a four-span membrane protein NS2B. A natural form of dengue protease containing full-length NS2B and NS3 protease domain NS2BFL-NS3pro will be useful for dengue drug discovery. In current study, detergents that can be used for protease purification were tested. Using a water soluble protease construct, 39 detergents were selected for both NS2B and NS2BFL-NS3pro purification. The results showed that 18 detergents were able to sustain the activity of the natural dengue protease and 11 detergents could be used for NS2B purification. The results obtained in this study will be useful for biochemical and biophysical studies on dengue protease.

Melting of Duplex DNA in the Absence of ATP by the NS3 Helicase Domain through Specific Interaction with a Single-Strand/Double-Strand Junction

Helicases unwind double-stranded nucleic acids, remove secondary structures from single-stranded nucleic acids, and remove proteins bound to nucleic acids. For many helicases, the mechanisms for these different functions share the ability to translocate with a directional bias as a result of ATP binding and hydrolysis. Nonstructural protein 3 (NS3) is an essential enzyme expressed by the hepatitis C virus (HCV) and is known to catalyze the unwinding of both DNA and RNA substrates in a 3'-to-5' direction. We investigated the role of nucleic acid binding in the unwinding mechanism by examining ATP-independent unwinding. We observed that even in the absence of ATP, the NS3 helicase domain (NS3h) unwound duplexes only when they contained a 3'-tail (i.e., 3'-to-5' directionality). Blunt-ended duplexes and 5'-tailed duplexes were not melted even in the presence of a large excess concentration of the protein. NS3h was found to diffuse rapidly along single-stranded DNA at a rate of 30 nucleotides(2) s(-1). Upon encountering an appropriate single-strand/double-strand (ss/ds) junction, NS3h slowly melted the duplex under conditions with an excess protein concentration relative to DNA concentration. When a biotin-streptavidin block was placed into the ssDNA region, no melting of DNA was observed, suggesting that NS3h must diffuse along the ssDNA, and that the streptavidin blocked the diffusion. We conclude that the specific interaction between NS3h and the ss/dsDNA junction, coupled with diffusion, allows binding energy to melt duplex DNA with a directional bias. Alternatively, we found that the full-length NS3 protein did not exhibit strict directionality and was dependent on duplex DNA length. NS3 was able to unwind the duplex even in the presence of the biotin-streptavidin block. We propose a noncanonical model of unwinding for NS3 in which the enzyme binds directly to the duplex via protein-protein interactions to melt the substrate.

Modulation of enzymatic activity of dengue virus nonstructural protein NS3 nucleoside triphosphatase/helicase by poly(U)

The nonstructural protein 3 (NS3) appears to be the most promising target for anti-flavivirus therapy because of its multiple enzymatic activities that are indispensable for virus replication. NS3 of dengue virus type 2 (DEN2) is composed of two domains, a serine protease in the N-terminal domain (NS3pro) and RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicase at the C-terminus (NS3h). NS3 plays an important role in viral replication and the coordinated regulation of all the catalytic activities in the full-length NS3 protein. In this study, a plasmid harboring the NS3 helicase domain (NS3h) was constructed by PCR. The 56.5 kDa NS3h protein was purified by metal-chelate affinity chromatography followed by renaturation, mediated by artificial chaperone-assisted refolding, which yielded the active helicase. NTPase activity was assayed with Malachite Green. The NTPase activity in the presence of poly(U) showed a higher turnover number (kcat) and a lower Km value than without poly(U). The activity increased approximately fourfold in the presence of polynucleotides. This indicates that NTPase activity of dengue NS3 can be stimulated by polynucleotides. A helicase assay based on internal fluorescence quenching was conducted using short internally quenched DNA oligonucleotides as substrates. Significant fluorescence signaling increase was observed in the absence of polynucleotides such as poly(U). No unwinding activity was observed with addition of poly(U). The approach we describe here is useful for the further characterization of substrate specificity and for the design of high-throughput assays aimed at discovery of inhibitors against NS3 NTPase/helicase activities.

Active Participation of Cellular Chaperone Hsp90 in Regulating the Function of Rotavirus Nonstructural Protein 3 (NSP3)

Heat shock protein 90 (Hsp90) has been reported to positively regulate rotavirus replication by modulating virus induced PI3K/Akt and NFκB activation. Here, we report the active association of Hsp90 in the folding and stabilization of rotavirus nonstructural protein 3 (NSP3). In pCD-NSP3-transfected cells, treatment with Hsp90 inhibitor (17-N,N-dimethylethylenediamine-geldanamycin (17DMAG)) resulted in the proteasomal degradation of NSP3. Sequence analysis and deletion mutations revealed that the region spanning amino acids 225-258 within the C-terminal eIF4G-binding domain of NSP3 is a putative Hsp90 binding region. Co-immunoprecipitation and mammalian two-hybrid experiments revealed direct interaction of the C-terminal 12-kDa domain of Hsp90 (C90) with residues 225-258 of NSP3. NSP3-Hsp90 interaction is important for the formation of functionally active mature NSP3, because full-length NSP3 in the presence of the Hsp90 inhibitor or NSP3 lacking the amino acid 225-258 region did not show NSP3 dimers following in vitro coupled transcription-translation followed by chase. Disruption of residues 225-258 within NSP3 also resulted in poor RNA binding and eIF4G binding activity. In addition, inhibition of Hsp90 by 17DMAG resulted in reduced nuclear translocation of poly(A)-binding protein and translation of viral proteins. These results highlight the crucial role of Hsp90 chaperone in the regulation of assembly and functionality of a viral protein during the virus replication and propagation in host cells.

Visualizing ATP-Dependent RNA Translocation by the NS3 Helicase from HCV

The structural mechanism by which nonstructural protein 3 (NS3) from the hepatitis C virus (HCV) translocates along RNA is currently unknown. HCV NS3 is an ATP-dependent motor protein essential for viral replication and a member of the superfamily 2 helicases. Crystallographic analysis using a labeled RNA oligonucleotide allowed us to unambiguously track the positional changes of RNA bound to full-length HCV NS3 during two discrete steps of the ATP hydrolytic cycle. The crystal structures of HCV NS3, NS3 bound to bromine-labeled RNA, and a tertiary complex of NS3 bound to labeled RNA and a non-hydrolyzable ATP analog provide a direct view of how large domain movements resulting from ATP binding and hydrolysis allow the enzyme to translocate along the phosphodiester backbone. While directional translocation of HCV NS3 by a single base pair per ATP hydrolyzed is observed, the 3′ end of the RNA does not shift register with respect to a conserved tryptophan residue, supporting a “spring-loading” mechanism that leads to larger steps by the enzyme as it moves along a nucleic acid substrate.

High Affinity Human Antibody Fragments to Dengue Virus Non-Structural Protein 3

BackgroundThe enzyme activities catalysed by flavivirus non-structural protein 3 (NS3) are essential for virus replication. They are distributed between the N-terminal protease domain in the first one-third and the C-terminal ATPase/helicase and nucleoside 5′ triphosphatase domain which forms the remainder of the 618-aa long protein.Methodology/Principal FindingsIn this study, dengue full-length NS3 protein with residues 49 to 66 of NS2B covalently attached via a flexible linker, was used as bait in biopanning with a naïve human Fab phage-display library. Using a range of truncated constructs spanning the NS2B cofactor region and the full-length NS3, 10 unique Fab were identified and characterized. Of these, monoclonal Fab 3F8 was shown to bind α3″ (residues 526 through 531) within subdomain III of the helicase domain. The antibody inhibits the ATPase and helicase activites of NS3 in biochemical assays and reduces DENV replication in HEK293 cells that were previously transfected with Fab 3F8 compared with mock transfected cells.Conclusions/SignificanceAntibodies such as 3F8 are valuable tools for studying the molecular mechanisms of flaviviral replication and for the monospecific detection of replicating dengue virus in vivo.

Hepatitis C Virus NS3 Protease Is Activated by Low Concentrations of Protease Inhibitors

The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) is a bifunctional enzyme with a protease and a helicase functionality located in each of the two domains of the single peptide chain. There is little experimental evidence for a functional role of this unexpected arrangement since artificial single domain forms of both enzymes are catalytically competent. We have observed that low concentrations of certain protease inhibitors activate the protease of full-length NS3 from HCV genotype 1a with up to 100%, depending on the preincubation time and the inhibitor used. The activation was reduced, but not eliminated, by increased ionic strength, lowered glycerol concentration, or lowered pH. In all cases, it was at the expense of a significant loss of activity. Activation was not seen with the artificial protease domain of genotype 1b NS3 fused with a fragment of the NS4A cofactor. This truncated and covalently modified enzyme form was much less active and exhibited fundamentally different catalytic properties to the full-length NS3 protease without the fused cofactor. The most plausible explanation for the activation was found to involve a slow transition between two enzyme conformations, which differed in their catalytic ability and affinity for inhibitors. Equations derived based on this assumption resulted in better fits to the experimental data than the equation for simple competitive inhibition. The mechanism may involve an inhibitor-induced stabilization of the helicase domain in a conformation that enhances the protease activity, or an improved alignment of the catalytic triad in the protease. The proposed mnemonic mechanism and derived equations are viable for both these explanations and can serve as a basic framework for future studies of enzymes activated by inhibitors or other ligands.

Crystal Structure of a Novel Conformational State of the Flavivirus NS3 Protein: Implications for Polyprotein Processing and Viral Replication

The flavivirus genome comprises a single strand of positive-sense RNA, which is translated into a polyprotein and cleaved by a combination of viral and host proteases to yield functional proteins. One of these, nonstructural protein 3 (NS3), is an enzyme with both serine protease and NTPase/helicase activities. NS3 plays a central role in the flavivirus life cycle: the NS3 N-terminal serine protease together with its essential cofactor NS2B is involved in the processing of the polyprotein, whereas the NS3 C-terminal NTPase/helicase is responsible for ATP-dependent RNA strand separation during replication. An unresolved question remains regarding why NS3 appears to encode two apparently disconnected functionalities within one protein. Here we report the 2.75-A-resolution crystal structure of full-length Murray Valley encephalitis virus NS3 fused with the protease activation peptide of NS2B. The biochemical characterization of this construct suggests that the protease has little influence on the helicase activity and vice versa. This finding is in agreement with the structural data, revealing a single protein with two essentially segregated globular domains. Comparison of the structure with that of dengue virus type 4 NS2B-NS3 reveals a relative orientation of the two domains that is radically different between the two structures. Our analysis suggests that the relative domain-domain orientation in NS3 is highly variable and dictated by a flexible interdomain linker. The possible implications of this conformational flexibility for the function of NS3 are discussed.

NS3 Helicase from the Hepatitis C Virus Can Function as a Monomer or Oligomer Depending on Enzyme and Substrate Concentrations

Hepatitis C virus NS3 helicase can unwind double-stranded DNA and RNA and has been proposed to form oligomeric structures. Here we examine the DNA unwinding activity of monomeric NS3. Oligomerization was measured by preparing a fluorescently labeled form of NS3, which was titrated with unlabeled NS3, resulting in a hyperbolic increase in fluorescence anisotropy and providing an apparent equilibrium dissociation constant of 236 nm. To evaluate the DNA binding activity of individual subunits within NS3 oligomers, two oligonucleotides were labeled with fluorescent donor or acceptor molecules and then titrated with NS3. Upon the addition of increasing concentrations of NS3, fluorescence energy transfer was observed, which reached a plateau at a 1:1 ratio of NS3 to oligonucleotides, indicating that each subunit within the oligomeric form of NS3 binds to DNA. DNA unwinding was measured under multiple turnover conditions with increasing concentrations of NS3; however, no increase in specific activity was observed, even at enzyme concentrations greater than the apparent dissociation constant for oligomerization. An ATPase-deficient form of NS3, NS3(D290A), was prepared to explore the functional consequences of oligomerization. Under single turnover conditions in the presence of excess concentration of NS3 relative to DNA, NS3(D290A) exhibited a dominant negative effect. However, under multiple turnover conditions in which DNA concentration was in excess to enzyme concentration, NS3(D290A) did not exhibit a dominant negative effect. Taken together, these data support a model in which monomeric forms of NS3 are active. Oligomerization of NS3 occurs, but subunits can function independently or cooperatively, dependent upon the relative concentration of the DNA.

Characterization of classical swine fever virus (CSFV) nonstructural protein 3 (NS3) helicase activity and its modulation by CSFV RNA-dependent RNA polymerase

Classical swine fever virus (CSFV) nonstructural protein 3 (NS3) is believed to possess three enzyme activities that are likely to be essential for virus replication: a serine protease located in the N-terminus and NTPase as well as helicase activities located in the C-terminus. In this report, we expressed NS3 helicase domain (NS3h) in E. coli and characterized its helicase activity. The NS3h helicase activity was dependent on the presence of NTP and divalent cations, with a preference for ATP and Mn 2+, and required the substrates possessing a 3′ un-base-paired region on the RNA template strand. The NS3h helicase activity was proportional to increasing lengths of the 3′ un-base-paired regions up to 16 nucleotides of the RNA substrates. We also investigated the modulation of NS3 NTPase/helicase activities by NS3 protease domain and NS5B, an RNA-dependent RNA polymerase (RdRp). Our data showed that the NS3 protease domain enhanced the helicase activity of NS3 but had no effect on its NTPase activity. For the truncated NS3 (helicase domain, NS3h), both NTPase and helicase activities were up-regulated by NS5B. However, for the full-length NS3 (NS3FL), the NTPase activity, but not the helicase activity, was stimulated by NS5B. Maltose-binding protein (MBP) pull-down as well as enzyme-linked immunosorbent assays confirmed the specific interaction between NS3 and NS5B.

Establishing a Mechanistic Basis for the Large Kinetic Steps of the NS3 Helicase

The NS3 helicase from hepatitis C virus is a prototypical DEx(H/D) RNA helicase. NS3 has been shown to unwind RNA in a discontinuous manner, pausing after long apparent steps of unwinding. We systematically examined the effects of duplex stability and ionic conditions on the periodicity of the NS3 unwinding cycle. The kinetic step size for NS3 unwinding was examined on diverse substrate sequences. The kinetic step size (16 bp/step) was found to be independent of RNA duplex stability and composition, but it exhibited strong dependence on monovalent salt concentration, decreasing to approximately 11 bp/step at low [NaCl]. We addressed this behavior by analyzing the oligomeric state of NS3 at various salt concentrations. Whereas only NS3 oligomers are capable of processive unwinding, we found that monomeric NS3 is an active helicase that unwinds with low processivity. We demonstrate that low salt conditions enhance unwinding by monomeric NS3, which is likely to account for the reduction in apparent step size under low salt conditions. Based on results reported here, as well as available structural and single molecule data, we present an unwinding mechanism that addresses the apparent periodicity of NS3 unwinding, the magnitude of the step size, and that integrates the various stepwise motions observed for NS3. We propose that the large kinetic step size of NS3 unwinding reflects a delayed, periodic release of the separated RNA product strand from a secondary binding site that is located in the NTPase domain (Domain II) of NS3. These findings suggest that the mechanism of product release represents an important and unexplored feature of helicase mechanism.

Crystal structure of the NS3 protease-helicase from dengue virus

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

Crystal Structure of the NS3 Protease-Helicase from Dengue Virus

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

Identification and characterization of the NTPase activity of classical swine fever virus (CSFV) nonstructural protein 3 (NS3) expressed in bacteria

The nonstructural protein 3 (NS3) of members of the family Flaviviridae possesses multiple enzyme activities that are likely to be essential for viral replication. Here, we cloned and expressed full-length CSFV NS3 protein (NS3FL) and its N-terminal truncated version (ntNS3) in E. coli. NTPase activities of the purified NS3FL and ntNS3 proteins and their reaction conditions were investigated. The results showed that CSFV NS3FL and ntNS3 proteins contained a specific polynucleotide-stimulated NTPase acitivity. Characterization of ntNS3 NTPase activity showed that optimal reaction conditions with respect to pH, MgCl2 and monovalent cations were similar to those of bovine viral diarrhea virus (BVDV) and hepatitis C virus (HCV). Site-directed mutagenesis analysis demonstrated that the GxGK(232)T to GxGAT mutation in the conserved motif I abolished the NTPase activity of ntNS3, whereas substitution of TATPA(354) for TATPV in the motif III had no effect on the enzyme activity. Moreover, the kinetic properties (K(m) and k(cat)) of CSFV NS3 were more similar to those of BVDV. Our results provide insight into the structure-function relationship of CSFV NS3 and facilitate our understanding of its role in the replication cycle of CSFV.

Functional Characterization of cis and trans Activity of the Flavivirus NS2B-NS3 Protease

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

Modulation of the Nucleoside Triphosphatase/RNA Helicase and 5′-RNA Triphosphatase Activities of Dengue Virus Type 2 Nonstructural Protein 3 (NS3) by Interaction with NS5, the RNA-dependent RNA Polymerase

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

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.