NS3 Protease Activity Research Articles

H1-A, a compound isolated from Fusarium oxysporum inhibits hepatitis C virus (HCV) NS3 serine protease

The present study was aimed to isolate the active compounds from the fermentation products of Fusarium oxysporum, which had hepatitis C virus (HCV) NS3 protease inhibitory activity. A bioactive compound was isolated by reverse-phase silica-gel column chromatography, silica-gel column chromatography, semi-preparative reverse-phase High Performance Liquid Chromatography (HPLC), and then its molecular structure was elucidated based on the spectrosopic analysis. As a result, the compound (H1-A, 1) Ergosta-5, 8 (14), 22-trien-7-one, 3-hydroxy-,(3β, 22E) was isolated and identified. To the best of our knowledge, this was the first report on the isolation of H1-A from microorganisms with the inhibitory activity of NS3 protease.

Sulforaphane Suppresses Hepatitis C Virus Replication by Up-Regulating Heme Oxygenase-1 Expression through PI3K/Nrf2 Pathway.

Hepatitis C virus (HCV) infection-induced oxidative stress is a major risk factor for the development of HCV-associated liver disease. Sulforaphane (SFN) is an antioxidant phytocompound that acts against cellular oxidative stress and tumorigenesis. However, there is little known about its anti-viral activity. In this study, we demonstrated that SFN significantly suppressed HCV protein and RNA levels in HCV replicon cells and infectious system, with an IC50 value of 5.7 ± 0.2 μM. Moreover, combination of SFN with anti-viral drugs displayed synergistic effects in the suppression of HCV replication. In addition, we found nuclear factor erythroid 2-related factor 2 (Nrf2)/HO-1 induction in response to SFN and determined the signaling pathways involved in this process, including inhibition of NS3 protease activity and induction of IFN response. In contrast, the anti-viral activities were attenuated by knockdown of HO-1 with specific inhibitor (SnPP) and shRNA, suggesting that anti-HCV activity of SFN is dependent on HO-1 expression. Otherwise, SFN stimulated the phosphorylation of phosphoinositide 3-kinase (PI3K) leading Nrf2-mediated HO-1 expression against HCV replication. Overall, our results indicated that HO-1 is essential in SFN-mediated anti-HCV activity and provide new insights in the molecular mechanism of SFN in HCV replication.

A Novel in Silico 4D Geometrical Measure of the Active Site Correlates with the Enzymatic Activity of HCV NS3 Protease; Implications in Catalysis and Drug Design

We have previously developed and improved on a 4D computational methodology, based on 3D structural modeling coupled with molecular dynamics simulations, to analyze, pan-genotypic, the active sites of the NS3 proteases of HCV, in relation to their catalytic activity and drug susceptibility. The 4D analysis of the interactions between the catalytic triad residues (His57, Asp81, and Ser139) yielded a divergent, gradual and genotype- dependent, 4D conformational instability measures, which correlate well the with known altered catalytic activities.

Development and application of a fast, reproducible assay to measure HCV NS3 protease activity using Escherichia coli lysate

The hepatitis C virus (HCV) NS3/4A protease is a key target of efforts to develop direct-acting antiviral inhibitors for treatment of chronic HCV infection. In vitro analyses of the effects of NS3/4A mutations and polymorphisms on protease inhibitor (PI) susceptibility are essential to nonclinical and clinical compound characterization, but can be hampered by time and technical limitations of current in vitro methods using replicon or purified protein systems. We have developed a fast and simple method utilizing full-length NS3/4A protease inducibly expressed in Escherichia coli cells. Minimally processed E. coli whole cell lysate was used for analyzing NS3/4A protease activity and inhibition by antiviral compounds. Assay conditions were optimized to develop a reproducible assay that can be used for efficient analysis of NS3 protease mutants with poor replication capacity in the replicon system. IC50 fold-changes for NS3 mutants relative to their wild-types generated by this NS3 assay are comparable to those observed in the replicon system, with an R2 of 0.82 for the values obtained by the two methods. In addition, we demonstrate that this assay can be successfully used for population and clonal phenotyping of patient samples and characterization of PIs against the NS3/4A protease from HCV genotypes 1–6.

Discovery of a series of novel compounds with moderate anti-hepatitis C virus NS3 protease activity in vitro

The hepatitis C virus (HCV) NS3/4A protease that plays an important role in the viral life cycle has been proven to be an excellent target for the discovery of anti-HCV drugs. Enlightened by some P2-triazole and amide compounds, which had been found as HCV NS3 protease inhibitors, we designed and synthesized a series of novel compounds by incorporating different amino acid residues in P1/P1′ and P3/P3′ position to develop novel antiviral agents. The result of enzyme inhibition assay indicated that all the designed compounds showed moderate anti-HCV NS3 protease activity. On the basis of the biological result, a detailed structure–activity relationship (SAR) was derived and discussed.

Human pegivirus (GB virus C) NS3 protease activity inhibits induction of the type I interferon response and is not inhibited by HCV NS3 protease inhibitors

We previously found that human pegivirus (HPgV; formerly GBV-C) NS3 protease activity inhibits Human Immunodeficiency Virus (HIV) replication in a CD4+ T cell line. Given the protease׳s similarity to the Hepatitis C virus (HCV) NS3 protease, we characterized HPgV protease activity and asked whether it affects the type I interferon response or is inhibited by HCV protease antagonists. We characterized the activity of proteases with mutations in the catalytic triad and demonstrated that the HCV protease inhibitors Telaprevir, Boceprevir, and Danoprevir do not affect HPgV protease activity. HPgV NS3 protease cleaved MAVS but not TRIF, and it inhibited interferon responses sufficiently to enhance growth of an interferon-sensitive virus. Therefore, HPgV׳s inhibition of the interferon response could help promote HPgV persistence, which is associated with clinical benefits in HIV-infected patients. Our results also imply that HCV protease inhibitors should not interfere with the beneficial effects of HPgV in HPgV/HCV/HIV infected patients.

Design and Docking Studies of Peptide Inhibitors as Potential Antiviral Drugs for Dengue Virus Ns2b/Ns3 Protease

Dengue virus (DENV), one of the members of genus Flavivirus is emerging as a global threat to human health. It had led to the emergence of dengue fever (flu-like illness), dengue shock syndrome, and the most severe dengue hemorrhagic fever (severe dengue with bleeding abnormalities). As Dengue hemorrhage diseases are the life-threatening ones, attempts are being made worldwide to design inhibitors for DENV-2 NS2B-NS3 protease. NS2B/NS3 protease plays a vital role in the replication of dengue virus. The trypsin-like serine protease domain of NS3 contains the functional catalytic triad His-51, Asp-75, and Ser-135 in the N-terminal region. Inhibition of the NS3 protease activity is expected to prevent the propagation of dengue virus. Current drug discovery methods are largely inefficient and thus relatively ineffective in tackling the growing threat to public health presented by emerging and remerging viral pathogens. Recently, there has been a need of interest in peptides and their mimetics as potential antagonists for dengue protease because these small peptides are unlikely to invoke an immune response since they fall below the immunogenic threshold. They are often potent and display fewer toxicity issues than small-molecule compounds as a result of high specificity. This study was conducted to design peptides as enzyme inhibitors of dengue virus NS3 protease through computational approach. Crystallographic structure of dengue protease was retrieved from Protein Data Bank (PDBID: 2FOM) and docked with the peptides and the results are analyzed. From the docking studies reported in this paper, tetrapeptide (Lys-Gly-Pro-Glu), pentapeptide (Ser-Ile-Lys-Phe-Ala) and hexapeptide (Ala-Ile-Lys-Lys-Phe-Ser) with glide energy -70.0 kcal/mol, -72.2 kcal/mol and - 80.4 kcal/mol respectively show promising results which can be considered for further optimization and in vitro studies.

Antiviral potential of 4-hydroxypanduratin A, secondary metabolite of Fingerroot, Boesenbergia pandurata (Schult.), towards Japanese Encephalitis virus NS2B/NS3 prote

4-hydroxypanduratin A is a secondary metabolite of Boesenbergia pandurata Schult. (Fingerroot) plant with various pharmacological activities such as neuroprotective, potent antioxidant, antibacterial and antifungal. Flaviviral NS2B/NS3 protease activity is essential for polyprotein processing and viral replication for Japanese Encephalitis Virus (JEV), a major cause of Acute Encephaltis in Asia. Inhibition of formation of this complex by arresting the binding of NS2B with NS3 would reduce the enzyme's activity to meager proportions and hence would prevent further viral proliferation. The automated 3D structure of NS2B protein of the JEV GP78 was predicted based on the sequence-to-structure-to-function paradigm using I-TASSER and the function of NS2B protein was inferred by matching to other known proteins. The stereochemical quality of predicted structure was checked by PROCHECK. The antiviral activity of 4-hydroxypanduratin A against NS2B protein as a potential drug has been elucidated in this paper. Docking simulation analysis showed 4-hydroxypanduratin A as potential inhibitor of NS2B protein/cofactor which is necessary for NS3 protease activity. 220 derivatives of 4-hydroxypanduratin A were virtually screened with rigid criteria of Lipinski's rule of 5 using Autodock4.2. 4-hydroxypanduratin A was found interacting with target hydrophilic domain in NS2B protein by two Hbonds (Gly80 and Asp81) with active residues, several hydrophobic interactions, Log P value of 5.6, inhibition constant (Ki) of 51.07nM and lowest binding energy of -9.95Kcal/Mol. Hence, 4-hydroxypanduratin A targeted to Site 2 will have sufficient profound effect to inhibit protease activity to abrogate viral replication. It could be a promising potential drug candidate for JEV infections using NS2B Site 2 as a Drug target.

Proteomic analysis of cell suspension cultures of Boesenbergia rotunda induced by phenylalanine: identification of proteins involved in flavonoid and phenylpropanoid biosynthesis pathways

Boesenbergia rotunda belongs to the Zingiberaceae family. It is widely found throughout Southeast Asia and is commonly used as a food ingredient and in folk medicine. Extracts from this plant contain a number of important bioactive compounds such as boesenbergin, cardamonin, pinostrobin, pinocembrin, panduratin A and 4-hydroxypanduratin A. These compounds have been shown to exhibit anti-HIV protease, anti-dengue NS2B/ NS3 protease, antibacterial, antifungal, anti-inflammatory, anticancer, and antioxidant activity. Here we report the use of proteomic approaches to identify proteins that may be involved in the biosynthesis of these compounds. Protein expressions of B. rotunda suspension cultures for phenylalanine-treated and normal callus were compared by two-dimensional gel electrophoresis. Following image analysis, protein spots whose expressions were found to be regulated were identified using Matrix Assisted Laser Desorption-Ionization tandem mass spectrometry. In all, thirty four proteins were identified. These proteins were categorized into nine functional categories—defence mechanism, protein biosynthesis, metabolism, terpenoid biosynthesis, cell division, cell organization, energy-related, signaling processes and proteins of unknown function. Eleven of the proteins involved in the phenylpropanoid biosynthetic pathway are related to the biosynthesis of cyclohexenyl chalcone derivatives.

Potent and selective inhibition of hepatitis C virus replication by novel phenanthridinone derivatives

A number of novel phenanthridinone derivatives were examined for their inhibitory effect on hepatitis C virus (HCV) replication in Huh-7 cells harboring self-replicating subgenomic viral RNA replicons with a luciferase reporter (LucNeo#2). The activity of compounds was further confirmed by inhibition of viral RNA copy number in different subgenomic and full-genomic replicon cells using real-time reverse transcription polymerase chain reaction. Among the compounds, 4-butyl-11-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)-7-methoxy-[1,3]dioxolo[4,5- c]phenanthridin-5(4 H)-one (HA-719) was found to be the most active with a 50% effective concentration of 0.063 ± 0.010 μM in LucNeo#2 cells. The compound did not show apparent cytotoxicity to the host cells at concentrations up to 40 μM. Western blot analysis demonstrated that HA-719 reduced the levels of NS3 and NS5A proteins in a dose-dependent fashion in the replicon cells. Interestingly, the phenanthridinone derivatives including HA-719 were less potent inhibitors of JFH1 strain (genobtype 2a HCV) in cell-free virus infection assay. Although biochemical assays revealed that HA-719 proved not to inhibit NS3 protease or NS5B RNA polymerase activity at the concentrations capable of inhibiting viral replication, their molecular target (mechanism of inhibition) remains unknown. Considering the fact that most of the anti-HCV agents currently approved or under clinical trials are protease and polymerase inhibitors, the phenanthridinone derivatives are worth pursuing for their mechanism of action and potential as novel anti-HCV agents.

Monoclonal antibodies against dengue NS2B and NS3 proteins for the study of protein interactions in the flaviviral replication complex

The replication of dengue virus (DENV) RNA requires at least two viral non-structural (NS) proteins, NS3 and NS5. To facilitate the study of the DENV replication complex, human monoclonal IgG that are specific for NS proteins have been generated and characterised. The anti-NS3 IgG, 3F8, binds a conserved epitope (aa526-531) in the NS3 helicase domain, and cross-reacts with NS3 from all four DENV serotypes and the related yellow fever virus. The anti-NS2B IgG, 3F10, binds aa49-66 of NS2B (CF18), which forms part of the 47 aa hydrophilic cofactor region required for NS3 protease activity. The specificity of the IgG for their respective non-structural proteins has been demonstrated by immunofluorescence of cells infected with DENV and Western blotting. 3F8 is able to co-immunoprecipitate NS3 and NS5 from BHK-21 cells infected with DENV2, and 3F10 is able to detect an interaction between recombinant NS2B(CF18)NS3 full-length protein and the NS5 RNA-dependent RNA polymerase (RdRp) domain in an ELISA-based binding assay. The assay is specific and highly reproducible, with a clear binding curve seen when RdRp is incubated with increasing amounts of full-length NS3, but not the NS3 protease domain. The NS3 helicase domain competes with NS3 full-length for NS5 RdRp binding, with a K(d.) of 2.5μM. Since NS3 and NS5 are required for DENV replication, this fascile assay could be used to screen for non-nucleoside, allosteric inhibitors that disrupt the interaction between the two proteins.

Suppression of hepatitis C virus by the flavonoid quercetin is mediated by inhibition of NS3 protease activity

Phytochemicals exert antiviral activity and may play a potential therapeutic role in hepatitis C virus (HCV) infection. In this work, we aimed to isolate NS3 inhibitors from traditional Indian medicinal plants that were found, in our earlier study, to inhibit HCV NS3 protease activity and to evaluate their potential to inhibit HCV replication. A potent inhibitory effect of NS3 catalytic activity was obtained with Embelia ribes plant extracts. Quercetin, a ubiquitous plant flavonoid, was identified as the active substance in the fractioned extract. It was found to inhibit NS3 activity in a specific dose-dependent manner in an in vitro catalysis assay. Quercetin inhibited HCV RNA replication as analysed in the subgenomic HCV RNA replicon system. It also inhibited HCV infectious virus production in the HCV infectious cell culture system (HCVcc), as analysed by the focus-forming unit reduction assay and HCV RNA real-time PCR. The inhibitory effect of quercetin was also obtained when using a model system in which NS3 engineered substrates were introduced in NS3-expressing cells, providing evidence that inhibition in vivo could be directed to the NS3 and do not involve other HCV proteins. Our work demonstrates that quercetin has a direct inhibitory effect on the HCV NS3 protease. These results point to the potential of quercetin as a natural nontoxic anti-HCV agent reducing viral production by inhibiting both NS3 and heat shock proteins essential for HCV replication.

Engineered toxins "zymoxins" are activated by the HCV NS3 protease by removal of an inhibitory protein domain.

The synthesis of inactive enzyme precursors, also known as “zymogens,” serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV) as a model, we designed two HCV NS3 protease-activated “zymogenized” chimeric toxins (which we denote “zymoxins”). In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA) and Ricin A chain (RTA), respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the “zymoxin” approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected.

Anthracene-based inhibitors of dengue virus NS2B–NS3 protease

Dengue virus (DENV) is a mosquito-borne flavivirus that has strained global healthcare systems throughout tropical and subtropical regions of the world. In addition to plaguing developing nations, it has re-emerged in several developed countries with recent outbreaks in the USA ( CDC, 2010), Australia ( Hanna et al., 2009), Taiwan ( Kuan et al., 2010) and France ( La Ruche et al., 2010). DENV infection can cause significant disease, including dengue fever, dengue hemorrhagic fever, dengue shock syndrome, and death. There are no approved vaccines or antiviral therapies to prevent or treat dengue-related illnesses. However, the viral NS2B–NS3 protease complex provides a strategic target for antiviral drug development since NS3 protease activity is required for virus replication. Recently, we reported two compounds with inhibitory activity against the DENV protease in vitro and antiviral activity against dengue 2 (DEN2V) in cell culture ( Tomlinson et al., 2009a). Analogs of one of the lead compounds were purchased, tested in protease inhibition assays, and the data evaluated with detailed kinetic analyses. A structure activity relationship (SAR) identified key atomic determinants (i.e. functional groups) important for inhibitory activity. Four “second series” analogs were selected and tested to validate our SAR and structural models. Here, we report improvements to inhibitory activity ranging between ∼2- and 60-fold, resulting in selective low micromolar dengue protease inhibitors.

Inhibition of protease-inhibitor-resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies

Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and a serious threat to human health. The HCV NS3/4A serine protease is necessary for viral replication and innate immune evasion, and represents a well-validated target for specific antiviral therapy. We previously reported the isolation of single-chain antibodies (scFvs) that inhibit NS3/4A protease activity in vitro. Expressed intracellularly (intrabodies), these scFvs blocked NS3-mediated proliferation of NS3-transfected cells. Here we show that anti-NS3 scFvs suppress HCV RNA replication when expressed intracellularly in Huh7 hepatoma cells bearing either subgenomic or genome-length HCV RNA replicons. The expression of intrabodies directed against NS3 inhibited the autonomous amplification of HCV replicons resistant to small-molecule inhibitors of the NS3/4A protease, and replicons derived from different HCV genotypes. The combination of intrabodies and interferon-α had an additive inhibitory effect on RNA replication in the replicon model. Intrabody expression also inhibited production of infectious HCV in a cell culture system. The NS3 protease activity was inhibited by the intrabodies in NS3-expressing cells. In contrast, cell-free synthesis of HCV RNA by preformed replicase complexes was not inhibited by intrabodies, suggesting that the major mode of inhibition of viral replication is inhibition of NS3/4A protease activity and subsequent suppression of viral polyprotein processing.

Comparison of HCV NS3 protease and NS5B polymerase inhibitor activity in 1a, 1b and 2a replicons and 2a infectious virus

The hepatitis C virus infection system represents an important new tool for drug discovery. In this study, we compared the in vitro antiviral efficacy of several NS3 and NS5B inhibitors in genotype 1a, 1b, and 2a replicons and in the 2a infectious virus system. The nucleoside inhibitor 2′-C-methyl adenosine showed similar efficacy in each system tested. Three non-nucleoside inhibitors had small differences in potency between genotype 1a and 1b. In contrast, there was a dramatic loss of potency for these non-nucleoside inhibitors in the genotype 2a replicon, 2a infectious virus, and 2a NS5B biochemical assays. The protease inhibitor BILN-2061 had similar efficacy against 1a and 1b replicons but was 61–109-fold less potent against the 2a replicon and virus, respectively. VX-950, a covalent protease inhibitor, had similar efficacy (<3-fold changes in EC 50) regardless of genotype or subtype. Importantly, we observed a significant correlation ( p < 0.0001) in antiviral potency between the 2a replicon and 2a infectious virus for all classes of compounds tested.

Identification of Amino Acid Variants in the Hepatitis C Virus Non-Structural Protein 4A

Chronic infections by hepatitis C virus (HCV) are a major cause of cirrhosis and hepatic cancer. The replication of HCV involves translation and proteolytic processing of polyproteins. The HCV single-stranded RNA encodes a single polyprotein of C/E1/E2/p7/NS2/NS3/NS4A/NS4B/NS5A/NS5B. The structural proteins, C, E1, E2, and p7, arise from the viral polyprotein by host proteases. Cleavage at the non-structural NS2/NS3 junction is performed by the NS2 protease. NS3 forms a complex with NS4A to cleave the rest of the viral polyprotein. The central 12-amino-acid sequence of NS4A, 21-GSVVIVGRIILS-32 (NS4Awt) is a determinant to enhance the NS3 protease activity at the NS5A/5B junction. We found that, from 13 blood donors infected with HCV, one sample showed five amino acid changes in the NS4A central region at V23I, I25C, I30S, L31T, and S32L, and another sample showed three changes at V23I, I25C, and I30V in this region. The other 11 samples showed the NS4Awt sequence. The effect of such amino acid variations on the NS3 proteolytic activity was evaluated in vitro using the central 12-amino-acid NS4Awt sequence with specific changes joined to NS3, and NS5A/5B as a substrate. Our results indicate that the amino acid changes of NS4A at V23I and I25C do not enhance the protease activity of NS3, whereas the amino acid changes at I30S, L31T, and S32L, as well as the NS4Awt sequence, enhance NS3 activity. Our results confirm that protease cofactor, encoded in NS4A, is of major regulatory relevance for the replication cycles of HCV.

Inhibition of hepatitis C virus RNA replicons by peptide aptamers

Background/aims Hepatitis C virus infection is a major worldwide health problem, causing chronic hepatitis, cirrhosis and primary liver cancer. In addition to its role in the viral polyprotein-processing, the viral NS3 serine protease has been implicated in interactions with various cell constituents resulting in phenotypic changes including malignant transformation. NS3 is currently regarded a prime target for anti-viral drugs thus specific inhibitors of its activities should be important. With the aim of inhibiting NS3 protease activity as a means to inhibit HCV replication we used a novel bacterial genetic screen to isolate NS3-inhibiting peptide aptamers. Methods We have isolated and characterized seven NS3-inhibiting peptide aptamers. We investigated the phenotypic changes that SEAP-secreting subgenomic RNA replicons undergo upon intracellular expression of these peptide aptamers, assayed by real-time RT-PCR and inhibition of SEAP secretion by transfected replicon cells. Results and conclusions The peptide aptamers inhibited NS3 protease activity in vitro with an IC 50 in the low micromolar range. Upon transfection, aptamers inhibited the replication of SEAP-secreting genotype 1b subgenomic RNA replicons. Aptamer-based intracellular immunization may emerge as a promising antiviral approach to interfere with the life cycle and pathogenicity of HCV.

Differential Requirements of NS4A for Internal NS3 Cleavage and Polyprotein Processing of Hepatitis C Virus

The NS3 protein of hepatitis C virus (HCV) possesses protease activity responsible for the proteolytic cleavage of the viral polyprotein at the junctions of nonstructural proteins downstream of NS3. The NS3 protein was also found to be internally cleaved. In this study, we demonstrated that internal cleavages occurred on the NS3 protein of genotype 1b in the presence of NS4A, both in culture cells and with a mouse model system. No internal cleavage products were detected with the NS3 and NS4A proteins of genotype 2a. Three potential cleavage sites were detected in the NS3 protein (genotype 1b), with IPT(402)|S being the major one. The internal cleavage requires the polyprotein processing activity of NS3 protease, but when supplemented in trans, the internal cleavage efficiency is reduced. In addition, several mutations in NS4A disrupted the internal cleavage of NS3 but did not affect polyprotein processing, indicating that NS4A contributes differently to these two proteolytic activities. Furthermore, Ile-25, Val-26, and Ile-29 of the NS4A protein, important for the NS4A-dependent internal cleavages, were also shown to be critical for the transforming activity of NS3, but mutations at these critical residues resulted only in a slight increase of HCV replicating efficiency. The internal cleavage-associated enhancement of the transforming activity of NS3 was reduced when a T402A substitution at the major internal cleavage site was introduced. The multiple roles of NS4A in viral multiplication and pathogenesis make NS4A an ideal molecular target for HCV therapy.

Functional determinants of NS2B for activation of Japanese encephalitis virus NS3 protease

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus, causing severe central nerve system diseases without specific treatments. The NS2B–NS3 protease of flaviviruses mediates several cleavages on the flavivirus polyprotein, being believed to be a target for antiviral therapy. NS2B is the cofactor of the viral serine protease, correlating with stabilization and substrate recognition of the NS3 protease. In this study, we investigate the functional determinants in the JEV NS2B for the activation of the NS3 protease. Cis- and trans-cleavage assays of the deletions at the N-terminal of NS2B demonstrated that the NS2B residues Ser 46 to Ile 60 were the essential region required for both cis and trans activity of the NS3 protease. In addition, alanine substitution at the residues Trp53, Glu55, and Arg56 in NS2B significantly reduced the cis- and trans-cleavage activities of the NS3 protease. Sequence alignment and modeled structures suggested that functional determinants at the JEV NS2B residues Ser46 to Ile60, particularly in Trp53, Glu55 and Arg56 could play an important configuration required for the activity of the flavivirus NS3 protease. Our results might be useful for development of inhibitors that block the interaction between NS2B and NS3.