NS2B/NS3 protease: allosteric effect of mutations associated with the pathogenicity of tick-borne encephalitis virus

The sequences of the protease domain of the tick-borne encephalitis (TBE) virus NS3 protein have two amino acid substitutions, 16 R→K and 45 S→F, in the highly pathogenic and poorly pathogenic strains of the virus, respectively. Two models of the NS2B-NS3 protease complex for the highly pathogenic and poorly pathogenic strains of the virus were constructed by homology modeling using the crystal structure of West Nile virus NS2B-NS3 protease as a template; 20?ns molecular dynamic simulations were performed for both models, the trajectories of the dynamic simulations were compared, and the averaged distance between the two models was calculated for each residue. Conformational differences between two models were revealed in the identified pocket. The different conformations of the pocket resulted in different orientations of the NS2B segment located near the catalytic triad. In the model of the highly pathogenic TBE virus the identified pocket had a more open conformation compared to the poorly pathogenic model. We propose that conformational changes in the active protease center, caused by two amino acid substitutions, can influence enzyme functioning and the virulence of the virus.     

Mutagenesis of the NS3 Protease of Dengue Virus Type 2

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

Cleavage of the dengue virus polyprotein at the NS3/NS4A and NS4B/NS5 junctions is mediated by viral protease NS2B-NS3, whereas NS4A/NS4B may be processed by a cellular protease.

The cleavage mechanism utilized for processing of the NS3-NS4A-NS4B-NS5 domain of the dengue virus polyprotein was studied by using the vaccinia virus expression system. Recombinant vaccinia viruses vNS2B-NS3-NS4A-NS4B-NS5, vNS3-NS4A-NS4B-NS5, vNS4A-NS4B-NS5, and vNS4B-NS5 were constructed. These recombinants were used to infect cells, and the labeled lysates were analyzed by immunoprecipitation. Recombinant vNS2B-NS3-NS4A-NS4B-NS5 expressed the authentic NS3 and NS5 proteins, but the other recombinants produced uncleaved polyproteins. These findings indicate that NS2B is required for processing of the downstream nonstructural proteins, including the NS3/NS4A and NS4B/NS5 junctions, both of which contain a dibasic amino acid sequence preceding the cleavage site. The flavivirus NS4A/NS4B cleavage site follows a long hydrophobic sequence. The polyprotein NS4A-NS4B-NS5 was cleaved at the NS4A/NS4B junction in the absence of other dengue virus functions. One interpretation for this finding is that NS4A/NS4B cleavage is mediated by a host protease, presumably a signal peptidase. Although vNS3-NS4A-NS4B-NS5 expressed only the polyprotein, earlier results demonstrated that cleavage at the NS4A/NS4B junction occurred when an analogous recombinant, vNS3-NS4A-84%NS4B, was expressed. Thus, it appears that uncleaved NS3 plus NS5 inhibit NS4A/NS4B cleavage presumably because the putative signal sequence is not accessible for recognition by the responsible protease. Finally, recombinants that expressed an uncleaved NS4B-NS5 polyprotein, such as vNS4A-NS4B-NS5 or vNS4B-NS5, produced NS5 when complemented with vNS2B-30%NS3 or with vNS2B plus v30%NS3. These results indicate that cleavage at the NS4B/NS5 junction can be mediated by NS2B and NS3 in trans.     

Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro

Dengue virus type 2 NS3, a multifunctional protein, has a serine protease domain (NS3pro) that requires the conserved hydrophilic domain of NS2B for protease activity in cleavage of the polyprotein precursor at sites following two basic amino acids. In this study, we report the expression of the NS2B-NS3pro precursor in Escherichia coli as a fusion protein with a histidine tag at the N terminus. The precursor was purified from insoluble inclusion bodies by Ni(2+) affinity and gel filtration chromatography under denaturing conditions. The denatured precursor was refolded to yield a purified active protease complex. Biochemical analysis of the protease revealed that its activity toward either a natural substrate, NS4B-NS5 precursor, or the fluorogenic peptide substrates containing two basic residues at P1 and P2, was dependent on the presence of the NS2B domain. The peptide with a highly conserved Gly residue at P3 position was 3-fold more active as a substrate than a Gln residue at this position. The cleavage of a chromogenic substrate with a single Arg residue at P1 was NS2B-independent. These results suggest that heterodimerization of the NS3pro domain with NS2B generates additional specific interactions with the P2 and P3 residues of the substrates.     

Homology modeling and molecular dynamics simulations of Dengue virus NS2B/NS3 protease: insight into molecular interaction

The pathogenic West Nile virus (WNV) and Dengue virus (DV) are growing global threats for which there are no specific treatments. Both viruses possess a two component NS2B/NS3 protease which cleaves viral precursor proteins. Whereas for the WNV protease two crystal structures in complex with an inhibitor have been solved recently, no such information is available for the DV protease. Here, we report the generation of a homology model of DV NS2B/NS3 protease. Since it is known from the related WNV protease that it adopts a distinct conformation in free and in inhibitor‐complexed form, a special emphasis was given to the analysis of the protease flexibility. Therefore, several models of DV NS2B/NS3 protease complexed with the peptidic inhibitor (Bz‐Nle(P4)‐Lys(P3)‐Arg(P2)‐Arg(P1)‐H) were generated. The first DV protease model (DV‐1) was constructed using the available crystal structure of the apo DV NS2B/NS3 protease. The second model (DV‐2) was built taking the WNV NS3/NS2B protease in the inhibitor‐complexed form as the template structure. Molecular dynamics simulations which were carried out for the WNV crystal structures as well as for the DV models provided an understanding of the role of NS2B for maintaining the protease in the active conformation. It was also demonstrated that NS2B is not only important for maintaining NS3 in the active form, but is also essential for establishing the interaction between residues from the S2 pocket and the peptidic inhibitor. The DV NS2B/NS3 model in the productive conformation can now be used for structure‐based design purposes. Copyright © 2009 John Wiley & Sons, Ltd.     

Discovery of novel cyclic peptide inhibitors of dengue virus NS2B-NS3 protease with antiviral activity

NS2B-NS3 protease is an essential enzyme for the replication of dengue virus (DENV), which continues to be a serious threat to worldwide public health. We designed and synthesized a series of cyclic peptides mimicking the substrates of this enzyme, and assayed their activity against the DENV-2 NS2B-NS3 protease. The introduction of aromatic residues at the appropriate positions and conformational restriction generated the most promising cyclic peptide with an IC₅₀ of 0.95 μM against NS2B-NS3 protease. Cyclic peptides with proper positioning of additional arginines and aromatic residues exhibited antiviral activity against DENV. Furthermore, replacing the C-terminal amide bond of the polybasic amino acid sequence with an amino methylene moiety stabilized the cyclic peptides against hydrolysis by NS2B-NS3 protease, while maintaining their enzyme inhibitory activity and antiviral activity.     

Analysis of cellular proteome changes in response to ZIKV NS2B-NS3 protease expression

The recent emergence of Zika virus (ZIKV) has caused global concern as a result of the association with neurological disorders, and brain development dysfunction in fetuses of mothers who become infected with ZIKV during pregnancy. The NS2B-NS3 protease is important for viral replication and offers an attractive drug target. In addition to processing the viral polypeptide, evidence has shown that the NS2B-NS3 protease also targets cellular proteins as part of the viral replication process. This study sought to determine new host cell protein targets of ZIKV NS2B-NS3 (zNS2B-NS3). Plasmids encoding the protease domains of zNS2B-NS3pro and an inactive zNS2B-NS3(S135A) were transfected into HEK293T/17 cells and differentially expressed proteins were detected by 2D gel electrophoresis. A total of 18 protein spots were observed as differentially expressed between zNS2B-NS3pro and zNS2B-NS3(S135A), of which 7 were selected for identification by mass spectrometry. Four proteins (protein disulfide-isomerase A3 (PDIA3), heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1), voltage-dependent anion-selective channel (VDAC) and aldolase A (ALDOA)) were selected for validation by independent transient expression and western blot analysis. Three proteins (PDIA3, hnRNP A2/B1 and ALDOA) were successfully validated, but only two proteins (PDIA3 and ALDOA) were shown to be regulated in ZIKV infection in agreement with the results of the transfection experiments. This study has identified two proteins, PDIA3 an ALDOA whose expression is modulated by the ZIKV NS2B-NS3 protease, and these proteins are involved in the ER stress response and glycolysis respectively, two critical cellular processes in ZIKV infection.     

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.     

Mechanism of NS2B-mediated activation of NS3pro in dengue virus: molecular dynamics simulations and bioassays

The NS2B cofactor is critical for proteolytic activation of the flavivirus NS3 protease. To elucidate the mechanism involved in NS2B-mediated activation of NS3 protease, molecular dynamic simulation, principal component analysis, molecular docking, mutagenesis, and bioassay studies were carried out on both the dengue virus NS3pro and NS2B-NS3pro systems. The results revealed that the NS2B-NS3pro complex is more rigid than NS3pro alone due to its robust hydrogen bond and hydrophobic interaction networks within the complex. These potent networks lead to remodeling of the secondary and tertiary structures of the protease that facilitates cleavage sequence recognition and binding of substrates. The cofactor is also essential for proper domain motion that contributes to substrate binding. Hence, the NS2B cofactor plays a dual role in enzyme activation by facilitating the refolding of the NS3pro domain as well as being directly involved in substrate binding/interactions. Kinetic analyses indicated for the first time that Glu92 and Asp50 in NS2B and Gln27, Gln35, and Arg54 in NS3pro may provide secondary interaction points for substrate binding. These new insights on the mechanistic contributions of the NS2B cofactor to NS3 activation may be utilized to refine current computer-based search strategies to raise the quality of candidate molecules identified as potent inhibitors against flaviviruses.     

黄病毒NS2B-NS3pro蛋白酶的结构研究进展

黄病毒能引起严重的人类疾病,但是并无特定药物来治疗病毒感染。黄病毒非结构蛋白NS3的N端区域及其辅因子NS2B构成蛋白酶,该酶切割病毒的多聚蛋白形成成熟的结构蛋白和非结构蛋白来帮助病毒完成增殖过程。NS2B-NS3pro蛋白酶在黄病毒生命周期中起关键的作用,使之成为抗病毒药物研发的重要靶标。本文综述了黄病毒属中寨卡病毒、登革热病毒、西尼罗病毒的NS2B-NS3pro蛋白酶结构的研究进展,并介绍了相关抑制剂与蛋白酶形成的复合物结构,以期为研发抗黄病毒药物提供必要的参考。     

Structural characterization and polymorphism analysis of the NS2B-NS3 protease from the 2017 Brazilian circulating strain of Yellow Fever virus

BackgroundThe Yellow Fever virus (YFV) is transmitted by mosquitos and causes an infection with symptoms including fever, headaches and nausea. In 20–50% of the cases, the disease may evolve to a visceral stage, reaching high mortality rates. YFV NS2B-NS3 protease has been identified as an important drug target.MethodsHerein, we describe the crystal structure of the NS2B-NS3 protease from the 2017 YFV Brazilian circulating strain using X-ray crystallography. Furthermore, we used a combination of biochemical and biophysical assays to characterize the enzyme and investigate the impact of the polymorphisms observed in different YFV circulating strains.ResultsSurprisingly, the crystal structure of YFV protease seems to adopt the closed conformation without the presence of a binding partner. Although D88E and K121R mutants exhibited a lower affinity for the substrate, both revealed to be more processive, resulting in a similar catalytic efficiency in relation to the WT protease. Still, both mutants showed an accentuated decrease in stability when compared with the WT.ConclusionsThe crystal structure of YFV NS2B-NS3 in closed conformation might be an important tool for the development of new drugs, as well as understanding the activation mechanism of viral proteases. Biochemical analyses indicate that the NS2B-NS3 protease of the circulating strain of YFV is more stable than previous strains.General significanceThe YFV NS2B-NS3 protease is the first flaviviral structure described in its closed conformation when in a free form, implying that external factors might induce the activation of the enzyme.     

The serine protease and RNA-stimulated nucleoside triphosphatase and RNA helicase functional domains of dengue virus type 2 NS3 converge within a region of 20 amino acids

NS3 protein of dengue virus type 2 has a serine protease domain within the N-terminal 180 residues. NS2B is required for NS3 to form an active protease involved in processing of the viral polyprotein precursor. The region carboxy terminal to the protease domain has conserved motifs present in several viral RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicases. To define the functional domains of protease and NTPase/RNA helicase activities of NS3, full-length and amino-terminal deletion mutants of NS3 were expressed in Escherichia coli and purified. Deletion of 160 N-terminal residues of NS3 (as in NS3del.2) had no detrimental effect on the basal and RNA-stimulated NTPase as well as RNA helicase activities. However, mutagenesis of the conserved P-loop motif of the RNA helicase domain (K199E) resulted in loss of ATPase activity. The RNA-stimulated NTPase activity was significantly affected by deletion of 20 amino acid residues from the N terminus or by substitutions of the cluster of basic residues, 184RKRK-->QNGN, of NS3del.2, although both mutant proteins retained the conserved RNA helicase motifs. Furthermore, the minimal NS3 protease domain, required for cleavage of the 2B-3 site, was precisely defined to be 167 residues, using the in vitro processing of NS2B-NS3 precursors. Our results reveal that the functional domains required for serine protease and RNA-stimulated NTPase activities map within the region between amino acid residues 160 and 180 of NS3 protein and that a novel motif, the cluster of basic residues 184RKRK, plays an important role for the RNA-stimulated NTPase activity.     

NMR and MD Studies Reveal That the Isolated Dengue NS3 Protease Is an Intrinsically Disordered Chymotrypsin Fold Which Absolutely Requests NS2B for Correct Folding and Functional Dynamics

Dengue genome encodes a two component protease complex (NS2B-NS3pro) essential for the viral maturation/infectivity, thus representing a key drug target. Previously, due to its “complete insolubility”, the isolated NS3pro could not be experimentally studied and it remains elusive what structure it adopts without NS2B and why NS2B is indispensable. Here as facilitated by our previous discovery, the isolated NS3pro has been surprisingly deciphered by NMR to be the first intrinsically-disordered chymotrypsin-like fold, which exists in a loosely-packed state with non-native long-range interactions as revealed by paramagnetic relaxation enhancement (PRE). The disordered NS3pro appears to be needed for binding a human host factor to trigger the membrane remodeling. Moreover, we have in vitro refolded the NS3pro in complex with either NS2B (48–100) or the full-length NS2B (1–130) anchored into the LMPC micelle, and the two complexes have similar activities but different dynamics. We also performed molecular dynamics (MD) simulations and the results revealed that NS2B shows the highest structural fluctuations in the complex, thus providing the dynamic basis for the observation on its conformational exchange between open and closed states. Remarkably, the NS2B cofactor plays a central role in maintaining the correlated motion network required for the catalysis as we previously decoded for the SARS 3CL protease. Indeed, a truncated NS2B (48–100;Δ77–84) with the flexible loop deleted is able to trap the NS2B-NS3pro complex in a highly dynamic and catalytically-impotent state. Taken together, our study implies potential strategies to perturb the NS2B-NS3pro interface for design of inhibitors for treating dengue infection.     

Mutagenesis of D80-82 and G83 residues in West Nile Virus NS2B: Effects on NS2B-NS3 activity and viral replication

Flaviviral NS2B is a required cofactor for NS3 serine protease activity and plays an important role in promoting functional NS2B-NS3 protease configuration and maintaining critical interactions with protease catalysis substrates. The residues D⁸⁰DDG in West Nile virus (WNV) NS2B are important for protease activity. To investigate the effects of D⁸⁰DDG in NS2B on protease activity and viral replication, the negatively charged region D⁸⁰DD and the conserved residue G83 of NS2B were mutated (D⁸⁰DD/E⁸⁰EE, D⁸⁰DD/K⁸⁰KK, D⁸⁰DD/A⁸⁰AA, G83F, G83S, G83D, G83K, and G83A), and NS3 D75A was designated as the negative control. The effects of the mutations on NS2B-NS3 activity, viral translation, and viral RNA replication were analyzed using kinetic analysis of site-directed enzymes and a transient replicon assay. All substitutions resulted in significantly decreased enzyme activity and blocked RNA replication. The negative charge of D⁸⁰DD is not important for maintaining NS2B function, but side chain changes in G83 have dramatic effects on protease activity and RNA replication. These results demonstrate that NS2B is important for viral replication and that D⁸⁰DD and G83 substitutions prevent replication; they will be useful for understanding the relationship between NS2B and NS3.     

Mutation and docking studies on NS2b-NS3 complex from yellow fever virus towards drug discovery

Yellow fever virus is the causative agent of Yellow fever. The genome of the virus contains three structural and seven non-structural proteins. Of these seven nonstructural proteins, NS2B-NS3 protein complex has protease activity required for viral replication. Predicting the 3D structure of this complex and studying the interaction of residues at the recognized catalytic triad of the complex is an integral part to understand the virus replication mechanism. In the present study, the structure was determined for NS2B-NS3 complex by Homology modeling and modeled structure was validated for its stability. Mutation studies at the residues His94, Asp118 and Ser176 revealed that Asp118-His94 bond played an important role in the structural stability of NS2B-NS3 complex. This indicates site-directed mutagenesis, controlling YFV replication, as one mechanism to design vaccine strains. Docking studies of the bioactive compounds at the active site of NS2B-NS3 complex also indicated 4-hydroxypanduratin A as potential lead compound for drug development. The theoretical models will further pave way to experimentally verify our mutation and docking studies, thus taking a lead in pharmacogenomics and drug development.

Abbreviations

YFV - Yellow Fever Virus, WNV - West Nile Virus, H-bonds - hydrogen bonds, SNP - Single nucleotide polymorphism.     

Processing and localization of Dengue virus type 2 polyprotein precursor NS3-NS4A-NS4B-NS5.

Processing of dengue virus type 2 polyprotein precursor NS3-NS4A-NS4B-NS5 could be mediated by the catalytically active NS3 protease domain and NS2B in trans at the dibasic sites NS3-NS4A and NS4B-NS5. Subcellular localization of the unprocessed precursor NS3-NS4A-NS4B-NS5 showed that it was confined to a distinct subcellular organelle in the cytoplasm, which was distinct from the distribution of the mature NS5.     

Structural basis for the activation of flaviviral NS3 proteases from dengue and West Nile virus

The replication of flaviviruses requires the correct processing of their polyprotein by the viral NS3 protease (NS3pro). Essential for the activation of NS3pro is a 47-residue region of NS2B. Here we report the crystal structures of a dengue NS2B-NS3pro complex and a West Nile virus NS2B-NS3pro complex with a substrate-based inhibitor. These structures identify key residues for NS3pro substrate recognition and clarify the mechanism of NS3pro activation.     

In vitro determination of dengue virus type 2 NS2B-NS3 protease activity with fluorescent peptide substrates

The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1 M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2BNS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.     

MD simulations reveal alternate conformations of the oxyanion hole in the Zika virus NS2B/NS3 protease

Recent crystallography studies have shown that the binding site oxyanion hole plays an important role in inhibitor binding, but can exist in two conformations (active/inactive). We have undertaken molecular dynamics (MD) calculations to better understand oxyanion hole dynamics and thermodynamics. We find that the Zika virus (ZIKV) NS2B/NS3 protease maintains a stable closed conformation over multiple 100-ns conventional MD simulations in both the presence and absence of inhibitors. The S1, S2, and S3 pockets are stable as well. However, in two of eight simulations, the A132-G133 peptide bond in the binding pocket of S1' spontaneously flips to form a 3₁₀-helix that corresponds to the inactive conformation of the oxyanion hole, and then maintains this conformation until the end of the 100-ns conventional MD simulations without inversion of the flip. This conformational change affects the S1' pocket in ZIKV NS2B/NS3 protease active site, which is important for small molecule binding. The simulation results provide evidence at the atomic level that the inactive conformation of the oxyanion hole is more favored energetically when no specific interactions are formed between substrate/inhibitor and oxyanion hole residues. Interestingly, however, transition between the active and inactive conformation of the oxyanion hole can be observed by boosting the valley potential in accelerated MD simulations. This supports a proposed induced-fit mechanism of ZIKV NS2B/NS3 protease from computational methods and provides useful direction to enhance inhibitor binding predictions in structure-based drug design.     

Upregulation of signalase processing and induction of prM-E secretion by the flavivirus NS2B-NS3 protease: roles of protease components.

Recently, we have shown that the ability of the flavivirus NS2B-NS3 protease complex to promote efficient signalase processing of the C-prM precursor, as well as secretion of prM and E, does not appear to depend strictly on cleavage of the precursor at its Lys-Arg-Gly dibasic site by the protease. We suggested that the association of the protease with the precursor via NS2B may be sufficient by itself for the above effects. To study the proposed association in more detail, we have developed an assay in which processing at the C-prM dibasic cleavage site is abolished by Lys-->Gly conversion. We constructed deletion mutants and chimeras of the West Nile (WN) flavivirus NS2B protein and expressed them in the context of [5'-C-->NS3(243)] containing either wild-type C-prM or its cleavage site mutant. All NS2B variants were able to form active protease complexes. Deletion of the carboxy-terminal cluster of hydrophobic amino acids in NS2B had no apparent effect on the formation of prM and prM-E secretion for the cassettes containing either wild-type or mutated C-prM precursor. Deletion of the amino-terminal hydrophobic cluster in NS2B did not affect prM-E secretion for the cassettes with wild-type C-prM but abrogated prM-E secretion for the cassettes with the mutated dibasic cleavage site in C-prM. Similarly, the NS2B-NS3(178) protease of Japanese encephalitis (JE) virus, when substituted for the WN virus NS2B-NS3(243) protease, was able to promote prM-E secretion for the cassette with the wild-type C-prM precursor but not with the mutated one. Replacement of the deleted amino-terminal hydrophobic cluster in the WN virus NS2B protein with an analogous JE virus sequence restored the ability of the protease to promote prM-E secretion. On the basis of these observations, roles of individual protease components in upregulation of C-prM signalase processing are discussed.