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。     

The active essential CFNS3d protein complex

The NS2B-NS3 protease complex is essential for the replication of dengue virus, which is the etiologic agent of dengue and hemorrhagic fevers, diseases that are a burden for the tropical and subtropical areas of the world. The active form of the NS3 protease linked to the 40 residues of the NS2B cofactor shows highly flexible and disordered region(s) that are responsible for its high propensity to aggregate at the concentrations necessary for NMR spectroscopy studies or for crystallization. Limited proteolysis of this active form of the protease enabled us to obtain a folded and new essential form of the NS2B-NS3 protease complex. We found that the region from residues D50 to E80 of NS2B interacts directly and strongly with the NS3 protease domain. The proteolytic activity of the noncovalently binding complex was determined by a rapid and continuous fluorescence resonance energy transfer activity assay using a depsipeptide substrate. The new protein-cofactor complex obtained, encompassing the NS2B fragment (D50-E80) and the NS3 protease, shows proteolytic activity. The (1)H-(15)N-heteronuclear single quantum coherence spectrum of the isotopically enriched protein complex shows good cross-peak dispersion; this is indicative of a stable folded state. Our results significantly complement the X-ray structure of the NS2B-NS3pro complex published recently. Moreover, these results open the way to performing direct structural and interaction studies in solution on a new active NS2B-NS3pro complex with libraries of substrates and inhibitors in order to identify new drugs that prevent viral polyprotein processing.     

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.     

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.     

Switching the substrate specificity of the two-component NS2B-NS3 flavivirus proteinase by structure-based mutagenesis

The flavivirus NS2B-NS3(pro)teinase is an essential element in the proteolytic processing of the viral precursor polyprotein and therefore a potential drug target. Recently, crystal structures and substrate preferences of NS2B-NS3pro from Dengue and West Nile viruses (DV and WNV) were determined. We established that the presence of Gly-Gly at the P1'-P2' positions is optimal for cleavage by WNV NS3pro, whereas DV NS3pro tolerates well the presence of bulky residues at either P1' or P2'. Structure-based modeling suggests that Arg(76) and Pro(131)-Thr(132) limit the P1'-P2' subsites and restrict the cleavage preferences of the WNV enzyme. In turn, Leu(76) and Lys(131)-Pro(132) widen the specificity of DV NS3pro. Guided by these structural models, we expressed and purified mutant WNV NS2B-NS3pro and evaluated cleavage preferences by using positional scanning of the substrate peptides in which the P4-P1 and the P3'-P4' positions were fixed and the P1' and P2' positions were each randomized. We established that WNV R76L and P131K-T132P mutants acquired DV-like cleavage preferences, whereas T52V had no significant effect. Our work is the first instance of engineering a viral proteinase with switched cleavage preferences and should provide valuable data for the design of optimized substrates and substrate-based selective inhibitors of flaviviral proteinases.     

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.     

Structure activity relationship (SAR) and quantitative structure activity relationship (QSAR) studies showed plant flavonoids as potential inhibitors of dengue NS2B-NS3 protease

Background

Due to dengue virus disease, half of the world population is at severe health risk. Viral encoded NS2B-NS3 protease complex causes cleavage in the nonstructural region of the viral polyprotein. The cleavage is essentially required for fully functional viral protein. It has already been reported that if function of NS2B-NS3 complex is disrupted, viral replication is inhibited. Therefore, the NS2B-NS3 is a well-characterized target for designing antiviral drug.

Results

In this study docking analysis was performed with active site of dengue NS2B-NS3 protein with selected plant flavonoids. More than 100 flavonoids were used for docking analysis. On the basis of docking results 10 flavonoids might be considered as the best inhibitors of NS2B-NS3 protein. The interaction studies showed resilient interactions between ligand and receptor atoms. Furthermore, QSAR and SAR studies were conducted on the basis of NS2B-NS3 protease complex docking results. The value of correlation coefficient (r) 0.95 shows that there was a good correlation between flavonoid structures and selected properties.

Conclusion

We hereby suggest that plant flavonoids could be used as potent inhibitors of dengue NS2B-NS3 protein and can be used as antiviral agents against dengue virus. Out of more than hundred plant flavonoids, ten flavonoid structures are presented in this study. On the basis of best docking results, QSAR and SAR studies were performed. These flavonoids can directly work as anti-dengue drug or with little modifications in their structures.

     

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.     

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.     

Steady-state cleavage kinetics for dengue virus type 2 ns2b-ns3(pro) serine protease with synthetic peptides

The N-terminal part of the NS3 protein from dengue virus contains a trypsin-like serine protease responsible for processing the nonstructural region of the viral polyprotein. Enzymatic activity of the NS2B-NS3(pro) precursor incorporating a full-length NS2B cofactor of dengue virus type 2 was examined by using synthetic dodecamer peptide substrates encompassing native cleavage sequences of the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 polyprotein junctions. Cleavage of the dansylated substrates was monitored by a HPLC-based assay and kinetic parameters for K(1M), k(cat) and k(cat)/K(m) were obtained. The data presented here show that NS2B-NS3(pro) expressed in recombinant E. coli can be renatured to an active protease which reacts in the absence of microsomal membranes with all 4 substrate peptides, albeit the molecule does not exhibit autoproteolytic processing at the NS2B/NS3 site. A marked difference in cleavage efficiency was found for the NS2B/NS3 substrate and the remaining 3 peptides based on the NS2A/NS2B, NS3/NS4A and NS4A/NS5 cleavage sites.     

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.     

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.     

Structural evidence for regulation and specificity of flaviviral proteases and evolution of the Flaviviridae fold

Pathogenic members of the flavivirus family, including West Nile Virus (WNV) and Dengue Virus (DV), are growing global threats for which there are no specific treatments. The two-component flaviviral enzyme NS2B-NS3 cleaves the viral polyprotein precursor within the host cell, a process that is required for viral replication. Here, we report the crystal structure of WNV NS2B-NS3pro both in a substrate-free form and in complex with the trypsin inhibitor aprotinin/BPTI. We show that aprotinin binds in a substrate-mimetic fashion in which the productive conformation of the protease is fully formed, providing evidence for an "induced fit" mechanism of catalysis and allowing us to rationalize the distinct substrate specificities of WNV and DV proteases. We also show that the NS2B cofactor of WNV can adopt two very distinct conformations and that this is likely to be a general feature of flaviviral proteases, providing further opportunities for regulation. Finally, by comparing the flaviviral proteases with the more distantly related Hepatitis C virus, we provide insights into the evolution of the Flaviviridae fold. Our work should expedite the design of protease inhibitors to treat a range of flaviviral infections.     

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.     

A straightforward experimental approach to expression, purification, refolding, and enzymatic analysis of recombinant dengue virus NS2B(H)-NS3pro protease

Dengue virus threatens around 2.5 billion people worldwide; about 50 million become infected every year, and yet no vaccine or drug is available for prevention and/or treatment. The flaviviral NS2B-NS3pro complex is indispensable for flaviviral replication and is considered to be an important drug target. The aim of this study was to develop a simple and generally applicable experimental strategy to construct, purify, and assay a highly active recombinant NS2B(H)-NS3pro complex that would be useful for high-throughput screening of potential inhibitors. The sequence of NS2B(H)-NS3pro was generated by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro complex was expressed in E. coli predominantly as insoluble protein and purified to >95% purity by single-step immobilized metal affinity chromatography. SDS-PAGE followed by immunoblotting of the purified enzyme demonstrated the presence of the NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 37, 21, and 10 kDa bands, respectively. Kinetic parameters, K _m, k _cat, and k _cat/K _m for the fluorophore-linked protease model substrate Ac-nKRR-amc were obtained using inner-filter effect correction. The kinetic parameters K _m, k _cat, and k _cat/K _m for Ac-nKRR-amc substrate were 100 μM, 0.112 s^?1, and 1120 M^?1·s^?1, respectively. A simplified procedure for the cloning, overexpression, and purification of the NS2B(H)-NS3pro complex was applied, and a highly active recombinant NS2B(H)-NS3pro complex was obtained that could be useful for the design of high-throughput assays aimed at flaviviral inhibitor discovery.     

Structural platform for the autolytic activity of an intact NS2B-NS3 protease complex from dengue virus

Dengue virus completes its protein synthesis inside human cells on the endoplasmic reticulum membrane by processing the single-chain polyprotein precursor into 10 functional proteins. This vital process relies on the two-component virus-encoded protease complex; nonstructural protein 3 (NS3) possesses the proteolytic activity in its N-terminus, and NS2B acts as a fundamental activator and membrane-anchoring subunit. The membrane-associated NS2B-NS3 complex has essentially not yet been isolated or studied. We describe here a useful protocol for the preparation of the full-length NS2B-NS3 complex from dengue serotype 2 virus by utilizing a Mistic-fusion expression cassette in Escherichia coli. The protease complex was successfully solubilized and stabilized from the bacterial membrane and purified with the use of fos-choline-14 detergent. The detergent-solubilized protease complex retained autolytic activity and, intriguingly, exists as a robust trimer, implying a molecular assembly in the membrane. We further conducted a random mutagenesis study to efficiently scan for entire residues and motifs contributing to autocleavage and provide evidence of the importance of the two distal β-hairpins in the activity of the viral protease. Our results provide the first comprehensive view of an active dengue protease in the membrane-bound form.     

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.     

Expression, purification, and initial structural characterization of nonstructural protein 2B, an integral membrane protein of Dengue-2 virus, in detergent micelles

Dengue virus causes serious diseases affecting people in tropical and sub-tropical regions. The nonstructural (NS) protein 2B is an integral membrane protein and important for the regulation of viral protease NS3, which is significant for virus replication. The NS2B-NS3 complex is an important drug target for treating dengue fever. However, little is known about the structure of NS2B in its entirety. Herein, we describe the expression and purification of this integral membrane protein from cell membrane and inclusion bodies of Escherichia coli cells. The initial nuclear magnetic resonance (NMR) and circular dichroism (CD) results indicate that the purified protein adopts alpha-helical structures in LMPG and TDPC micelles.     

NS3 protease from flavivirus as a target for designing antiviral inhibitors against dengue virus

The development of novel therapeutic agents is essential for combating the increasing number of cases of dengue fever in endemic countries and among a large number of travelers from non-endemic countries. The dengue virus has three structural proteins and seven non-structural (NS) proteins. NS3 is a multifunctional protein with an N-terminal protease domain (NS3pro) that is responsible for proteolytic processing of the viral polyprotein, and a C-terminal region that contains an RNA triphosphatase, RNA helicase and RNA-stimulated NTPase domain that are essential for RNA replication. The serine protease domain of NS3 plays a central role in the replicative cycle of dengue virus. This review discusses the recent structural and biological studies on the NS2B-NS3 protease-helicase and considers the prospects for the development of small molecules as antiviral drugs to target this fascinating, multifunctional protein.