Mutations at KFRDI and VGK domains of enterovirus 71 3C protease affect its RNA binding and proteolytic activities

The 3C proteases (3C^pro) of enterovirus 71 (EV71) is a good molecular target for drug discovery. Notably, this protease was found to possess RNA-binding activity. The regions responsible for RNA binding were classified as KFRDI (positions 82–86) and VGK (positions 154–156) in 3C^pro by mutagenesis study. Although the RNA-binding regions are structurally distinct from the catalytic site of EV71 3C^pro, mutations in the RNA-binding regions influenced 3C^pro proteolytic activity. In contrast, mutations at the catalytic site had almost no influence on RNA binding ability. We identified certain mutations within 3C^pro which abrogated both the RNA-binding activity of the expressed, recombinant, protease and the ability to rescue virus from an infectious full-length clone of EV71 (pEV71). Interestingly, mutation at position 84 from Arg(R) to Lys(K) was found to retain good RNA binding and proteolytic activity for the recombinant 3C^pro; however, no virus could be rescued when pEV71 with the R84K mutation was introduced into the infectious copy. Together, these results may provide useful information for using 3C^pro as the molecular target to develop anti-EV71 agents.The second and the third authors contributed equally to this work.     

Optimize the interactions at S4 with efficient inhibitors targeting 3C proteinase from enterovirus 71

Enterovirus 71 (EV71) is the causative agent of hand, foot and mouth disease and can spread its infections to the central nervous and other systems with severe consequences. The replication of EV71 depends on its 3C proteinase (3C^pro), a significant drug target. By X‐ray crystallography and functional assays, the interactions between inhibitors and EV71 3C^pro were evaluated. It was shown that improved interactions at S4 for the substrate binding could significantly enhance the potency. A new series of potent inhibitors with high ligand efficiency was generated for developing antivirals to treat and control the EV71‐associated diseases. Copyright © 2016 John Wiley & Sons, Ltd.     

EV71 3C protease cleaves host anti-viral factor OAS3 and enhances virus replication

The global spread of enteroviruses (EVs) has become more frequent, severe and life-threatening. Intereron (IFN) I has been proved to control EVs by regulating IFN-stimulated genes (ISG) expression. 20-50-oligoadenylate synthetases 3 (OAS3) is an important ISG in the OAS/RNase L antiviral system. The relationship between OAS3 and EVs is still unclear. Here, we reveal that OAS3, superior to OAS1 and OAS2, significantly inhibited EV71 replication in vitro. However, EV71 utilized autologous 3C protease (3C^pro) to cleave intracellular OAS3 and enhance viral replication. Rupintrivir, a human rhinovirus 3C protease inhibitor, completely abolished the cleavage of EV71 3C^pro on OAS3. And the proteolytically deficient mutants H40G, E71A, and C147G of EV71 3C^pro also lost the ability of OAS3 cleavage. Mechanistically, the Q982-G983 motif in C-terminal of OAS3 was identified as a crucial 3C^pro cutting site. Further investigation indicated that OAS3 inhibited not only EV71 but also Coxsackievirus B3 (CVB3), Coxsackievirus A16 (CA16), Enterovirus D68 (EVD68), and Coxsackievirus A6 (CA6) subtypes. Notably, unlike other four subtypes, CA16 3C^pro could not cleave OAS3. Two key amino acids variation Ile36 and Val86 in CA16 3C^pro might result in weak and delayed virus replication of CA16 because of failure of OAS and 3AB cleavage. Our works elucidate the broad anti-EVs function of OAS3, and illuminate a novel mechanism by which EV71 use 3C^pro to escape the antiviral effect of OAS3. These findings can be an important entry point for developing novel therapeutic strategies for multiple EVs infection.     

Insights into Cleavage Specificity from the Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease Complexed with a Peptide Substrate

Picornavirus replication is critically dependent on the correct processing of a polyprotein precursor by 3C protease(s) (3C^pro) at multiple specific sites with related but non-identical sequences. To investigate the structural basis of its cleavage specificity, we performed the first crystallographic structural analysis of non-covalent complexes of a picornavirus 3C^pro with peptide substrates. The X-ray crystal structure of the foot-and-mouth disease virus 3C^pro, mutated to replace the catalytic Cys by Ala and bound to a peptide (APAKQ|LLNFD) corresponding to the P5-P5′ region of the VP1-2A cleavage junction in the viral polyprotein, was determined up to 2.5 Å resolution. Comparison with free enzyme reveals significant conformational changes in 3C^pro on substrate binding that lead to the formation of an extended interface of contact primarily involving the P4-P2′ positions of the peptide. Strikingly, the deep S1′ specificity pocket needed to accommodate P1′-Leu only forms when the peptide binds. Substrate specificity was investigated using peptide cleavage assays to show the impact of amino acid substitutions within the P5-P4′ region of synthetic substrates. The structure of the enzyme-peptide complex explains the marked substrate preferences for particular P4, P2 and P1 residue types, as well as the relative promiscuity at P3 and on the P′ side of the scissile bond. Furthermore, crystallographic analysis of the complex with a modified VP1-2A peptide (APAKE|LLNFD) containing a Gln-to-Glu substitution reveals an identical mode of peptide binding and explains the ability of foot-and-mouth disease virus 3C^pro to cleave sequences containing either P1-Gln or P1-Glu. Structure-based mutagenesis was used to probe interactions within the S1′ specificity pocket and to provide direct evidence of the important contribution made by Asp84 of the Cys-His-Asp catalytic triad to proteolytic activity. Our results provide a new level of detail in our understanding of the structural basis of polyprotein cleavage by 3C^pro.     

Enterovirus 71 Protease 2Apro Targets MAVS to Inhibit Anti-Viral Type I Interferon Responses

Enterovirus 71 (EV71) is the major causative pathogen of hand, foot, and mouth disease (HFMD). Its pathogenicity is not fully understood, but innate immune evasion is likely a key factor. Strategies to circumvent the initiation and effector phases of anti-viral innate immunity are well known; less well known is whether EV71 evades the signal transduction phase regulated by a sophisticated interplay of cellular and viral proteins. Here, we show that EV71 inhibits anti-viral type I interferon (IFN) responses by targeting the mitochondrial anti-viral signaling (MAVS) protein—a unique adaptor molecule activated upon retinoic acid induced gene-I (RIG-I) and melanoma differentiation associated gene (MDA-5) viral recognition receptor signaling—upstream of type I interferon production. MAVS was cleaved and released from mitochondria during EV71 infection. An in vitro cleavage assay demonstrated that the viral 2A protease (2A^pro), but not the mutant 2A^pro (2A^pro-110) containing an inactivated catalytic site, cleaved MAVS. The Protease-Glo assay revealed that MAVS was cleaved at 3 residues between the proline-rich and transmembrane domains, and the resulting fragmentation effectively inactivated downstream signaling. In addition to MAVS cleavage, we found that EV71 infection also induced morphologic and functional changes to the mitochondria. The EV71 structural protein VP1 was detected on purified mitochondria, suggesting not only a novel role for mitochondria in the EV71 replication cycle but also an explanation of how EV71-derived 2A^pro could approach MAVS. Taken together, our findings reveal a novel strategy employed by EV71 to escape host anti-viral innate immunity that complements the known EV71-mediated immune-evasion mechanisms.     

Autoprocessing: an essential step for expression and purification of enterovirus 71 3Cpro in Escherichia coli

A gene encoding the 3BC of human enterovirus 71 (EV71) was cloned and inserted into a derivative of plasmid pET-32a(+) driven by T7 promoter. The expressed 3C protease (3C^pro) autocatalytically cleaved itself from the recombinant protein Trx-3BC and the mature 3C^pro partitioned in the soluble fraction of bacterial lysate. The 13-amino-acid peptide substrates with the junction of 3B/3C were used to verify the proteolysis activity of the purified 3C^pro. The EV71 3C^pro had a K_m value of 63 μM (measured by a continuous fluorescence assay). The other solid-phase activity assay of the EV71 3C^pro was developed using HPLC to analyze the proteolytic products. The combination of two activity assays contributes to promote the identification of the specific inhibitors targeted to the EV71 3C^pro.     

以EV71 3Cpro为靶标的抗病毒药物筛选模型的建立及抗3Cpro化合物筛选

建立一种以EV71 3C蛋白酶为靶标的抗肠病毒药物筛选模型,并应用于小分子化合物库筛选具有抗EV71活性的化合物.从临床手足口病例标本中分离肠道病毒进行PCR鉴定及基因组测序.通过插入突变在黄色荧光YFP编码框合适位点处引入EV71 3C酶切位点,构建对3C蛋白酶敏感的报告质粒pc DNA3-m YFP,然后将其与表达3C的质粒共转293A细胞,在3C抑制剂Rupintrivir存在与否的情况下通过荧光显微镜和酶标仪检测Ex(500nm)/Em(535nm)荧光信号的变化,判断建模是否成功;利用建好的筛选模型在高通量药物筛选平台对小分子化合物库进行初筛和复筛;再利用空斑分析检测筛选出的活性化合物是否对临床分离的EV71毒株具有抑制作用.m YFP在293A细胞中表达良好,3C的表达使荧光信号下降80%,Rupintrivir的存在则几乎不影响荧光表达,说明以3C为靶位的筛选模型构建成功.经过高通量初筛和复筛从26 000多种小分子化合物中获得26种能够显著回复m YFP表达的活性化合物;空斑分析显示其中2种化合物具有较为明显的抑制EV71复制的活性.因此,我们所构建的3C-m YFP共表达系统是一种简便有效的、可用于高通量筛选抗EV71 3C~(pro)药物的筛选模型.     

Enterovirus 71 3C Protease Cleaves a Novel Target CstF-64 and Inhibits Cellular Polyadenylation

Identification of novel cellular proteins as substrates to viral proteases would provide a new insight into the mechanism of cell–virus interplay. Eight nuclear proteins as potential targets for enterovirus 71 (EV71) 3C protease (3C^pro) cleavages were identified by 2D electrophoresis and MALDI-TOF analysis. Of these proteins, CstF-64, which is a critical factor for 3′ pre-mRNA processing in a cell nucleus, was selected for further study. A time-course study to monitor the expression levels of CstF-64 in EV71-infected cells also revealed that the reduction of CstF-64 during virus infection was correlated with the production of viral 3C^pro. CstF-64 was cleaved in vitro by 3C^pro but neither by mutant 3C^pro (in which the catalytic site was inactivated) nor by another EV71 protease 2A^pro. Serial mutagenesis was performed in CstF-64, revealing that the 3C^pro cleavage sites are located at position 251 in the N-terminal P/G-rich domain and at multiple positions close to the C-terminus of CstF-64 (around position 500). An accumulation of unprocessed pre-mRNA and the depression of mature mRNA were observed in EV71-infected cells. An in vitro assay revealed the inhibition of the 3′-end pre-mRNA processing and polyadenylation in 3C^pro-treated nuclear extract, and this impairment was rescued by adding purified recombinant CstF-64 protein. In summing up the above results, we suggest that 3C^pro cleavage inactivates CstF-64 and impairs the host cell polyadenylation in vitro, as well as in virus-infected cells. This finding is, to our knowledge, the first to demonstrate that a picornavirus protein affects the polyadenylation of host mRNA.     

A picornaviral loop-to-loop replication complex

Picornaviruses replicate their RNA genomes through a highly conserved mechanism that involves an interaction between the principal viral protease (3C^pro) and the 5′-UTR region of the viral genome. The 3C^pro catalytic site is the target of numerous replication inhibitors. This paper describes the first structural model of a complex between a picornaviral 3C^pro and a region of the 5′-UTR, stem-loop D (SLD). Using human rhinovirus as a model system, we have combined NMR contact information, small-angle X-ray scattering (SAXS) data, and previous mutagenesis results to determine the shape, position and relative orientation of the 3C^pro and SLD components. The results clearly identify a 1:1 binding stoichiometry, with pronounced loops from each molecule providing the key binding determinants for the interaction. Binding between SLD and 3C^pro induces structural changes in the proteolytic active site that is positioned on the opposite side of the protease relative to the RNA/protein interface, suggesting that subtle conformational changes affecting catalytic activity are relayed through the protein.     

Fragment-wise design of inhibitors to 3C proteinase from enterovirus 71

BackgroundEnterovirus 71 (EV71) is a causative agent of hand, foot and mouth disease (HFMD), which can spread its infection to central nervous and other systems with severe consequence. A key factor in the replication of EV71 is its 3C proteinase (3C^pro), a significant drug target. Peptidomimetics were employed as inhibitors of this enzyme for developing antivirals. However, the peptide bonds in these peptidomimetics are a source of low bioavailability due to their susceptibility to protease digestion. To produce non-peptidomimetic inhibitors by replacing these peptide bonds, it would be important to gain better understanding on the contribution of each component to the interaction and potency.MethodsA series of compounds of different lengths targeting 3C^pro and having an α,β-unsaturated ester as the warhead were synthesized and their interactions with the enzyme were evaluated by complex structure analyses and potency assays for a better understanding on the relationship between potency and evolution of interaction.ResultsThe P2 moiety of the compound would need to be oriented to interact in the S2 site in the substrate binding cleft and the P3–P4 moieties were required to generate sufficient potency. A hydrophobic terminal group will benefit the cellular uptake and improve the activity in vivo.Conclusions and general significanceThe data presented here provide a basis for designing a new generation of non-peptidomimetics to target EV71 3C^pro.     

siRNA Targeting the 2Apro Genomic Region Prevents Enterovirus 71 Replication In Vitro

Enterovirus 71 (EV71) is the most important etiological agent of hand, foot, and mouth disease (HFMD) in young children, which is associated with severe neurological complications and has caused significant mortalities in recent HFMD outbreaks in Asia. However, there is no effective antiviral therapy against EV71. In this study, RNA interference (RNAi) was used as an antiviral strategy to inhibit EV71 replication. Three small interfering RNAs (siRNAs) targeting the 2A^pro region of the EV71 genome were designed and synthesized. All the siRNAs were transfected individually into rhabdomyosarcoma (RD) cells, which were then infected with strain EV71-2006-52-9. The cytopathic effects (CPEs) in the infected RD cells, cell viability, viral titer, and viral RNA and protein expression were examined to evaluate the specific viral inhibition by the siRNAs. The results of cytopathogenicity and MTT tests indicated that the RD cells transfected with the three siRNAs showed slight CPEs and significantly high viability. The 50% tissue culture infective dose (TCID₅₀) values demonstrated that the viral titer of the groups treated with three siRNAs were lower than those of the control groups. qRT–PCR and western blotting revealed that the levels of viral RNA and protein in the RD cells treated with the three siRNAs were lower than those in the controls. When RD cells transfected with siRNAs were also infected with strain EV71-2008-43-16, the expression of the VP1 protein was significantly inhibited. The levels of interferon α (IFN-α) and IFN-β did not differ significantly in any group. These results suggest that siRNAs targeting the 2A^pro region of the EV71 genome exerted antiviral effects in vitro.     

Homology modeling, docking, molecular dynamics simulation, and structural analyses of coxsakievirus B3 2A protease: an enzyme involved in the pathogenesis of inflammatory myocarditis

2A protease of the pathogenic coxsackievirus B3 is key to the pathogenesis of inflammatory myocarditis and, therefore, an attractive drug target. However lack of a crystal structure impedes design of inhibitors. Here we predict 3D structure of CVB3 2A^pro based on sequence comparison and homology modeling with human rhinovirus 2A^pro. The two enzymes are remarkably similar in their core regions. However they have different conformations at the N-terminal. A large number of N-terminal hydrophobic residues reduce the thermal stability of CVB3 2A^pro, as we confirmed by fluorescence, western blot and turbidity measurement. Molecular dynamic simulation revealed that elevated temperature induces protein motion that results in frequent movement of the N-terminal coil. This may therefore induce successive active site changes and thus play an important role in destabilization of CVB3 2A^pro structure.     

Structural basis for antiviral inhibition of the main protease, 3C, from human enterovirus 93

Members of the Enterovirus genus of the Picornaviridae family are abundant, with common human pathogens that belong to the rhinovirus (HRV) and enterovirus (EV) species, including diverse echo-, coxsackie- and polioviruses. They cause a wide spectrum of clinical manifestations ranging from asymptomatic to severe diseases with neurological and/or cardiac manifestations. Pandemic outbreaks of EVs may be accompanied by meningitis and/or paralysis and can be fatal. However, no effective prophylaxis or antiviral treatment against most EVs is available. The EV RNA genome directs the synthesis of a single polyprotein that is autocatalytically processed into mature proteins at Gln↓Gly cleavage sites by the 3C protease (3C^pro), which has narrow, conserved substrate specificity. These cleavages are essential for virus replication, making 3C^pro an excellent target for antivirus drug development. In this study, we report the first determination of the crystal structure of 3C^pro from an enterovirus B, EV-93, a recently identified pathogen, alone and in complex with the anti-HRV molecules compound 1 (AG7404) and rupintrivir (AG7088) at resolutions of 1.9, 1.3, and 1.5 Å, respectively. The EV-93 3C^pro adopts a chymotrypsin-like fold with a canonically configured oxyanion hole and a substrate binding pocket similar to that of rhino-, coxsackie- and poliovirus 3C proteases. We show that compound 1 and rupintrivir are both active against EV-93 in infected cells and inhibit the proteolytic activity of EV-93 3C^pro in vitro. These results provide a framework for further structure-guided optimization of the tested compounds to produce antiviral drugs against a broad range of EV species.     

Antiviral effects of two Ganoderma lucidum triterpenoids against enterovirus 71 infection

Enterovirus 71 (EV71) is a major causative agent for hand, foot and mouth disease (HFMD), and fatal neurological and systemic complications in children. However, there is currently no clinical approved antiviral drug available for the prevention and treatment of the viral infection. Here, we evaluated the antiviral activities of two Ganoderma lucidum triterpenoids (GLTs), Lanosta-7,9(11),24-trien-3-one,15;26-dihydroxy (GLTA) and Ganoderic acid Y (GLTB), against EV71 infection. The results showed that the two natural compounds display significant anti-EV71 activities without cytotoxicity in human rhabdomyosarcoma (RD) cells as evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay. The mechanisms by which the two compounds affect EV71 infection were further elucidated by three action modes using Ribavirin, a common antiviral drug, as a positive control. The results suggested that GLTA and GLTB prevent EV71 infection through interacting with the viral particle to block the adsorption of virus to the cells. In addition, the interactions between EV71 virion and the compounds were predicated by computer molecular docking, which illustrated that GLTA and GLTB may bind to the viral capsid protein at a hydrophobic pocket (F site), and thus may block uncoating of EV71. Moreover, we demonstrated that GLTA and GLTB significantly inhibit the replication of the viral RNA (vRNA) of EV71 replication through blocking EV71 uncoating. Thus, GLTA and GLTB may represent two potential therapeutic agents to control and treat EV71 infection.     

Peptidomimetic ethyl propenoate covalent inhibitors of the enterovirus 71 3C protease: a P2–P4 study

Enterovirus 71 (EV71) is a highly infectious pathogen primarily responsible for Hand, Foot, and Mouth Disease, particularly among children. Currently, no approved antiviral drug has been developed against this disease. The EV71 3C protease is deemed an attractive drug target due to its crucial role in viral polyprotein processing. Rupintrivir, a peptide-based inhibitor originally developed to target the human rhinovirus 3C protease, was found to inhibit the EV71 3C protease. In this communication, we report the inhibitory activities of 30 Rupintrivir analogs against the EV71 3C protease. The most potent inhibitor, containing a P2 ring-constrained phenylalanine analog (compound 9), was found to be two-fold more potent than Rupintrivir (IC₅₀ value 3.4?±?0.4 versus 7.3?±?0.8?μM). Our findings suggest that employing geometrically constrained residues in peptide-based protease inhibitors can potentially enhance their inhibitory activities.     

Virucidal activity and the antiviral mechanism of acidic polysaccharides against Enterovirus 71 infection in vitro

Enterovirus 71 (EV71) is the predominant pathogen for severe hand, foot, and mouth disease (HFMD) in children younger than 5 years, and currently no effective drugs are available for EV71. Thus, there is an urgent need to develop new drugs for the control of EV71 infection. In this study, LJ04 was extracted from Laminaria japonica using diethylaminoethyl cellulose-52 with 0.4 mol/l NaCl as the eluent, and its virucidal activity was evaluated based on its cytopathic effects on a microplate. LJ04 is composed of fucose, galactose, and mannose and mainly showed good virucidal activity against EV71. The antiviral mechanisms of LJ04 were the direct inactivation of the virus, the blockage of virus binding, disruptions to viral entry, and weak inhibitory activity against the nonstructural protein 3C. The two most important findings from this study were that LJ04 inhibited EV71 proliferation in HM1900 cells, which are a human microglia cell line, and that LJ04 can directly inactivate EV71 within 2 hr at 37°C. This study demonstrates for the first time the ability of a polysaccharide from L. japonica to inhibit viral and 3C activity; importantly, the inhibition of 3C might have a minor effect on the antiviral effect of LJ04. Consequently, our results identify LJ04 as a potential drug candidate for the control of severe EV71 infection in clinical settings.