Crystal structure of foot-and-mouth disease virus 3C protease. New insights into catalytic mechanism and cleavage specificity

Foot-and-mouth disease virus (FMDV) causes a widespread and economically devastating disease of domestic livestock. Although FMDV vaccines are available, political and technical problems associated with their use are driving a renewed search for alternative methods of disease control. The viral RNA genome is translated as a single polypeptide precursor that must be cleaved into functional proteins by virally encoded proteases. 10 of the 13 cleavages are performed by the highly conserved 3C protease (3C(pro)), making the enzyme an attractive target for antiviral drugs. We have developed a soluble, recombinant form of FMDV 3C(pro), determined the crystal structure to 1.9-angstroms resolution, and analyzed the cleavage specificity of the enzyme. The structure indicates that FMDV 3C(pro) adopts a chymotrypsin-like fold and possesses a Cys-His-Asp catalytic triad in a similar conformation to the Ser-His-Asp triad conserved in almost all serine proteases. This observation suggests that the dyad-based mechanisms proposed for this class of cysteine proteases need to be reassessed. Peptide cleavage assays revealed that the recognition sequence spans at least four residues either side of the scissile bond (P4-P4') and that FMDV 3C(pro) discriminates only weakly in favor of P1-Gln over P1-Glu, in contrast to other 3C(pro) enzymes that strongly favor P1-Gln. The relaxed specificity may be due to the unexpected absence in FMDV 3C(pro) of an extended beta-ribbon that folds over the substrate binding cleft in other picornavirus 3C(pro) structures. Collectively, these results establish a valuable framework for the development of FMDV 3C(pro) inhibitors.     

Structural and mutagenic analysis of foot-and-mouth disease virus 3C protease reveals the role of the beta-ribbon in proteolysis

The 3C protease (3C(pro)) from foot-and-mouth disease virus (FMDV), the causative agent of a widespread and economically devastating disease of domestic livestock, is a potential target for antiviral drug design. We have determined the structure of a new crystal form of FMDV 3C(pro), a chymotrypsin-like cysteine protease, which reveals features that are important for catalytic activity. In particular, we show that a surface loop which was disordered in previous structures adopts a beta-ribbon structure that is conformationally similar to equivalent regions on other picornaviral 3C proteases and some serine proteases. This beta-ribbon folds over the peptide binding cleft and clearly contributes to substrate recognition. Replacement of Cys142 at the tip of the beta-ribbon with different amino acids has a significant impact on enzyme activity and shows that higher activity is obtained with more hydrophobic side chains. Comparison of the structure of FMDV 3C(pro) with homologous enzyme-peptide complexes suggests that this correlation arises because the side chain of Cys142 contacts the hydrophobic portions of the P2 and P4 residues in the peptide substrate. Collectively, these findings provide compelling evidence for the role of the beta-ribbon in catalytic activity and provide valuable insights for the design of FMDV 3C(pro) inhibitors.     

The leader proteinase of foot-and-mouth disease virus negatively regulates the type I interferon pathway by acting as a viral deubiquitinase

The leader proteinase (L(pro)) of foot-and-mouth disease virus (FMDV) is a papain-like proteinase that plays an important role in FMDV pathogenesis. Previously, it has been shown that L(pro) is involved in the inhibition of the type I interferon (IFN) response by FMDV. However, the underlying mechanisms remain unclear. Here we demonstrate that FMDV Lb(pro), a shorter form of L(pro), has deubiquitinating activity. Sequence alignment and structural bioinformatics analyses revealed that the catalytic residues (Cys51 and His148) are highly conserved in FMDV Lb(pro) of all seven serotypes and that the topology of FMDV Lb(pro) is remarkably similar to that of ubiquitin-specific protease 14 (USP14), a cellular deubiquitylation enzyme (DUB), and to that of severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLpro), a coronaviral DUB. Both purified Lb(pro) protein and in vivo ectopically expressed Lb(pro) removed ubiquitin (Ub) moieties from cellular substrates, acting on both lysine-48- and lysine-63-linked polyubiquitin chains. Furthermore, Lb(pro) significantly inhibited ubiquitination of retinoic acid-inducible gene I (RIG-I), TANK-binding kinase 1 (TBK1), TNF receptor-associated factor 6 (TRAF6), and TRAF3, key signaling molecules in activation of type I IFN response. Mutations in Lb(pro) that ablate the catalytic activity (C51A or D163N/D164N) or disrupt the SAP (for SAF-A/B, Acinus, and PIAS) domain (I83A/L86A) abrogated the DUB activity of Lb(pro) as well as its ability to block signaling to the IFN-β promoter. Collectively, these results demonstrate that FMDV Lb(pro) possesses DUB activity in addition to serving as a viral proteinase and describe a novel mechanism evolved by FMDV to counteract host innate antiviral responses.     

Cleavage of eukaryotic translation initiation factor 4GII within foot-and-mouth disease virus-infected cells: identification of the L-protease cleavage site in vitro

Foot-and-mouth disease virus (FMDV) induces a very rapid inhibition of host cell protein synthesis within infected cells. This is accompanied by the cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI). The cleavage of the related protein eIF4GII has now been analyzed. Within FMDV-infected cells, cleavage of eIF4GI and eIF4GII occurs with similar kinetics. Cleavage of eIF4GII is induced in cells and in cell extracts by the FMDV leader protease (L(pro)) alone, generating cleavage products similar to those induced by enterovirus and rhinovirus 2A protease (2A(pro)). By the use of a fusion protein containing residues 445 to 744 of human eIF4GII, it was demonstrated that the FMDV L(pro) specifically cleaves this protein between residues G700 and S701, immediately adjacent to the site (V699/G700) cleaved by rhinovirus 2A(pro) in vitro. The G700/S701 cleavage site does not correspond, by amino acid sequence alignment, to that cleaved in eIF4GI by the FMDV L(pro) in vitro. Knowledge of the cleavage sites and the three-dimensional structures of the FMDV L(pro) and rhinovirus 2A(pro) enabled mutant forms of the eIF4GII sequence to be generated that are differentially resistant to either one of these proteases. These results confirmed the specificity of each protease and showed that the mutant forms of the fusion protein substrate retained their correct sensitivity to other proteases.     

Inactivation of Foot-and-Mouth Disease Virus with Ethylenimine

Foot-and-mouth disease virus (FMDV) was inactivated by ethylenimine (EI) at three concentrations and two temperatures. Comparison of inactivation kinetics and the antigenic and immunogenic potency of EI and N-acetylethylenimine (AEI)-inactivated FMDV indicates that EI has nearly optimal characteristics as an inactivant for FMDV vaccine preparation. Although AEI-inactivated FMDV has proved to be a potent specific immunogen, an equivalent percentage of EI inactivated FMDV at substantially faster rates and produced an equally potent immunogen. In addition, EI inactivated FMDV at rates that were essentially linear throughout the loss of nearly all measurable infectivity.     

The binding of foot-and-mouth disease virus leader proteinase to eIF4GI involves conserved ionic interactions

The leader proteinase (L(pro)) of foot-and-mouth disease virus (FMDV) initially cleaves itself from the polyprotein. Subsequently, L(pro) cleaves the host proteins eukaryotic initiation factor (eIF) 4GI and 4GII. This prevents protein synthesis from capped cellular mRNAs; the viral RNA is still translated, initiating from an internal ribosome entry site. L(pro) cleaves eIF4GI between residues G674 and R675. We showed previously, however, that L(pro) binds to residues 640-669 of eIF4GI. Binding was substantially improved when the eIF4GI fragment contained the eIF4E binding site and eIF4E was present in the binding assay. L(pro) interacts with eIF4GI via residue C133 and residues 183-195 of the C-terminal extension. This binding domain lies about 25 A from the active site. Here, we examined the binding of L(pro) to eIF4GI fragments generated by in vitro translation to narrow the binding site down to residues 645-657 of human eIF4GI. Comparison of these amino acids with those in human eIF4GII as well as with sequences of eIF4GI from other organisms allowed us to identify two conserved basic residues (K646 and R650). Mutation of these residues was severely detrimental to L(pro) binding. Similarly, comparison of the sequence between residues 183 and 195 of L(pro) with those of other FMDV serotypes and equine rhinitis A virus showed that acidic residues D184 and E186 were highly conserved. Substitution of these residues in L(pro) significantly reduced eIF4GI binding and cleavage without affecting self-processing. Thus, FMDV L(pro) has evolved a domain that specifically recognizes a host cell protein.     

Comparative genomics of foot-and-mouth disease virus

Here we present complete genome sequences, including a comparative analysis, of 103 isolates of foot-and-mouth disease virus (FMDV) representing all seven serotypes and including the first complete sequences of the SAT1 and SAT3 genomes. The data reveal novel highly conserved genomic regions, indicating functional constraints for variability as well as novel viral genomic motifs with likely biological relevance. Previously undescribed invariant motifs were identified in the 5' and 3' untranslated regions (UTR), as was tolerance for insertions/deletions in the 5' UTR. Fifty-eight percent of the amino acids encoded by FMDV isolates are invariant, suggesting that these residues are critical for virus biology. Novel, conserved sequence motifs with likely functional significance were identified within proteins L(pro), 1B, 1D, and 3C. An analysis of the complete FMDV genomes indicated phylogenetic incongruities between different genomic regions which were suggestive of interserotypic recombination. Additionally, a novel SAT virus lineage containing nonstructural protein-encoding regions distinct from other SAT and Euroasiatic lineages was identified. Insights into viral RNA sequence conservation and variability and genetic diversity in nature will likely impact our understanding of FMDV infections, host range, and transmission.     

Residue L143 of the foot-and-mouth disease virus leader proteinase is a determinant of cleavage specificity

The foot-and-mouth disease virus (FMDV) leader proteinase (L(pro)) self-processes inefficiently at the L(pro)/VP4 cleavage site LysLeuLys*GlyAlaGly (* indicates cleaved peptide bond) when the leucine at position P2 is replaced by phenylalanine. Molecular modeling and energy minimization identified the L(pro) residue L143 as being responsible for this discrimination. The variant L(pro) L143A self-processed efficiently at the L(pro)/VP4 cleavage site containing P2 phenylalanine, whereas the L143M variant did not. L(pro) L143A self-processing at the eIF4GII sequence AspPheGly*ArgGlnThr was improved but showed more-extensive aberrant processing. Residue 143 in L(pro) is occupied only by leucine and methionine in all sequenced FMDV serotypes, implying that these bulky side chains are one determinant of the restricted specificity of L(pro).     

Inoculation of swine with foot-and-mouth disease SAP-mutant virus induces early protection against disease

Foot-and-mouth disease virus (FMDV) leader proteinase (L(pro)) cleaves itself from the viral polyprotein and cleaves the translation initiation factor eIF4G. As a result, host cell translation is inhibited, affecting the host innate immune response. We have demonstrated that L(pro) is also associated with degradation of nuclear factor κB (NF-κB), a process that requires L(pro) nuclear localization. Additionally, we reported that disruption of a conserved protein domain within the L(pro) coding sequence, SAP mutation, prevented L(pro) nuclear retention and degradation of NF-κB, resulting in in vitro attenuation. Here we report that inoculation of swine with this SAP-mutant virus does not cause clinical signs of disease, viremia, or virus shedding even when inoculated at doses 100-fold higher than those required to cause disease with wild-type (WT) virus. Remarkably, SAP-mutant virus-inoculated animals developed a strong neutralizing antibody response and were completely protected against challenge with WT FMDV as early as 2 days postinoculation and for at least 21 days postinoculation. Early protection correlated with a distinct pattern in the serum levels of proinflammatory cytokines in comparison to the levels detected in animals inoculated with WT FMDV that developed disease. In addition, animals inoculated with the FMDV SAP mutant displayed a memory T cell response that resembled infection with WT virus. Our results suggest that L(pro) plays a pivotal role in modulating several pathways of the immune response. Furthermore, manipulation of the L(pro) coding region may serve as a viable strategy to derive live attenuated strains with potential for development as effective vaccines against foot-and-mouth disease.     

Expression of foot and mouth disease virus nonstructural polyprotein 3ABC with inactive 3C(pro) in Escherichia coli

Nonstructural 3ABC protein of foot and mouth disease virus (FMDV) was widely used to differentiate vaccinated from natural FMDV-infected animals. 3ABC is a polyprotein which is auto-processed to 3A, three copies of 3B and 3C(pro) by 3C(pro) protease. The 3ABC gene was cloned and expressed in Escherichia coli as native or mutated 3ABC (mu3ABC) forms. Cysteine residues 142 and 163 of the catalytic triad within the 3C(pro) of mu3ABC were changed to serine and glycine, respectively, to inhibit its protease activity. Both native and mutated 3ABC ORFs were cloned into BamHI and HindIII restriction sites of an expression vector, pQE80L. The expression of the recombinant native 3ABC and mu3ABC genes in E. coli BL21 was induced with 0.2mM isopropyl-beta-d-thiogalactopyranoside at 37 °C for 5h. SDS-PAGE and Western blot analysis revealed that the full length 3ABC was present in the lysate from mu3ABC but not native 3ABC transformed cells. The recombinant mu3ABC was expressed mainly in the inclusion body and presented as monomer and dimer. In addition, the mu3ABC reacted strongly with a convalescent serum from a natural FMDV-infected cattle but very weakly with a serum from vaccinated cattle. This study clearly demonstrates that successful expression of the full length 3ABC occurs only when the protease active sites within the 3C(pro) were completely abolished. This information would accelerate in house development of the 3ABC-based diagnostic test that can distinguish between vaccinated and FMDV-infected animals.     

VP1 of serotype C foot-and-mouth disease viruses: long-term conservation of sequences.

The nucleotide sequences of the VP1-coding regions of several isolates of serotype C3 foot-and-mouth disease virus (FMDV) were determined. The deduced amino acid sequences were compared with those of serotype C1 FMDV. The results provide evidence for two different lineages of FMDV C3 and document the potential for both long-term conservation and rapid evolution of FMDV.     

Resiquimod and polyinosinic–polycytidylic acid formulation with aluminum hydroxide as an adjuvant for foot-and-mouth disease vaccine

Background

Toll-like receptor (TLR) agonists reportedly have potent antiviral and antitumor activities and may be a new kind of adjuvant for enhancing immune efficacy. Resiquimod (R848) is an imidazoquinoline compound with potent antiviral activity and functions through the TLR7/TLR8 MyD88-dependent signaling pathway. Polyinosinic-polycytidylic acid [poly(I:C)] is a synthetic analog of double-stranded RNA that induces the production of pro-inflammatory cytokines by the activation of NF-κB through TLR3. This study investigated the potential of R848 and poly(I:C) as an adjuvant 146S foot-and-mouth disease virus (FMDV) vaccine formulated with aluminum hydroxide (Al(OH)₃).

Results

Antibody titers to FMDV and CD8⁺ T cells were markedly enhanced in mice immunized to 146S FMDV?+?Al(OH)₃?+?R848?+?poly(I:C) compared with mice immunized to FMDV?+?ISA206. IFN-γ secretion substantially increased compared with IL-4 secretion by splenic T cells stimulated with FMDV antigens in vitro, suggesting that R848, poly(I:C), and with Al(OH)₃ together biased the immune response toward a Th1-type direction.

Conclusions

These results indicated that the R848 and poly(I:C) together with Al(OH)₃ enhanced humoral and cellular immune responses to immunization with 146S FMDV antigens. Thus, this new vaccine formulation can be used for FMDV prevention.     

Foot-and-mouth disease virus, but not bovine enterovirus, targets the host cell cytoskeleton via the nonstructural protein 3Cpro

Foot-and-mouth disease virus (FMDV), a member of the Picornaviridae, is a pathogen of cloven-hoofed animals and causes a disease of major economic importance. Picornavirus-infected cells show changes in cell morphology and rearrangement of cytoplasmic membranes, which are a consequence of virus replication. We show here, by confocal immunofluorescence and electron microscopy, that the changes in morphology of FMDV-infected cells involve changes in the distribution of microtubule and intermediate filament components during infection. Despite the continued presence of centrosomes in infected cells, there is a loss of tethering of microtubules to the microtubule organizing center (MTOC) region. Loss of labeling for gamma-tubulin, but not pericentrin, from the MTOC suggests a targeting of gamma-tubulin (or associated proteins) rather than a total breakdown in MTOC structure. The identity of the FMDV protein(s) responsible was determined by the expression of individual viral nonstructural proteins and their precursors in uninfected cells. We report that the only viral nonstructural protein able to reproduce the loss of gamma-tubulin from the MTOC and the loss of integrity of the microtubule system is FMDV 3C(pro). In contrast, infection of cells with another picornavirus, bovine enterovirus, did not affect gamma-tubulin distribution, and the microtubule network remained relatively unaffected.     

Generation of monoclonal antibodies against non-structural protein 3AB of foot-and-mouth disease virus

To identify linear epitopes on the non-structural protein 3AB of foot-and-mouth disease virus (FMDV), BABL/c mice were immunized with the 3AB protein and splenocytes of BALB/c mice were fused with myeloma Sp2/0 cells. Two hybridoma monoclonal antibodies (mAbs) cell lines against the 3AB protein of foot-and-mouth disease virus (FMDV) were obtained, named C6 and E7 respectively. The microneutralization titer was 1:1024 for mAb C6, and 1:512 for E7. Both mAbs contain kappa light chains, and were of subclass IgG2b. In order to define the mAbs binding epitopes, the reactivity of these mAbs against FMDV were examined by indirect ELISA. The results showed that both mAbs can react with FMDV, but had no cross-reactivity with Swine Vesicular Disease (SVD) antigens. The titers in abdomen liquor were 1:5×10⁶ for C6 and 1:2×10⁶ for E7. In conclusion, the mAbs obtained from this study are specific for the detection of FMDV, can be used for etiological and immunological researches on FMDV, and have potential use in diagnosis and future vaccine designs.     

Intranasal delivery of cationic PLGA nano/microparticles-loaded FMDV DNA vaccine encoding IL-6 elicited protective immunity against FMDV challenge

Mucosal vaccination has been demonstrated to be an effective means of eliciting protective immunity against aerosol infections of foot and mouth disease virus (FMDV) and various approaches have been used to improve mucosal response to this pathogen. In this study, cationic PLGA (poly(lactide-co-glycolide)) nano/microparticles were used as an intranasal delivery vehicle as a means administering FMDV DNA vaccine encoding the FMDV capsid protein and the bovine IL-6 gene as a means of enhancing mucosal and systemic immune responses in animals. Three eukaryotic expression plasmids with or without bovine IL-6 gene (pc-P12A3C, pc-IL2AP12A3C and pc-P12AIL3C) were generated. The two latter plasmids were designed with the IL-6 gene located either before or between the P12A and 3C genes, respectively, as a means of determining if the location of the IL-6 gene affected capsid assembly and the subsequent immune response. Guinea pigs and rats were intranasally vaccinated with the respective chitosan-coated PLGA nano/microparticles-loaded FMDV DNA vaccine formulations. Animals immunized with pc-P12AIL3C (followed by animals vaccinated with pc-P12A3C and pc-IL2AP12A3C) developed the highest levels of antigen-specific serum IgG and IgA antibody responses and the highest levels of sIgA (secretory IgA) present in mucosal tissues. However, the highest levels of neutralizing antibodies were generated in pc-IL2AP12A3C-immunized animals (followed by pc-P12AIL3C- and then in pc-P12A3C-immunized animals). pc-IL2AP12A3C-immunized animals also developed stronger cell mediated immune responses (followed by pc-P12AIL3C- and pc-P12A3C-immunized animals) as evidenced by antigen-specific T-cell proliferation and expression levels of IFN-γ by both CD4+ and CD8+ splenic T cells. The percentage of animals protected against FMDV challenge following immunizations with pc-IL2AP12A3C, pc-P12AIL3C or pc-P12A3C were 3/5, 1/5 and 0/5, respectively. These data suggested that intranasal delivery of cationic PLGA nano/microparticles loaded with various FMDV DNA vaccine formulations encoding IL-6 as a molecular adjuvant enhanced protective immunity against FMDV, particularly pc-IL2AP12A3C with IL-6 gene located before P12A3C gene.     

Role of RNA structure and RNA binding activity of foot-and-mouth disease virus 3C protein in VPg uridylylation and virus replication

The uridylylation of the VPg peptide primer is the first stage in the replication of picornavirus RNA. This process can be achieved in vitro using purified components, including 3B (VPg) with the RNA dependent RNA polymerase (3Dpol), the precursor 3CD, and an RNA template containing the cre/bus. We show that certain RNA sequences within the foot-and-mouth disease virus (FMDV) 5' untranslated region but outside of the cre/bus can enhance VPg uridylylation activity. Furthermore, we have shown that the FMDV 3C protein alone can substitute for 3CD, albeit less efficiently. In addition, the VPg precursors, 3B(3)3C and 3B(123)3C, can function as substrates for uridylylation in the absence of added 3C or 3CD. Residues within the FMDV 3C protein involved in interaction with the cre/bus RNA have been identified and are located on the face of the protein opposite from the catalytic site. These residues within 3C are also essential for VPg uridylylation activity and efficient virus replication.     

共表达口蹄疫病毒衣壳前体蛋白P1-2A基因和蛋白酶3C基因牛肾细胞株的筛选及稳定性

[目的]筛选稳定表达口蹄疫病毒衣壳蛋白的牛肾细胞(Madin-Darby bovinekidney,MDBK)株.[方法]采用聚合酶链式反应(Polymerase chain reaction,PCR)方法从重组质粒pMD-P1-2A和pMD-3C中分别扩增口蹄疫病毒衣壳前体蛋白P1-2A基因和蛋白酶3C基因,将两基因依次插入逆转录病毒载体pBABE-puro.重组逆转录病毒载体pBABE-puro/P1-2A-3C和pVSV-G质粒载体用脂质体介导共转染GP2-293包装细胞.产生的重组逆转录病毒感染MDBK细胞后使用嘌呤霉素筛选抗性细胞.利用克隆环套取法得到单克隆细胞.经间接免疫荧光和酶联免疫吸附测定(Enzyme-linkedimmunosorbent assay,ELISA)方法检测MDBK细胞中衣壳蛋白的表达,并在电镜下观察口蹄疫病毒空衣壳.[结果]成功筛选到稳定表达口蹄疫病毒衣壳蛋白的MDBK细胞株,衣壳前体蛋白P1-2A在蛋白酶3C裂解作用下正确组装成空衣壳.[结论]该研究为口蹄疫亚单位疫苗的研制提供了实验材料.