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The shutoff of host protein synthesis by certain picornaviruses is mediated, at least in part, by proteolytic cleavage of eIF4G-1. Previously, we developed a cleavage site variant of eIF4G-1, termed eIF4G-1(SM), that was 100-fold more resistant to in vitro cleavage by Coxsackievirus 2A protease (2A(Pro)) than wild-type eIF4G-1 (eIF4G-1(WT)), but it was still digested at high protease concentrations. Here we identified a secondary cleavage site upstream of the primary site. We changed Gly at the P1'-position of the secondary site to Ala to produce eIF4G-1(DM). eIF4G-1(DM) was 1,000-10,000-fold more resistant to cleavage in vitro than eIF4G-1(WT). Full functional activity of eIF4G-1(DM) was demonstrated in vitro by its ability to restore cap-dependent translation to a 2A(Pro)-pretreated rabbit reticulocyte system. An isoform containing the binding site for poly(A)-binding protein, eIF4G-1e(DM), was more active in this assay than an isoform lacking it, eIF4G-1a(DM), but only with polyadenylated mRNA. Functional activity was also demonstrated in vivo with stably transfected HeLa cells expressing eIF4G-1(DM) from a tetracycline-regulated promoter. Cap-dependent green fluorescent protein synthesis was drastically inhibited by 2A(Pro) expression, but synthesis was almost fully restored by induction of either eIF4G-1a(DM) or eIF4G-1e(DM). By contrast, encephalomyocarditis virus internal ribosome entry site-dependent green fluorescent protein synthesis was stimulated by 2A(Pro); stimulation was suppressed by eIF4G-1e(DM) but not eIF4G-1a(DM).

Background: Infection with enteroviruses like coxsackievirus B3 (CVB3) as well as genetic dystrophin deficiency can cause dilated cardiomyopathy. We recently identified cleavage and functional impairment of dystrophin by the viral protease 2A during CVB3-infection as a molecular mechanism that may contribute to the pathogenesis of enterovirus-induced cardiomyopathy. Nitric oxide (NO) is elevated in human dilated cardiomyopathy, but the relevance of this finding is unknown. In mice, NO inhibits CVB3 myocarditis. Therefore, we investigated the effects of NO on the coxsackieviral protease 2A., Methods and Results: In vitro, NO donors like PAPA-NONOate inhibited the cleavage of human and mouse dystrophin by recombinant coxsackievirus B protease 2A in a dose-dependent manner (IC(50), 51 micromol/L). In CVB3-infected HeLa cells, addition of the NO donor SNAP inhibited protease 2A catalytic activity on dystrophin. Because this inhibitory effect was reversed by the thiol-protecting agent DTT, we investigated whether NO S:-nitrosylates the protease 2A. In vitro, NO nitrosylated the active-site cysteine (C110) of the coxsackieviral protease 2A, as demonstrated by site-directed mutagenesis. Within living COS-7 cells, SNAP-induced S:-nitrosylation of this site was confirmed with electron spin resonance spectroscopy., Conclusions: These data demonstrate inactivation of a coxsackieviral protease 2A by NO through active-cysteine S:-nitrosylation in vitro and intracellularly. Given that the enteroviral protease 2A cleaves mouse and human dystrophin, NO may be protective in human heart failure with an underlying enteroviral pathogenesis through inhibition of dystrophin proteolysis.

Enteroviruses such as Coxsackievirus B3 can cause dilated cardiomyopathy through unknown pathological mechanism(s). Dystrophin is a large extrasarcomeric cytoskeletal protein whose genetic deficiency causes hereditary dilated cardiomyopathy. In addition, we have recently shown that dystrophin is proteolytically cleaved by the Coxsackievirus protease 2A leading to functional impairment and morphological disruption. However, the mechanism of dystrophin cleavage and the exact cleavage site remained to be identified. Antibody epitope mapping of endogenous dystrophin indicated protease 2A-mediated cleavage at the site in the hinge 3 region predicted by a neural network algorithm (human, amino acid 2434; mouse, amino acid 2427). Using site-directed mutagenesis, peptide sequencing, and fluorescence resonance energy transfer assays with recombinant dystrophin, we demonstrate that this putative site in mouse and human dystrophin is a direct substrate for the Coxsackieviral protease 2A both in vitro and in vivo. The substrate analogue protease inhibitor z-LSTT-fmk was designed based on the dystrophin sequence that interacts with the protease 2A and was found to have an IC(50) of 550 nM in vitro. Dystrophin is the first cellular substrate of the enteroviral protease 2A that was identified using by a bioinformatic approach and for which the cleavage site was molecularly mapped within living cells.

Enteroviruses such as Coxsackievirus B3 can cause dilated cardiomyopathy, but the mechanism of this pathology is unknown. Mutations in cytoskeletal proteins such as dystrophin cause hereditary dilated cardiomyopathy, but it is unclear if similar mechanisms underlie acquired forms of heart failure. We demonstrate here that purified Coxsackievirus protease 2A cleaves dystrophin in vitro as predicted by computer analysis. Dystrophin is also cleaved during Coxsackievirus infection of cultured myocytes and in infected mouse hearts, leading to impaired dystrophin function. In vivo, dystrophin and the dystrophin-associated glycoproteins alpha-sarcoglycan and beta-dystroglycan are morphologically disrupted in infected myocytes. We suggest a molecular mechanism through which enteroviral infection contributes to the pathogenesis of acquired forms of dilated cardiomyopathy.

Infection of cells by picornaviruses of the rhinovirus, aphthovirus, and enterovirus groups results in the shutoff of host protein synthesis but allows viral protein synthesis to proceed. Although considerable evidence suggests that this shutoff is mediated by the cleavage of eukaryotic translation initiation factor eIF4G by sequence-specific viral proteases (2A protease in the case of coxsackievirus), several experimental observations are at variance with this view. Thus, the cleavage of other cellular proteins could contribute to the shutoff of host protein synthesis and stimulation of viral protein synthesis. Recent evidence indicates that the highly conserved 70-kDa cytoplasmic poly(A)-binding protein (PABP) participates directly in translation initiation. We have now found that PABP is also proteolytically cleaved during coxsackievirus infection of HeLa cells. The cleavage of PABP correlated better over time with the host translational shutoff and onset of viral protein synthesis than did the cleavage of eIF4G. In vitro experiments with purified rabbit PABP and recombinant human PABP as well as in vivo experiments with Xenopus oocytes and recombinant Xenopus PABP demonstrate that the cleavage is catalyzed by 2A protease directly. N- and C-terminal sequencing indicates that cleavage occurs uniquely in human PABP at 482VANTSTQTM downward arrowGPRPAAAAAA500, separating the four N-terminal RNA recognition motifs (80%) from the C-terminal homodimerization domain (20%). The N-terminal cleavage product of PABP is less efficient than full-length PABP in restoring translation to a PABP-dependent rabbit reticulocyte lysate translation system. These results suggest that the cleavage of PABP may be another mechanism by which picornaviruses alter the rate and spectrum of protein synthesis.

Previously, the purified recombinant 2A proteases (2Apro) of coxsackievirus B4 (CVB4) and human rhinovirus type 2 (HRV2) were shown to cleave synthetic peptides derived from human or rabbit elF4G as well as elF4G protein purified from rabbit reticulocytes. These results were in contrast to previous evidence which supported the view that elF4G cleavage activity in poliovirus-infected HeLa cells required a cellular factor(s) activated by poliovirus (PV) 2Apro. In the present study, recombinant PV 2Apro was shown to cleave either rabbit or human elF4G or their derived peptides in direct cleavage reactions, but cleaved the 4G-derived peptides with 100-fold lower efficiency than with a peptide derived from the poliovirus polyprotein. In these experiments, up to 25-fold molar excess of 2Apro over elF4G protein was required to cause greater than 50% cleavage. CVB4 2Apro was also tested in peptide cleavage assays under the same conditions as PV 2Apro and was found to cleave all elF4G substrates with efficiencies similar to PV 2Apro. Finally, cleavage reactions utilizing recombinant elF4G containing a G486E substitution at the cleavage site for CVB4 and HRV2 proteases resulted in drastically reduced cleavage by PV 2Apro, similar to the reduction previously seen with HRV2 and CVB4 2Apro, confirming that all three viral 2A proteases recognize the same cleavage site on elF4G. These data show that PV 2Apro can directly cleave elF4G in vitro with efficiencies similar to those of CVB 2Apro, but cleavage efficiency of elF4G is approximately 1000-fold lower than cleavage of a peptide derived from the authentic 2A cleavage site on the poliovirus polyprotein.

Infection of cells with picornaviruses of the rhino-, aphtho-, and enterovirus groups causes a shut-off in cap-dependent translation of cellular mRNAs but permits cap-independent viral RNA translation to proceed. This shut-off is thought to be mediated in part by the proteolytic cleavage of eukaryotic initiation factor 4G (eIF4G), although there is evidence to the contrary. Cleavage of eIF4G results in the separation of the eIF4E-binding domain from the ribosome- and helicase-binding domains of the factor, thereby limiting the ability of eIF4G to function in cap-dependent recruitment of mRNAs. Previously we determined the cleavage site within eIF4G targeted by the 2A proteases from human coxsackievirus serotype B4 and human rhinovirus serotype 2 using highly purified eIF4F and recombinant proteases. To examine further the role proteolysis of eIF4G plays in shut-off of translation, we altered the 2A cleavage site in human eIF4G by site-directed mutagenesis. Strikingly, the replacement of one amino acid at the 2A cleavage site resulted in a protein that is approximately 100-fold resistant to cleavage by coxsackievirus 2A protease and 10-50-fold for rhinovirus 2A. Alteration of the cleavage site had no effect on factor activity since the variant was just as active as wild-type eIF4G in restoring cap-dependent translation to an in vitro translation system depleted of endogenous eIF4G. Furthermore, the presence of the variant form of eIF4G rendered in vitro translation reactions resistant to the 2A protease-mediated inhibition of cap-dependent translation initiation. These results support the model that 2A proteases inhibit cap-dependent translation through direct proteolysis of eIF4G.

Cap-dependent binding of mRNA to the 40 S ribosomal subunit during translational initiation requires the association of eukaryotic initiation factor 4G (eIF4G; formerly eIF-4 gamma and p220) with other initiation factors, notably eIF4E, eIF4A, and eIF3. Infection of cells by picornaviruses results in proteolytic cleavage of eIF4G and generation of a cap-independent translational state. Rhinovirus 2A protease and foot-and-mouth-disease virus L protease were used to analyze the association of eIF4G with eIF4A, eIF4E, and eIF3. Both proteases bisect eIF4G into N- and C-terminal fragments termed cpN and cpC. cpN was shown to contain the eIF4E-binding site, as judged by retention on m7GTP-Sepharose, whereas cpC was bound to eIF3 and eIF4A, based on ultracentrifugal co-sedimentation. Further proteolysis of cpN by L protease produced an 18-kDa polypeptide termed cpN2 which retained eIF4E binding activity and corresponded to amino acid residues 319-479 of rabbit eIF4G. Further proteolysis of cpC yielded several smaller fragments. cpC2 (approximately 887-1402) contained the eIF4A binding site, whereas cpC3 (approximately 480-886) contained the eIF3 binding site. These results suggest that cleavage by picornaviral proteases at residues 479-486 separates eIF4G into two domains, one required for recruiting capped mRNAs and one for attaching mRNA to the ribosome and directing helicase activity. Only the latter would appear to be necessary for internal initiation of picornaviral RNAs.

The rate-limiting step of eukaryotic protein synthesis is the binding of mRNA to the 40 S ribosomal subunit, a step which is catalyzed by initiation factors of the eIF-4 (eukaryotic initiation factor 4) group: eIF-4A, eIF-4B, eIF-4E, and eIF-4 gamma. Infection of cells with picornaviruses of the rhino- and enterovirus groups causes a shut-off in translation of cellular mRNAs but permits viral RNA translation to proceed. This change in translational specificity is thought to be mediated by proteolytic cleavage of eIF-4 gamma, which is catalyzed, directly or indirectly, by the picornaviral 2A protease. In this report we have used highly purified recombinant 2A protease from either human Coxsackievirus serotype B4 or rhinovirus serotype 2 to cleave eIF-4 gamma in vitro in the eIF-4 complex purified from rabbit reticulocytes. Neither the rate of cleavage nor fragment sizes were affected by addition of eIF-3. The NH2- and COOH-terminal fragments of eIF-4 gamma were separated by reverse phase HPLC and identified with specific antibodies, and the NH2-terminal sequence of the COOH-terminal fragment was determined by automated Edman degradation. The cleavage site for both proteases is 479GRPALSSR decreases GPPRGGPG494 in rabbit eIF-4 gamma, corresponding to 478GRTTLSTR decreases GPPRGGPG493 in human eIF-4 gamma.

Translation of tobacco vein mottling virus (TVMV) RNA in a wheat germ system resulted in two products that were not observed in a rabbit reticulocyte system. One of these was the N-terminal protein, based on its being the most abundant product and its migration on SDS-PAGE at about 34 kDa. The second product was similar or identical to helper component (HC) isolated from TVMV-infected plants, based on co-migration with HC on SDS-PAGE and immunoprecipitation with anti-HC antibodies. The N-terminus of this product was determined by radiochemical Edman degradation to be Ser-257 of the polyprotein. This assignment was supported by peptide mapping with a tryptophan-specific reagent. A similar cleavage was observed when tobacco etch virus was translated in a wheat germ system. Comparison with homologous regions in five other potyviruses indicated conservation of amino acid residues on both sides of the proposed cleavage site. Conversion of Phe-256 to Met, Pro, Arg, His, or Trp by site-directed mutagenesis of a TVMV RNA transcription template inhibited cleavage in the wheat germ system. These results suggest that in vitro cleavage occurs between Phe-256 and Ser-257 and that this cleavage is the same as the in vivo cleavage which liberates the N-terminus of HC.