Hydrolysis of a Series of Synthetic Peptide Substrates by the Human Rhinovirus 14 3C Proteinase, Cloned and Expressed in Escherichia coli

Abstract

SUMMARYThe 3C proteins of several picornaviruses, including poliovirus, foot-and-mouth disease virus (FMDV) and encephatomyocarditis virus (EMCV), have been demonstrated to be cysteine-type proteinases, involved in the processing of the respective polyproteins expressed by the monocistronic RNA genome. Nucleotide sequencing data have indicated that the human rhinovirus 14 (HRV-14) RNA genome encodes a homologous 3C protein. The HRV-14 3C protein was purified to homogeneity from Escherichia coli expressing the cloned 3C genomic fragment. The enzyme was assayed against peptides corresponding to those residues, predicted (by nucleotide sequencing data) to occur at authentic cleavage sites within the polyprotein. The peptides representing the 1B/1C, 2A/2B, 2C/3A, 3A/3B, 3B/3C and 3C/3D cleavage sites, where proteolysis was predicted to occur at a Gln-Gly junction, were all cleaved by the 3C proteinase. The hydrolysis was shown (by reverse phase fast protein liquid chromatography and amino acid analysis) to occur specifically at the Gln-Gly bond in each of the peptides. The ready availability of such convenient substrates facilitated the further characterization of the 3C proteinase. By contrast, peptides corresponding to the predicted 2B/2C and 1C/1D cleavage sites, where the processing was presumed to occur at a Gln-Ala or Glu-Gly bond respectively, were not cleaved by the 3C proteinase. The ability of the HRV-14 3C proteinase to hydrolyse the synthetic peptides was inhibited if a Cys~Ser(146) mutation was introduced into the protein. Studies with known proteinase inhibitors substantiated the conclusion that the HRV-14 3C protein appears to be a cysteine proteinase and that the Cys residue at position 146 may be the active site nucleophile. The HRV-14 3C proteinase probably plays an important role, analogous to that implied for the poliovirus 3C proteinase, in the replication of the virus and thus represents a potential target for antiviral chemotherapy.

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“…This may partially explain the preference shown in cleavage junction sequences for serine and threonine residues in the S2 position. The HRV14 enzyme, in contrast, has a larger cavity in this area that is consistent with the demonstrated tolerance for larger side chains in S2 (Cordingley et al, 1989(Cordingley et al, , 1990Orr et al, 1989;Long et al, 1989).In both the HAV and HRV enzymes, the side chain of the S3 residue appears to point away from the active site toward solvent, which would suggest that little or no specificity is obtained from this residue. This is consistent with studies that showed that susceptibility of a peptide substrate to proteolytic cleavage by HAV3C was unaffected by replacement of an arginine residue in S3 by alanine (Jewell et ai., 1992).…”

“…In addition, in vitro transcriptiontranslation systems have been developed to express picornaviral RNA, further facilitating investigations into polyprotein processing in trans by recombinant 3C proteolytic activities (Parks et al, 1986;Vakharia et al, 1987;Ypma-Wong & Semler, 1987;Nicklin et al, 1988;Kusov et al, 1992;Schultheiss et al, 1994). Finally, the advancement in recent years of solidphase peptide synthesis and the development of high throughput assays have permitted detailed comparisons of proteolytic behaviors of 3C proteinases from different picornaviruses (Cordingley et al, 1989(Cordingley et al, , 1990Long et al, 1989;Orr et al, 1989;Pallai et al, 1989;Petithory et al, 1991; Jewel1 et al, 1992). These approaches have generated a wealth of data concerning 3C proteinase specificity and mechanism for each system that are at times difficult to reconcile.…”

“…All 3C proteinases cleave after glutamine residues, although this is clearly not the only determinant of specificity (Dewalt et al, 1989; Kean et al, 1990;Mirzayan et al, 1991;Petithory et al, 1991; Jewel1 et al, 1992). In the cases of the enteroviruses and rhinoviruses, synthetic peptide studies have shown that the glutamine residue should be followed by a glycine and proline residue in the P; and P i subsites, respectively (notation of Schechter & Berger, 1967) to ensure efficient cleavage, suggest-1442 ing that a type I1 p turn may be an important structural recognition element (Cordingley et al, 1989(Cordingley et al, , 1990Long et al, 1989;Orr et al, 1989;Pallai et al, 1989). Studies on HAV-3C, on the other hand, show that it is, in general, less discriminating, effectively cleaving peptides in which any small amino acid (G, A, or S) is in the P; position and virtually any amino acid is in the Pi position with the exception of proline and arginine, arguing against the turn motif for this 3C proteinase (Petithory et al, 1991).…”

The protease of adenovirus serotype 2 requires cysteine residues for both activation and catalysis

“…The sequence of the 23 kDa protein contains no recognizable protease motifs (Anderson, 1990;Houde & Weber, 1990); nor do searches against the databases reveal similarities to any known proteases (Webster et al, 1989a) although the protease sequence is well conserved in the serotypes for which sequence information is available (Cai et al, 1992). Although initially classed as a serine protease , extensive inhibitor studies (Webster et al, 1989a) have suggested that it is a member of the cysteine class, albeit of unusual mechanism, resembling most closely in its inhibition profile the 3C protease of rhinoviruses (Orr et al, 1989).…”

The active adenovirus protease is the intact L3 23K protein

“…There is more similarity between the Ad2 protease and the picornaviral proteases, which are cysteine proteases. In addition to inhibition by zinc, both proteases are inhibited by thiol-directed reagents such as iodoacetate, pCMB and NEM (Orr et al, 1989;Webster et al, 1989b) but not by cystatin and E-64 (Orr et al, 1989). Definite assignment of the Ad2 protease as a cysteine centre enzyme, however, must await more conclusive evidence from site-directed mutagenesis and active site labelling.…”