Which residues around the Gln/Gly scissile bond a picornavirus 3C protease needs was poorly defined. Competition cleavage of synthetic HRV-14 peptides defined TLFQ/GP as the minimal substrate, and Val at P4 raised cleavage 4.8-fold over the native Thr.
Purified human rhinovirus type 14 (HRV-14) 3C protease cleaves the six-residue peptide TLFQ/GP (P4-P2', from the 2C-3A cleavage site) between Gln and Gly, with a relative specificity constant (kcat/Km) of 0.033 against the longer reference peptide DSLETLFQ/GPVYKDL (100 nM protease, 100 µM peptide, pH 7.2, competition cleavage assay). Six residues are the minimum substrate, and the seven-residue peptide ETLFQ/GP, which retains the P5 Glu, has a relative kcat/Km of 0.4, about ten-fold higher.
02
HRV-14 3C protease needs the P4 residue of its substrate. The peptide LFQ/GPVY, which lacks the P4 Thr of the 2C-3A site, is not cleaved even with excess protease over long incubations, whereas TLFQ/GPVY (P4-P4') is cleaved at 0.048 relative to the longer reference peptide DSLETLFQ/GPVYKDL and ETLFQ/GPVY (P5-P4') at 0.55.
03
HRV-14 3C protease needs two residues on the carboxyl-terminal side of the scissile bond, while P3' and P4' contribute little. The seven-residue peptide ETLFQ/GP (relative kcat/Km 0.4) is cleaved about as well as the nine-residue ETLFQ/GPVY (0.55), but replacing the P2' Pro with an amide (ETLFQ/G-NH2) lowers cleavage below 0.001, so that product is detectable only with excess protease.
04
Substituting the P4 Thr of the HRV-14 2C-3A peptide ETLFQ/GPVY changes cleavage by HRV-14 3C protease by more than two orders of magnitude. Lys at P4 lowers the relative kcat/Km to 0.014 (70-fold), Asp and Phe lower it to 0.47 each, and the small uncharged residues Ala and Val raise it to 2.8 and 4.8 (relative to the Thr peptide, set at 1.00), so short uncharged P4 side chains are preferred.
05
The identity of the P5 residue of the HRV-14 2C-3A peptide ETLFQ/GPVY is not critical for cleavage by HRV-14 3C protease. Replacing the P5 Glu with Asp leaves cleavage unchanged (relative kcat/Km 1.00), and replacing it with Gln, Arg, Ile, His or Phe lowers cleavage only to 0.23-0.5, while the presence of a P5 residue itself raises cleavage about ten-fold over a peptide that starts at P4.
06
HRV-14 3C protease strictly requires Gly at P1'. The peptide ETLFQ/APVY, in which the P1' Gly of the 2C-3A site is replaced by Ala, is not cleaved at a measurable rate, in line with the lack of cleavage of the HRV-14 2B-2C site peptide that has Gln/Ala at the scissile bond.
07
Pro at P2' (next to the P1' Gly) contributes strongly to cleavage by HRV-14 3C protease. Replacing the P2' Pro of ETLFQ/GPVY with Ala (ETLFQ/GAVY) lowers the relative kcat/Km to 0.09, about ten-fold, and the most efficiently cleaved HRV-14 peptides have Pro at P2'.
08
Residues flanking the core sequence can change how well HRV-14 3C protease cleaves a given site. The 16-residue 3A-3B site peptide YKLFAQTQ/GPYSGNPP has a relative kcat/Km of 0.47, about 20-fold higher than the 9-residue FAQTQ/GPYS (0.021), whereas for the 2C-3A site a 9-residue peptide (ETLFQ/GPVY, 0.55) is cleaved about as well as the 16-residue peptide.
09
HRV-14 3C protease cleaves the putative 2C-3A site peptides of coxsackie B virus type 3 (CBV-3) and poliovirus type 1 (PV-1) 3- to 10-fold more efficiently than the HRV-14 2C-3A peptide (relative kcat/Km 3.7 for CBV-3 and 9.4 for PV-1). Both heterologous peptides share the HRV-14 core sequence but carry Ala instead of Thr at P4, which matches the P4 Ala preference seen in HRV-14 peptide substitutions.
10
HRV-14 3C protease cleaves the putative 3A-3B site peptides of CBV-3 and PV-1 poorly compared with the HRV-14 3A-3B peptide (relative kcat/Km 0.03 for CBV-3 and 0.004 for PV-1, against 0.47 for the HRV-14 peptide). The CBV-3 and PV-1 peptides carry Ala instead of Pro at P2', and the PV-1 peptide, which differs from the CBV-3 peptide at P2 (His instead of Phe) and P6', is cleaved about ten-fold less than the CBV-3 peptide.
11
HRV-14 3C protease cleaves a peptide representing the putative 2C-3A site of human rhinovirus type 2 (DVMTAIFQ/GPIDMKNP) with near-equivalent efficiency to the HRV-14 2C-3A peptide, but cleavage of a peptide representing the putative 2C-3A site of hepatitis A virus (FMELWSQ/GISDDDND) is not detected.
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Abstract
A series of synthetic peptides representing authentic proteolytic cleavage sites of human rhinovirus type 14 were assayed as substrates for purified 3C protease. Competition cleavage assays were employed to determine the relative specificity constants (Kcat/Km) for substrates with sequences related to the viral 2C-3A cleavage site. Variable length peptides representing the 2C-3A cleavage site were cleaved with comparable efficiency. These studies defined a minimum substrate of 6 amino acids (TLFQ/GP), although retention of the residue at position P5 (ETLFQ/GP) resulted in a better substrate by an order of magnitude. Amino acid substitutions at position P5, P4, P1', or P2' indicated that the identity of the residue at position P5 was not critical, whereas substitutions at position P4, P1' or P2' resulted in substrates with Kcat/Km values varying over 2 orders of magnitude. In contrast to the 2C-3A cleavage site, small peptide derivatives representative of the 3A-3B cleavage site were relatively poor substrates, which suggested that residues flanking the minimum core sequence may influence susceptibility to cleavage. The 3C protease of rhinovirus type 14 was also capable of cleaving peptides representing comparable cleavage sites predicted for coxsackie B virus and poliovirus.
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