DETERMINANTS OF THE P-28 CLEAVAGE SITE RECOGNIZED BY THE FIRST PAPAIN-LIKE CYSTEINE PROTEINASE OF MURINE CORONAVIRUS

The murine coronavirus polymerase gene is 22 kb in length with the pot ential to encode a polyprotein of approximately 750 kDa. The polyprote in has been proposed to encode three proteinase domains which are resp onsible for the processing of the polyprotein into mature proteins. Th e proteolytic activity of the first proteinase domain has been charact erized and resembles the papain family of cysteine proteinases. This p roteinase domain acts autoproteolytically to cleave the amino terminal portion of the polymerase polyprotein, releasing a 28-kDa protein des ignated p28. To identify the cleavage site of this papain-like cystein e proteinase, we isolated the peptide adjacent to p28 and determined t he amino terminus sequence by Edman degradation reaction. We report th at proteolysis occurs between the Gly-247 and Val-248 dipeptide bond. To determine the role of the amino acid residues surrounding the cleav age site, we introduced a total of 42 site-specific mutations at the r esidues spanning the P5 to P3' positions and assessed the effects of t he mutations on the processing of p28 in an in vitro transcription and translation system. The substitutions of Gly-247 at the P1 position o r Arg-246 at the P2 position resulted in a dramatic decrease of proteo lytic activity, and the mutations of Arg-243 at P5 position also led t o considerable reduction in p28 cleavage. In contrast, the substitutio ns of amino acids Gly-244 (P4), Tyr-245 (P3), Val-248 (P1'), Lys-249 ( P2'), and Pro-250 (P3') had little or no effect on the amount of p28 t hat was released. This work has identified Gly-247-Val-248 as the clea vage site for the release of p28, the amino-terminal protein of the mu rine coronavirus polymerase polyprotein. Additionally, we conclude tha t the Gly-247 and Arg-246 are the major determinants for the cleavage site recognition by the first papain-like cysteine proteinase of murin e coronavirus. (C) 1994 Academic Press, Inc.