IDENTIFICATION OF THE MURINE CORONAVIRUS P-28 CLEAVAGE SITE

Mouse hepatitis virus strain A59 encodes a papain-like cysteine protei nase (PLP-1) that, during translation of ORF1a, cleaves p28 from the a mino terminus of the growing polypeptide chain. In order to determine the amino acid sequences surrounding the p28 cleavage site, the first 4.6 kb of murine hepatitis virus strain A59 ORF1a was expressed in a c ell-free transcription-translation system. Amino terminal radiosequenc ing of the resulting downstream cleavage product demonstrated that cle avage occurs between Gly-247 and Val-248. Site-directed mutagenesis of amino acids surrounding the p28 cleavage site revealed that substitut ions of Arg-246 (P2) and Gly-247 (P1) nearly eliminated cleavage of p2 8. Single-amino-acid substitutions of other residues between P7 and P2 ' were generally permissive for cleavage, although a few changes did g reatly reduce proteolysis. The relationship between the p28 cleavage s ite and other viral and cellular papain proteinase cleavage sites is d iscussed.