IDENTIFICATION AND CHARACTERIZATION OF A SERINE-LIKE PROTEINASE OF THE MURINE CORONAVIRUS MHV-A59

Gene 1 of the murine coronavirus, MHV-A59, encodes approximately 800 k Da of protein products within two overlapping open reading frames (ORF s 1a and 1b), The gene is expressed as a polyprotein that is processed into individual proteins, presumably by virus-encoded proteinases, OR F 1a has been predicted to encode proteins with similarity to viral an d cellular proteinases, such as papain, and to the 3C proteinases of t he picornaviruses (A. E. Gorbalenya, A. P. Donchenko, V. M. Blinov, an d E. V. Koonin, FEES Lett, 243:103-114, 1989; A. E. Gorbalenya, E. V. Koonin, A. P. Donchenko, and V. M. Blinov, Nucleic Acids Res, 17:4847- 4861, 1989), We have cloned into a T7 transcription vector a cDNA frag ment containing the putative 3C-like proteinase domain of MHV-A59, alo ng with portions of the flanking hydrophobic domains, The construct wa s used to express a polypeptide in a combined in vitro transcription-t ranslation system. Major polypeptides with molecular masses of 38 and 33 kDa were detected at early times, whereas polypeptides with molecul ar masses of 32 and 27 kDa were predominant after 30 to 45 min and app eared to be products of specific proteolysis of larger precursors, Mut ations at the putative catalytic histidine and cysteine residues aboli shed the processing of the 27-kDa protein, Translation products of the pGpro construct were able to cleave the 27-kDa protein in trans from polypeptides expressed from the noncleaving histidine or cysteine muta nts, The amino terminal cleavage of the 27-kDa protein occurred at a g lutamine-serine dipeptide as previously predicted, This study provides experimental confirmation that the coronaviruses express an active pr oteinase within the 3C-like proteinase domain of gene 1 ORF 1a and tha t this;proteinase utilizes at least one canonical QS dipeptide as a cl eavage site in vitro.