Determinants of Mouse Hepatitis Virus 3C-like Proteinase Activity

Abstract The coronavirus, mouse hepatitis virus strain A59 (MHV), expresses a chymotrypsin-like cysteine proteinase (3CLpro) within the gene 1 polyprotein. The MHV 3CLpro is similar to the picornavirus 3C proteinases in the relative location of confirmed catalytic histidine and cysteine residues and in the predicted use of Q/(S, A, G) dipeptide cleavage sites. However, less is known concerning the participation of aspartic acid or glutamic acid residues in catalysis by the coronavirus 3C-like proteinases or of the precise coding sequence of 3CLpro within the gene 1 polyprotein. In this study, aspartic acid residues in MHV 3CLpro were mutated and the mutant proteinases were tested for activity in anin vitro transcleavage assay. MHV 3CLpro was not inactivated by substitutions at Asp3386(D53) or Asp3398(D65), demonstrating that they were not catalytic residues. MHV 3CLpro was able to cleave at a glutamine–glycine (QG3607-8) dipeptide within the 3CLpro domain upstream from the predicted carboxy-terminal QS3635-6cleavage site of 3CLpro. The predicted full-length 3CLpro (S3334to Q3635) had an apparent mass of 27 kDa, identical to the p27 3CLpro in cells, whereas the truncated proteinase (S3334to Q3607) had an apparent mass of 24 kDa. This 28-amino-acid carboxy-terminal truncation of 3CLpro rendered it inactive in atranscleavage assay. Thus, MHV 3CLpro was able to cleave at a site within the putative full-length proteinase, but the entire predicted 3CLpro domain was required for activity. These studies suggest that the coronavirus 3CL-proteinases may have a substantially different structure and catalytic mechanism than other 3C-like proteinases.

[1]  Xiaotao Lu,et al.  Intracellular andin Vitro-Translated 27-kDa Proteins Contain the 3C-like Proteinase Activity of the Coronavirus MHV-A59 , 1996, Virology.

[2]  I. Brierley,et al.  Characterization in vitro of an autocatalytic processing activity associated with the predicted 3C-like proteinase domain of the coronavirus avian infectious bronchitis virus , 1996, Journal of virology.

[3]  E. Domingo,et al.  Effect of expression of the aphthovirus protease 3C on viral infection and gene expression. , 1995, Virology.

[4]  J. Ziebuhr,et al.  Characterization of a human coronavirus (strain 229E) 3C-like proteinase activity , 1995, Journal of virology.

[5]  T. Brown,et al.  Characterisation and Mutational Analysis of an ORF 1a-Encoding Proteinase Domain Responsible for Proteolytic Processing of the Infectious Bronchitis Virus 1a/1b Polyprotein , 1995, Virology.

[6]  Xiaotao Lu,et al.  Identification and characterization of a serine-like proteinase of the murine coronavirus MHV-A59 , 1995, Journal of virology.

[7]  S. Weiss,et al.  Identification and Characterization of a 65-kDa Protein Processed from the Gene 1 Polyprotein of the Murine Coronavirus MHV-A59 , 1995, Virology.

[8]  H. Laude,et al.  Complete Sequence (20 Kilobases) of the Polyprotein-Encoding Gene 1 of Transmissible Gastroenteritis Virus , 1995, Virology.

[9]  D. Matthews,et al.  Structure of human rhinovirus 3C protease reveals a trypsin-like polypeptide fold, RNA-binding site, and means for cleaving precursor polyprotein , 1994, Cell.

[10]  Marc Allaire,et al.  Picornaviral 3C cysteine proteinases have a fold similar to chymotrypsin-like serine proteinases , 1994, Nature.

[11]  A. Gorbalenya,et al.  Mouse Hepatitis Virus Strain A59 RNA Polymerase Gene ORF 1a: Heterogeneity among MHV Strains , 1994, Virology.

[12]  T. Raabe,et al.  Nucleotide Sequence of the Human Coronavirus 229E RNA Polymerase Locus , 1993, Virology.

[13]  T. Raabe,et al.  Characterization of the human coronavirus 229E (HCV 229E) gene 1. , 1993, Advances in experimental medicine and biology.

[14]  B. Semler,et al.  Alternate poliovirus nonstructural protein processing cascades generated by primary sites of 3C proteinase cleavage. , 1992, Virology.

[15]  E. Wimmer,et al.  Mutational analysis of the proposed FG loop of poliovirus proteinase 3C identifies amino acids that are necessary for 3CD cleavage and might be determinants of a function distinct from proteolytic activity , 1992, Journal of virology.

[16]  E. Koonin,et al.  The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase , 1991, Virology.

[17]  S. Weiss,et al.  The primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus MHV-A59; a highly conserved polymerase is expressed by an efficient ribosomal frameshifting mechanism. , 1990, Nucleic acids research.

[18]  V. Blinov,et al.  Coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis. , 1989, Nucleic acids research.

[19]  T. Brown,et al.  Completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus. , 1987, The Journal of general virology.