The 5' end of the equine arteritis virus replicase gene encodes a papainlike cysteine protease.
The presence of a papainlike cysteine protease (PCP) domain in the N-terminal region of the equine arteritis virus (EAV) replicase, which had been postulated on the basis of limited sequence similarities with cellular and viral thiol proteases, was confirmed by in vitro translation and mutagenesis studies. The EAV protease was found to direct an autoproteolytic cleavage at its C terminus which leads to the production of an approximately 30-kDa N-terminal replicase product (nsp1) containing the PCP domain. Amino acid residues Cys-164 and His-230 of the EAV replicase polyprotein were identified as the most likely candidates for the role of PCP catalytic residues. By means of N-terminal sequence analysis of a PCP cleavage product, derived from a bacterial expression system, it was shown that cleavage occurs between Gly-260 and Gly-261. No evidence for PCP-directed cleavages at other positions in the EAV replicase was obtained. In cotranslational and posttranslational trans-cleavage assays, neither EAV nsp1 nor its precursor was able to process the PCP cleavage site in trans.
Snijder
E J
EJ
Department of Virology, Faculty of Medicine, Leiden University, The Netherlands.
Wassenaar
A L
AL
Spaan
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics*; D007240:Equartevirus / Q000201:enzymology* / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016366:Open Reading Frames; D010206:Papain / Q000235:genetics*; D010957:Plasmids; D014176:Protein Biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D012324:RNA-Dependent RNA Polymerase; D003546:Cysteine Endopeptidases; D010206:Papain