The arterivirus nsp4 protease is the prototype of a novel group of chymotrypsin-like enzymes, the 3C-like serine proteases.
The replicase of equine arteritis virus, an arterivirus, is processed by at least three viral proteases. Comparative sequence analysis suggested that nonstructural protein 4 (Nsp4) is a serine protease (SP) that shares properties with chymotrypsin-like enzymes belonging to two different groups. The SP was predicted to utilize the canonical His-Asp-Ser catalytic triad found in classical chymotrypsin-like proteases. On the other hand, its putative substrate-binding region contains Thr and His residues, which are conserved in viral 3C-like cysteine proteases and determine their specificity for (Gln/Glu) downward arrow(Gly/Ala/Ser) cleavage sites. The replacement of the members of the predicted catalytic triad (His-1103, Asp-1129, and Ser-1184) confirmed their indispensability. The putative role of Thr-1179 and His-1199 in substrate recognition was also supported by the results of mutagenesis. A set of conserved candidate cleavage sites, strikingly similar to junctions cleaved by 3C-like cysteine proteases, was identified. These were tested by mutagenesis and expression of truncated replicase proteins. The results support a replicase processing model in which the SP cleaves multiple Glu downward arrow(Gly/Ser/Ala) sites. Collectively, our data characterize the arterivirus SP as a representative of a novel group of chymotrypsin-like enzymes, the 3C-like serine proteases.
Snijder
E J
EJ
Department of Virology, Institute of Medical Microbiology, Leiden University, The Netherlands.
Wassenaar
A L
AL
van Dinten
L C
LC
Spaan
W J
WJ
Gorbalenya
A E
AE
D000595:Amino Acid Sequence; D000818:Animals; D018018:Arterivirus / Q000201:enzymology; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D007240:Equartevirus / Q000201:enzymology* / Q000235:genetics; D016679:Genome, Viral; D006736:Horses; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry*; D013552:Swine
D011994:Recombinant Proteins; C023140:DNA replicase; D004259:DNA-Directed DNA Polymerase; D012697:Serine Endopeptidases; D002918:Chymotrypsin