Alternative proteolytic processing of the arterivirus replicase ORF1a polyprotein: evidence that NSP2 acts as a cofactor for the NSP4 serine protease.
The C-terminal half of the replicase ORF1a polyprotein of the arterivirus equine arteritis virus is processed by a chymotrypsinlike serine protease (SP) (E. J. Snijder et al., J. Biol. Chem. 271:4864-4871, 1996) located in nonstructural protein 4 (nsp4). Three probable SP cleavage sites had previously been identified in the ORF1a protein. Their proteolysis explained the main processing products generated from the C-terminal part of the ORF1a protein in infected cells (E. J. Snijder et al., J. Virol. 68:5755-5764, 1994). By using sequence comparison, ORF1a expression systems, and site-directed mutagenesis, we have now identified two additional SP cleavage sites: Glu-1430 / Gly and Glu-1452 / Ser. This means that the ORF1a protein can be cleaved into eight processing end products: nsp1 to nsp8. By microsequence analysis of the nsp5 and nsp7 N termini, we have now formally confirmed the specificity of the SP for Glu / (Gly/Ser) substrates. Importantly, our studies revealed that the C-terminal half of the ORF1a protein (nsp3-8) can be processed by the SP following two alternative pathways, which appear to be mutually exclusive. In the majority of the nsp3-8 precursors the SP cleaves the nsp4/5 site, yielding nsp3-4 and nsp5-8. Subsequently, the latter product is cleaved at the nsp7/8 site only, whereas the newly identified nsp5/6 and nsp6/7 sites appear to be inaccessible to the protease. In the alternative proteolytic cascade, which is used at a low but significant level in infected cells, it is the nsp4/5 site which remains uncleaved, while the nsp5/6 and nsp6/7 sites are processed to yield a set of previously unnoticed processing products. Coexpression studies revealed that nsp3-8 has to interact with cleaved nsp2 to allow processing of the nsp4/5 junction, the first step of the major processing pathway. When the nsp2 cofactor is absent, the nsp4/5 site cannot be processed and nsp3-8 is processed following the alternative, minor pathway.
Wassenaar
A L
AL
Department of Virology, Leiden University Medical Center, The Netherlands.
Spaan
W J
WJ
Gorbalenya
A E
AE
Snijder
E J
EJ
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003067:Coenzymes / Q000378:metabolism; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D007240:Equartevirus / Q000201:enzymology*; D006736:Horses; D016366:Open Reading Frames; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D017421:Sequence Analysis; D012694:Serine; D012697:Serine Endopeptidases / Q000378:metabolism*
D003067:Coenzymes; D011506:Proteins; D012694:Serine; D012324:RNA-Dependent RNA Polymerase; D012697:Serine Endopeptidases; D003546:Cysteine Endopeptidases; C078306:nsP2 proteinase