ALTERNATIVE PROTEOLYTIC PROCESSING OF THE ARTERIVIRUS REPLICASE ORF1APOLYPROTEIN - EVIDENCE THAT NSP2 ACTS AS A COFACTOR FOR THE NSP4 SERINE-PROTEASE

The C-terminal half of the replicase ORF1a polyprotein of the arterivi rus equine arteritis virus is processed by a chymotrypsinlike serine p rotease (SP) (E. J. Snijder et al., J. Biol. Chem. 271:4864-4871, 1996 ) located in nonstructural protein 4 (nsp4). Three probable SP cleavag e sites had previously been identified in the ORF1a protein. Their pro teolysis explained the main processing products generated from the C-t erminal part of the ORF1a protein in infected cells (E. J. Snijder et al., J. Virol. 68:5755-5764, 1994). By using sequence comparison, ORF1 a expression systems, and site-directed mutagenesis, we have now ident ified two additional SP cleavage sites: Glu-1430 down arrow Gly and Gl u-1452 down arrow Ser. This means that the ORF1a protein can be cleave d into eight processing end products: nsp1 to nsp8. By microsequence a nalysis of the nsp5 and nsp7 N termini, we have now formally confirmed the specificity of the SP for Glu down arrow (Gly/Ser) substrates. Im portantly, 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 th e nsp3-8 precursors the SP cleaves the nsp4/5 site, yielding nsp3-4 an d nsp5-8. Subsequently, the latter product is cleaved at the nsp7/8 si te only, whereas the newly identified nsp5/6 and nsp6/7 sites appear t o be inaccessible to the protease. In the alternative proteolytic casc ade, which is used at a low but significant level in infected cells, i t is the nsp4/5 site which remains uncleaved, while the nsp5/6 and nsp 6/7 sites are processed to yield a set of previously unnoticed process ing products. Coexpression studies revealed that nsp3-8 has to interac t with cleaved nsp2 to allow processing of the nsp4/5 junction, the fi rst step of the major processing pathway. When the nsp2 cofactor is ab sent, the nsp4/5 site cannot be processed and nsp3-8 is processed foll owing the alternative, minor pathway.