Proteolytic maturation of replicase polyprotein pp1a by the nsp4 main proteinase is essential for equine arteritis virus replication and includes internal cleavage of nsp7

The positive-stranded RNA genome of the arterivinus Equine arteritis virus (order Nidovirales) encodes the partially overlapping replicase polyproteins pp1a (1727 aa) and ppl ab (3175 aa). Previously, three viral proteinases were reported to cleave these large polyproteins into 12 non-structural proteins (nsps). The chymotrypsin-like viral main proteinase residing in nsp4 is responsible for eight of these cleavages. Processing of the C-terminal half of ppl a (the nsp3-8 region) was postulated to occur following either of two alternative proteolytic pathways (the 'major' and 'minor' pathways). Here, the importance of these two pathways was investigated by using a reverse-genetics system and inactivating each of the cleavage sites by site-directed mutagenesis. For all of these ppl a cleavage sites, mutations that prevented cleavage by the nsp4 proteinase were found to block or severely inhibit EAV RNA synthesis. Furthermore, our studies identified a novel nsp4 cleavage site (Glu-1575/Ala-1576) that is located within nsp7 and is conserved in arteriviruses. The N-terminal nsp7 fragment (nsp7 alpha) derived from this cleavage was detected in lysates of both EAV-infected cells and cells transiently expressing ppla. Mutagenesis of the novel cleavage site in the context of an EAV full-length cDNA clone proved to be lethal, underlining the fact that the highly regulated, nsp4-mediated processing of the C-terminal half of ppl a is a crucial event in the arterivirus life cycle.