Proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and is essential for virus replication

The open reading frame (ORF) 1b-encoded part of the equine arteritis virus (EAV) replicase is expressed by ribosomal frameshifting during genome trans lation, which results in the production of an ORF1ab fusion protein (345 kD a). Four ORF1b-encoded processing products, nsp9 (p80), nsp10 (p50), nsp11 (p26), and nsp12 (p12), have previously been identified in EAV-infected cel ls (L. C. van Dinten, A. L. M. Wassenaar, A. E. Gorbalenya, W. J. M. Spaan, and E. J. Snijder, J. Virol. 70:6625-6633, 1996). In the present study, th e generation of these four nonstructural proteins was shown to be mediated by the nsp4 serine protease, which is the main viral protease (E. J. Snijde r, A. L. M. Wassenaar, L. C. van Dinten, W. J. M. Spaan, and A. E. Gorbalen ya, J. Biol. Chem. 271:4864-4871, 1996). Mutagenesis of candidate cleavage sites revealed that Glu-2370/Ser, Gln-2837/Ser, and Glu-3056/Gly are the pr obable nsp9/10, nsp10/11, and nsp11/12 junctions, respectively. Mutations w hich abolished ORF1b protein processing were introduced into a recently dev eloped infectious cDNA clone (L. C. van Dinten, J. A. den Boon, A. L. M. Wa ssenaar, W. J. M. Spaan, and E. J. Snijder, Proc. Natl. Acad. Sci. USA 94:9 91-997, 1997). An analysis of these mutants showed that the selective block age of ORF1b processing affected different stages of EAV reproduction. In p articular, the mutant with the nsp10/11 cleavage site mutation Gln-2837-->P ro displayed an unusual phenotype, since it was still capable of RNA synthe sis but was incapable of producing infectious virus.