Characterization of an equine arteritis virus replicase mutant defective in subgenomic mRNA synthesis

Equine arteritis virus (EAV) is a positive-stranded RNA virus that synthesi zes a 5'- and 3'-coterminal nested set of six subgenomic mRNAs. These mRNAs all contain a common leader sequence which is derived from the 5' end of t he genome. Subgenomic mRNA transcription and genome replication are directe d by the viral replicase, which is expressed in the form of two polyprotein s and subsequently processed into smaller nonstructural proteins (nsps), Du ring the recent construction of an EAV infectious cDNA clone (pEAV030 [L. C . van Dinten, J. A. den Boon, A. L. M. Wassenaar, W.J. M. Spaan, and E, J. Snijder, Proc. Nail. Acad, Sci. USA 94:991-996, 1997]), a mutant cDNA clone (pEAV030F) which carries a single replicase point mutation was obtained, T his substitution (Ser-2429-->Pro) is located in the nsp10 subunit and rende rs the EAV030F virus deficient in subgenomic mRNA synthesis. To obtain more insight into the role of nsp10 in transcription and the nature of the tran scriptional defect, we have now analyzed the EAV030F mutant in considerable detail. The Ser-2429-->Pro mutation does not affect the proteolytic proces sing of the replicase but apparently affects the function of nsp10 in trans cription. Furthermore, our study showed that EAV030F still produces subgeno mic positive and negative strands, albeit at a very low level. Both subgeno mic positive-strand synthesis and negative-strand synthesis are equally aff ected by the Ser-2429-->Pro mutation, suggesting that nsp10 plays an import ant role in an early step of EAV mRNA transcription.