AN INFECTIOUS ARTERIVIRUS CDNA CLONE - IDENTIFICATION OF A REPLICASE POINT MUTATION THAT ABOLISHES DISCONTINUOUS MESSENGER-RNA TRANSCRIPTION

Equine arteritis virus (EAV) is a positive- strand RNA virus that uses a discontinuous transcription mechanism to generate a nested set of s ix subgenomic mRNAs from which its structural genes are expressed, A s table bacterial plasmid (pEAV030) containing a full-length cDNA copy o f the 12.7-kb EAV genome was constructed, After removal of a single po int mutation in the replicase gene, RNA transcripts generated in vitro from pEAV030 were shown to be infectious upon electroporation into BH K-21 cells. A genetic marker mutation was introduced at the cDNA level and recovered from the genome of the progeny virus, The potential of pEAV030 as a tool to express foreign genes was demonstrated by the eff icient expression of the chloramphenicol acetyltransferase (CAT) repor ter gene from two different subgenomic mRNAs. The point mutation that initially rendered the full-length clone noninfectious was found to re sult in a particularly intriguing phenotype: RNA carrying this mutatio n can replicate efficiently but does not produce the subgenomic mRNAs required for structural protein expression. To our knowledge, this mut ant provides the first evidence that the requirements for arterivirus genome replication and discontinuous mRNA synthesis are, at least part ially, different and that these processes may be separated experimenta lly.