Isolation and characterisation of mutants of cowpea mosaic virus
A nitrous acid-induced, temperature sensitive mutant of cowpea mosaic virus (CPMV) known as 8-14, (Evans 1985, Virology 1985, 141, 275-282), was characterised. The phenotypic defect in 8 -14 was shown not to affect translation of the RNA or the first proteolytic cleavage of the B RNA-encoded polyprotein. The defect is probably at the level of genome replication. The technique of two dimensional RNA fingerprinting showed the mutant genome to be similar to the parental wild-type but did not resolve the genetic alteration(s) specific for the mutation. The mechanism of CPMV translation was investigated by site-directed mutagenesis of a full-length cDNA clone of CPMV M RNA from which infectious RNA could be generated by in vitro transcription. The results obtained confirm the AUG at position 161 is used to direct the synthesis of the 105K protein in vitro. The detection of a 58K protein in infected protoplasts suggests that it is also used in vivo. The synthesis of the 95K protein can be initiated from either of the AUGs at positions 512 and 524. Synthesis of this protein is not essential for CPMV replication in protoplasts. Several deletion mutations were created in the M RNA cDNA clone in order to determine the regions of M RNA essential for replication of M RNA. Analysis of one mutant indicated that sequences between 1446 and 1620 are probably not required for replicase recognition. However, the accumulation of this mutant in protoplasts was reduced, presumably as a result of lack of encapsidation of the RNA as this mutant is thought not to synthesise functional coat protein. Data from several mutants showed that alterations of M RNA around nucleotides 161 and 189 prevent transcript accumulation in protoplasts possibly owing to a severe reduction in replicability of the input RNA.