Infectious RNA transcripts derived from full-length DNA copies of the genomic RNAs of cowpea mosaic virus

Abstract

A set of full-length DNA copies of both M and B RNA of cowpea mosaic virus (CPMV) was cloned downstream of a phage T7 promoter. Upon in vitro transcription using T7 RNA polymerase, M and B RNA-like transcripts were obtained from these DNA copies with only two additional nucleotides at the 5' end and five extra nucleotides at the 3' end in comparison to natural viral RNA. In cowpea protoplasts the transcripts of several cDNA clones of B RNA were able to replicate leading to detectable synthesis of viral RNA and proteins. Transcripts of M cDNA clones inoculated together with these B RNA transcripts were also expressed, although the number of protoplasts in which both transcripts were expressed was very low. Preliminary infectivity tests with mutagenized RNA transcripts indicate essential roles of the B RNA-encoded 24K and 32K polypeptides in viral RNA replication.