Red clover necrotic mosaic virus infectious transcripts synthesized in vitro

Abstract

The red clover necrotic mosaic dianthovirus (RCNMV) genome is split between two essentially nonhomologous ssRNAs of 3.9 kb (RNA-1) and 1.45 kb (RNA-2) which are each capped at the 5' terminus with m7GpppA. cDNA clones short of full length by several nucleotides at both termini have been generated to both RNAs. Oligonucleotide-directed mutagenesis was employed to generate a series of RNA-1 and -2 transcription vectors in which the bacteriophage T7 RNA polymerase promoter was fused to full-length cDNA clones. Yields of in vitro transcripts initiating with wild-type viral 5'-terminal adenosine were extremely low. Efficient transcription was achieved only when one, or alternatively two, nonviral guanosines were engineered 5' to the authentic viral sequence at the transcription start site. m7GpppG-capped or -uncapped RCNMV RNA-1 and RNA-2 transcripts were infectious and induced symptoms identical to those of wild-type virus infection when coinoculated on the systemic hosts Nicotiana benthamiana and N. clevelandii, and on the local lesion host Chenopodium amaranticolor. Uncapped in vitro transcripts were somewhat less infectious. Progeny virus derived from infectious transcript inoculum was as infectious as wild-type virus. Primer extension analysis indicated that the 5'-terminal nonviral guanosine residues were not maintained in the progeny virus.