Effects of Extra 5' Non-Viral Bases on the Infectivity of Transcripts from a cDNA Clone of Satellite RNA (Strain Y) of Cucumber Mosaic Virus

Abstract

Full-length cDNA of a satellite RNA (Strain Y) which induces bright yellow symptoms on tobacco plants infected with cucumber mosaic virus (CMV) was cloned and sequenced. The published sequence of the satellite RNA was revised with three possible differences (residues 161, 167, and 173) and a nucleotide insertion at residue 234. The satellite cDNA was then inserted into a commercially available transcription vector. In vitro transcription products from the recombinant plasmid harbored 24 non-viral bases at their 5' ends and had very low infectivity when coinoculated with CMV. After removal of the extra 5' sequence of the transcripts with RNase H, the infectivity of the transcripts increased markedly. Analysis of the effects of extra 5' sequences of several lengths confirmed the importance of natural 5' ends for biological activity of the satellite. Trimming down to 6-9 extra bases at the 5' end enhanced the infectivity of the transcripts by 10-fold, although the specific activity of the natural satellite is still 100-fold higher. Dideoxynucleotide sequence analysis proved that the progeny satellite RNA did not retain the 24 non-viral bases atthe 5' end of the transcript from pIBI 31-MC.