Construction of full-length cDNA clones of cucumber mosaic virus RNAs 1, 2 and 3: generation of infectious RNA transcripts.

Full-length cDNA copies of cucumber mosaic virus (CMV) RNAs 1 and 2 of the Fny strain were constructed from partial cDNA clones and were cloned downstream of bacteriophage T7 promoters. In one pair of clones, transcription proceeded from an unaltered T7 promoter such that in vitro transcripts representing RNAs 1 and 2 contained an additional 17 nucleotides at their 5' termini. In a second pair of clones, the T7 promoter/cDNA junction was altered by oligonucleotide-directed mutagenesis such that the in vitro transcripts contained only an additional G residue at their 5' ends. In addition, a full-length cDNA copy of Fny-CMV RNA 3 was constructed from two overlapping cDNA clones and was cloned downstream of an altered T7 promoter such that the resultant in vitro transcripts also contained only an additional G residue at their 5' ends. In vitro transcripts derived from all clones contained an additional C residue at their 3' ends. In vitro transcripts representing RNAs 1, 2 and 3 which contained an additional residue at each terminus were shown to be infectious together in several hosts of CMV.