Infectious in vitro transcripts from amplified cDNAs of the Y and Kin strains of cucumber mosaic virus.
Using a method based on the polymerase chain reaction (PCR) with primers that include the phi 10 promoter of bacteriophage T7, we obtained cDNA clones of the three RNA genomes of two different strains of cucumber mosaic virus (CMV; Kin and Y strains) from which infectious in vitro transcripts were generated, and demonstrated that the same primers could be used for amplification of at least two other strains of CMV (O and Py). This method is rapid and requires only limited nucleotide (nt) sequence data (16-18 nt) from the termini of the RNA species. Either viral RNA or unpurified RNA samples from infected plants can be used as template for first-strand cDNA synthesis. For cDNAs of RNA1 and RNA2 of the Y strain, the transcription efficiency was substantially lower than with the Kin strain, unless the primer sequence included transcribed G residues on the 5' side of the viral cDNA, so that the promoter for T7 RNA polymerase resembled more closely the canonical sequence from the bacteriophage T7 phi 10 promoter. The lower specific infectivity of transcripts of the modified cDNAs was more than compensated for by increased transcription efficiency. The possibility that the PCR process may introduce deleterious mutations into the viral cDNA was investigated by re-amplification of a functional cloned cDNA of RNA2: all six cDNA clones of the re-amplified cDNA produced transcripts as infectious as those from the progenitor cDNA.
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Sainsbury Laboratory, Norwich Research Park, UK.
Baulcombe
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D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000235:genetics* / Q000472:pathogenicity; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D014026:Nicotiana / Q000382:microbiology
D004279:DNA, Viral; D012367:RNA, Viral