In vitromutagenesis of the putative replicase genes of tobacco mosaic virus

We have established an in vitro transcription system to produce infectious tobacco mosaic virus (TMV) RNA from a cloned cDNA copy. Using this system, several TMV mutants were transcribed in vitro from cDNA clones mutagenized at or near the leaky amber termination codon of the 130K protein gene, and their infectivity was assayed on tobacco plants. Three (two frame-shift and one non-sense) mutants with an intact 130K but a defective 180K protein gene were not infectious, while two mutants with a one-amino-acid insertion in the 180K protein gene were infectious. When the amber codon of the 130K protein gene was deleted, infectivity was lost. However, when the amber termination codon was replaced with ochre or tyrosine codon, infectivity was retained. Sequence analyses revealed that introduced mutations were retained in progeny viral sequences except in the progeny of the amber-to-tyrosine mutant, which was a mixture of the parental mutagenized virus and a pseudo-revertant with ochre codon.