Low level of genetic drift in foreign sequences replicating in an RNA virus in plants.

The accumulation of mutations was measured in foreign sequences constituting a portion of a hybrid virus derived from the 6.4-kb (+) RNA virus, tobacco mosaic tobamovirus (TMV). Neither of the two foreign sequences tested (dihydrofolate reductase and neomycin phosphotransferase II) are functionally required by the virus, so they should be free of selective pressures and should be a true measure of viral sequence drift in whole plants. Four hybrid virus populations, two of each foreign sequence, were taken through 9-10 passages in whole plants of Nicotiana benthamiana. Sequences were sampled from these populations by conversion to cDNA, amplification by the polymerase chain reaction, and sequencing resulting bacterial clones. The background mutation rate contributed by the enzymes of this assay system allowed viral mutation rates greater than 10(-4) mutations per base per passage to be measured. Surprisingly, all native and foreign genes accumulated mutations at a very low rate, lower than could be detected by the assay procedure. This low mutation accumulation rate of < or = 10(-4) mutations per base per passage may be due to replicase fidelity or populational "bottlenecking." Sequence drift should not be a practical limitation to most uses of TMV as a vector, although deletion phenomena observed in this study may present difficulties.