SPONTANEOUS MUTAGENESIS OF A PLANT POTYVIRUS GENOME AFTER INSERTION OF A FOREIGN GENE

The RNA genome of tobacco etch potyvirus (TEV) was engineered to expre ss bacterial beta-glucuronidase (GUS) fused to the virus helper compon ent proteinase (HC-Pro). It was shown previously that prolonged period s (approximately 1 month) of TEV-GUS propagation in plants resulted in the appearance of spontaneous deletion variants. Nine deletion mutant s were identified by nucleotide sequence analysis of 40 cDNA clones ob tained after polymerase chain reaction amplification. The mutants were missing between 1,741 and 2,074 nucleotides from TEV-GUS, including t he sequences coding for most of GUS and the N-terminal region of HC-Pr o. This region of HC-Pro contains determinants involved in helper comp onent activity during aphid transmission, as well as a highly conserve d series of cysteine residues. The deletion variants were shown to rep licate and move systemically without the aid of a helper virus. Infect ious viruses harboring the two largest HC-Pro deletions (termed TEV-2d el and TEV-7del) were reconstructed by subcloning the corresponding mu tated regions into full-length DNA copies of the TEV genome. Character ization of these and additional variants derived by site-directed muta genesis demonstrated that deletion of sequences coding for the HC-Pro N-terminal domain had a negative effect on accumulation of viral RNA a nd coat protein. The TEV-2del variant possessed an aphid-nontransmissi ble phenotype that could be rescued partially by prefeeding of aphids on active HC-Pro from another potyvirus. These data suggest that the N -terminal domain of HC-Pro or its coding sequence enhances virus repli cation or genome expression but does not provide an activity essential for these processes. The function of this domain, as well as a propos ed deletion mechanism involving nonhomologous recombination, is discus sed.