Point Mutation in the 30-K Open Reading Frame of TMV Implicated in Temperature-sensitive Assembly and Local Lesion Spreading of Mutant Ni 2519

Tobacco mosaic virus mutant Ni 2519 forms local lesions on tobacco cultivars carrying the N gene which, unlike wild-type lesions, do not enlarge at elevated temperature. This may reflect temperature sensitivity of a viral gene product required for cell to cell spreading of infectivity. Ni 2519 also carries an unselected cis-dominant lesion in viral assembly. Peptide mapping of in vitro translation products of Ni 2519 RNA reveals at least one, and possibly two changes in p30 and p19, two products of the 30-K open reading frame, compared with its parental strain A14. An A to G transition at position 5332 in Ni 2519 RNA accounts for the altered mobility of the major variable peptide. The corresponding A14 peptide itself differs from the wild-type due to another A to G transition at residue 5329. These residues are close to the viral assembly origin. A revertant virus population which could assemble at the restrictive temperature regained the wild-type sequence in place of the point mutation specific to Ni 2519 at position 5332, and formed wild-type local lesions as efficiently as the parental strain. This result implicates mutation of residue 5332 in the temperature sensitivity of viral assembly (by altering the structure of the RNA close to the assembly origin) and/or local lesion spreading (via a radical Arg to Gly substitution in p30 or its derivatives). The mutation occurs in a position where the predicted amino acid sequence shows homology with a group of proteins encoded by yeast mitochondrial introns.