Replication, stability, and gene expression of tobacco mosaic virus mutants with a second 30K ORF

A series of tobacco mosaic virus (TMV)-hybrids containing a second 30K open reading frame (ORF) inserted into different positions of the genome 3' region were constructed. These insertional mutants were used to evaluate the effects of a modified viral genome organization on replication and gene expression. They were evaluated for stability upon systemic infection and subsequent host passage using RNase protection assays. A mutant with the second 30K ORF fused in frame to two-thirds of the coat protein reading frame replicated as a free-RNA virus and produced increased amounts of the hybrid protein compared to the wild-type 30K protein, but substantially reduced amounts compared to the wild-type coat protein. A mutant with the second 30K ORF inserted between the native 30K and coat protein ORFs produced reduced amounts of 30K protein but replicated efficiently and was maintained for weeks of systemic infection before the population gradually shifted to progeny wild-type TMV. Mutants with the second 30K ORF fused behind different lengths of the coat protein subgenomic RNA promoter/leader region and inserted between the coat protein gene and the 3' nontranslated sequences replicated poorly and the mutations were not maintained during continued replication in plants.