Insertion of sequences containing the coat protein subgenomic RNA promoter and leader in front of the tobacco mosaic virus 30K ORF delays its expression and causes defective cell-to-cell movement.
The regulation of the internal open reading frame (ORF) of tobacco mosaic virus (TMV) that encodes the 30K movement protein was examined by constructing mutants in vitro with the putative coat protein subgenomic RNA promoter and leader sequences inserted upstream of the 30K ORF. A mutant with a 49-nucleotide fragment of the promoter region inserted replicated only transiently before being overtaken by a progeny wild-type virus with the insert deleted. A mutant with a 253-nucleotide promoter region fragment inserted replicated stably, and the inserted promoter was active in its new location. The production of 30K protein was not enhanced by this promoter/leader insertion to a level similar to that of coat protein. However, the accumulation of 30K protein was delayed, suggesting that different promoters/leader sequences determine the time of expression of the genes. This mutant was deficient in movement. A similar mutant, but with increased production of 30K protein, overcame the movement deficiency, suggesting that 30K protein is needed during the early stages of infection for efficient cell-to-cell movement of the virus.