Second-site reversion of a dysfunctional mutation in a conserved region of the tobacco mosaic tobamovirus movement protein

Abstract

The N-terminal two-thirds of tobamovirus movement proteins (MPs) contain two well conserved regions. Within region I (amino acids 56-96) is an area predicted by computer analysis to have loop secondary structure (amino acids 76-87). A single or two double amino acid mutations were introduced into the loop in region I of the TMV MP to destabilize the structure. The three mutant MPs were defective in movement function. The single amino acid mutation resulted in a Pro81-->Ser substitution. The mutant virus, TP81S, containing the Pro81-->Ser substitution, was propagated on a transgenic line of Nicotiana tabacum that expresses the sunn-hemp mosaic tobamovirus MP. Inoculation of virus progeny from the transgenic plants onto hypersensitive N. tabacum indicated the presence of infectious virus at a low frequency. Necrotic lesions were detected at 4 days postinoculation, 2 days later than those induced by wild-type TMV. Inoculation of virus extracted from necrotic lesions onto N. tabacum resulted in a delayed and attenuated systemic infection relative to that induced by TMV, indicating that a second-site mutation restored movement function rather than a reversion of the original mutation. Sequence analysis revealed that the revertant MP gene had two additional amino acid substitutions, a Thr104-->Ile and a Arg167-->Lys. Introduction of the amino acid substitutions individually or in combination into the MP of TP81S indicated that both substitutions were required for the revertant phenotype. The data indicate that structure within region I is important in maintaining an active conformation for functional MP, that changes outside region I can compensate for alterations within the region, and suggest that region I may interact with a distal portion of the protein.