2ND-SITE REVERSION OF A DYSFUNCTIONAL MUTATION IN A CONSERVED REGION OF THE TOBACCO MOSAIC TOBAMOVIRUS MOVEMENT PROTEIN

The N-terminal two-thirds of tobamovirus movement proteins (MPs) conta in two well conserved regions. Within region I (amino acids 56-96) is an area predicted by computer analysis to have loop secondary structur e (amino acids 76-87). A single or two double amino acid mutations wer e introduced into the loop in region 1 of the TMV MP to destabilize th e 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, wa s 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 pr esence of infectious virus at a low frequency. Necrotic lesions were d etected at 4 days postinoculation, 2 days later than those induced by wild-type TMV. Inoculation of virus extracted from necrotic lesions on to N. tabacum resulted in a delayed and attenuated systemic infection relative to that induced by TMV, indicating that a second-site mutatio n restored movement function rather than a reversion of the original m utation. Sequence analysis revealed that the revertant MP gene had two additional amino acid substitutions, a Thr104-->IIe and a Arg 167-->L ys. Introduction of the amino acid substitutions individually or in co mbination 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 fo r functional MP, that changes outside region I can compensate for alte rations within the region, and suggest that region I may interact with a distal portion of the protein. (C) 1997 Academic Press.