Essential features of the assembly origin of tobacco mosaic virus RNA as studied by directed mutagenesis

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Abstract
The assembly origin of tobacco mosaic virus RNA contains three stable hairpin loops. Coat protein disks bind first to loop 1 (the 3' most) during virus assembly, but the whole region is coated in a concerted fashion even in conditions of limiting protein. It is shown by in vitro packaging assays using mutant assembly origin transcripts that rapid and specific assembly initiation occurs in the absence of loops 2 and 3, but is abolished on removal of loop 1. Deletion or alteration of the unpaired AAGAAGUCG sequence at the apex of loop 1 also abolishes rapid packaging; this sequence is therefore instrumental in disk binding. Alteration of this sequence to (A)9 leads to packaging at a very low rate (half time 12 hours) which is apparently non-sequence specific. Substitution of (CCG)3 evokes packaging with a half time of 3 hours, as compared to 15 seconds for the wild type assembly origin. These results suggest that the three-base G periodicity within this sequence element is an important feature in assembly nucleation.