An internal RNA element in the P3 cistron of Wheat streak mosaic virus revealed by synonymous mutations that affect both movement and replication

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Abstract
Multiple synonymous substitution mutations in the Wheat streak mosaic virus P3 cistron did not affect translation in vitro but rendered the virus incapable of systemic infection. Multiple synonymous substitutions in the cylindrical inclusion cistron did not alter infectivity or in vitro translation. To assess replication and movement phenotypes, P3 mutations were placed in context with a GUS reporter gene. GUS activity measured in barley protoplasts 36 h post-transfection indicated that mutants with synonymous substitutions in P3 retained the ability to replicate at 22–80 % of wild-type levels. Almost no GUS activity was detected in protoplasts transfected with a P3 frame-shift mutant. Histochemical GUS assays conducted 3 days post-inoculation (p.i.) revealed genomes with multiple synonymous substitutions in P3, which were able to establish infection foci limited to small clusters of cells that increased in size only slightly by 5 days p.i. Infection foci produced by wild-type Wheat streak mosaic virus-expressing GUS were much larger at 3 days p.i. and had coalesced by 5 days p.i. No GUS activity was detected in plants inoculated with the frame-shift mutant bearing GUS. Three of four mutants, each with a single synonymous substitution in the 3′-proximal half of the P3 cistron, were wild-type with respect to systemic infectivity. A model RNA secondary structure obtained for the region was disrupted by the debilitating single mutation but not by the other three single mutations. Collectively, these results identify an internal RNA sequence element in the P3 cistron that affects both replication and movement of the viral genome.