Submitted to: Journal of General Virology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 6/6/2005
Publication Date: 9/1/2005
Citation: Choi, I., Horken, K.M., Stenger, D.C., French, R.C. 2005. An internal rna element in the wheat streak mosaic virus p3 cistron revealed by synonymous mutations that affect both movement and replication.. Journal of General Virology. v. 86. p 2605-2614. Interpretive Summary: Alterations within the P3 gene of the wheat streak mosaic virus (WSMV) genome that do not affect encoded protein sequence or expression were found to reduce viral RNA replication and block systemic infection of virus in wheat plants. Use of a reporter gene (GUS) placed within the genome of these WSMV mutants indicated that the inability of P3 mutants to infect plants was due to localization of infection foci to small clusters of cells at the site of inoculation. Thus, lack of systemic infection by the P3 mutants was due to cessation of viral movement prior to entry of phloem tissue such that long-distance movement was precluded. Collectively, these results indicate that the primary RNA sequence of the WSMV P3 gene contains a previously unrecognized and essential sequence element required for virus infection of wheat.
Technical Abstract: Mutants bearing multiple synonymous substitutions in the Wheat streak mosaic virus (WSMV) P3 cistron were unable to systemically infect wheat but did not differ from wild type when translated in vitro. Multiple synonymous substitutions in the CI cistron did not alter infectivity or in vitro translation, whereas a frame shift mutation in the P3 cistron abolished infectivity and altered translation in vitro. To assess replication and movement phenotypes, P3 mutations were placed in a WSMV genome containing the GUS reporter gene. GUS activity measured in barley protoplasts 36 hours post-transfection indicated that genomes bearing multiple synonymous substitutions in P3 retained the ability to replicate, albeit at 22-80% of wild type levels. Almost no GUS activity was detected in protoplasts transfected with the frame shift mutant, even though the reporter gene was inserted upstream of the frame shift. Histochemical GUS assays conducted 3 days post inoculation (d.p.i.) revealed genomes with multiple synonymous substitutions in P3 were able to establish subliminal infections in inoculated wheat leaves with infection foci limited to small clusters of cells that increased in size only slightly by 5 d.p.i. Infection foci produced by wild type WSMV expressing GUS were much larger at 3 d.p.i. and had coalesced by 5 d.p.i. No GUS activity was detected in plants inoculated with the frame shift mutant bearing GUS. Three of four mutants with a single synonymous substitution in the P3 cistron were wild type with respect to systemic infectivity, indicating that only some of the synonymous substitutions affected movement. Collectively, these results identify an internal RNA sequence element in the P3 cistron that affects both replication and movement of the viral genome.