Role of brome mosaic virus and cucumber mosaic virus coat proteins in viral movement

Brome mosaic bromovirus (BMV) and cucumber mosaic cucumovirus (CMV) are genetically related members of the plant virus family Bromoviridae . While these two viruses are very similar in their genome organization, they exhibit differences in viral-host interactions and their requirements for viral spread. BMV has adapted to infecting mainly monocots and a few dicotyledenous hosts. CMV has a very wide host range and equally infects both monocots and dicots. To evaluate the extent to which BMV coat protein (CP) gene is involved in cell-to-cell movement, Chenopodium quinoa plants were inoculated with BMV wild type (wt) RNAs 1 and 2 and a RNA3 variant containing either the beta-glucoronidase (GUS) reporter gene in place of its CP gene or a large deletion in the CP open reading frame (ORF). GUS expression from the BMV CP variant was at all times restricted to initially infected single cells. Application of fluorescence in situ hybridization (FISH) to leaves inoculated with the BMV CP deletion variant demonstrated that resulting infections were subliminal. FISH proved a suitable technique for differentiating subliminal infection sites from cell-to-cell spread. Observing FISH-treated leaves with confocal laser scanning microscopy (CLSM) showed that subliminal infection sites resulting from inoculations with the BMV CP deletion variant did not expand beyond a single cell type, whereas a wt BMV infection could spread to several. These results show that CP, probably in an encapsidation-competent form, along with movement protein (MP), is required for cell-to-cell spread of BMV. Both, BMV and CMV require their CPs for systemic spread, however the form in which CP is involved in cell-to-cell movement is distinct. The N-terminal basic arm of CMV CP contains an arginine-rich cluster, characteristic of RNA binding proteins, which is conserved among BMV and other plant and non-plant viruses. To identify regions of the CMV CP N-terminus that are essential for viral RNA interactions, a set of CMV CP variants was constructed and the behavior of each variant with respect to infectivity, packaging, and spread was examined. Biological assays in C. quinoa and Nicotiana benthamiana and progeny analysis revealed that a major portion of the CMV CP N-terminus is dispensable for CP-RNA interactions and established that CMV can move cell-to-cell in a non-virion form, while encapsidation is obligatory for long distance spread.