Residues R(^192) and K(^225) in RNA-Binding Pocket of Tobacco Vein Banding Mosaic Virus CP Control Virus Cell-to-Cell Movement and Replication

Abstract

Potyviruses move to neighboring cells in the form of virus particles or a coat protein (CP)-containing ribonucleoprotein complex. However, the precise roles of RNA-binding residues in potyviral CP in viral cell-to-cell movement remain to be elucidated. In this study, we predicted the three-dimensional model of tobacco vein banding mosaic virus (TVBMV)-encoded CP and found nine residues presumably located in the CP RNA-binding pocket. Substitutions of the two basic residues at positions 192 and 225 (R(^192) and K(^225)) with either alanine, cysteine, or glutamic acid abolished TVBMV cell-to-cell and systemic movement in Nicotiana benthamiana plants. These substitutions also reduced the replication of the mutant viruses. Results from the electrophoretic mobility shift assay showed that the RNA-binding activity of mutant CPs derived from R(^192) or K(^225) substitutions was significantly lower than that of wild-type CP. Analysis of purified virus particles showed that mutant viruses with R(^192) or K(^225) substitutions formed RNA-free virus-like particles. Mutations of R(^192) and K(^225) did not change the CP plasmodesmata localization. The wild-type TVBMV CP could rescue the deficient cell-to-cell movement of mutant viruses. Moreover, deletion of any of the other seven residues also abolished TVBMV cell-to-cell movement and reduced the CP RNA-binding activity. The corresponding nine residues in watermelon mosaic virus CP were also found to play essential roles in virus cell-to-cell movement. In conclusion, residues R(^192) and K(^225) in the CP RNA-binding pocket are critical for viral RNA binding and affect both virus replication and cell-to-cell movement.