Experimental and bioinformatic evidence that raspberry leaf blotch emaravirus P4 is a movement protein of the 30K superfamily

Emaravirus is a recently described genus of negative-strand RNA plant viruses. Emaravirus P4 protein localized to plasmodesmata, suggesting that it could be a viral movement protein (MP). In the current study, we showed that the P4 protein of raspberry leaf blotch emaravirus (RLBV) rescued the cell-to-cell movement of a defective potato virus X (PVX) that had a deletion mutation in the triple gene block 1 movement-associated protein. This demonstrated that RLBV P4 is a functional MP. Sequence analyses revealed that P4 is a distant member of the 30K superfamily of MPs. All MPs of this family contain two highly conserved regions predicted to form beta-strands, namely beta 1 and beta 2. We explored by alanine mutagenesis the role of two residues of P4 (Ile106 and Asp127) located in each of these strands. We also made the equivalent substitutions in the 29K MP of tobacco rattle virus, another member of the 30K superfamily. All substitutions abolished the ability to complement PVX movement, except for the l106A substitution in the beta 1 region of P4. This region has been shown to mediate membrane association of 30K MPs; our results show that it is possible to make non-conservative substitutions of a well-conserved aliphatic residue within beta 1 without preventing the membrane association or movement function of P4.