Identification of residues or motif(s) of the rice stripe virus NS3 protein required for self-interaction and for silencing suppressor activity

Rice stripe virus (RSV) is an important pathogen of rice. The RSV genome consists of four single-stranded RNA segments that encode seven viral proteins. A previous report found that NS3 is a viral suppressor of RNA silencing and self interacts. Using a model that predicts protein structure, we identified amino acid residues or motifs, including four α-helix motifs, required for NS3 self-interaction. We then used yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays to study the interactions between full-length NS3 and its truncated and alanine substitution mutants. Y2H and BiFC results showed that the N-terminal region of NS3 is essential for self-interaction. All α-helix deletion mutants and substitution mutants lost the ability to self-interact. To identify the relationship between NS3 self-interaction and silencing suppressor activity, we used a GFP silencing system in Nicotiana benthamiana with Agrobacterium-mediated transient overexpression of each mutated NS3 protein. All of the deletion and the α-helix substitution mutants that had lost the ability to self-interact also lost their silencing suppressor ability. The substitution of amino acids with alanine at positions 70-75, 76-83, and 173-177, however, resulted in mutants that were able to self-interact but were unable to function as silencing suppressors. These results suggest that RSV requires NS3 self-interaction to suppress RNA silencing and to thereby counter host defenses.