Mutations in the WG and GW motifs of the three RNA silencing suppressors of grapevine fanleaf virus alter their systemic suppression ability and affect virus infectivity

Viral suppressors of RNA silencing (VSRs) encoded by grapevine fanleaf virus (GFLV), one of the most economically consequential viruses of grapevine (<i>Vitis</i> spp.), were recently identified. GFLV VSRs include the RNA1-encoded protein 1A and the putative helicase protein 1B^Hel, as well as their fused form (1AB^Hel). Key characteristics underlying the suppression function of the GFLV VSRs are unknown. In this study, we explored the role of the conserved tryptophan-glycine (WG) motif in protein 1A and glycine-tryptophan (GW) motif in protein 1B^Hel in their systemic RNA silencing suppression ability by co-infiltrating <i>Nicotiana benthamiana</i> 16c line plants with a <i>GFP</i> silencing construct and a wildtype or a mutant GFLV VSR. We analyzed and compared wildtype and mutant GFLV VSRs for their (i) efficiency at suppressing RNA silencing, (ii) ability to limit siRNA accumulation, (iii) modulation of the expression of six host genes involved in RNA silencing, (iv) impact on virus infectivity <i>in planta</i>, and (v) variations in predicted protein structures using molecular and biochemical assays, as well as bioinformatics tools such as AlphaFold2. Mutating W to alanine (A) in WG of proteins 1A and 1AB^Hel abolished their ability to induce systemic RNA silencing suppression, limit siRNA accumulation, and downregulate <i>NbAGO2</i> expression by 1AB^Hel. This mutation in the GFLV genome resulted in a non-infectious virus. Mutating W to A in GW of proteins 1BHel and 1ABHel reduced their ability to suppress systemic RNA silencing and abolished the downregulation of <i>NbDCL2</i>, <i>NbDCL4,</i>, and <i>NbRDR6</i> expression by 1B^Hel. This mutation in the GFLV genome delayed infection at the local level and inhibited systemic infection <i>in planta</i>. Double mutations of W to A in WG and GW of protein 1ABHel abolished its ability to induce RNA silencing suppression, limit siRNA accumulation, and downregulate <i>NbDCL2</i> and <i>NbRDR6</i> expression. Finally, <i>in silico</i> protein structure prediction indicated that a W to A substitution potentially modifies the structure and physicochemical properties of the three GFLV VSRs. Together, this study provided insights into the specific roles of WG/GW not only in GFLV VSR functions but also in GFLV biology.