Plants utilize RNA silencing as a conserved and effective defense mechanism against viruses. To counteract this antiviral defense pathway, plant viruses have evolved to encode one or multiple proteins known as viral suppressors of RNA silencing (VSRs) that interfere with specific steps of the RNA silencing pathways. Although VSRs have been identified for numerous plant viruses, none is known for grapevine fanleaf virus (GFLV; genus Nepovirus, family Secoviridae). GFLV causes fanleaf degeneration, the most destructive viral disease of grapevine (Vitis spp.) in most vineyards worldwide. This disease causes devastating yield loss up to 80% and substantially reduces the lifespan of vineyards. My dissertation aims to enhance our understanding of how this economically concerning virus counteracts host antiviral RNA silencing defense by (i) identifying GFLV VSRs and characterizing their suppression activities, (ii) pinpointing GFLV VSRs amino acids essential for suppression of RNA silencing and host gene expression modulation abilities, and (iii) determining the subcellular localization and temporal translocation of GFLV VSRs in planta. I agroinfiltrated transgenic Nicotiana benthamiana expressing enhanced green fluorescent protein (EGFP) with a hairpin construct to trigger systemic silencing of EGFP. Then, I evaluated GFLV proteins for their ability to induce systemic suppression of EGPF silencing in apical leaves using fluorescence imaging, quantitative assessment of EGFP fluorescence intensity, measurements of EGFP transcript levels and changes in host gene expression by RT-qPCR. My results revealed that GFLV encodes three VSRs, 1A, 1B, and 1AB, that either individually or in combination, suppress systemic RNA silencing and differentially modify the expression of host genes involved in RNA silencing. Next, I identified the conservation of a tryptophan-glycine and glycine-tryptophan (WG/GW) motif in VSRs across different GFLV isolates. Using a reverse genetics approach, I determined that mutating W to A in the WG/GW motif of GFLV VSRs either abolishes or reduces their systemic RNA silencing ability and impairs their capacity to modulate key plant RNA silencing genes. Finally, I explored subcellular localization patterns and temporal translocation trends of GFLV VSRs tagged with yellow fluorescent protein using a live plant cell imaging via confocal microscopy. I showed that 1A localizes in the nucleus, while 1B and 1AB exhibit dynamic subcellular translocation across the nucleus, endoplasmic reticulum, cytoplasm, and cell periphery. Together, my findings suggest that GFLV deploys multiple distinct strategies, specific to each VSR, to counteract plant antiviral immunity. In conclusion, my dissertation research deepened our understanding of the counterdefense strategies employed by GFLV to evade plant antiviral immunity and shed light on the complex molecular interactions between GFLV and its plant host.