Grapevine fanleaf virus (GFLV) is one of the most adverse viral pathogens of grapevine worldwide. GFLV is a species of the genus Nepovirus in the family Secoviridae in the order Picornavirales and is the causative agent of fanleaf degeneration of grapevines. This disease is characterized by symptoms including developmental abnormalities such as double nodes, shortened internodes, fasciations, zigzag growth; foliar symptoms include cupping, asymmetrical leaf shape, and the namesake “fanleaf” consisting of abnormally open petiolar sinuses and aberrant vein spacing in a radial pattern; and also poor and uneven berry set, leading to yield losses. This degeneration may lead to premature vine death. The molecular mechanisms underlying the development of GFLV symptoms are not well studied. In the herbaceous systemic host Nicotiana benthamiana, most GFLV isolates produce an asymptomatic infection, while a recently described isolate, GFLV strain GHu, induces distinct vein clearing symptoms on the apical leaves of N. benthamiana. Using a reverse genetics approach, I determined that symptom production in N. benthamiana by GFLV-GHu is modulated by a single residue of protein 1EPol, the RNA-dependent RNA polymerase. Mutation of GFLV-GHu 1EPol residue 802 (lysine) modulates symptom development. While some substitutions (e.g. glycine, alanine) abolish symptoms in GFLV-GHu, other substitutions (arginine) have no effect on symptoms. Some mutations (e.g. proline, serine, threonine) produce intermediate, faint vein clearing that is distinct from symptoms produced by wild type GFLV-GHu. Lysine 802 is not sufficient for symptom production in N. benthamiana and this residue appears to be flanked by motifs or structures that are highly conserved among GFLV isolates. I also probed the protein interaction network of 1EPol through affinity purification of epitope-tagged 1EPol from systemically infected N. benthamiana tissue, followed by protein identification by high-resolution tandem mass spectrometry. I found that 1EPol, regardless of strain, appears to be in complex with GFLV proteins 1DPro and 1BHel. GFLV-GHu 1EPol affinity purification products were specifically enriched in proteins with various cellular functions, most notably a number of proteins associated with chloroplasts. These data lay the groundwork for mapping the 1EPol protein interaction network and for identifying the mechanism by which GFLV-GHu induces vein clearing in N. benthamiana. These insights into the molecular biology of GFLV symptom development will aid efforts to develop GFLV management strategies in grapevine by providing information on virus-host interactions that play a central role in GFLV biology.