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To understand why transgenic Nicotiana occidentalis plants expressing a functional movement protein (MP) of Apple chlorotic leaf spot virus (ACLSV) show specific resistance to Grapevine berry inner necrosis virus (GINV), the MPs of ACLSV (50KP) and GINV (39KP) were fused to green, yellow, or cyan fluorescent proteins (GFP, YFP, or CFP). These fusion proteins were transiently expressed in leaf cells of both transgenic (50KP) and nontransgenic (NT) plants, and the intracellular and intercellular trafficking and tubule-inducing activity of these proteins were compared. The results indicate that in epidermal cells and protoplasts from 50KP plant leaves, the trafficking and tubule-inducing activities of GINV-39KP were specifically blocked while those of ACLSV-50KP and Apple stem grooving virus MP (36KP) were not affected. Additionally, when 39KP-YFP and 50KP-CFP were coexpressed in the leaf epidermis of NT plants, the fluorescence of both proteins was confined to single cells, indicating that 50KP-CFP interferes with the cell-to-cell trafficking of 39KP-YFP and vice versa. Mutational analyses of 50KP showed that the deletion mutants that retained the activities described above still blocked cell-to-cell trafficking of 39KP, but the dysfunctional 50KP mutants could no longer impede cell-to-cell movement of 39KP. Transgenic plants expressing the functional 50KP deletion mutants showed specific resistance against GINV. In contrast, transgenic plants expressing the dysfunctional 50KP mutants did not show any resistance to the virus. From these results, we conclude that the specific resistance of 50KP plants to GINV is due to the ability of the 50KP to block intracellular and intercellular trafficking of GINV 39KP.