Targeting phase separation of TSWV nucleocapsid protein with novel methyl vanillate derivative A10 to inhibit biomacromolecule aggregate formation.

Phase separation (PS)-driven biomolecular condensates have recently emerged as crucial elements in the life cycles of numerous phytoviruses and their interactions with host plants. While inhibiting the formation of these aggregates presents a promising plant protection strategy, research into discovering such inhibitors remains scarce. Here, we report a novel achievement in this field. Through biological screening of a series of structurally designed methyl vanillate derivatives incorporating N-Acetyl-D-glucosamines, a novel compound A10 is discovered to possess excellent antiviral potency against tomato spotted wilt virus (TSWV). Compared with the commercial control ribavirin, A10 demonstrates superior curative (56.9%), protective (50.5%), and inactivating (62.2%) activities, with its EC50 based on inactivation effect (181.7 μg/mL) being lower than that of ribavirin (EC50 = 748.5 μg/mL). A10 treatment significantly reduces the formation of TSWV nucleocapsid (N) protein condensates. Molecular docking, Molecular dynamics simulations, and Bio-layer interferometry analyses identify Arg94, Tyr184, and Lys192 as key residues on the N protein for A10 binding. Combining Agrobacterium-mediated infiltration assays and confocal microscopy observations allows A10 to mechanistically inhibit PS and aggregate generation of the N protein, thereby significantly suppressing viral RNP proliferation and systemic infection in host plants. This study contributes a valuable new lead compound for phytoviral management by targeting PS.