Plant negative-strand RNA virus phosphoprotein condensates exploit host trafficking and lipid synthesis for viral factory assembly.

RNA viruses often remodel host intracellular membranes to establish specialized replication compartments through viral protein-induced phase separation. However, the mechanisms underlying membrane remodeling and the characteristics that render these sites conducive to replication remain poorly understood, particularly in plant negative-strand RNA viruses. Here, we demonstrate that the phosphoprotein (P) of rice stripe mosaic virus (RSMV) forms biomolecular condensates via liquid-liquid phase separation (LLPS) to recruit essential components for viral replication factories (VFs). We identify a direct interaction between RSMV P and adenosine diphosphate (ADP) ribosylation factor 1 (OsARF1C), a crucial regulator of the coatomer protein I (COP I) vesicle transport pathway that is vital for viral replication. This interaction indirectly recruits OsARF1C's partner, phosphatidylinositol 4-kinase beta (OsPI4KB), which drives localized phosphatidylinositol-4 phosphate (PI4P) synthesis. Concurrently, the P protein modulates its aggregates and LLPS droplets through PI4P, thereby expanding the replication site and enhancing viral replication.