In vitro liquid-liquid phase separation induced by respiratory syncytial virus proteins and RNA.

Respiratory syncytial virus (RSV) causes severe respiratory infections, with viral replication occurring in cytoplasmic membraneless viral factories formed by liquid-liquid phase separation (LLPS). The interactions among the RSV nucleoprotein N, phosphoprotein P, transcription factor M2-1, and RNA drive these condensates. Here, we used a microfluidic PhaseScan platform, together with biochemical and cellular assays, to systematically characterize LLPS involving RSV proteins and RNA. We identified optimal concentrations of oligomeric N and P tetramers for condensate formation in vitro without crowding agents, demonstrated that monomeric N inhibits LLPS and revealed that M2-1 enhances condensate formation by increasing multivalency. Notably, we found that M2-1 preferentially binds 5' capped RNA, distinguishing it from N, which binds uncapped RNA. These findings elucidate molecular determinants of RSV viral factory assembly and subcompartmentalization, providing insights into viral replication mechanisms and informing potential antiviral strategies targeting LLPS processes.