Phosphorylation tunes strain-specific protein condensation during rotavirus replication organelle assembly

In many viruses, intrinsically disordered proteins (IDPs) drive the formation of replicative organelles via liquid–liquid phase separation (LLPS). In species A rotaviruses, the disordered protein NSP5 forms condensates with NSP2, but its high sequence diversity raises questions about whether this mechanism is conserved across strains. Using a machine learning approach, we show that NSP5 variants differ significantly in LLPS propensity. We engineered an NSP5 variant with features derived from strains with low-LLPS propensity (low-LLPS). Despite lacking the ability to phase separate in vitro unless phosphorylated, this variant nevertheless supported condensate formation and viral replication in cells. We found that low-LLPS variants require phosphorylation to nucleate phase separation, whereas high-LLPS variants do not, suggesting distinct nucleation mechanisms between viral strains. Hydrogen–deuterium exchange mass spectrometry revealed a phosphorylation-driven allosteric switch that alters NSP2 interactions depending on the NSP5 variant. These findings suggest that phosphorylation plays a context-dependent role in condensate formation, tuning NSP5-NSP2 interactions in a strain-specific manner and highlighting the mechanistic diversity underpinning replicative organelle formation among viral strains.