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

Journal article

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.

Authors: Acker, J; Wang, X; Pardal, AJ; Desirò, D; Agarwal, T

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: EMBO Press
• Journal: The EMBO Journal
• Publication date: 2026-05-26
• DOI: 10.1038/s44318-026-00814-z
• EISSN: 1460-2075
• ISSN: 0261-4189
• Language: English
• Keywords: Sequence (biology); Nucleation; Phosphorylation; Scaffold protein; Intrinsically disordered proteins; Organelle; Replication (statistics); Rotavirus; Viral replication