Name
Hidden rules in apparent disorder: diverse rotavirus A variants use distinct mechanisms to nucleate viral factories
Presenter
Alexander Borodavka, University of Cambridge
Co-Author(s)
Alex Borodavka
Abstract Category
Virus Replication: Entry, Exit and Everything in Between
Abstract
Intrinsically disordered proteins (IDPs) are increasingly recognised as key drivers of viral replication by forming membraneless organelles via liquid-liquid phase separation (LLPS). In rotavirus A, the disordered protein NSP5 forms such condensates, but its striking sequence variability across strains raises a fundamental question: Is LLPS a universal mechanism for NSP5 function, or do different variants use distinct molecular strategies?
To address this, we combined machine learning with experimental reconstitution. Our predictive models revealed significant differences in LLPS propensity among NSP5 variants. Strikingly, a synthetic variant designed with features from low-propensity strains was unable to phase separate in vitro yet supported condensate formation and viral replication in cells. This prompted a deeper mechanistic investigation. We found that low-propensity NSP5 variants require phosphorylation to initiate phase separation, whereas high-propensity variants do not. Hydrogen-deuterium exchange mass spectrometry further uncovered a phosphorylation-dependent allosteric switch in high-propensity NSP5 that regulates the accessibility of interaction sites.
Together, our findings reveal that rotaviruses have evolved distinct phosphorylation-tuned mechanisms for nucleating replication factories despite using the same disordered protein. This work redefines how we understand NSP5 function across strains and illustrates the evolutionary plasticity of viral replication strategies, highlighting opportunities for targeting phase separation processes as an antiviral strategy.
To address this, we combined machine learning with experimental reconstitution. Our predictive models revealed significant differences in LLPS propensity among NSP5 variants. Strikingly, a synthetic variant designed with features from low-propensity strains was unable to phase separate in vitro yet supported condensate formation and viral replication in cells. This prompted a deeper mechanistic investigation. We found that low-propensity NSP5 variants require phosphorylation to initiate phase separation, whereas high-propensity variants do not. Hydrogen-deuterium exchange mass spectrometry further uncovered a phosphorylation-dependent allosteric switch in high-propensity NSP5 that regulates the accessibility of interaction sites.
Together, our findings reveal that rotaviruses have evolved distinct phosphorylation-tuned mechanisms for nucleating replication factories despite using the same disordered protein. This work redefines how we understand NSP5 function across strains and illustrates the evolutionary plasticity of viral replication strategies, highlighting opportunities for targeting phase separation processes as an antiviral strategy.