Acker Julia, Wang Xinyu, Desirò Daniel, Agarwal Tanushree, Colyer Alice, Haller Cyril, Scrutton Rob, Sherry Lee, Saar Kadi L, Murray Rosie, Fominykh Ksenia, Chong Sai Hou, Schmit Jeremy D, Calabrese Antonio N, Knowles Tuomas P J, Borodavka Alexander
Department of Chemical Engineering and Biotechnology, University of Cambridge, CB3 0AS, Cambridge, UK.
Department of Chemistry, University of Cambridge, Cambridge, UK.
bioRxiv. 2025 May 24:2025.05.24.655949. doi: 10.1101/2025.05.24.655949.
In many viruses, intrinsically disordered proteins (IDPs) drive the formation of replicative organelles essential for viral production. In species A rotaviruses, the disordered protein NSP5 forms condensates in cells via liquid-liquid phase separation (LLPS). Yet the sequence diversity of NSP5 raises the question of whether condensate formation is conserved across all strains and if distinct variants employ alternative mechanisms for nucleating phase separation. Using a machine learning approach, we demonstrate that NSP5 variants differ significantly in their propensity to phase-separate. We engineered a variant incorporating amino acid signatures from strains with low LLPS tendency, which failed to phase separate in vitro yet supported the formation of replicative condensates in recombinant viruses in cells. Low-tendency LLPS strains require phosphorylation of NSP5 to nucleate phase separation, whereas high-tendency strains do not, suggesting distinct nucleation mechanisms. Furthermore, hydrogen-deuterium exchange mass spectrometry revealed a phosphorylation-driven allosteric switch between binding sites on the high-propensity variant. These findings establish that phosphorylation plays a context-dependent role in the formation of replicative organelles across diverse rotaviruses.
在许多病毒中，内在无序蛋白（IDP）驱动着病毒产生所必需的复制细胞器的形成。在A种轮状病毒中，无序蛋白NSP5通过液-液相分离（LLPS）在细胞中形成凝聚物。然而，NSP5的序列多样性引发了一个问题，即凝聚物的形成是否在所有菌株中都保守，以及不同的变体是否采用不同的机制来启动相分离。使用机器学习方法，我们证明NSP5变体在其相分离倾向方面存在显著差异。我们设计了一个包含低LLPS倾向菌株氨基酸特征的变体，该变体在体外未能发生相分离，但在细胞中的重组病毒中支持复制凝聚物的形成。低倾向LLPS菌株需要NSP5磷酸化才能启动相分离，而高倾向菌株则不需要，这表明存在不同的成核机制。此外，氢-氘交换质谱揭示了高倾向变体结合位点之间由磷酸化驱动的变构开关。这些发现表明，磷酸化在不同轮状病毒复制细胞器形成中发挥着依赖于背景的作用。
