Convergent evolution of the G3BP1-binding motif in betacoronavirus nucleocapsid proteins

Betacoronaviruses (β-CoVs) display divergent mechanisms to evade host antiviral responses, yet the evolutionary origin and functional relevance of their strategies remain unclear. Stress granules (SGs), central to host defenses, are disrupted by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein via its interaction with G3BP1 mediated by an ¹⁵ITFG¹⁸ motif. This interaction inhibits SG assembly, enhancing viral replication and immune evasion. Here, we analyzed 179 N protein sequences across β-CoVs subgenera and identified the ITFG motif in sarbecoviruses but not in MERS-CoV or embecoviruses. Among tested CoVs, only SARS-CoV-2 N showed robust G3BP1 binding in vitro and in infected cells. Phylogenetic analyses revealed that the ΦxFG motif emerged independently in sarbecoviruses and a bat-infecting merbecovirus clade. The VGTF motif in these merbecoviruses also binds to G3BP1, suggesting convergent evolution in viral evasion strategies. The emergence of this motif was unrelated to 4a protein inactivation, another viral protein that inhibits SG formation.