On-virion structural dynamics reveal temperature- and receptor-coordinated activation of an alphacoronavirus spike

Abstract Human alphacoronaviruses primarily infect the human upper respiratory tract, yet how physiological temperature and receptor-binding coordinate viral spike (S) protein activation remain unclear. Using cryo-electron tomography and subtomogram averaging, we directly visualized the structural dynamics of S on human coronavirus 229E (HCoV-229E) virions under physiologically relevant temperatures and in the presence of the host receptor human aminopeptidase N (hAPN). We identified two prefusion S2 conformations, compact and loose, that interconvert reversibly by temperature, revealing intrinsic S2 “breathing” on virions. Receptor-binding domain (RBD)-up conformations are rare on apo virions but are promoted at physiological temperature and stabilized upon receptor binding. hAPN can bind bivalently to neighboring S, forming Gemini S-hAPN complexes. Receptor incubation further promotes conversion from prefusion to postfusion S, an effect enhanced at 33. Together, our results demonstrate an optimized temperature for the HCoV-229E viral fitness, establish temperature and receptor binding as synergistic regulators of alphacoronavirus S activation and define an integrated, on-virion model of S conformational transitions during viral entry. These findings provide a structural framework for understanding alphacoronavirus infectivity.