Molecular basis for the acid initiated uncoating of human enterovirus D68
Enterovirus D68 (EV-D68) belongs to a group of enteroviruses that contain a single positive-sense RNA genome surrounded by an icosahedral capsid. Like common cold viruses, EV-D68 mainly causes respiratory infections and is acid labile. The molecular mechanism by which the acid sensitive EV-D68 virions uncoat and deliver their genome into a host cell is unknown. Using cryo-electron microscopy (cryo-EM), we have determined the structures of the full native virion and an uncoating intermediate (the A(altered)-particle) of EV-D68 at 2.2 [A] and 2.7 [A] resolution. These structures showed that acid treatment of EV-D68 leads to particle expansion, externalization of the viral protein VP1 N-termini from the capsid interior, and formation of pores around the icosahedral two-fold axes through which the viral RNA can exit. Moreover, because of the low stability of EV-D68 at neutral pH, cryo-EM analyses of a mixed population of particles demonstrated the involvement of multiple structural intermediates during virus uncoating. Among these, a previously undescribed state, the expanded (\"E1\") particle, shows a majority of internal regions (e.g, the VP1 N-termini) to be ordered as in the full native virion. Thus, the E1 particle acts as an intermediate in the transition from full native virions to A-particles. Molecular determinants, including a histidine-histidine pair near the two-fold axes, were identified that facilitate this transition under acidic conditions. Thus, the present work delineates the pathway of EV-D68 uncoating and provides the molecular basis for the acid lability of EV-D68 and of the related common cold viruses.\n\nSignificance StatementEnterovirus D68 (EV-D68) is an emerging pathogen that primarily causes childhood respiratory infections and is linked to neurological diseases. It was unclear how the virus uncoats and delivers its genome into a host cell to establish viral replication. Using high resolution cryo-electron microscopy, we showed that acid induces structural rearrangements of EV-D68 to initiate genome release from the virus. Structural analyses delineated a viral uncoating pathway that involves multiple distinct conformational states. Particularly, the structure of a previously unknown uncoating intermediate enabled the identification of molecular determinants that facilitate EV-D68 uncoating in an acidic environment. These results advance the knowledge of cell entry of EV-D68 and open up possibilities for developing antiviral therapeutics that impede structural rearrangements of the virus.