Cryo-EM structures reveal two distinct conformational states in a picornavirus cell entry intermediate

The virions of enteroviruses such as poliovirus undergo a global conformational change after binding to the cellular receptor, characterized by a 4% expansion, and by the opening of holes at the two and quasi-three-fold symmetry axes of the capsid. The resultant particle is called a 135S particle or A-particle and is thought to be on the pathway to a productive infection. Previously published studies have concluded that the membrane-interactive peptides, namely VP4 and the N-terminus of VP1, are irreversibly externalized in the 135S particle. However, using established protocols to produce the 135S particle, and single particle cryo-electron microscopy methods, we have identified at least two unique states that we call the early and late 135S particle. Surprisingly, only in the "late" 135S particles have detectable levels of the VP1 N-terminus been trapped outside the capsid. Moreover, we observe a distinct density inside the capsid that can be accounted for by VP4 that remains associated with the genome. Taken together our results conclusively demonstrate that the 135S particle is not a unique conformation, but rather a family of conformations that could exist simultaneously. Author summary Nonenveloped viruses need to provide mechanisms that allow their genomes to be delivered across membranes. This process remains poorly understood. For enteroviruses such as poliovirus, genome delivery involves a program of conformational changes that include expansion of the particle and externalization of two normally internal peptides, VP4 and the VP1 N-terminus, which then insert into the cell membrane, triggering endocytosis and the creation of pores that facilitate the transfer of the viral RNA genome across the endosomal membrane. This manuscript describes five high-resolution cryo-EM structures of altered poliovirus particles that represent a number of intermediates along this pathway. The structures reveal several surprising findings, including the discovery of a new intermediate that is expanded, but has not yet externalized the membrane interactive peptides; the clear identification of a unique exit site for the VP1 N-terminus; the demonstration that the externalized VP1 N-terminus partitions between two different sites in a temperature-dependent fashion; direct visualization of an amphipathic helix at the N-terminus of VP1 in an ideal position for interaction with cellular membranes; and the observation that a significant portion of VP4 remains inside the pa