In-situ Visualization of Non-enveloped Virus Cell Entry by Cryo-ET

Escape into the cytosol from a membrane-bound endocytic compartment is a barrier faced by non-enveloped viruses and more generally by any macromolecular complex with an intracellular target. We were able to visualize by using cryogenic electron tomography (cryo-ET) the full course of rotavirus entry, from cell attachment and inward budding of the virion to arrival of a subviral particle in the cytosol. Rotaviruses deliver into the cytosol a transcriptionally active double-layer particle (DLP), which does not undergo further uncoating. We used subtomogram averaging, live-cell fluorescence microscopy, and single-liposome assays, to study the molecular details of rotavirus entry. The results allowed us to define the molecular mechanism of the step that releases DLPs into the cytosol. 

We observed directly sequential stages of entry, from virus attachment and membrane invagination to membrane perforation and cytosolic release of the DLP. Cryo-ET shows that uptake is driven by a conformational transition of the spike protein VP4, in which the VP5* domain inserts a coiled-coil and foot domains into the plasma membrane, generating tight membrane contacts and promoting inward budding. This insertion permeabilizes the membrane to Ca2+. Loss of Ca2+ within the closed vesicle induces dissociation of the outer-layer protein VP7, initiating uncoating distal to the membrane contact sites. We found that dissociated VP7 monomers are responsible for membrane perforation. Cryo-ET captures the formation of a single, large pore in the vesicular membrane through which the DLP escapes into the cytosol. In vitro, VP7 monomers, but not trimers, reproduce this activity by perforating liposomes by exposing an amphipathic C-terminal helix. Our results define a two-step membrane penetration/perforation mechanism for rotavirus entry and show how VP7 becomes a pore-forming protein, with potential implications for entry pathways of other non-enveloped viruses. 

This work was supported by National Institutes of Health, https://ror.org/01cwqze88, R01 CA13202, National Institutes of Health, https://ror.org/01cwqze88, R25 GM130386, National Institutes of Health, https://ror.org/01cwqze88, R01 AI163019.