The rotavirus VP5*/VP8* conformational transition permeabilizes membranes to Ca ²⁺

Abstract

Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca ²⁺ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca ²⁺-stabilized trimer; loss of Ca ²⁺ releases both VP7 and the other outer-layer protein, VP4, from the particle. VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca ²⁺ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca ²⁺ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry.

Author summary

To infect a cell, non-enveloped viruses must have a mechanism to transfer their genomes across a membrane. The double-strand RNA (dsRNA) viruses deliver into the cytosol not just their genomes but an intact subviral particle (diameter ~70 nm). Rhesus rotavirus (RRV) particles attach through a spike-like outer-layer protein, VP4 (cleaved to VP5* and VP8*), then wrap themselves in membrane at the cell surface, producing a small, cytosolic vesicle surrounding each virion, from which the subviral particles escape directly. Loss of Ca ²⁺ from the entering virion always precedes escape. In previous work we found that a conformational change in VP5*/VP8* projects the C-terminal "foot" domain of VP5* outward. We now show, by combining cryo-EM and single-particle optical microscopy of RRV particles interacting with liposomes, that the outward-projected foot penetrates the lipid bilayer, permeabilizing it to Ca ²⁺. The new experiments thus recapitulate the initial step in particle escape and show that the conformational change must occur while the spike protein is membrane-attached, to couple the VP5* conformational change with membrane insertion. These findings bring us closer to a full molecular description how a large, subviral particle (about 70 nm diameter) can be delivered into a target host cell.