Acid-induced movements in the glycoprotein shell of an alphavirus turn the spikes into membrane fusion mode

Abstract

In the icosahedral (T = 4) Semliki Forest virus, the envelope protomers, i.e. E1±E2 heterodimers, make one-to-one interactions with capsid proteins below the viral lipid bilayer, transverse the membrane and form an external glycoprotein shell with projections. The shell is organized by protomer domains interacting as hexamers and pentamers around shell openings at icosahedral 2-and 5-fold axes, respectively, and the projections by other domains associating as trimers at 3-and quasi 3-fold axes. We show here, using cryoelectron microscopy, that low pH, as occurs in the endosomes during virus uptake, results in the relaxation of protomer interactions around the 2-and the 5-fold axes in the shell, and movement of protomers towards 3-and quasi 3-fold axes in a way that reciprocally relocates their putative E1 and E2 domains. This seemed to be facilitated by a trimerization of transmembrane segments at the same axes. The alterations observed help to explain several key features of the spike-mediated membrane fusion reaction, including shell dissolution, heterodimer dissociation, fusion peptide exposure and E1 homotrimerization.

The Dynamic Envelope of a Fusion Class II Virus

“…Virus Purification by Tartrate Gradient Centrifugation and Quality Control-The SFV4 was propagated and purified, essentially as described previously (29,39). The SFV SQL was activated for infection by chymotrypsin treatment but otherwise handled in the same way as the wild type virus.…”

“…The SFV WT and mutant SQL were purified using tartrate gradient ultracentrifugation, as described above (29). Equal amounts (ϳ85 ng/well) of purified SFV WT and SQL were coated overnight at 4°C onto 96-well ELISA plates (high binding enzyme immunoassay/radioimmunoassay plate; Corning Glass).…”

“…At the final stages of the extensive purification of SFV WT particles for cryo-EM, the glycoprotein E3 was essentially removed. Such E3-depleted particles have been the source for our recently presented structure of SFV (29,31,42). As medium for the density gradient centrifugation, tartrate has been preferred to sucrose, since it is easier to remove and therefore allows a better situation for the imaging.…”

“…In the whole virus structure, revealed by electron cryomicroscopy (cryo-EM), the trimeric spikes protrude above a protein shell layer that surrounds the virus at some distance above its membrane (25)(26)(27)(28)(29)(30)(31). Modeling of the solved crystal structure of the ectodomain of fusion protein E1 (32,33) into cryo-EMderived whole virion structures implies that E1 constitutes the protein shell by intermolecular E1-E1 interactions and internal interactions between its domain III and I (31)(32)(33)(34).…”

Characterization of an early-stage fusion intermediate of Sindbis virus using cryoelectron microscopy

“…The structures of these viruses at pH values ∼5.0−5.9 have been examined using cryoelectron microscopy (cryo-EM) and 3D reconstruction techniques (22)(23)(24)(25); however, low-pH-triggered conformational rearrangements of the virus in the context of a target membrane are rarely investigated via these techniques (26). Here we present a cryo-EM study of SINV TE12 in complex with a liposomal membrane at pH 6.4.…”