During the structural transition the FD the PHD and

During the structural transition, the FD, the PHD and the TrD retain their tertiary structure. Nevertheless, they undergo large rearrangements in their relative orientation due to secondary changes in hinge segments (R1 to R5) which refold during the low-pH induced conformational change. Particularly, in the post-fusion state, the core of the trimer is made by the three TrD central helices (extended by the refolding of segment R4) whose grooves accommodate three lateral helices resulting in the refolding of R5 (Fig. 1). This six-helix bundle organization is very similar to that of the trimeric core of the post-fusion state of some class I fusion mi2 such as HIV-1 gp41 or Ebola gp2 [29], [30]. In this review, we will present the recent crystalline structures of monomeric intermediates [31] obtained for CHAV G ectodomain and how they fit with numerous historical articles showing the existence of G monomers. We will also discuss the ability of G monomers to form flat antiparallel dimers and the experimental data indicating that those dimers play a role during the fusion process [31]. Finally, we will discuss if this mode of operating is specific to Vesiculovirus glycoproteins. Particularly, we will review EM data indicating that the initial interaction between class II fusion glycoproteins and the target membrane is mediated by monomers and examine some non-trimeric structures of influenza hemagglutinin present in the Protein Data Bank (PDB) but whose oligomerization state and protomer conformation have never been discussed thoroughly before.
Monomeric states of VSV G The first attempt to characterize the oligomeric status of VSV G revealed that a soluble ectodomain obtained by treatment of virions with cathepsin D is monomeric at high pH [32]. Later, it was shown that G solubilized from membranes with detergent behaves as a monomer at high pH whereas it forms a stable trimer at low pH [11]. In addition, the existence of a thermodynamic equilibrium between monomers and trimers of VSV G solubilized by octyl-glucoside was demonstrated [33]. This equilibrium was also demonstrated to exist in vivo[34], [35]. From those pioneering works, it was clear that VSV G trimer was much less stable than HA trimer, which was the best characterized viral fusion glycoprotein at that time [36]. The behavior of VSV Gth (residues 1–422, generated by thermolysin-limited proteolysis of viral particles in solution, which has been crystallized) was further characterized using several biophysical techniques, including analytical ultracentrifugation, circular dichroism, EM and small angle X-ray scattering. While the post-fusion trimer was the major species detected at low pH, the pre-fusion trimer was never detected in solution. Indeed, at high pH, Gth appeared to be a flexible monomer exploring a large conformational space and adopting more elongated conformations when pH decreases [37]. The oligomeric status of VSV G has also been analyzed at the surface of the viral particle by EM and tomography. Below pH 6, the only structure which is observed is the trimeric post-fusion state which has a tendency to reorganize into regular arrays [38]. Above pH 7, although few pre-fusion trimers are observed [38], the vast majority seems to be flexible monomers [37]. At intermediate pH (pH 6.7), the shape of the spikes was not homogeneous and several forms of G could be observed [37]. In some regions, G seemed to have kept its high pH organization. Some spikes also had the typical post-fusion shape. Some elongated monomeric rod-like shape structures could be distinguished. The sequential appearance of the different species when lowering the pH suggested that the monomers were intermediates during the conformational change [37]. Indeed, independent refolding of monomers before re-association to form the post-fusion trimer overcomes some topological problems encountered if G remains trimeric all along the structural transition [39].
Structure of monomeric intermediates