Our structural characterization demonstrates that Gth
is in a monomeric state in solution at high pH. We also provide evidences for the presence of monomers at the viral surface both at pH 7.5 and 6.7. At pH 7.5, in agreement with previous reconstructions from EM tomography 
, we show that only a few pre-fusion trimers can be detected and that most glycoproteins are likely in a monomeric state. At pH 6.7, rod-shaped elongated structures are also observed, the dimensions of which are consistent with a monomeric state.
We propose that in its monomeric state, G explores a manifold of conformations that are intermediates along the structural transition from the pre- to the post-fusion trimeric state. The following findings support this view.
First, both the pre- and post-fusion trimeric states can be generated and crystallized from the same high pH monomeric form. Indeed, the pre-fusion trimer is observed in the crystal 
, while the post-fusion trimer can be obtained by lowering the pH in solution (this study) and subsequently crystallized 
in perfect agreement with what is expected from a bona fide
Second, SAXS data establish that a significant fraction of monomers adopt a range of conformations that differ from that of the protomers in both the pre- and post-fusion trimeric structures.
Third, there are large differences between the pre- and post-fusion trimeric interfaces 
Fourth and last, as previously mentioned 
, it is topologically impossible for pre-fusion trimeric Gth
to reach the post-fusion state unless the threefold symmetry is lost, for example through dissociation into monomers.
The conformational space explored by monomeric Gth in solution exhibits a marked pH-dependence summarized in . Indeed, the analysis of our SAXS data demonstrates that a higher fraction of Gth molecules adopts elongated conformations at pH 7.5 than at pH 8.8. Comparison with CD data reveals that these elongated states (significantly populated at pH 7.5) are detected in absence of significant change in the secondary structure content of Gth. This indicates that large movements of domains can occur without elongation of the central helix nor formation of the lateral one that together make the 6-helix bundle structure found in the trimeric post-fusion conformation.
Pathway for the pH-dependent structural transition of G in solution and at the viral surface.
The flexible high-pH monomeric form is unable to bind target membranes and does not form rosettes in solution. In contrast, at intermediate pH (6.7), rosettes are observed that exhibit a different appearance from those formed by the post-fusion trimer at pH 5.7. They are visible at low protein concentration at which no trimer is detected, suggesting that they result from monomer association. This interpretation is consistent with the fact that Gth
segments leading to the center of these aggregates are much thinner than those in the rosettes observed at pH 5.7. However, no side-chain is present at the tip of the fusion domain whose protonation state can change significantly within the pH range 8.8-6.7 
. Thus, the hydrophobicity of the loop region should remain unaltered over this pH range, which raises the question of the origin of rosette formation from monomers at pH 6.7. We propose that, at this pH, monomeric Gth
explores preferentially very extended conformations that might resemble intermediates a6 to a9 () with the C-terminal part and fusion domain located at opposite extremities of the molecule. In such conformations the two hydrophobic fusion loops at the tip of the fusion domain reach out far into the solvent, and their unrestricted exposure makes mutual contact highly probable and energetically favorable, leading finally to rosette formation (, pH 6.7). This restricted conformational space would be favored by the protonation of histidine residues H60, H162 (located in the fusion domain part distal from the fusion loops) and H407 (located in the C-terminal part of the molecule) that are in close proximity in the pre-fusion conformation and have been proposed to play the role of pH-sensitive molecular switch 
Most probably, target membranes interact with the fusion loops whose access is essentially unhindered in proteins that adopt more elongated conformations. The presence of target membranes thereby shifts the equilibrium between the various forms toward the more elongated conformations and, as a consequence, affects the pH-dependence of the population landscape.
The major part of this work has been performed on a soluble form of G. One might argue that the absence of the transmembrane (TM) domain in Gth
could affect the pathway of the structural transition and that the trimer of full-length G within the viral membrane could be stabilized through inter-TM domain interactions, by the increase in G local concentration or by optimization of G orientation for trimer interaction at the viral surface. However, when solubilized from the viral or cellular membranes at pH 7.4, full-length G has also been reported to be monomeric 
and there is no evidence for any interaction between the TM domains of the three protomers within a pre-fusion trimer. In addition, our EM studies on viral particles show, in agreement with previous data 
, that at pH 7.5 most G molecules at the viral surface are not in the trimeric pre-fusion state and that at pH 6.7 monomeric elongated structures, oblique to the viral membrane, can also be observed. We may therefore confidently conclude that VSV G ectodomains can fully dissociate at the viral surface during the structural transition.
From these results, a plausible pathway for the structural transition is suggested in . At high pH, G molecules at the viral surface are in equilibrium between the pre-fusion trimer and flexible monomers. Lowering the pH results in monomers adopting, in increasing number, elongated conformations with the fusion loops exposed at the top of the glycoprotein, thereby favoring the initial interaction with the target membrane. Finally, these monomers complete their refolding process to re-associate and form post-fusion trimers.
The topology of the structural transition of class I fusion glycoproteins is identical to that of VSV G 
. For this class of fusion proteins, the initial steps leading to fusion peptide exposure and its interaction with the target membrane may maintain strict trimeric symmetry but the folding back of the C-terminal part of the molecule requires to break the three-fold symmetry of the molecule 
. Remarkably, paramyxovirus F proteins (a representative of the class I fusion proteins) also exhibit large differences between their pre- and post-fusion trimeric interfaces 
. Thus, here again, a plausible scheme for the structural transition could also involve a manifold of transient short-lived monomeric intermediates. The scheme established here for a representative of class III fusion proteins could thus be a general feature of the structural transition of other viral fusion proteins.