Here, we report a new crystal structure of CHAV G th . The crystal contains two distinct conformations, apparently corresponding to early and late refolding states of G, arranged in a flat dimer of heterodimers exposing fusion loops. At the viral surface and in the absence of target membrane, under appropriate pH and temperature conditions, electron microscopy (EM) and tomography revealed monomeric spikes that are in a conformation similar to one of the protomers present in the crystalline structure. Through native mass spectrometry (MS), we provide evidences that dimeric assemblies of both VSV and CHAV G th are present in solution at intermediate pH. Furthermore, mutations of residues located at a crystalline dimeric interface, formed by two FDs laterally associated in an antiparallel manner, affect the fusion properties. The location of the compensatory mutations restoring the fusion activity strongly suggests that this interface is functionally relevant. Taken together, these data offer a detailed pathway for the conformational transition from pre‐ to post‐fusion trimers. They further suggest that flat dimers, associated through their FDs, play a role at some early stage of the fusion process. Therefore, this work provides the molecular basis of a more detailed model for vesiculovirus membrane fusion.

In the trimers (Fig EV1 ), G th pre‐ and post‐fusion states of a protomer (respectively referred to as PRE and POST hereafter) are related by flipping both FD and the C‐terminal segment around CD (Roche et al , 2007 ) thanks to refolding of segments R1 to R5. The complex conformational change involves monomeric intermediates (Albertini et al , 2012a ), but the pathway of the transition remains unknown.

Data information: Domains and segments are colored as indicated in Table 1 . Hydrophobic residues of the fusion loops are in pink sticks. FD and the C‐terminal part of the ectodomain are flipped around the CD (which has the same orientation in B, C, and D) during the structural transition.

The polypeptide chain of G ectodomain folds into three distinct domains (Baquero et al , 2013 ) (Table 1 and Fig EV1 ). The fusion domain (FD, in yellow) is made of an extended β‐sheet structure at the tip of which are located the two fusion loops (in pink). FD is inserted in a loop of a pleckstrin homology domain (PHD, in orange) that is itself inserted into a central domain (CD, in red), which is involved in trimerization of the molecule in both the pre‐ and post‐fusion states. The CD connects to the segment R5 (in purple), which itself connects to the C‐terminal TM domain in the full‐length glycoprotein. Segments R1 and R4 (in cyan) connect CD to PHD, while R2 and R3 (in green) connect PHD to FD.

The trimeric structures of both the pre‐ and post‐fusion states of a soluble form of the ectodomain of VSV G (VSV G th , amino acid (aa) residues 1–422, generated by thermolysin‐limited proteolysis of viral particles) (Roche et al , 2006 , 2007 ) and of the post‐fusion state of CHAV G ectodomain (CHAV G th , aa residues 1–419, generated the same way as VSV G th ) (Baquero et al , 2015b ) have been determined. The structures of post‐fusion trimers for herpesvirus glycoprotein gB (Heldwein et al , 2006 ; Backovic et al , 2009 ; Burke & Heldwein, 2015 ; Chandramouli et al , 2015 ) and baculovirus glycoprotein gp64 (Kadlec et al , 2008 ) show that they and vesiculovirus G define the class III of viral fusion glycoproteins (Baquero et al , 2015a ).

The Rhabdoviridae are enveloped bullet‐shaped viruses that are widespread among a great variety of organisms including plants, insects, crustaceans, fishes, reptiles, and mammals (Rose & Whitt, 2001 ). This family includes VSV, the prototype of the vesiculovirus genus, as well as notable human pathogens, such as Chandipura virus (CHAV), a vesiculovirus responsible for deadly encephalopathies (Rao et al , 2004 ), and rabies virus (RABV), the prototype of the lyssavirus genus. In their membrane, rhabdoviruses have a single glycoprotein (G) that mediates both virus attachment to specific receptors and, after virion endocytosis and acidification of the endosome lumen, fusion between viral and endosomal membranes (Albertini et al , 2012b ). It has been demonstrated that the low‐pH‐induced structural transition of rhabdovirus G is reversible (Doms et al , 1987 ; Gaudin et al , 1993 ) in contrast to what has been shown for fusion glycoproteins from other viral families (Gaudin, 2000 ). The reversibility is the consequence of the existence of a pH‐dependent thermodynamic equilibrium between different states of G (pre‐fusion trimer, flexible monomers, and post‐fusion trimer). At pH above 7.5, both pre‐fusion trimers and flexible monomers can be observed at the viral surface, whereas the equilibrium is shifted toward the post‐fusion trimer at low pH (Roche & Gaudin, 2002 ; Libersou et al , 2010 ; Albertini et al , 2012a ). EM studies performed on VSV revealed that fusion is driven by two successive events involving the glycoprotein. First, fusion is initiated at the flat base of the virion. Second, glycoproteins located in the cylindrical part of the particle reorganize into regular arrays to complete the fusion reaction (Libersou et al , 2010 ).

Entry of enveloped viruses into their host cell requires fusion of the viral membrane with a cellular membrane. This step is mediated by viral glycoproteins, anchored in the viral membrane by a transmembrane (TM) domain, that undergo structural rearrangements from a pre‐ to a post‐fusion state upon interaction with specific triggers (e.g. a low pH environment and/or cellular receptors). During this conformational change, hydrophobic motifs (the so‐called fusion peptides or fusion loops) are exposed and interact with one or both of the participating membranes, resulting in their destabilization and merger. At the end of the refolding process, the fusion proteins are in a hairpin‐like post‐fusion structure, in which the fusion loops (or peptides) and TM domain are at the same extremity of the molecule and in the same membrane (Harrison, 2015 ). Viral fusion glycoproteins have been classified based on common structural motifs (Li & Modis, 2014 ; Harrison, 2015 ). Experimental data suggest that the membrane fusion pathway is very similar for all the enveloped viruses studied so far whatever the organization of their fusion machinery (Chernomordik & Kozlov, 2008 ).

Results

Evidence for dimeric species for both CHAV G th and VSV G th In the crystal asymmetric unit, CHAV G th molecules form a flat dimer of heterodimers (Fig 1A and B, Appendix Fig S3), whereas the oligomeric species of class III fusion proteins identified so far are symmetric protruding trimers (Heldwein et al, 2006; Roche et al, 2006, 2007; Kadlec et al, 2008; Backovic et al, 2009; Baquero et al, 2015b; Burke & Heldwein, 2015; Chandramouli et al, 2015; Zeev‐Ben‐Mordehai et al, 2016). This unexpected crystalline organization prompted us to investigate the oligomeric states of CHAV G th in our preparation. For this, we used native MS (Boeri Erba & Petosa, 2015; Mehmood et al, 2015). CHAV G th sample solutions were first desalted and buffer‐exchanged in 200 mM ammonium acetate at pH values 6.0, 7.5, and 8.8 (final protomer concentration: 7 μM). At pH 8.8, only monomers were detected. At pH 6.0, only trimers were observed. However, at pH 7.5, both monomeric and dimeric species were present (Fig 3A). Figure 3.Evidence for dimers of CHAV G th and VSV G th in solution Native mass spectra of CHAV G th at three pH values (pH 6.0, 7.5 and 8.8). In acidic conditions, CHAV G th is a trimer. At pH 7.5, both dimers and monomers were observed; at pH 8.8, only monomers were detected. Native mass spectra of VSV G th . At pH 6.0, VSV G th is detected as trimers and hexamers. At pH 7.5, trimers, dimers, and monomers are present; the trimeric ions with 24 charges (24+) have the same m/z of dimeric ions with 16 charges (16+) (m/z 6,462.6). At pH 8.8, VSV G th forms monomers and dimers. A similar analysis performed on VSV G th revealed an analogous behavior (Fig 3B). A dimeric species was observed (along with a subpopulation of trimers) together with monomers at pH 7.5 and to a lesser extent at pH 8.8. In contrast, at pH 6.0, only trimers and hexamers (likely corresponding to two post‐fusion trimers associated through their fusion loops) were detected. Therefore, the native MS experiments revealed the ability of the vesiculovirus glycoprotein to form dimeric species around pH 7.5.