This electron microscopy study provides new insight into the molecular organization of VSV and the functioning of its fusion machinery.
Tomograms of negatively stained virions obtained at low pH confirmed the presence of material within the coiled nucleocapsid (Barge et al., 1993
). However, due to the low resolution of the tomograms, we cannot draw firm conclusions about the nature of this material, which was not observed in the recent virus reconstruction by cryo-EM (Ge et al., 2010
). However, this internal material did not display the helical symmetry imposed by Ge et al. (2010)
in their reconstruction of the viral nucleocapsid.
Both crystal structures of Gth
were observed at the surface of the virion. At pH values of 6.6 and below, all G spikes were in the elongated, post-fusion conformation. At pH 7.5, we observed only a few spikes with the tripod-like pre-fusion conformation. We cannot exclude the possibility that the oligomeric pre-fusion structure was disrupted during the preparation of the sample for observation by electron microscopy. However, the small number of G trimers observed at the surface of the virus suggests that at least one other conformation of G, different from both the structures determined by crystallography, is also present on the surface of the virion at pH values above 7. We suggest that this reflects the equilibrium between monomeric and trimeric forms of G. The existence of such an equilibrium has been demonstrated for detergent-solubilized G (Lyles et al., 1990
). As previously suggested (Roche et al., 2008
), the monomeric conformation of G may be an intermediate in the transition pathway.
Our data suggest that the flat base of the virion is the preferred site for fusion. This may be due to the particular curvature of the membrane at the base of the virion, a particular glycoprotein density and/or organization at this site, or both. Consistent with a role for glycoprotein density and organization, under pre-fusion conditions at pH 7.5, the flat base often seemed to be devoid of spikes, suggesting a lower abundance or a different structure and/or packing of spikes on this part of the virion. Indeed, a dense layer of protein (such as that on the cylindrical part and the round tip of the viral particle) would probably impede the formation of the initial lipid structures connecting the viral and target membranes.
Our findings demonstrate that the structural transition from the pre- to post-fusion conformation is not sufficient to drive viral fusion with a target membrane. Indeed, no fusion was detected at pH 6.6, despite most of the spikes present at the viral surface having undergone the conformational change. Indeed, lateral interactions (probably requiring a second protonation step) between G ectodomains in the post-fusion conformation were required for fusion. At pH 6, these lateral interactions, which are exclusively local (see , , and ), did not deform the viral particle. However, at this pH the arrays formed by Gth induced the deformation of liposomes to form tubular structures.
At pH values of 5.5 or below, both G and Gth formed extensive helical networks on the surfaces of virions and liposomes, respectively. These networks disrupted the viral membrane and induced the formation of elongated rigid protein–lipid tubes, respectively.
Thus, the VSV fusion pathway seems to involve several distinct stages. The first stage is the association of the virion with the target membrane via its flat base. This is followed by local membrane deformations, leading to the formation of one or several stalks, and the formation of one or several initial fusion pores. Interactions between glycoproteins, in their low pH conformation, outside the contact zone, then lead to the enlargement of these initial pores, completing the fusion of the membranes. Pore enlargement is probably driven by the membrane tension induced by local reorganization of the glycoprotein network on the lateral side of the virion. This step did not require complete helical network formation: as pore enlargement requires less energy than membrane disruption, the formation of local arrays is probably sufficient. Indeed, premature formation of the helical network leads to membrane disruption, RNP release, and viral inactivation, accounting for the lower fusion efficiency at lower pH values.
A role for network formation at a late stage of the fusion process is consistent with published data for rabies virus showing that, at low temperature and at pH 6.4, fusion is arrested, probably at the stage of initial pore formation (Gaudin, 2000
). In these conditions, the structural transition toward the post-fusion state is known to be blocked in rabies virus (Roche and Gaudin, 2002
), precluding formation of the network outside the contact zone and preventing membrane fusion.
These observations are also consistent with the model of Kozlov and Chernomordik (2002)
, according to which, fusion proteins outside the contact zone generate the driving force for fusion by forming a coat around the fusion site. It remains unclear whether this mechanism can be generalized to other enveloped viruses. Nevertheless, it has been suggested that influenza hemagglutinins outside the contact zone are involved in late stages of the fusion process (Leikina et al., 2004
). Furthermore, for paramyxovirus PIV5, micrographs indicate that F proteins in their post-fusion conformation tend to cluster at the surface of the virus (Ludwig et al., 2008
). Finally, the spikes of class II fusion glycoproteins, in their post-fusion conformation, self-associate to form networks of various degrees of regularity, different from the icosahedral organization observed before fusion (Gibbons et al., 2003
; Stiasny et al., 2004
; Sánchez-San Martín et al., 2008
). It is thus tempting to speculate that similar mechanisms are used by different fusion machineries.