Characterization of Monomeric Intermediates during VSV Glycoprotein Structural Transition

Abstract

Entry of enveloped viruses requires fusion of viral and cellular membranes, driven by conformational changes of viral glycoproteins. Crystal structures provide static pictures of pre- and post-fusion conformations of these proteins but the transition pathway remains elusive. Here, using several biophysical techniques, including analytical ultracentrifugation, circular dichroïsm, electron microscopy and small angle X-ray scattering, we have characterized the low-pH-induced fusogenic structural transition of a soluble form of vesicular stomatitis virus (VSV) glycoprotein G ectodomain (Gth, aa residues 1–422, the fragment that was previously crystallized). While the post-fusion trimer is the major species detected at low pH, the pre-fusion trimer is not detected in solution. Rather, at high pH, Gth is a flexible monomer that explores a large conformational space. The monomeric population exhibits a marked pH-dependence and adopts more elongated conformations when pH decreases. Furthermore, large relative movements of domains are detected in absence of significant secondary structure modification. Solution studies are complemented by electron micrographs of negatively stained viral particles in which monomeric ectodomains of G are observed at the viral surface at both pH 7.5 and pH 6.7. We propose that the monomers are intermediates during the conformational change and thus that VSV G trimers dissociate at the viral surface during the structural transition.

Mechanism of membrane fusion induced by vesicular stomatitis virus G protein

“…On the surface of a virion at neutral pH and above, our results together with published data indicate that full-length G is in equilibrium between the prefusion trimer conformation and flexibly extended monomers (10,(20)(21)(22). In the absence of a target membrane, irreversible transition of virion G to its conformation at the end of a complete fusion reaction would require that three subunits come together, fold back, and insert their fusion loops into the viral membrane (Fig.…”

“…G th was cleaved and purified from virus particles as described in ref. 21 with the following modifications. In the cleavage reaction, the concentration of WT virus was 10 mg/mL, of G-W72A virus 11.1 mg/mL, and of thermolysin 0.6 mg/mL.…”

Characterization of pH-Sensitive Molecular Switches That Trigger the Structural Transition of Vesicular Stomatitis Virus Glycoprotein from the Postfusion State toward the Prefusion State

“…In the case of WT G, the prefusion trimer, although detected at the viral surface (16), is not stable in solution and only monomers are detected at high pH (14,17). On the other hand, at low pH, the postfusion trimer is stable.…”

“…For both RABV and VSV, it has been demonstrated that low-pH-induced structural transition of G is reversible (14,15). In fact, at the viral surface, there is a pH-dependent thermodynamic equilibrium between different states of G, which is shifted toward the trimeric postfusion form at low pH (16)(17)(18). This is the main difference between rhabdoviral G and other viral fusion glycoproteins activated at low pH for which the prefusion conformation is metastable and, as a consequence, the low-pH-induced structural transition is irreversible (4).…”

Chimeric rabies SADB19-VSVg-pseudotyped lentiviral vectors mediate long-range retrograde transduction from the mouse spinal cord

“…Both have a single glycoprotein that forms trimeric structures in the viral envelope. 5 A key difference between these viruses is the ability of rabies to undergo retrograde transport. Rabies virus infects nerves in the peripheral nervous system, and subsequently the central nervous system by trans-synaptic spread.…”