Physical properties of a soluble form of the glycoprotein of vesicular stomatitis virus at neutral and acidic pH

Abstract

We have analyzed a soluble form of the glycoprotein (G) obtained from vesicular stomatitis virus (VSV) by treatment of intact virions with cathepsin D. This form lacks the carboxy-terminal and membrane-spanning domains and thus is analogous to the previously described secreted form of G, Gs. The molecular weight of the cathepsin D produced G, G(Cath D), measured by sedimentation equilibrium in the analytical ultracentrifuge is 57 600, indicating that it is a monomer. Intact G protein extracted from virions by octyl beta-D-glucoside also is monomeric, based on sedimentation equilibrium analysis. These results suggest that G may be monomeric in virions. The Stokes radii (Rs) of the two forms of G were obtained from their migration in nondenaturing polyacrylamide gradient gels. The Rs of G(Cath D) in the absence of nonionic detergent was 37 A; in the presence of nonionic detergent, it increased to 55 A. The Rs of detergent-extracted intact G was 63 A in nonionic detergent. From the molecular weight and Rs of G(Cath D), we calculated a sedimentation coefficient of 3.8 S; the value determined by centrifugation in a sucrose gradient was 3.7 S. Viruses such as VSV fuse with cell membranes at low pH [White, J., Matlin, K., & Helenius, A. (1981) J. Cell Biol. 89, 674-679]. We have used the fluorescent probe cis,trans,trans,cis-9,11,13,15-parinaric acid (cis-PnA) to detect a reversible conformational change in G(Cath D) when the protein was exposed to an acidic environment close to pH 5. cis-PnA binds to hydrophobic regions of protein, causing a quenching of the intrinsic tryptophan fluorescence and an increase in the fluorescence of the probe.

VESICULAR STOMATITIS VIRUS-MEDIATED CELL FUSION SUBSEQUENT TO VIRUS ADSORPTION AT DIFFRENT pH VALUES

“…The fusion activity of VSV resides in the G protein, a single envelope glycoprotein (7), which undergoes conformational changes after exposure to low pH (8)(9)(10). These changes in the conformation of G protein are thought to associate with the fusion event (2,11,12), but the molecular mechanisms underlying VSV-induced cell fusion remain uncertain (11,(13)(14)(15).…”

Cytoplasmic domains of cellular and viral integral membrane proteins substitute for the cytoplasmic domain of the vesicular stomatitis virus glycoprotein in transport to the plasma membrane.

“…Thus, the HA protein may rely more heavily than G protein on extracytoplasmic signals to promote its rapid transport to the cell surface. Because the HA protein is known to be a trimer (63) while G protein may be a monomer (14), an alternative explanation is that the extracellular and transmembrane domains of G protein are not structurally compatible with the HA cytoplasmic domain. For example, perhaps the cytoplasmic domain of HA is efficiently recognized in a trimeric configuration, and since such a conformation is unexpected in the hybrid protein, the rate of transport observed for GHA was reduced as compared with either parent molecule.…”

Viral membrane fusion: is glycoprotein G of rhabdoviruses a representative of a new class of viral fusion proteins?

“…Rabies virus and VSV G proteins are organized as trimers anchored to the viral membrane via a single transmembrane sequence close to the C-terminus (21)(22)(23)(24). The trimeric structure of VSV G protein is stabilized at mild acidic pH (22) but both rabies and VSV G protein trimers seem to be less stable than the other trimeric viral glycoproteins (24,25).…”

“…It has been shown that the incubation of the protein at mild acidic pH leads to an exposure of a hydrophobic region (25). Binding of the fluorescent probe bis-ANS revealed that the exposure of hydrophobic domains was maximal at pH 6.2 (40).…”