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. 1980 Feb 1;84(2):421–429. doi: 10.1083/jcb.84.2.421

Reconstituted G protein-lipid vesicles from vesicular stomatitus virus and their inhition of VSV infection

DK Miller
PMCID: PMC2110555  PMID: 6247354

Abstract

The single glycoprotein (G) of vesiclar stomatitis virus (VSV) was isolated in nearly quantitative yield by extraction of the purified virions with 0.05 M octyl-β-D- glucoside (OG) in 0.01 M sodium phosphate, pH 8.0. The extract contained essentially all of the viral phospholipids and glycolipids, and was free of other essentially all of the viral phospholipids and glycolipids, and was free of other viral proteins. Dialysis to remove OG resulted in the formation of G protein-viral lipid vesicles having a lipid-G protein ratio similar to that of the intact virions. The vesicles were 250-1,000 A in diameter, with a “fuzzy” external layer also similar to that of intact virions. The vesicles were predominantly unilamellar and sealed, with both phosphatidyl ethanolamine and gangliosides symmetrically distributed in the bilayer. G protein was asymmetrically oriented, with about 80 percent accessible to exogenous protease. Addition of soybean phospholipid to the viral extract before dialysis resulted in vesicles that incorporated viral proteins and lipids quantitatively, but that were markedly decreased in buoyant density. The G neutralized protein-lipid vesicles were effective in eliciting specific anti-G antibodies that neutralized viral infectivity. Competitive radioimmunoassay showed that both reconstituted vesicles and a soluble form of G protein (Gs) were indistinguishable from purified VSV in their antibody binding properties. Addition of G protein-lipid vesicles of BHK-21 cells before, or simultaneously with, infection by VSV inhibited viral infectivity, as measured by two independent techniques (viral RNA production in the presence of actinomycin D and a neutral red assay of cell viability). The total inhibitory activity of G protein in the vesicular form was, however, less than 5 percent of that found for intact virus particles that have been inactivated by ultraviolet light irradiation. Gs was inactive as an inhibitor as determined by the RNA production assay.

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Selected References

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