In an assay measuring virus-directed RNA synthesis, infection of BHK cells by a standard test dose of vesicular stomatitis virus (VSV) was inhibited by ultraviolet light-irradiated wt VSV and by is 045, one of a number of thermolabile, temperature-sensitive G protein mutants of VSV . After heat treatment for 1 h at 45°C, the thermolabile mutants were no longer able to inhibit the VSV infection . In contrast, the thermolabile M protein mutant is G31 and the nonthermolabile G protein mutant is 044 could still inhibit the test VSV dose . Thus,the presence of G protein in its native conformation was necessary for inhibition of infection . There was little difference in the binding to cells or the internalization to a trypsinresistant state of is 045 or wt VSV before and after heat treatment, and there was no evidence of specific saturable receptors on the cell surface . None of the irradiated virions at concentrations that gave maximal inhibition of infection could prevent binding of infectious VSV to, or internalization by, BHK cells . The G protein-specific inhibition, therefore, did not occur at the cell surface but must have occurred at some intracellular site, which has been suggested to be the lysosome . The lysosomal inhibitor chloroquine, when added with the infecting virus, completely inhibited VSV infection at all multiplicities of infection tested, and it gave 50% inhibition when added 1 .5 h after infection . The possible importance of the lysosome in the intracellular pathway of infection is discussed. KEY WORDS vesicular stomatitis virus temperature-sensitive mutants -G protein thermolability virus binding -virus internalization chloroquineThe spike glycoprotein G of vesicular stomatitis virus (VSV)' has been shown to be essential to the ' Abbreviations used in this paper: VSV, vesicular stomatitis virus ; PBS, Dulbecco's phosphate-buffered saline ; MEM, Eagle's minimal essential medium; BES, N,Ninfectivity of VSV in BHK cells : both removal of the G protein by proteolysis (1) and coincubation of the cells with purified G protein in vesicular form (13) inhibit viral infection. It has been widely accepted, but not directly shown, that the importance of the G protein rests in its ability to enable VSV to attach to cells (2) . In this study, we have bis(2-hydroxyethyl)-2-aminoethane sulfonic acid; SFV, Semliki Forest virus ; BSA, bovine serum albumin; FCS, fetal calf serum ; m.o .i ., multiplicity of infection .J . CELL BIOLOGY
The single glycoprotein (G) of vesicular stomatitis virus (VSV) was isolated in nearly quantitative yield by extraction of the purified virions with 0.05 M octyl-,ß-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 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% 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 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 proteinlipid 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% of that found for intact virus particles that had been inactivated by ultraviolet light irradiation. Gs was inactive as an inhibitor as determined by the RNA production assay. J. CELL BIOLOGY
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