Inhibition of vesicular stomatitis virus infection by spike glycoprotein. Evidence for an intracellular, G protein-requiring step.

Abstract

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

Host Cell Factors and Functions Involved in Vesicular Stomatitis Virus Entry

“…The internalization of VSV thus occurred rapidly and synchronously. Why only one-third of the virus was internalized is not clear, but the inefficient internalization was consistent with previous observations in MDCK and BHK-21 cells (41,50).…”

“…VSV binding to the cell surface was quite inefficient and sensitive to external factors, such as pH and cell type (41,50). Internalization was also relatively inefficient, with only onethird of surface-bound virus particles entering the cell.…”

Entry of Mouse Hepatitis Virus 3 into Cells

“…Internalization of virus particles by endocytosis is accomplished by the uncoating of viral nucleocapsids after fusion with primary lysosomes (Helenius et al, 1980;Matlin et al, 1982). A low pH in these organelles seems to trigger a fusion between viral envelopes and lysosomal membranes (Helenius et al, 1980;White et al, 1980), whereas the increase of intralysosomal pH with lysosomotropic agents such as NH,C1 (Ohkuma & Poole, 1978) or chloroquine (Miller & Lenard, 1980) was shown to block the release of the viral genome into the cytoplasm (Matlin et al, 1982). Mallucci (1966) reported a differential effect of chloroquine on MHV3 replication in mouse macrophages, which was thought to be due to enzymes escaping from the lysosomes.…”

Cytopathic Effects in Mouse Neuroblastoma Cells during a Non-permissive Infection with a Mutant of Vesicular Stomatitis Virus

“…The effect of chloroquine, a lysosomotropic agent (Ohkuma & Poole, 1978), was also examined. A previous report using chloroquine during VSV infection suggested that lysosomes were required for uncoating of the virus, and that only 50% of the viral RNA was made when chloroquine was added 1.5 h after infection (Miller & Lenard, 1980). Therefore, we added chloroquine to ts G31-infected N-18 cells 2 h after infection to allow initiation of infection but to block subsequent lysosomal enzyme activity.…”