Acidic pH triggers LCMV membrane fusion activity and conformational change in the glycoprotein spike

Membrane fusion activity of the glycoprotein (GP) complex of LCMV was determined using the R18 fluorescent dequenching assay. Utilization of an endosomal entry route by LCMV and acidic activation of the membrane fusion activity were indicated by the inhibition of LCMV infection at an early stage by the lysosomal weak base chloroquine and the ionophore monensin. When LCMV was mixed with R18-labeled liposomes with a lipid composition mimicking that of the endosome, dequenching occurred only at acidic pH (< or = 6.0). The measured dequenching was inhibited by protease treatment of the LCMV, indicative of protein-mediated membrane fusion. The initial rate of fusion was measured at pH values between 5.3 and 7.0 and was found to decrease rapidly and linearly between pH 5.3 (0.177%/sec) and pH 6.0 (0.027%/sec). Binding and fusion of R18-labeled LCMV with BHK cells were also examined. No difference was found between R18-labeled and unlabeled LCMV in binding to or infection of BHK cells. Dequenching was observed in labeled LCMV endocytosed by BHK cells. With BHK cells neither LCMV fusion with the plasma membrane nor LCMV-induced cell-cell fusion was observed, even under acidic conditions, and examination of the sequence of LCMV GP did not reveal a likely candidate sequence for a "fusion peptide." The binding of conformationally dependent monoclonal antibodies to GP was measured at neutral and acidic pH in order to seek evidence of pH-dependent conformational change in GP. Dissociation of the GP-1 from the complex was measured by sucrose gradients run on purified virus. These experiments revealed that after exposure to acid pH the LCMV glycoprotein spike complex underwent irreversible conformational change in which GP-1 was dissociated from the virion, conformational epitopes on GP-1 were lost, and sequestered epitopes on GP-2 became exposed. Further, LCMV infectivity was irreversibly inactivated by exposure to acidic pH (< 6.0), likely due to the loss of GP-1 and conformation changes in GP-2.