Kinetics and pH dependence of acid-induced structural changes in the lymphocytic choriomeningitis virus glycoprotein complex.

The NBD-PE/Rd-PE fluorescent membrane fusion assay was used to measure the pH dependence and kinetics of the fusion activity of lymphocytic choriomeningitis virus with liposomes designed to mimic the composition of the endosomal membrane. Fusion activity was only observed at pH values less than 6.3 and showed a greater rate and extent at lower pH values. Pronounced kinetic fusion curves were observed at pH values below 5.8. When equivalent lipid amounts of target liposomes and virus were mixed at pH 5.3 the dequenching activity had a t1/2 of 45 +/- 10 sec. In addition to catalyzing membrane fusion after acidification the glycoprotein complex was previously found to undergo conformational change (C. Di Simone, M. A. Zandonatti, and M. J. Buchmeier, 1994, Virology 198, 455-465), including loss of the GP-1 polypeptide from the virion surface. The pH dependence and kinetics of this acid-induced GP-1 release were quantitated using centrifugal separation of solubilized GP-1 from pelleted virions. A pH-dependent elution curve was determined with progressively more GP-1 released at pH values below 6.3 and reaching nearly 100% dissociation at pH 5.5 after 30 min at 37 degrees. At pH 5.3 the GP disassembly proceeded with a t1/2 of 7 +/- 2 min. The t1/2 of virus inactivation was also measured at pH 5.3 and 7.0 and found to be 7.9 +/- 1 and 150 min, respectively. Fusion, GP dissociation, and inactivation kinetics data suggest a mechanism in which GP is activated to a fusion active state where membrane lipid exchange occurs and then undergoes an irreversible conformational change which includes the loss of GP-1 from the spike complex.

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.

Membrane fusion of mumps virus with ghost erythrocytes and CV-1 cells.

The octadecyl rhodamine (R18) fluorescent dequenching assay was used to examine membrane fusion between mumps virus and mammalian cells. Rapid fluorescent dequenching, indicative of membrane fusion, was observed when labeled mumps virus was mixed with either ghost erythrocytes or CV-1 cells. After 15 min a saturation limit of 18 virus per erythrocyte ghost and 6400 virus per CV-1 cell was observed. Fetuin was found to inhibit virus fusion, suggesting a role for sialic acid in virus binding to the cells. Two dequenching processes were observed of which the faster process is thought to be membrane fusion and the second process is thought to be probe proximal transfer.