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 compositio n of the endosomal membrane. Fusion activity was only observed at pH v alues less than 6.3 and showed a greater rate and extent at lower pH v alues. Pronounced kinetic fusion curves were observed at pH values bel ow 5.8. When equivalent lipid amounts of target liposomes and virus we re mixed at pH 5.3 the dequenching activity had a t(1/2) of 45 +/- 10 sec. In addition to catalyzing membrane fusion after acidification the glycoprotein complex was previously found to undergo conformational c hange (C. Di Simone, M. A. Zandonatti, and M. J. Buchmeier, 1994, Viro logy 198, 455-465), including loss of the GP-1 polypeptide from the vi rion 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 determine d with progressively more GP-1 released at pH values below 6.3 and rea ching nearly 100% dissociation al pH 5.5 after 30 min at 37 degrees. A t pH 5.3 the GP disassembly proceeded with a t(1/2) of 7 +/- 2 min. Th e t(1/2) 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 dissociat ion, and inactivation kinetics data suggest a mechanism in which GP is activated to a fusion active state where membrane lipid exchange occu rs and then undergoes an irreversible conformational change which incl udes the loss of GP-1 from the spike complex. (C) 1995 Academic Press, Inc.