Biochemical consequences of a mutation that controls the cholesterol dependence of Semliki Forest virus fusion

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a low -pH-triggered membrane fusion reaction that requires cholesterol and sphing olipid in the target membrane. Cholesterol-depleted insect cells are highly resistant to alphavirus infection and were used to select srf-3, an SFV mu tant that is similar to 100-fold less cholesterol dependent for infection d ue to a single amino acid change in the E1 spike subunit, proline 226 to se rine. Sensitive lipid-mixing assays here demonstrated that the in vitro fus ion of srf-3 and wild-type (wt) virus with cholesterol-containing liposomes had comparable kinetics, activation energies, and sphingolipid dependence. In contrast, srf-3 fusion with sterol-free liposomes was significantly mor e efficient than that of wt virus. Thus, the srf-3 mutation does not affect its general fusion properties with purified lipid bilayers but causes a ma rked and specific reduction in cholesterol dependence. Upon exposure to low pH, the E1 spike subunit undergoes distinct conformational changes, result ing in the exposure of an acid conformation-specific epitope and formation of an E1 homotrimer. These conformational changes were strongly cholesterol and sphingolipid dependent for wt SFV and strikingly less cholesterol depe ndent for srf-3. Our results thus demonstrate the functional importance of fusogenic E1 conformational changes in the control of SFV cholesterol depen dence.