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 sphingolipid in the target membrane. Cholesterol-depleted insect cells are highly resistant to alphavirus infection and were used to select srf-3, an SFV mutant that is approximately 100-fold less cholesterol dependent for infection due to a single amino acid change in the E1 spike subunit, proline 226 to serine. Sensitive lipid-mixing assays here demonstrated that the in vitro fusion 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 more 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 marked and specific reduction in cholesterol dependence. Upon exposure to low pH, the E1 spike subunit undergoes distinct conformational changes, resulting 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 dependent for srf-3. Our results thus demonstrate the functional importance of fusogenic E1 conformational changes in the control of SFV cholesterol dependence.
Chatterjee
P K
PK
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Vashishtha
M
M
Kielian
M
M
D000818:Animals; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D006224:Cricetinae; D009033:Culicidae / Q000166:cytology; D006863:Hydrogen-Ion Concentration; D008081:Liposomes; D008561:Membrane Fusion / Q000502:physiology*; D017354:Point Mutation; D011487:Protein Conformation; D012672:Semliki forest virus / Q000235:genetics / Q000378:metabolism*; D013107:Sphingolipids / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D008081:Liposomes; D013107:Sphingolipids; D014759:Viral Envelope Proteins; D002784:Cholesterol