title: Multistep regulation of membrane insertion of the fusion peptide of Semliki Forest virus.
abstract: A prevailing model for virus membrane fusion proteins has been that the hydrophobic fusion peptide is hidden in the prefusion conformation, becomes exposed once the fusion reaction is triggered, and then either inserts into target membranes or is rapidly inactivated. This model is in general agreement with the structure and mechanism of class I fusion proteins, such as the influenza virus hemagglutinin. We here describe studies of the class II fusion protein E1 from the alphavirus Semliki Forest virus (SFV). SFV fusion is triggered by low pH, which releases E1 from its heterodimeric interaction with the E2 protein and induces the formation of a stable E1 homotrimer. The exposure and target membrane interaction of the E1 fusion peptide (residues 83 to 100) were followed using a monoclonal antibody (MAb E1f) mapping to E1 residues 85 to 95. In agreement with the known structure of SFV and other alphaviruses, the fusion peptide was shielded in native SFV particles and exposed when E1-E2 dimer dissociation was triggered by acidic pH. In contrast, the fusion peptide on purified E1 ectodomains (E1(*)) was fully accessible at neutral pH. Functional assays showed that MAb E1f binding at neutral pH prevented subsequent low-pH-triggered E1(*) interaction with target membranes and trimerization. E1(*) was not inactivated by low pH when treated either in the absence of target membranes or in the presence of fusion-inactive cholesterol-deficient liposomes. Thus, the membrane insertion of the E1 fusion peptide is regulated by additional low-pH-dependent steps after exposure, perhaps involving an E1-cholesterol interaction.
authors.affiliation: Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: Don L
authors.initials: DL
authors.lastname: Gibbons
authors.forename: Anna
authors.initials: A
authors.lastname: Ahn
authors.forename: Maofu
authors.initials: M
authors.lastname: Liao
authors.forename: Lena
authors.initials: L
authors.lastname: Hammar
authors.forename: R Holland
authors.initials: RH
authors.lastname: Cheng
authors.forename: Margaret
authors.initials: M
authors.lastname: Kielian
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D019556:COS Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D002784:Cholesterol / Q000378:metabolism; D006224:Cricetinae; D018604:Epitope Mapping; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000378:metabolism; D008561:Membrane Fusion / Q000187:drug effects; D020836:Protein Structure, Quaternary / Q000187:drug effects; D012672:Semliki forest virus / Q000187:drug effects / Q000502:physiology*; D014760:Viral Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D008081:Liposomes; D014760:Viral Fusion Proteins; D002784:Cholesterol