title: Novel mutations that control the sphingolipid and cholesterol dependence of the Semliki Forest virus fusion protein.
abstract: The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a membrane fusion reaction mediated by the E1 membrane protein. Efficient SFV-membrane fusion requires the presence of cholesterol and sphingolipid in the target membrane. Here we report on two mutants, srf-4 and srf-5, selected for growth in cholesterol-depleted cells. Like the previously isolated srf-3 mutant (E1 proline 226 to serine), the phenotypes of the srf-4 and srf-5 mutants were conferred by single-amino-acid changes in the E1 protein: leucine 44 to phenylalanine and valine 178 to alanine, respectively. Like srf-3, srf-4 and srf-5 show striking increases in the cholesterol independence of growth, infection, membrane fusion, and exit. Unexpectedly, and unlike srf-3, srf-4 and srf-5 showed highly efficient fusion with sphingolipid-free membranes in both lipid- and content-mixing assays. Both srf-4 and srf-5 formed E1 homotrimers of decreased stability compared to the homotrimers of the wild type and the srf-3 mutant. All three srf mutations lie in the same domain of E1, but the srf-4 and srf-5 mutations are spatially separated from srf-3. When expressed together, the three mutations could interact to produce increased sterol independence and to cause temperature-sensitive E1 transport. Thus, the srf-4 and srf-5 mutations identify novel regions of E1 that are distinct from the fusion peptide and srf-3 region and modulate the requirements for both sphingolipid and cholesterol in virus-membrane fusion.
authors.affiliation: Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
authors.forename: Prodyot K
authors.initials: PK
authors.lastname: Chatterjee
authors.forename: Christina H
authors.initials: CH
authors.lastname: Eng
authors.forename: Margaret
authors.initials: M
authors.lastname: Kielian
mesh_terms: D002784:Cholesterol / Q000502:physiology*; D008561:Membrane Fusion / Q000502:physiology; D009154:Mutation; D012672:Semliki forest virus / Q000502:physiology*; D013107:Sphingolipids / Q000502:physiology*; D014760:Viral Fusion Proteins / Q000502:physiology*
chemical_list: D013107:Sphingolipids; D014760:Viral Fusion Proteins; D002784:Cholesterol