title: Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein.
abstract: Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactions in vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particles in vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interaction in vitro, either by directly contacting acidic lipids or by promoting Gag multimerization.
  
  
  OBJECTIVE
  Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt a folded-over conformation on a lipid bilayer, implicating both the N and C termini in membrane binding. In addition, binding of Gag to membranes is diminished when either terminal domain is truncated. RSV Gag membrane association is significantly less sensitive than HIV Gag membrane association to lipid acyl chain saturation. These findings shed light on Gag assembly and membrane binding, critical steps in the viral life cycle and an untapped target for antiretroviral drugs.
authors.affiliation: Department of MolecularBiology and Genetics, Cornell University, Ithaca, New York, USA rad82@cornell.edu dattasi@mail.nih.gov.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Dick
authors.affiliation: HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, USA rad82@cornell.edu dattasi@mail.nih.gov.
authors.forename: Siddhartha A K
authors.initials: SA
authors.lastname: Datta
authors.affiliation: NIST Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, Maryland, USA Department of Physics, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.
authors.forename: Hirsh
authors.initials: H
authors.lastname: Nanda
authors.affiliation: Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, National Cancer Institute, National Institutes of Health, Frederick, Maryland, USA.
authors.forename: Xianyang
authors.initials: X
authors.lastname: Fang
authors.affiliation: Department of MolecularBiology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wen
authors.affiliation: Department of Physics, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.
authors.forename: Marilia
authors.initials: M
authors.lastname: Barros
authors.affiliation: Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, National Cancer Institute, National Institutes of Health, Frederick, Maryland, USA.
authors.forename: Yun-Xing
authors.initials: YX
authors.lastname: Wang
authors.affiliation: HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, USA.
authors.forename: Alan
authors.initials: A
authors.lastname: Rein
authors.affiliation: Department of MolecularBiology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: Volker M
authors.initials: VM
authors.lastname: Vogt
mesh_terms: D002462:Cell Membrane / Q000737:chemistry*; D002784:Cholesterol / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015683:Gene Products, gag / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D015497:HIV-1 / Q000737:chemistry; D057446:Hydrodynamics; D008051:Lipid Bilayers / Q000737:chemistry*; D009994:Osmolar Concentration; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D052736:Rous sarcoma virus / Q000737:chemistry* / Q000648:ultrastructure; D014771:Virion / Q000737:chemistry* / Q000648:ultrastructure
chemical_list: D015683:Gene Products, gag; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D019269:Phosphatidylinositol 4,5-Diphosphate; D011994:Recombinant Proteins; C057561:1-palmitoyl-2-oleoylphosphatidylethanolamine; D002784:Cholesterol; C017251:1,2-oleoylphosphatidylcholine; C028694:1-palmitoyl-2-oleoylphosphatidylcholine