title: Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation.
abstract: By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals--electrostatic, hydrophobic, and lipid-specific-to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities.
  
  
  OBJECTIVE
  Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane-bounded protein lattice that recruits genomic RNA into the virus and forms the shell of a budding immature viral capsid. In binding studies of HIV-1 Gag MA to model membranes with well-controlled lipid composition, we dissect the multiple interactions of the MA domain with its target membrane. This results in a detailed understanding of the thermodynamic aspects that determine membrane association, preferential lipid recruitment to the viral shell, and those aspects of Gag assembly into the membrane-bound protein lattice that are determined by MA.
authors.affiliation: Department of Physics, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.
authors.forename: Marilia
authors.initials: M
authors.lastname: Barros
authors.affiliation: Department of Physics, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.|NIST Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, Maryland, USA.
authors.forename: Frank
authors.identifier: http://orcid.org/0000-0002-8579-553X
authors.initials: F
authors.lastname: Heinrich
authors.affiliation: HIV Dynamics and Replication Program, Center for Cancer Research, National Institute of Cancer, Frederick, Maryland, USA.
authors.forename: Siddhartha A K
authors.identifier: http://orcid.org/0000-0002-4098-7490
authors.initials: SAK
authors.lastname: Datta
authors.affiliation: HIV Dynamics and Replication Program, Center for Cancer Research, National Institute of Cancer, Frederick, Maryland, USA reina@mail.nih.gov quench@cmu.edu.
authors.forename: Alan
authors.initials: A
authors.lastname: Rein
authors.affiliation: Biomolecular Measurement Division, National Institute of Standards and Technology, Gaithersburg, Maryland, USA.|Institute for Bioscience and Biotechnology Research, Rockville, Maryland, USA.
authors.forename: Ioannis
authors.identifier: http://orcid.org/0000-0002-2799-766X
authors.initials: I
authors.lastname: Karageorgos
authors.affiliation: Department of Physics, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.|NIST Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, Maryland, USA.
authors.forename: Hirsh
authors.initials: H
authors.lastname: Nanda
authors.affiliation: Department of Physics, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA reina@mail.nih.gov quench@cmu.edu.|Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA.|NIST Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, Maryland, USA.
authors.forename: Mathias
authors.identifier: http://orcid.org/0000-0001-6666-916X
authors.initials: M
authors.lastname: Lösche
mesh_terms: D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism; D015488:HIV Antigens / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D058635:Lipid-Linked Proteins / Q000378:metabolism; D008055:Lipids / Q000737:chemistry; D021962:Membrane Microdomains / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D011485:Protein Binding; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism*
chemical_list: D015488:HIV Antigens; D008051:Lipid Bilayers; D058635:Lipid-Linked Proteins; D008055:Lipids; D008565:Membrane Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D054301:gag Gene Products, Human Immunodeficiency Virus; C054858:p17 protein, Human Immunodeficiency Virus Type 1; D002784:Cholesterol