title: Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes.
abstract: The structural protein of HIV-1, Pr55(Gag) (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an essential step for virus particle formation, since a defect in Gag membrane binding results in severe impairment of viral particle production. To gain mechanistic details of Gag-lipid membrane interactions, in vitro methods based on NMR, protein footprinting, surface plasmon resonance, liposome flotation centrifugation, or fluorescence lipid bead binding have been developed thus far. However, each of these in vitro methods has its limitations. To overcome some of these limitations and provide a complementary approach to the previously established methods, we developed an in vitro assay in which interactions between HIV-1 Gag and lipid membranes take place in a "cell-like" environment. In this assay, Gag binding to lipid membranes is visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique. Here we describe the background and the protocols to obtain myristoylated full-length Gag proteins and GUV membranes necessary for the assay and to detect Gag-GUV binding by microscopy.
authors: Department of Microbiology and Immunology, University of Michigan Medical School.
authors: Balaji
authors: B
authors: Olety
authors: Department of Biophysics, University of Michigan.
authors: Sarah L
authors: SL
authors: Veatch
authors: Department of Microbiology and Immunology, University of Michigan Medical School; akiraono@umich.edu.
authors: Akira
authors: A
authors: Ono
mesh_terms: D001426:Bacterial Proteins; D002462:Cell Membrane; D015683:Gene Products, gag / Q000378:metabolism*; D015497:HIV-1; D008164:Luminescent Proteins; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D053835:Unilamellar Liposomes / Q000378:metabolism*; D019065:Virus Assembly
chemical_list: D001426:Bacterial Proteins; D015683:Gene Products, gag; D008164:Luminescent Proteins; D053835:Unilamellar Liposomes; C054562:yellow fluorescent protein, Bacteria