title: Methods to Study Determinants for Membrane Targeting of HIV-1 Gag In Vitro.
abstract: Assembly of HIV-1 viral particles is a critical step of the HIV-1 life cycle; yet many details of this complex process are unknown. The Gag polyprotein drives viral particle assembly at the plasma membrane via three different types of interactions: protein-protein, protein-RNA, and protein-membrane interactions. As an approach to tease apart the importance of these interactions during viral particle assembly, in particular at the step of Gag membrane binding, we have developed an in vitro liposome-binding assay. Below we describe how to prepare liposomes, which serve as model membranes, and how to assess their interaction with Gag by liposome flotation centrifugation. Additionally, we outline extensions of this basic assay that can be used to address the role of RNA in regulating Gag-membrane interactions.
authors.affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, 5736 Medical Science Building II, 1150 W. Medical Center Drive, Ann Arbor, MI, 48109, USA.
authors.forename: Gabrielle C
authors.initials: GC
authors.lastname: Todd
authors.affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, 5736 Medical Science Building II, 1150 W. Medical Center Drive, Ann Arbor, MI, 48109, USA. akiraono@umich.edu.
authors.forename: Akira
authors.initials: A
authors.lastname: Ono
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism* / Q000821:virology; D002498:Centrifugation / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D015683:Gene Products, gag / Q000378:metabolism*; D015658:HIV Infections / Q000378:metabolism* / Q000821:virology; D015497:HIV-1 / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008081:Liposomes / Q000378:metabolism*; D011485:Protein Binding; D012313:RNA / Q000378:metabolism; D011817:Rabbits; D019065:Virus Assembly
chemical_list: D015683:Gene Products, gag; D008081:Liposomes; D012313:RNA
keywords: Gag; HIV-1 assembly; Liposome flotation centrifugation; Membrane binding; RNA