title: Association of human immunodeficiency virus type 1 gag with membrane does not require highly basic sequences in the nucleocapsid: use of a novel Gag multimerization assay.
abstract: Human immunodeficiency virus type 1 (HIV-1) particle production, a process driven by the Gag polyprotein precursor, occurs on the plasma membrane in most cell types. The plasma membrane contains cholesterol-enriched microdomains termed lipid rafts, which can be isolated as detergent-resistant membrane (DRM). Previously, we and others demonstrated that HIV-1 Gag is associated with DRM and that disruption of Gag-raft interactions impairs HIV-1 particle production. However, the determinants of Gag-raft association remain undefined. In this study, we developed a novel epitope-based Gag multimerization assay to examine whether Gag assembly is essential for its association with lipid rafts. We observed that membrane-associated, full-length Gag is poorly detected by immunoprecipitation relative to non-membrane-bound Gag. This poor detection is due to assembly-driven masking of Gag epitopes, as denaturation greatly improves immunoprecipitation. Gag mutants lacking the Gag-Gag interaction domain located in the N terminus of the nucleocapsid (NC) were efficiently immunoprecipitated without denaturation, indicating that the epitope masking is caused by higher-order Gag multimerization. We used this assay to examine the relationship between Gag assembly and Gag binding to total cellular membrane and DRM. Importantly, a multimerization-defective NC mutant displayed wild-type levels of membrane binding and DRM association, indicating that NC-mediated Gag multimerization is dispensable for association of Gag with membrane or DRM. We also demonstrate that different properties of sucrose and iodixanol membrane flotation gradients may explain some discrepancies regarding Gag-raft interactions. This report offers new insights into the association of HIV-1 Gag with membrane and with lipid rafts.
authors.affiliation: Virus-Cell Interaction Section, HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, Maryland, USA. akiraono@umich.edu
authors.forename: Akira
authors.initials: A
authors.lastname: Ono
authors.forename: Abdul A
authors.initials: AA
authors.lastname: Waheed
authors.forename: Anjali
authors.initials: A
authors.lastname: Joshi
authors.forename: Eric O
authors.initials: EO
authors.lastname: Freed
mesh_terms: D001483:Base Sequence; D002462:Cell Membrane / Q000821:virology*; D003001:Cloning, Molecular; D015683:Gene Products, gag / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D021962:Membrane Microdomains / Q000821:virology; D019251:Nucleocapsid / Q000235:genetics*; D011489:Protein Denaturation; D014771:Virion / Q000235:genetics / Q000502:physiology
chemical_list: D015683:Gene Products, gag