title: Quantitative fluorescence resonance energy transfer microscopy analysis of the human immunodeficiency virus type 1 Gag-Gag interaction: relative contributions of the CA and NC domains and membrane binding.
abstract: The human immunodeficiency virus type 1 structural polyprotein Pr55(Gag) is necessary and sufficient for the assembly of virus-like particles on cellular membranes. Previous studies demonstrated the importance of the capsid C-terminal domain (CA-CTD), nucleocapsid (NC), and membrane association in Gag-Gag interactions, but the relationships between these factors remain unclear. In this study, we systematically altered the CA-CTD, NC, and the ability to bind membrane to determine the relative contributions of, and interplay between, these factors. To directly measure Gag-Gag interactions, we utilized chimeric Gag-fluorescent protein fusion constructs and a fluorescence resonance energy transfer (FRET) stoichiometry method. We found that the CA-CTD is essential for Gag-Gag interactions at the plasma membrane, as the disruption of the CA-CTD has severe impacts on FRET. Data from experiments in which wild-type (WT) and CA-CTD mutant Gag molecules are coexpressed support the idea that the CA-CTD dimerization interface consists of two reciprocal interactions. Mutations in NC have less-severe impacts on FRET between normally myristoylated Gag proteins than do CA-CTD mutations. Notably, when nonmyristoylated Gag interacts with WT Gag, NC is essential for FRET despite the presence of the CA-CTD. In contrast, constitutively enhanced membrane binding eliminates the need for NC to produce a WT level of FRET. These results from cell-based experiments suggest a model in which both membrane binding and NC-RNA interactions serve similar scaffolding functions so that one can functionally compensate for a defect in the other.
authors.affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.
authors.forename: Ian B
authors.initials: IB
authors.lastname: Hogue
authors.forename: Adam
authors.initials: A
authors.lastname: Hoppe
authors.forename: Akira
authors.initials: A
authors.lastname: Ono
mesh_terms: D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism* / Q000821:virology; D031541:Fluorescence Resonance Energy Transfer; D015658:HIV Infections / Q000378:metabolism* / Q000821:virology; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D019251:Nucleocapsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D019065:Virus Assembly; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D036022:Capsid Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus