title: Cell-based Fluorescence Complementation Reveals a Role for HIV-1 Nef Protein Dimerization in AP-2 Adaptor Recruitment and CD4 Co-receptor Down-regulation.
abstract: The HIV-1 Nef accessory factor enhances viral infectivity, immune evasion, and AIDS progression. Nef triggers rapid down-regulation of CD4 via the endocytic adaptor protein 2 (AP-2) complex, a process linked to enhanced viral infectivity and immune escape. Here, we describe a bimolecular fluorescence complementation (BiFC) assay to visualize the interaction of Nef with AP-2 and CD4 in living cells. Interacting protein pairs were fused to complementary non-fluorescent fragments of YFP and co-expressed in 293T cells. Nef interactions with both CD4 and AP-2 resulted in complementation of YFP and a bright fluorescent signal by confocal microcopy that localized to the cell periphery. Co-expression of the AP-2 α subunit enhanced the Nef·AP-2 σ2 subunit BiFC signal and vice versa, suggesting that the AP-2 α-σ2 hemicomplex interacts cooperatively with Nef. Mutagenesis of Nef amino acids Arg-134, Glu-174, and Asp-175, which stabilize Nef for AP-2 α-σ2 binding in a recent co-crystal structure, substantially reduced AP-2 interaction without affecting CD4 binding. A dimerization-defective mutant of Nef failed to interact with either CD4 or AP-2 in the BiFC assay, indicating that Nef quaternary structure is required for CD4 and AP-2 recruitment as well as CD4 down-regulation. A small molecule previously shown to bind the Nef dimerization interface also reduced Nef interactions with AP-2 and CD4 and restored CD4 expression to the surface of HIV-infected cells. Our findings provide a mechanistic explanation for previous observations that dimerization-defective Nef mutants fail to down-regulate CD4 and validate the Nef dimerization interface as a target site for antiretroviral drug development.
authors.affiliation: From the Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219.
authors.forename: Sherry T
authors.initials: ST
authors.lastname: Shu
authors.affiliation: From the Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219.
authors.forename: Lori A
authors.initials: LA
authors.lastname: Emert-Sedlak
authors.affiliation: From the Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15219 tsmithga@pitt.edu.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Smithgall
mesh_terms: D015704:CD4 Antigens / Q000378:metabolism*; D002460:Cell Line; D019281:Dimerization; D015536:Down-Regulation; D005453:Fluorescence; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D013347:Subcellular Fractions / Q000378:metabolism; D050656:Transcription Factor AP-2 / Q000378:metabolism*; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
chemical_list: D015704:CD4 Antigens; D050656:Transcription Factor AP-2; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1
keywords: AP-2; Nef; bimolecular fluorescence complementation; cluster of differentiation 4 (CD4); dimerization; endocytosis; human immunodeficiency virus (HIV); protein-protein interaction