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.
Highlights
The nef gene of primate lentiviruses encodes a small (25–30 kDa) pathogenic factor required for the high titer replication of both HIV and SIV2 in vivo [1]
We demonstrated that interfering with Nef homodimerization, either genetically or pharmacologically, destabilized interactions with both CD4 and adaptor protein-2 (AP-2), suggesting that Nef dimers are required for recruitment of CD4 and AP-2 and subsequent endocytosis
Visualization of Nef Interaction with CD4 and AP-2 by bimolecular fluorescence complementation (BiFC) Assay—To investigate the direct interaction of Nef with CD4 and AP-2 in a cell-based setting, we developed an assay based on structural complementation of non-fluorescent YFP fragments
Summary
The nef gene of primate lentiviruses encodes a small (25–30 kDa) pathogenic factor required for the high titer replication of both HIV and SIV2 in vivo [1]. Mutations based on the Nef homodimerization interface formed by the ␣-helices present in the crystal structure with SH3 (PDB code 1EFN) [30] resulted in partial or complete loss of fluorescence in the complementation assay, providing biological validation for this crystalline Nef dimer structure.
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