Dissection of the HIV Vif interaction with human E3 ubiquitin ligase

Abstract

The human immunodeficiency virus type 1 (HIV-1) protein Vif recruits the host E3 ubiquitin ligase, composed of cullin 5 (Cul5), Rbx2, Elongin B, and Elongin C (EloBC), to polyubiquitinate the antiviral protein APOBEC3G. Multiple regions in the C-terminal half of Vif interact with the E3 ligase. We have purified individual regions of Vif and investigated their thermodynamic contributions to the ligase assembly in vitro using isothermal titration calorimetry and fluorescence anisotropy. Our results quantify the high-affinity interactions between the Vif BC box and EloBC and between the Vif zinc finger and Cul5, as well as the modest interaction between the Vif cullin box and Cul5. Our purified Vif constructs also provide direct biochemical evidence that the Vif cullin box, containing the PPLP region, leads to the dimerization of Vif-EloBC complexes but not Cul5-Vif-EloBC complexes.

FIG. 1.

(A) A sequence schematic of Vif showing the regions that interact with A3G, A3F, EloBC, and Cul5. (B) An illustration of the assembly of the Vif-E3 ubiquitin ligase. (C) A homology model of Vif-Cul5-EloBC, where the Vif BC box-EloBC is actual structural data (PDB ID 3DCG).

FIG. 2.

(A) A surface representation of EloBC bound to FITC-Vif_140-154 (ribbon) (14). (B to D) ITC analysis of WT and mutant Vif_139-192 titrated into EloBC. (E) FP binding curve of FITC-Vif_140-154-EloBC.

FIG. 3.

(A to C) ITC analysis of Cul5 titrated into the Vif_100-192-EloBC, Vif_100-154-EloBC, and Vif_139-192-EloBC complexes, respectively. (D) ITC analysis of Cul5 titrated into EloBC. The upper panels show the Vif domains used in the experiments and schematics of the E3 ligase assembly.

FIG. 4.

(A) The relationship of molecular weight and elution volume for various Vif-EloBC and Vif-Cul5-EloBC complexes on a Superdex 75 (10/300 GL) size exclusion column. The expected molecular weight (in thousands) is plotted against predicted positions in the line calibrated using molecular weight standards. The Vif_100-154-EloBC is plotted as a monomer, and all other complexes are plotted as dimers. (B) Multiangle laser light scattering results of the molecular weights of Cul5-Vif_100-176-EloBC, Vif_139-176-EloBC, and Vif_100-154-EloBC. The measured molecular weights of Cul5-Vif_100-176-EloBC, Vif_139-176-EloBC, and Vif_100-154-EloBC (80,900, 50,500, and 32,500, respectively) match the theoretical values of monomers for Cul5-Vif_100-176-EloBC (76,200) and Vif_100-154-EloBC (29,700) and the theoretical value for a dimer of Vif_139-176-EloBC (56,500). (C) Size exclusion chromatogram of Vif_1-154-EloBC and an SDS-PAGE gel of the peak fraction showing a pure, monomeric complex.