Figure 1. AP–MS experiments identify CBF-β as a Vif-dependent component of the Vif–CUL5 ubiquitin ligase complex.

a, Flow-chart of the proteomic analysis performed during the study. b, Affinity-tagged versions of Vif, Vpu and Vpr were purified using 3×Flag from HEK293 and Jurkat cells, subjected to SDS–PAGE and stained with silver. Visible bands corresponding to interactions that are known for each accessory factor are labelled. Note Vif and CBF-β run at a similar place on the gel. Tagged versions of Vpr and Vpu were used as specificity controls. c, A network representation of Vif–host protein–protein interactions from both HEK293 (blue) and Jurkat T cells (red) after subjecting the data derived from the AP–MS analysis to the MiST scoring system¹⁵. The intensity of the node colours corresponds to the quantitative MiST score. Blue edges represent interactions derived during this work; black edges are previously described interactions between host factors; dashed edges correspond to previously described Vif–host interactions present in the database VirusMint. d, The double purification approach, which allows for the identification of stable, stoichiometric protein complexes. e, Double purifications were performed in triplicate using 3×Flag-tagged CUL5, A3G or CBF-β with 2×Strep-tagged Vif in HEK293 cells. Proteins that were identified in all three double purifications, after trypsin digestion and analysis by mass spectrometry, are represented. The coverage corresponds to the percentage of protein identified by tryptic peptides. f, Immunoblots showing that Vif recruits CBF-β to theCUL5/ELOBC/RBX2 ubiquitin ligase complex. HA-tagged ELOB or CUL5 were immunoprecipitated in the absence or presence of increasing amounts of Vif, and endogenous CBF-β was monitored by immunoblot. g, HIV and SIV Vif co-immunoprecipitate CBF-β and ELOC. GFP and HIV Nef were analysed in parallel as specificity controls.