Vif hijacks CBF-b to degrade APOBEC3G and promote HIV-1 infection

Restriction factors, such as the retroviral complementary DNA deaminase APOBEC3G, are cellular proteins that dominantly block virus replication. TheAIDSvirus, human immunodeficiency virus type 1 (HIV-1), produces the accessory factor Vif, which counteracts the host’s antiviral defence by hijacking a ubiquitin ligase complex, containing CUL5, ELOC, ELOB and a RING-box protein, and targeting APOBEC3G for degradation. Here we reveal, using an affinity tag/purification mass spectrometry approach, that Vif additionally recruits the transcription cofactor CBF-b to this ubiquitin ligase complex. CBF-b, which normally functions in concert with RUNX DNA binding proteins, allows the reconstitution of a recombinant six-protein assembly that elicits specific polyubiquitination activity with APOBEC3G, but not the related deaminase APOBEC3A. Using RNA knockdown and genetic complementation studies, we also demonstrate that CBF-b is required for Vif-mediated degradation of APOBEC3G and therefore for preserving HIV-1 infectivity. Finally, simian immunodeficiency virus (SIV) Vif also binds to and requires CBF-b to degrade rhesus macaque APOBEC3G, indicating functional conservation.Methods of disrupting theCBF-b–Vif interactionmight enableHIV-1 restriction and provide a supplement to current antiviral therapies that primarily target viral proteins. Mammals have evolved cellular proteins termed restriction factors that function to prevent the spread of mobile genetic elements including retroviruses. As a counter-defence, most retroviruses, including the human pathogen HIV-1, have developed mechanisms to prevent restriction, often throughsubversionof thehost’s ubiquitin–proteasome system. In eukaryotic cells, 8.6-kDa ubiquitin moieties are added to a target protein by sequential action of one of two ubiquitin-activating enzymes (E1), which transfer ubiquitin to a pool of dozens of ubiquitinconjugating enzymes (E2) that, in turn, collaborate with hundreds of ubiquitin ligases (E3) to catalyse transfer to specific substrates. If more than four ubiquitins are joined together throughK48 linkages, the target protein is usually degraded by the 26S proteasome. At least threeHIV1proteins,Vif,Vpu andVpr, hijack cullin-RINGE3 ligases consistingof CUL5,CUL1andCUL4Atopromoteubiquitinationanddegradationof APOBEC3 family members (for example, APOBEC3G, A3G), BST2/ tetherin and an unknown, putative restriction factor, respectively. Understanding the composition of cullin-RING E3 ligase complexes and the underlying cellular signalling components may provide therapeutic routes for treating a variety of human diseases, including infection by HIV-1. HIV-1 Vif is recruited to CUL5 by virtue of its SOCS box, which contains an elongin C binding helix (the BC-box), a conserved HCCH Zn bindingmotif and a short Cullin Box. Although a structure of the BC-box peptide in complex with the heterodimer of Elongin B and C (ELOBC) has been reported, the architecture of the full-length Vif in complex with host factors has remained elusive, in part because Vif complexes have poor solubility and activity. We therefore reasoned that Vif may bind an additional host factor and that such a factor may render it more tractable in vitro. We took an unbiased proteomic approach to identify host factors that bind all 18 HIV processed and polyproteins using an affinity tag/ purification mass spectrometry (AP–MS) approach. To this end, 23Strep and 33Flag was fused to the carboxy (C) terminus of these factors, including Vif. The tagged Vif construct was both transiently transfected into HEK293 cells and used to make a stable, tetracyclineinducible Vif–Strep–Flag Jurkat T cell line (Fig. 1a). Epitope-tagged Vif was purified from both cell types using antibodies specific to either Strep or Flag and aliquots of the co-purifying proteins were subjected to SDS–polyacrylamide gel electrophoresis (SDS–PAGE) (Fig. 1b). Materials from each step were analysed by mass spectrometry. Using a new scoring system for data derived from AP–MS studies, termed Mass Spectrometry Interaction Statistics (MiST), we identified 24 Vif–human protein–protein interactions with seven of them found in both cell types (Fig. 1c). Seventeen of these were verified independently by co-immunoprecipitation (Supplementary Fig. 1). Among these were the components of the E3 ubiquitin ligase complex, CUL5, ELOB and ELOC, known to interact with Vif and trigger A3G degradation. Although theRING-boxproteinRBX1was originally reported as part of this complex, only RBX2was above theMiST score threshold used consistent with recent work showing that it binds CUL5 (refs 16, 17).Wedidnot find endogenousA3G,probablybecause of its poor expression in HEK293 and Jurkat cell lines exacerbated by further depletion through Vif-mediated degradation. We did find Vif associating with two proteins that function in autophagy, AMRA1 and SQSTM, as well as with the transcriptional co-repressor complex NCOR1/HDAC3/GPS2/TBL1R (the last only in T cells) (Fig. 1c). Also, in both cell types, Vif was found to interact with the transcription cofactor CBF-b, which is known to heterodimerize with the RUNX family of transcription factors. To determine if any of the newly defined Vif interactors belong to the Vif–CUL5 complex, we performed double affinity purifications using cells co-transfected with Vif–23Strep and either A3G– or CUL5–33Flag (Fig. 1d, e). After purification first with Strep-Tactin and second with anti-Flag beads, mass spectrometry analysis of the final elution revealed the presence of CUL5, ELOB, ELOC, RBX2 and invariably CBF-b, strongly suggesting that this last protein may be a new component of the Vif E3 ubiquitin ligase complex (other factors from single Vif purifications depicted in Fig. 1c were not present). To confirm this interaction and the composition of the complex, we performed an additional double affinity purification experiment using