Human CBFβ has recently been identified as a critical regulator of HIV-1 Vif function 
. In the present study, we demonstrate that this host regulator directly interacts with Vif alone and in complex with E3 ligase components, in vitro
. CBFβ is the non-DNA-binding subunit of a heterodimeric transcription factor, including RUNX family proteins 
. CBFβ regulates the folding and DNA-binding activity of RUNX partners, which play important roles in the development and differentiation of diverse cell types, including T lymphocytes and macrophages 
. We have recently reported that CBFβ is critical for Vif-induced A3G polyubiquitination and degradation 
. Further clarification of the Vif-CBFβ-EloB/C-Cul5 interaction and complex assembly would provide key insights into how Vif recruits these E3 ligase components to degrade A3G/A3F.
Co-expression of HIV-1 Vif with CBFβ in the absence of all other human factors increased Vif solubility in E. coli
. Soluble Vif could be co-precipitated with both His-tagged full length or truncated CBFβ () In the absence of binding partners, previous research has suggested full length Vif appears to be unstructured and poorly soluble, in vitro
. Recently, Wolfe et al.
were able to obtain soluble C-terminal domain fragments of Vif in complex with EloB/C and Cul5 
. Attempts at characterizing full length Vif in complex with EloB/C and Cul5 were unsuccessful, suggesting that the N-terminus was responsible for Vif's poor solubility, in the absence of N-terminal binding partners. We have shown that CBFβ binds the N-terminal region of Vif, specifically requiring hydrophobic interactions at amino acids W21 and W38 
. We hypothesize that the exposure of the N-terminal hydrophobic surface may contribute to Vif insolubilty when expressed alone. In vivo
, CBFβ appears to be necessary for Vif-Cul5 binding, though CBFβ does not bind Cul5 directly 
. Thus, a possible role for CBFβ would be to stabilize Vif structure and promote the assembly of the Vif-Cul5 E3 ubiquitin ligase complex.
Vif and CBFβ co-fractionated in gel filtration analyses and appeared as a 1
1 ratio complex. Isoforms 1 and 2 as well as a truncated form (amino acids 1–140) of CBFβ all interacted with HIV-1 Vif. Thus, most, if not all, of the Vif binding activity is preserved within the first 140 amino acids of CBFβ. Of note, C-terminal truncation of CBFβ up to amino acids 1–135 have been reported to bind and act in complex with RUNX family proteins 
. In addition, we have confirmed that CBFβ binds to at least the first 140 amino acids of HIV-1 Vif. Thus, the known protein-binding domains in Vif, including the EloB/C binding BC-box, the cullin box containing the PPLP motif, are not essential for the Vif-CBFβ interaction. Vif forms homo-oligomers, and the PPLP motif has been suggested to be required for oligomerization 
. Since Vif140 still forms oligomers with CBFβ140, CBFβ182, and CBFβ187, our results suggest that regions in Vif in addition to PPLP may also participate in Vif oligomerization. This conclusion is consistent with the recent finding that the PPLP motif is not sufficient for Vif multimerization 
Biophysical and structural information for Vif has been limited as a result of its insolubility and strong tendency to oligomerize into high molecular weight aggregates. Of note, previous biochemical studies have employed full-length Vif protein obtained by the denaturing/refolding method 
or have used truncated tagged protein 
. Interestingly, when CBFβ and EloB/C were present, even untagged full-length Vif could be purified as a stable and soluble complex.
Association of Vif with CBFβ alone, and especially in combination with EloB/C, greatly increases the solubility of full-length Vif. We have shown that a stable complex containing Vif-CBFβ140-EloB/C can be purified in large quantities. This complex appeared to contain one subunit of each protein and did not dissociate upon RNase treatment. More importantly, the Vif-CBFβ140-EloB/C complexes we produced could interact with purified Cul5 and form stable Vif-CBFβ140-EloB/C-Cul5 complexes. This successful purification of monomeric Vif-E3 ligase complexes in high purity will greatly facilitate biochemical studies, structural determination, and functional analyses in this field.
Because CBFβ is a unique regulator of Vif's ability to hijack the cellular CRL5 E3 ligase, disrupting interactions within the Vif-CBFβ140-EloB/C-Cul5 complex represents an exciting drug strategy for targeting HIV-1. Inhibitors that prevent complex formation would be potential candidates for HIV-1 suppression, and purification of these Vif complexes in homogeneous form would provide the basis for screens to identify and evaluate inhibitor candidates. Thus, our strategy for purifying Vif-Cul5-CBFβ-EloB/C complexes may lead to useful screening approaches for identifying novel anti-HIV drug candidates.