Abstract
BackgroundHIV-1 Vif interacts with the cellular core-binding factor β (CBFβ) and counteracts the protective roles of certain human APOBEC3 (A3) proteins by targeting them for proteasomal degradation. Previous studies have identified some amino acids important for Vif–CBFβ interactions, and recently a co-crystal structure of a pentameric complex of HIV-1 Vif, CBFβ, Cul5, EloB, and EloC was resolved. However, a comprehensive analysis of Vif–CBFβ interactions that are important for Vif function has not been performed.ResultsHere, we carried out double-alanine scanning mutagenesis of the first 60 amino acids of Vif and determined their effects on interaction with CBFβ and their ability to induce A3G degradation as well as rescue HIV-1 replication in the presence of A3G. We found that multiple Vif residues are involved in the extensive N-terminal Vif–CBFβ interaction and that the 5WQVMIVW11 region of Vif is the major determinant. A minimum of three alanine substitutions are required to completely abrogate the Vif–CBFβ interaction and Vif’s ability to rescue HIV-1 infectivity in the presence of A3G. Mutational analysis of CBFβ revealed that F68 and I55 residues are important and participate in a tripartite hydrophobic interaction with W5 of Vif to maintain a stable and functional Vif–CBFβ complex. We also determined that CBFβ amino acids 73WQGEQR78, which are not resolved in the structure of the pentameric complex, are not involved in interaction with HIV-1 Vif.ConclusionsOur results provide detailed insight into the Vif–CBFβ interactions that are critical for Vif function and may contribute to the rational design of HIV-1 inhibitors that block Vif-mediated degradation of A3 proteins.
Highlights
HIV-1 Vif interacts with the cellular core-binding factor β (CBFβ) and counteracts the protective roles of certain human APOBEC3 (A3) proteins by targeting them for proteasomal degradation
Identification of HIV‐1 Vif determinants that are important for interaction with CBFβ To identify HIV-1 Vif determinants that are essential for interaction with CBFβ, we used a previously described panel of single or double-alanine substitution mutants of the first 60 amino acids of Vif [19]
The solvent accessible surface area (SASA; determined by using getarea program available at http:// curie.utmb.edu/getarea.html) for H28 is only 4.86 Å2, and W38 is not surface exposed (SASA is 0.0 Å2), suggesting that these amino acids are buried in the Vif protein, and substitution of these amino acids with alanines may have disrupted the overall structure of the α-domain of Vif
Summary
HIV-1 Vif interacts with the cellular core-binding factor β (CBFβ) and counteracts the protective roles of certain human APOBEC3 (A3) proteins by targeting them for proteasomal degradation. Previous studies have identified some amino acids important for Vif–CBFβ interactions, and recently a co-crystal structure of a pentameric complex of HIV-1 Vif, CBFβ, Cul, EloB, and EloC was resolved. Jager et al [4] and Zhang et al [5] recently reported that in addition to binding to cullin 5 (Cul5) and elongin B/C (EloB/C), Vif binds to the cellular core-binding factor β (CBFβ), and the Vif–CBFβ interaction is essential for inducing efficient degradation of A3 proteins, which inhibit HIV-1 replication in non-permissive cell types. CBFβ was proposed to play a critical role in stabilizing the intrinsically unstructured HIV-1 Vif and promoting formation of Desimmie et al Retrovirology (2017) 14:19 a well-ordered substrate receptor by facilitating local folding of the N-terminal Vif region [4, 5, 9,10,11]
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