Identification of a tripartite interaction between the N-terminus of HIV-1 Vif and CBFß that is critical for Vif function.

ABSTRACT

BACKGROUND: HIV-1 Vif interacts with the cellular core-binding factor ß (CBFß) 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-CBFß interactions, and recently a co-crystal structure of a pentameric complex of HIV-1 Vif, CBFß, Cul5, EloB, and EloC was resolved. However, a comprehensive analysis of Vif-CBFß interactions that are important for Vif function has not been performed. RESULTS: Here, we carried out double-alanine scanning mutagenesis of the first 60 amino acids of Vif and determined their effects on interaction with CBFß 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-CBFß 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-CBFß interaction and Vif's ability to rescue HIV-1 infectivity in the presence of A3G. Mutational analysis of CBFß 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-CBFß complex. We also determined that CBFß amino acids 73WQGEQR78, which are not resolved in the structure of the pentameric complex, are not involved in interaction with HIV-1 Vif. CONCLUSIONS: Our results provide detailed insight into the Vif-CBFß 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.