Fig. 1

Identification of Vif determinants that are critical for binding to CBFβ. a A schematic of Vif structure and its interaction domains with cellular proteins APOBEC3F (A3F), APOBEC3G (A3G), cullin 5 (Cul5) and elongin C (EloC). Double alanine mutants of the first 60 amino acids of Vif were analyzed. b Immunoblots of cell lysates to detect Flag-CBFβ, Vif, and tubulin (used as loading control). c Immunoblots of WT and mutant Vifs that co-immunoprecipitated with Flag-CBFβ. The average binding efficiency of Vif mutants to CBFβ from five independent transfection experiments is shown below each lane relative to WT Vif (set to 100%). Statistical significance was determined using t test; *P < 0.05. The mutants with significantly reduced Vif–CBFβ binding are shown in green; mutants with increased Vif–CBFβ binding are shown in red. The upper bands of the Vif IP blots of Q12A-V13A and WT (the last panel) are most likely the light chains of the anti-Flag antibody used for immunoprecipitation. d A ribbon diagram of HIV-1 Vif; the highlighted residues ⁵WQVMIVW¹¹, H27–H28, D37–W38, ⁴⁵ESTN⁴⁸, and ⁵³SEVHIP⁵⁸ decrease CBFβ binding efficiency as shown in (c). Vif ⁴⁵ESTNPKIS⁵² residues are in a loop and beginning of a β-strand; these Vif amino acids are near a loop of CBFβ that was not resolved in the structure (⁷⁵GEQRQPT⁸¹, purple dashed line). Vif is shown in red and CBFβ is shown in cyan (PDB:4N9F)