Fab-based inhibitors reveal ubiquitin independent functions for HIV Vif neutralization of APOBEC3 restriction factors
(A) In vitro ubiquitination of Myc-tagged A3F-CTD in the presence of increasing amounts of Flag-tagged 1D1. A3F-CTD and 1D1 were visualized by WB using anti-Myc and anti-Flag, respectively. The asterisk indicates non-reduced 1D1. (B) MBP-tagged VCBC was immobilized on amylose resin and incubated with 1D1 and/or CUL5-NTD. (C) MBP-tagged VCBC WT or VCBC containing ELOC T41Y were immobilized on amylose resin and incubated with CUL5-NTD. Input and final bead (FB) samples were resolved by SDS-PAGE gel and show proteins present before and after wash steps. (D-F) Env-deficient NL4-3 HIV (either Vif+ or Vif-) was co-transfected with HA-tagged A3G and Flag-tagged (D) CUL5-NTD, (E) ELOC WT (F) ELOC T41Y. A3G levels were measured in the cell and VLPs by WB with anti-HA.