FIG 4.

Mutation of the 174IR175 region in FIV Vif does not impair interaction with FcaA3s, ELOB, and ELOC. (A) The FIV Vif 174175IR-AA mutant has lost degradation activity against FcaA3Z2bZ3. Cells were transfected with expression plasmids for FcaA3Z2bZ3-HA and wild-type FIV Vif-V5 or the indicated FIV Vif mutants or with the empty pcDNA3.1 plasmid. Cells were harvested and analyzed by immunoblotting with anti-HA, anti-V5, and antitubulin antibodies, respectively. (B) The FIV Vif 174175IR-AA mutant does not antagonize FcaA3Z2bZ3 antiviral activity. Single-round FIVΔvif luciferase reporter virions were produced in the presence of feline A3 expression plasmids (FcaA3Z2bZ3) with wild-type FIV Vif or Vif mutants; pcDNA3.1(+) was added as a control for feline A3 (control) and FIV Vif (vector). The infectivity of reporter vectors was determined by quantification of luciferase activity in HEK293T cells transduced with normalized amounts of viral vector particles. (C and D) The FIV Vif 174175IR-AA mutant still has the capability to bind to FcaA3s, ELOB, and ELOC. HEK293T cells were transfected with expression plasmids for FcaZ2bZ3-HA, wild-type FIV Vif-V5, or the indicated FIV Vif mutants or with the empty pcDNA3.1 plasmid (C) or with wild-type FIV Vif-V5, the indicated FIV Vif mutants, T7-ELOC, or HA-ELOB and the empty pcDNA3.1 plasmid (D). Cells were harvested 48 h after transfection, and proteins of cell lysates (input) and immunoprecipitated complexes were analyzed by using Western blots stained with anti-V5 antibody for FIV Vif, anti-HA antibody for FcaZ2bZ3-HA and HA-Elongin B, and anti-T7 antibody for T7-Elongin C. ***, P value of <0.001.