Sensitivity of old and new APOBEC3G to degradation by HIV-1 Vif. (A) HeLa-A3G cells (5 × 10⁶) stably expressing human APOBEC3G (A3G; lanes 1 to 3) were transfected with 1 μg each of empty vector DNA (−), pNL-A1 (A1 Vif), or pcDNA-hVif (hVif), using Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA). In parallel, normal HeLa cells (lanes 4 to 6) were transfected with 0.5 μg of pcDNA-A3G together with 1 μg each of empty vector DNA, pNL-A1, or pcDNA-hVif. In this and all subsequent experiments, the total amount of transfected DNA was adjusted to 5 μg, using empty-vector DNA as appropriate. Cells were harvested 24 h posttransfection. Whole-cell lysates were separated by sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis and analyzed by immunoblotting, using an APOBEC3G-specific antibody (top panel), a Vif monoclonal antibody (middle panel), or an antitubulin antibody (bottom panel). (B) HeLa-A3G cells were transfected with 5 μg of pcDNA-hVif (lanes 2 to 4). Lane 1 is a control of mock-transfected cells. Cells were harvested at 24 (lanes 1 and 2), 48 (lane 3), or 72 (lane 4) h, and cell lysates were subjected to immunoblotting as in panel A. Bands were quantified and corrected for fluctuations in tubulin levels. Results are shown below the A3G panel. Values are expressed as percentages of the Vif⁻ [Vif(−)] control (lane 1). (C) HeLa-A3G cells were transfected with 0.5 μg of GFP-expressing plasmid (pEGFP), along with 0.5 μg of pcDNA-A3G-MycHis (A3G-Myc) in the absence or presence of 1 μg of pNL-A1 or pcDNA-hVif. Cells were harvested 24 h later and sorted for GFP-positive cells, using a FACSAria cell sorter (BD Biosciences Immunocytometry, San Jose, CA). Cell sorting was performed on live cells suspended in phosphate-buffered saline. The instrument setup was performed according to the manufacturer's instructions. All sorts were performed at 70 lb/in². GFP-positive cells were lysed in sample buffer. Lysates from an equal number of cells were analyzed by immunoblotting as in panel A.

Vif interacts more efficiently with new than with old APOBEC3G. HeLa cells were transiently transfected with pcDNA-A3G (A3G; 0.5 μg) in the absence of Vif (lanes 2 and 7) or together with 1.5 μg of pcDNA-hVif (A3G + Vif; lanes 3 and 8). In parallel, stable HeLa-A3G cells were transfected without (mock; lanes 4 and 9) or together with 1.5 μg of pcDNA-hVif (Vif; lanes 5 and 10). Mock-transfected HeLa cells were analyzed as controls (lanes 1 and 6). To prevent degradation of APOBEC3G, the dominant negative Cul5-Rbx mutant (1 μg) was included in all samples. The cells were lysed 24 h posttransfection, using NP-40 lysis buffer. Cell lysates were either analyzed directly (lanes 1 to 5) or were first immunoprecipitated, using an APOBEC3G-specific antibody (α-A3G; lanes 6 to 10). Samples were probed with antibodies to APOBEC3G (top panel) or Vif (α-Vif; lower panel). Proteins are identified on the right. IP, immunoprecipitation; WB, Western blot; IgG, immunoglobulin G.

Localization and characterization of old versus new APOBEC3G in HeLa cells. (A) HeLa cells (left panel) were transfected with 5 μg of pcDNA-A3G. Cell fractionation was performed 24 h later, using a ProteoExtract subcellular proteome extraction kit (EMD Chemicals Inc., Gibbstown, NJ) according to the manufacturer's instructions. HeLa-A3G cells (right panel) were analyzed in parallel. Proteins from individual fractions were analyzed by immunoblotting, using antibodies to APOBEC3G (A3G), calpain-1/2 as a cytoplasmic marker (EMD Chemicals Inc., Gibbstown, NJ), cytochrome P450 reductase (P 450) as a membrane marker (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), c-Jun as a nuclear marker (c-jun; BD Biosciences Immunocytometry, San Jose, CA), and vimentin as a cytoskeletal marker (Sigma Aldrich, Inc., St. Louis, MO). (B) APOBEC3G from stable HeLa-A3G cells and normal HeLa cells transiently transfected with pcDNA-A3G was fractionated on a 15 to 40% sucrose gradient as described previously (3). Nineteen fractions (550 μl each) were collected from the top of the gradient, and odd-numbered fractions were analyzed by immunoblotting, using an APOBEC3G-specific antibody.

(A) Cellular expression and packaging of old versus new APOBEC3G. HeLa-A3G cells were transfected with 0.5 μg of pcDNA-A3G-MycHis (even lane numbers) along with 3 μg of vif-defective pNL4-3 in the absence of Vif [Vif(−); lanes 1 and 2 and 7 and 8] or in the presence of 1 μg of pNL-A1 (A1 Vif; lanes 3 and 4 and 9 and 10) or pcDNA-hVif (hVif; lanes 5 and 6 and 11 and 12). Cells were harvested 24 h posttransfection and lysed in sample buffer. Virus-containing supernatant was concentrated by passing pellets through 20% sucrose. Cell and viral lysates were analyzed by immunoblotting with antibodies to APOBEC3G (A3G; top panel), Vif (middle panel), or an HIV-positive patient serum (CA; lower panel). Ctrl, control. (B) Virus-containing supernatants from transfected cultures were collected 24 h after transfection, normalized for reverse transcriptase activity, and used for the infection of LuSIV indicator cells. The relative infectivities of the viruses were determined by measuring the virus-induced expression of luciferase in the LuSIV cells 24 h later. The lane numbers correspond to those in panel A.