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Figure 7 | Retrovirology

Figure 7

From: Interactions between HIV-1 Vif and human ElonginB-ElonginC are important for CBF-β binding to Vif

Figure 7

EloB stabilizes Vif via the same region by which it interacts with EloC. (A, B) Vectors expressing untagged Vif (1 μg) or Vif-SLQ (1 μg) were transiently transfected into 293 T cells (106), either without (-EB/EC) or with co-transfection of 1 μg of EloB (+EB) or 2 μg of EloBΔC34 (+EBΔC34). Cells were treated with CHX and harvested as described in Figure 3. (C, D) Quantification of Vif/Vif-SLQ bands in (A, B). (E) Vectors expressing VifΔPPL-HA (1 μg) were transiently transfected into 293 T cells (106), either without (-EB/EC) or with co-transfection of 2 μg of EloBΔC34 (+EBΔC34). Cells were treated with CHX and harvested. (F) Quantification of VifΔPPL-HA bands in (F). All results in (C, D, F) are representative of three independent experiments. Error bars represent ± SD (n = 3). (G) 293 T cells (106) were co-transfected with 1 μg of VR-EB-HA or VR-EB-Myc and 2.5 μg VR-Vif-SLQ or 1 μg VR-Vif. Cell lysates were co-immunoprecipitated with an anti-HA antibody, followed by Western blotting with anti-HA, anti-EloB and anti-Vif antibodies. Results are representative of six independent experiments. (H) Schematic representation of the deletion mutagenesis of the EloC binding domain of EloB. (I) 293 T cells (106) were transfected with 1 μg of VR-Vif-SLQ, either without or with co-transfection of 1 μg VR-EloB-HA (+EB), VR-EloBΔ9-17-HA (+EBΔ9-17), VR-EloBΔI34-HA (+EBΔI34) or VR-EloBΔ69-72-HA (+EBΔ69-72). Cells were treated with CHX and harvested. Vif-SLQ were detected with anti-Vif antibody, and EloB was detected with an anti-HA antibody. Results are representative of three independent experiments. (J) 293 T cells (106) were transfected with 1 μg of VR-Vif SLQ with co-transfection of 1 μg VR-EloBΔ9-11-HA (+EBΔ9-11), VR-EloBΔ12-14-HA (+EBΔ12-14) or VR-EloBΔ15-17-HA (+EBΔ15-17). Cells were treated with CHX and harvested. Vif-SLQ and EloB was detected as described in (I). Results are representative of three independent experiments.

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