Figure 6.

Importance of βTrCP. (A) 293FT, βTrCP-1/2 KO, and add back cells were transfected with HIV-CMV-GFP (Vpu⁺, Vpu⁻, and Vpu^S52/56A) along with MLV Env or GaLV Env Δ8. After 48 h, media was transferred to 293T mCAT-1 cells and infectivity was measured by flow cytometry 48 h later. Infectious particle production from each data set was normalized to HIV-CMV-GFP (Vpu⁻). Shown is the average and standard deviation from six independent experiments. (B) The same cell lines were transfected with human CD4 with or without Vpu-IRES-GFP, surface stained for CD4 expression, and analyzed by flow cytometer 48 h later. (C) The same cell lines were tranfected with HA-tagged human CD4 with or without Vpu-IRES-GFP, collected after 2 days, and immunoblotted for HA-CD4. GAPDH was blotted as a loading control. (D) The same cells lines were transfected with HIV-CMV-GFP (Vpu⁺, Vpu⁻, and Vpu^S52/56A), along with VSV-G and varying amounts of HA-Tetherin (0–80 ng). After 48 h, media was transferred to 293T mCAT-1 cells and infectivity was measured by flow cytometry 48 h later. Infectious particle production from each data set was normalized to that of the well with 0 ng tetherin. Shown is the average and standard deviation of seven independent experiments. Students t test was performed comparing Vpu⁺ to Vpu^S52,56A. Asterisks indicate p < 0.01. NS indicates difference is not significant.