FIG 4.

MLN4924 inhibits Vpu-mediated downmodulation of CD4 and increases the surface expression of CD4-induced Env epitopes in primary CD4⁺ T cells. (A) Uninfected primary CD4⁺ T cells were treated with either DMSO alone or 200 nM MLN4924 in DMSO for 12 h and then stained for surface CD4 or BST2 and analyzed by flow cytometry. (B and C) Primary CD4⁺ T cells were infected with either wild-type HIV-1 or the indicated mutants. Four days later, the cells were incubated with either DMSO alone or 200 nM MLN4924 in DMSO for 12 h and then stained for surface CD4, BST2, and Env with the indicated antibodies and analyzed by flow cytometry, gating on the p24-positive cells. Black lines in the panels for A32 and 2G12 staining are the results for uninfected cells. Panel B shows a subset of the data displayed in panel C selected to show the effects of Nef and Vpu in the absence of MLN4924. (D) Independent data from two additional donors for CD4 and Env (detected by A32 and 2G12); the cells were infected either with wild-type HIV-1 or with the indicated mutants and treated or not with MLN4924 as indicated. (E) For the three preceding independent donors, the mean fluorescence intensities for CD4, BST2, A32, and 2G12 were averaged and normalized to Δvpu in the absence of MLN4924, which was set to 100%. −, open bars indicate the absence of MLN4924; +, filled bars indicate treatment with MLN4924. Error bars indicate ±standard deviations.