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Figure 5 | Retrovirology

Figure 5

From: HIV Nef and Vpu protect HIV-infected CD4+ T cells from antibody-mediated cell lysis through down-modulation of CD4 and BST2

Figure 5

Dissecting the potential involvement of BST2 in enhancing target cell lysis via ADCC. CEM.NKR CD4+ T cells were transduced with a lentivirus containing a non-targeting (NT) or a BST2-targeting (SH) shRNA as described in Methods. (A) BST2 and CD4 expression on NT and SH cells as examined by flow cytometry. Parallel staining with a rabbit PI was used as a control for BST2 staining. Shown next to the overlays are expression levels in MFI obtained for gated GFP+ infected cells from a representative analysis. (B) NT and SH T cells were infected with WT or ∆Vpu (U-) virus and assessed for HIV-1 viral release efficiency by Western blotting. Mock (M)-infected cells were used as control. Parallel virions and cell lysates were analyzed for total Gag proteins, GFP and Vpu. The histograms underneath the Western blots depict the average quantifications of the densitometric signals from two experiments. Virus release was determined to be the ratio of the virion-associated Gag signal (corresponding to the mature p24) over all cell-associated Gag signals (corresponding to p24 and precursor p55). Within the NT and SH cells, viral release by WT virus was considered to be 100% and that by ∆Vpu virus counterparts was expressed as % of the WT. (C-D) NT and SH CD4+ T cells were infected with WT, ∆Nef∆Vpu (N-U-), or ∆Nef∆Vpu-D368A Env (N-U-D368A) virus. Env staining by A32 and 2G12 was analyzed by flow cytometry (C). The histograms depict average fold increase (+/- SD) in Env recognition relative to WT virus-infected cells in three experiments. In parallel, target cells were evaluated for their susceptibility to A32-mediated cell lysis by ADCC as detailed in Figure 2 legend (D). Shown are average (+/-SD) net cell lyses by PBMC from six donors. See also Additional file 1: Figure S1 and Additional file 2: Figure S2.

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