Fig. 7.

Non-NL4.3 Vpu proteins downregulate cell surface CD28. CD4⁺ Sup-T1 and peripheral blood mononuclear cells (PBMCs) were infected with VSV-G pseudotyped NL4.3 viruses lacking Nef and encoding wild-type Vpu (dNef, blue), no Vpu (dNef dVpu, green) or three non-NL4.3 Vpu proteins (grey). Cells were analyzed by flow cytometry and mean geometric fluorescence intensities (MFI) of infected (GFP⁺) cells were determined. a Representative histograms illustrating cell surface CD28 on live infected (GFP⁺) Sup-T1 cells. MFIs are indicated. b Mean (± SE) relative cell surface CD28 on Sup-T1 cells infected with NL4.3 lacking a functional Nef protein (dNef), but encoding various Vpu proteins (n = 5). c Mean (± SE) relative total cellular CD28 of Sup-T1 cells infected with NL4.3 encoding various Vpu proteins (n ≥ 8). d Western blot illustrating expression of eGFP-tagged versions of the analyzed Vpu proteins from transfected HEK293T cells. e Representative dot plots illustrating infection level (GFP) and cell surface CD28 (APC) on primary CD4⁺ cells. f Representative histograms illustrating cell surface CD28 on either uninfected (isotype control, uninfected) or CD4⁺ infected (GFP⁺) cells. MFIs are indicated. g Mean (± SE) relative cell surface CD28 on CD4⁺ cells infected with viruses encoding various vpu genes (n = 8). Data were obtained from infection of PBMCs from two healthy donors. (Mr: molecular weight; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFP: green fluorescent protein; SE: standard error; UI: uninfected; *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001)