Effects of Vpu TM mutations on Vpu-mediated degradation and surface downregulation of CD4.

<p>(A) 293T cells were co-transfected with 100 ng CD4-HA expression plasmid along with 200 ng VR1012 control vector or VR1012 encoding Vpu TM variants. At 48 h post-transfection, the cells were harvested for immunoblotting analysis. CD4 and Vpu were detected with anti-HA and anti-myc antibodies, respectively. Tubulin was detected as a loading control. (B) CD4 levels were measured using Bandscan software and normalized by tubulin levels. Percentages of degraded CD4 were calculated by subtracting the densitometric intensity values of the indicated Vpu WT or mutant bands from that of the mock band to represent the different abilities of Vpu variants to mediate CD4 degradation. Results shown are the average of two independent experiments. (C) HeLa CD4 cells were co-transfected with 500 ng pEGFP-N3 along with 500 ng VR1012 control vector or VR1012 encoding Vpu TM variants. Cell surface CD4 was stained with CD4 antibodies followed by Alexa 633 goat anti-mouse IgG and analyzed by flow cytometry. Samples were gated on EGFP+ cells, and the surface CD4 levels are shown in the histograms with median values at the top right corner.