Effect of a BST2 GPI anchor mutant on Vpu-mediated control of IFN-I production by pDCs.

<p><b>(A-B)</b> HEK293T cells were transfected with either an empty plasmid or plasmids expressing BST2 or BST2-dGPI 48 h prior to co-culture with PBMCs. <b>(A)</b> Surface expression of BST2 was evaluated 48 h post transfection by flow cytometry in controls cells (shaded grey histogram), as well as in cell expressing BST2 (solid black histogram), or BST2-dGPI (dashed grey histogram). Mean fluorescence intensity (MFI) values are indicated for each sample <b>(B)</b> After 6 h of co-culture, samples were untreated or treated with Imiquimod (TLR7 agonist) and levels of bioactive IFN-I in supernatants were measured 18 h later. The amount of IFN-I released by PBMCs in contact with HEK293T cells transfected with the empty plasmid in presence of the TLR 7 agonist was set at 100% (n = 4). As a control, transfected HEK293T cells were treated with TLR7 agonist without PBMCs. <b>(C)</b> Percentage of IFN-I released after co-culture of infected SupT1-Empty with PBMCs normalized to the value obtained with dU HIV-infected SuptT1 cells (100%) (n = 4). <b>(D)</b> A representative example of absolute levels of IFN-I produced after co-culture of mock or infected-SupT1,-SupT1-BST2 or-SupT1-BST2-dGPI cells with PBMCs is shown. <b>(E)</b> Relative percentages of IFN-I produced after co-culture of infected-SupT1-BST2 or SupT1-BST2-dGPI cells with PBMCs are shown. The amount of IFN-I released by PBMCs in contact with dU HIV-infected SupT1-BST2 cells was set at 100% (n = 4). Two-tailed paired <i>t</i>-test was used in B and C (* p<0.05, ns not significant (p>0.05)). Repeated measures ANOVA with Bonferroni’s multiple comparison test was used in E (*** p<0.001, ns not significant (p>0.05)). Error bars represent SD.