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. 2012 Jan 17;7(1):e30130. doi: 10.1371/journal.pone.0030130

Figure 1. HIV-1 replication is enhanced in Dlg1-depleted T cells.

Figure 1

A. Dlg1 expression in Dlg1+ and Dlg1- Jurkat T cells obtained with two lentivirus-based shRNA vectors. The stable Dlg1-depleted Jurkat T cell lines (Dlg1-) were obtained using two lentivirus-based vectors that target two different sequences on Dlg1. Control Jurkat T cell lines (Dlg1+) were obtained using the control vectors. T cells transduced with the GFP-encoding vectors (pHIV-H1shRNADlg1 or pHIV-H1shRNACtl) or with the puromycin-encoding vectors (Mission shRNADlg1 or Mission shRNA Ctl) were analyzed by western blot, and Dlg1 levels were quantified using ImageJ software. B. Expression of surface molecules in Dlg1+ and Dlg1- Jurkat T cells. Cells were labeled with antibodies against CD3, CD4, CXCR4, LFA-1 and ICAM-1 and analyzed by flow cytometry. The data are representative of three independent experiments performed in triplicates. For LFA-1 the mean values of three independent experiments performed in triplicates is also presented. C. HIV-1 replication in Dlg1+ and Dlg1- Jurkat T cells infected with the NL4.3 HIV strain. Viral replication was measured by determining the fraction of HIV-1-infected cells in the two cultures by intracellular Gag labeling and flow cytometry. Cells were labeled with the anti-HIV-p24 mAb KC57-PE, 3, 5 and 7 dpi. The data are the means of four independent experiments performed in duplicate. P = 0.031 for 5 dpi. P<0.0001 for 7 dpi. D. Efficiency of early steps of the HIV-1 life cycle in cells expressing or not Dlg1. Dlg1+ and Dlg1- Jurkat T cells were infected with the single cycle pNL4.3-derived pNluc vector pseudotyped with the HIV-1 Env expression vector pIIINL4env and luciferase activity was measured 48 h post infection. The data are the means of three independent experiments performed in triplicate, using 100 ng to 1000 ng of p24 of virus produced by 293T cells co-transfected with 5 µg of pNLuc and 0.5, 0.75 or 1 µg of pIIINL4env. P = 0.59. Error bars represent standard error of the mean (SEM). ns  =  no statistically significant difference. *, P<0.05. ***, P<0.001. AU  =  arbitrary units.