Stability of HIV-1 envelope expressed on the plasma membrane of T cells cultured under shaking conditions.

<p><b>A.</b> Cell-surface level of HIV-1 envelope on CEM-213env1 cells cultured under static or shaking conditions. The T-cell line CEM-213env1 stably expressing the HIV-1 envelope glycoprotein was cultured for up to 7 days under static or shaking conditions. The cell-surface levels of the HIV-1 S envelope were determined by flow cytometry analysis using the anti-Env 5F7 antibodies. Percentage of Env-positive cells are shown in the top pannel and mean of fluorescence intensity (MFI) are shown in the bottom pannel. Two independent experiments were carried out in triplicate. Error bars represent standard deviations. <b>B. Western blot analysis and quantification of protein extracts of static and shaking CEM-213env1 T-cell cultures.</b> Equal amounts of total proteins from cell lysates recovered 1, 2, 4, and 7 days under static or shaking conditions were analyzed using antibodies against the HIV-1 envelope proteins. The envelope levels were estimated from the intensity of the signals on western blots using the ImageJ software and calculated as gp120/tubulin ratios. The results are from one representative experiment out of two independent experiments carried out in duplicate.</p

HIV-1 replication and particle infectivity are drastically reduced in mobile lymphocyte T cells.

<p><b>A.</b> HIV-1 replication in Jurkat T cells cultured under static or shaking conditions. Jurkat T cells infected with the NL4.3 HIV strain were cultured under static or shaking conditions. 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 days post infection (dpi). Data in top pannel are the means of three independent experiments performed in duplicate. FACS profiles in bottom pannel are of one representative experiment. P = 0.01 for 3 dpi. P = 0.006 for 5 dpi. P = 0.005 for 7 dpi. Error bars represent standard error of the mean (SEM). *, P<0.05. **, P<0.01. <b>B. HIV-1 yield of Jurkat T cells cultured under static or shaking conditions.</b> Supernatants were collected at 7 dpi, filtered and the p24 content was measured by ELISA. The values were normalized for protein content of extracts of cultured cells. The data are means of three independent experiments each carried out in duplicate. For each experiment the values were normalized taking as 100% the value obtained for one of the duplicates of cells under static culture at 7 dpi. P = 0.003 at 7 dpi. <b>C. Infectivity of HIV-1 particles produced by Jurkat T cells cultured under static or shaking conditions.</b> Viral supernatants of Jurkat T cells infected with HIV-1 NL4.3 were collected at 7 dpi, filtered and used to infect indicator HeLa P4.2 reporter cells. Equal amounts of virus determined by p24 quantification were used. β-galactosidase production was assessed by a colorimetric assay based on cleavage of CPRG. The data are means of three independent experiments carried out in triplicate. Normalization was performed as in <b>B.</b> P = 0.001. Error bars represent SEM. **, P<0.01.</p

Dlg1 depletion affects IS but not VS formation in T cells.

<p><b>A. MTOC polarization toward the nascent IS in Dlg1+ and Dlg1- Jurkat T cells.</b> Left panel: IS conjugates were formed between Jurkat and APC (Raji B) cells. Phosphotyrosine accumulation at the interface between two contacting cells served as marker of a productive IS contact that led to TCR activation and to activation of the downstream signaling cascade. Centrin was visualized simultaneously to localize the MTOC. A polarized MTOC in a Dlg1+ T cell and an unpolarized MTOC in a Dlg1- T cell are seen. The nuclei of Jurkat T cells are shown in blue, the MTOCs are shown in red and indicated by a white arrow. The phosphotyrosine (PTYR) is shown in magenta. The results shown were obtained with the pHIV-H1shRNADlg1 and pHIV-H1shRNACtl vectors expressing the GFP. Similar results were obtained with cells transduced with the lentivirus vectors pHIV-H1shRNADlg1 and Mission shRNADlg1. Right panel: <b>Quantification of MTOC polarization</b> observed in Dlg1+ (n = 131) and Dlg1- (n = 151) T cells in four independent experiments. P = 0.029. <b>B. Cellular localization of Gag in the presence and absence of Dlg1.</b> Dlg1+ and Dlg1- Jurkat T cells were immunostained for Gag and for Dlg1. White arrows indicate Gag accumulation at the VS. The images shown are representative of three independent experiments. <b>C. VS formation in Dlg1+ and Dlg1- Jurkat T cells.</b> Upper panel: HIV-1-infected Dlg1+ and Dlg1- Jurkat T cells were mixed with an equal number of non-infected primary CD4+ target cells and were allowed to establish contacts on poly-L-lysine-coated coverslips for 1 h at 37°C. The cells were fixed, permeabilized, immunostained for Gag and CD4 and analyzed by confocal microscopy. Monosynapses were defined as contacts between a single effector and a single target cell presenting co-localization (in white) of Gag on the infected cell and of CD4 on the CD4+ target cell at sites of contact. Polysynapses were defined as contacts between a single effector cell and multiple target cells. Lower panel: Quantification of cellular contacts, monosynapses and polysynapses between HIV-1-infected Dlg1+ or Dlg1- Jurkat T cells and CD4+ target cells. A total of 1603 Dlg1+ control T cells and 1616 Dlg1- T cells were counted. The data are the means of 3 independent experiments. P = 0.7 for contacts. P = 1.0 for monosynapses. P = 1.0 for polysynapses. Error bars represent SEM. ns  =  no statistically significant difference. *, P<0.05. Scale bar  =  5 µm.</p

The levels of HIV-1 Env incorporated into viral particles produced by Dlg1-depleted T cells do not correlate with increased infectivity.

<p><b>A. HIV-1 yield of Dlg1+ and Dlg1- T cells.</b> Supernatants were collected during the course of infection, filtered and the p24 content was measured by ELISA. The values were normalized for protein content of extracts of cultured cells. The data are the mean values of three independent experiments each carried out in duplicate. For each experiment the values were normalized taking as 100% the value obtained for one of the duplicates of Dlg1+ cells at 7 dpi. P = 0.007 for 5 dpi. P = 0.02 for 7 dpi. <b>B. Infectivity of HIV-1 particles produced by Dlg1+ and Dlg1- T cells.</b> Viral supernatants of Dlg1+ and Dlg1- Jurkat T cells infected with HIV-1 NL4.3 were collected 7 dpi, filtered and used to infect indicator HeLa P4.2 reporter cells. Equal amounts of virus determined by p24 quantification were used. ß-galactosidase production was assessed by a colorimetric assay based on cleavage of CPRG. The data are means of four independent experiments carried out in triplicate. P = 0.0004. <b>C. Summary of quantification of cell-associated Env in Dlg1+ and Dlg1- cells and of virus-associated Env in progeny viruses.</b> The cell-associated Env levels were estimated from the intensity of the signals on western blots and calculated as gp160+gp120/Gag in cell lysates. Equal amounts of total protein in lysates of Dlg1+ and Dlg1- HIV-1-infected cells recovered 5 dpi were analyzed using antibodies against HIV-1 Gag and Env. The virus-associated Env levels (gp120) were estimated from the intensity of the signals on the western blots; equal amounts of p24 of purified viruses from supernatants of Dlg1+ and Dlg1- HIV-1-infected cells were analyzed using antibodies against HIV-1 Gag and Env. The infectivity of these viruses was determined as described in B. The data are from four independent experiments. <b>D. Western blot analysis of protein extracts of Dlg1+ and Dlg1- cells and of purified viruses produced.</b> Results of experiment 4 are shown. Left panel: equal amounts of total protein in lysates of Dlg1+ and Dlg1- HIV-1-infected cells recovered 5 dpi were analyzed using antibodies against HIV-1 Gag and Env, against Dlg1 and against GAPDH. Right panel: equal amounts of p24 of purified viruses from supernatants of Dlg1+ and Dlg1- HIV-1-infected cells were analyzed using antibodies against HIV-1 Gag and Env. <b>E. Cell-surface level of HIV-1 Env on Dlg1+ and Dlg1- HIV-1-infected T cells.</b> The cell-surface level of HIV-1 Env was determined by flow cytometry analysis using the anti-Env 5F7 antibodies. HIV-1-infected Dlg1+ and Dlg1- Jurkat T cells were analyzed 5 dpi. Two independent experiments were carried out in duplicate. Error bars represent SEM. ns  =  no statistically significant difference. *, P<0.05. **, P<0.01. ***, P<0.001.</p

Budding of HIV-1 particles in Dlg1- Jurkat T cells occurs at the plasma membrane and in internal compartments.

<p><b>A. Budding of HIV-1 particles in Dlg1- Jurkat T cells from the plasma membrane.</b> Dlg1+ and Dlg1- Jurkat T cells infected with HIV-1 were examined 6 dpi, when infection was about 60%, by electron microscopy in cells not forced to form conjugates. A total of 320 Dlg1+ Jurkat T cells (out of 5300) showing viral particles and of 392 Dlg1- Jurkat T cells (out of 6500) showing viral particles were counted. Budding from plasma membrane was identical in Dlg1+ and Dlg1- cells; the image presented is of a Dlg1- Jurkat T cell. Two independent experiments were performed with both NL4.3 and LAI.2 HIV-1. <b>B. Budding of HIV-1 particles in Dlg1- Jurkat T cells from internal compartments.</b> Cells were treated as above. Budding and mature particles are seen in an internal compartment in a Dlg1- Jurkat cell. <b>C and D. HIV-1 particles in internal compartments of Dlg1- Jurkat T cells.</b> Mature particles are seen in the enlargement in D. <b>E and F. HIV-1 particles in compartments near the plasma membrane of Dlg1- Jurkat T cells.</b> Mature particles are seen in the enlargement in F. Same magnification was used in C and E and in D and F. P = 0.02 for NL4.3. P = 0.01 for LAI.2 infected cells. Scale is 0.5 µm for A, D and F, 2 µm for C and E and 100 nm for B.</p

HIV-1 particles produced by Dlg1-depleted cells accumulated higher total HIV-1 DNA amounts during the first hours of infection and have increased cholesterol content.

<p><b>A. Quantification of</b> t<b>otal viral DNA.</b> Total viral DNA was quantified by quantitative PCR in Jurkat T cells 6 h after infection with NL4.3 or LAI.2 HIV-1 particles produced by Dlg1+ and Dlg1- cells. The results are expressed as mean NRQ (normalized RQ described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030130#s2" target="_blank">materials and methods</a>). Values are from one representative experiment performed in triplicate for infection and in triplicate for qPCR. P = 0.001. <b>B. Viral cholesterol content of HIV-1 particles produced by 293T cells.</b> Dlg1- 293T cells were obtained by siRNA transfection. The cholesterol content of LAI.2 HIV-1 produced by Dlg1+ or Dlg1- 293T cells was determined. The data are the means of three independent experiments performed in duplicate. For each experiment the values were normalized to the value obtained with one of the duplicates of viruses from Dlg1+ cells taken as 100%. P = 0.0026. <b>C. Viral cholesterol content of HIV-1 particles produced by Jurkat T cells.</b> Cholesterol content of LAI.2 HIV-1 produced by Dlg1+ or Dlg1- Jurkat T cells was determined. The data are the means of one experiment performed in duplicate. The values were normalized to the value obtained for one of the duplicates of viruses from Dlg1+ cells taken as 100%. Error bars represent SEM. **, P<0.01.</p

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

<p><b>A. Dlg1 expression in Dlg1+ and Dlg1- Jurkat T cells obtained with two lentivirus-based shRNA vectors.</b> 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>B. Expression of surface molecules in Dlg1+ and Dlg1- Jurkat T cells.</b> 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. <b>C. HIV-1 replication in Dlg1+ and Dlg1- Jurkat T cells infected with the NL4.3 HIV strain.</b> 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. <b>D. Efficiency of early steps of the HIV-1 life cycle in cells expressing or not Dlg1.</b> 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.</p