HIV-1-infected cells modify the spatial distribution of LCs in inner foreskin.

<p>(A) Representative images of normal inner foreskin (top), and inner foreskin explants exposed to either non-infected (left) or HIV-1-infected (right) PBMCs for 1 h (middle) or 4 h (bottom). Explants were then stained with anti-langerin Ab and visualized with AEC (normal foreskin) or DAB (foreskin explants) peroxidase substrates. The black double-headed arrows denote individual distances of LCs from the apical surface for each condition and are marked as [a_i]-[e_i] respectively. Images are representative of three independent experiments. Scale bars = 20 µm. (B) Calculated mean±SEM distances covered by LCs, following exposure of inner foreskin explants to either non-infected (white bars) or HIV-1-infected (grey bars) PBMCs. For exposure to non-infected PBMCs, the distances after 1 h and 4 h were calculated as [b]-[a] and [c]-[b], respectively, where a, b, c are means of the individual [a_i], [b_i], [c_i] distances (measured for 106, 193 and 148 different cells in three independent explants). For exposure to HIV-1-infected PBMCs, the distances after 1 h and 4 h were calculated as [a]-[d] and [e]-[d], respectively, where d and e are mean of the individual [d_i] and [e_i] distances (measured for 186 and 108 different cells in three independent explants). *p<0.0001 infected vs. non-infected at 4 h; Student's t-test. (C) shows the actual distances from the apical surface measured for each experimental condition. *p<0.0001 vs. normal foreskin; Student's t-test.</p

(A, B) HIV-1-infected cells induce retention of LCs within the epidermis.

<p>Parallel inner (A) and outer (B) foreskin explants were exposed for 4 h to either HIV-1-infected (solid lines) or non-infected (broken lines) PBMCs. Explants were then stained with anti-langerin Ab and visualized with DAB peroxidase substrate. Shown are means±SEM langerin+ cell densities (cells/mm²) in epidermis derived from three independent explants. Cells were counted in a minimum of 10 fields/section in each experiment. In (A): p = 0.0322 non-infected epidermis 4 h vs. 0 h and p = 0.0415 HIV-1-infected vs. non-infected at 4 h; Student's t-test. (C, D) HIV-1 entry and capture by LCs in inner, but not outer, foreskin explants. Single sections observed by confocal microscopy of parallel inner (C) and outer (D) foreskin explants, following 4 h exposure to HIV-1-infected PBMCs. Horizontal and vertical lines represent the localization of the sectioned stacks along the z axis (25 sections, 0.2 µm apart) that are shown below and to the right of each image. Sections were double-stained with goat-anti-human langerin Ab followed by TRITC-conjugated anti-goat IgG Ab and a mixture of several human/mouse anti-HIV-1 mAbs followed by FITC-conjugated anti-human/mouse IgG Abs. In (C), langerin is detected around cell bodies and dendrites reaching the apical surface (red arrowheads); HIV-1 virions are detected as dots in the epidermis associated with epithelial cells or in dermis (green arrowheads), as well as co-localized with LCs (green arrows). Higher magnification image with the xyz planes rotated shows viral particles internalized into LCs. Cell nuclei were counterstained with DAPI. White dotted lines denote the epidermis/dermis interface. Scale bars = 10 µm. Representative images of n = 3 experiments.</p

HIV-1-infected cells affect secretion of chemokines by the inner foreskin.

<p>Inner foreskin explants were inoculated in a polarized manner for 4 h with either non-infected or HIV-1-infected PBMCs, washed, and further incubated in medium in a non-polarized manner. The levels of twelve chemokines and cytokines were measured the next day in the culture supernatants, using a custom multiplex bead immunoassay and quantified by flow cytometry or specific ELISA. Shown are mean folds±SEM secretion of each analyte (derived from two independent explants), calculated as [secretion after exposure to HIV-1-infected PBMCs/secretion after exposure to non-infected PBMCs], for either chemokines/cytokines not altered (A) or altered (B) following exposure to HIV-1-infected cells. In (B), p = 0.0105, 0.0308, 0.0166, 0.0045 for INF gamma, CCL3/MIP-1 alpha, CCL20/MIP-3 alpha and CCL5/RANTES, respectively, Student's t-test, n = 2.</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

CCL5/RANTES mediates T-cell recruitment into the epidermis.

<p>(A) Representative FACS profiles out of two independent experiments, showing epidermal single-cell suspensions derived from inner foreskin explants exposed for 4 h to either medium alone (left), non-infected PBMCs (middle), or HIV-1-infected PBMCs (right). Cells were stained with PE-conjugated anti-human CD3 mAb and numbers represent the percentages of CD3+ cells in the framed windows (determined based on the non-specific staining of a matched isotype control Ab). (B) Inner foreskin explants were exposed for 4 h to either non-infected PBMCs (white bar) or HIV-1-infected PBMCs: alone (black bar); in the presence of 40 µg/ml control goat IgG Ab (dark grey bar); in the presence of 20 or 80 µg/ml neutralizing goat Ab to CCL5/RANTES (light grey bars). Following infection, epidermal single-cell suspensions were prepared and stained for CD3 expression as described above. Shown are mean folds increase±SEM of the percentages of CD3+ cells, normalized against that following exposure to non-infected PBMCs and derived from two independent explants. p = 0.0129 infected vs. non-infected and p = 0.0128 infected+CCL5 Ab 80 µg/ml vs. infected; Student's t-test.</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

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

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