Cell-cell spread of human immunodeficiency virus type 1 overcomes tetherin/BST-2-mediated restriction in T cells - PubMed (original) (raw)
Cell-cell spread of human immunodeficiency virus type 1 overcomes tetherin/BST-2-mediated restriction in T cells
Clare Jolly et al. J Virol. 2010 Dec.
Abstract
Direct cell-to-cell spread of human immunodeficiency virus type 1 (HIV-1) between T cells at the virological synapse (VS) is an efficient mechanism of viral dissemination. Tetherin (BST-2/CD317) is an interferon-induced, antiretroviral restriction factor that inhibits nascent cell-free particle release. The HIV-1 Vpu protein antagonizes tetherin activity; however, whether tetherin also restricts cell-cell spread is unclear. We performed quantitative cell-to-cell transfer analysis of wild-type (WT) or Vpu-defective HIV-1 in Jurkat and primary CD4(+) T cells, both of which express endogenous levels of tetherin. We found that Vpu-defective HIV-1 appeared to disseminate more efficiently by cell-to-cell contact between Jurkat cells under conditions where tetherin restricted cell-free virion release. In T cells infected with Vpu-defective HIV-1, tetherin was enriched at the VS, and VS formation was increased compared to the WT, correlating with an accumulation of virus envelope proteins on the cell surface. Increasing tetherin expression with type I interferon had only minor effects on cell-to-cell transmission. Furthermore, small interfering RNA (siRNA)-mediated depletion of tetherin decreased VS formation and cell-to-cell transmission of both Vpu-defective and WT HIV-1. Taken together, these data demonstrate that tetherin does not restrict VS-mediated T cell-to-T cell transfer of Vpu-defective HIV-1 and suggest that under some circumstances tetherin might promote cell-to-cell transfer, either by mediating the accumulation of virions on the cell surface or by regulating integrity of the VS. If so, inhibition of tetherin activity by Vpu may balance requirements for efficient cell-free virion production and cell-to-cell transfer of HIV-1 in the face of antiviral immune responses.
Figures
FIG. 1.
Confirmation of the Vpu-mediated budding defect in Jurkat T cells. (A) Jurkat T cells were infected with pNL4.3ΔVpu or pNL4.3 WT virus, and at 3 days postinfection the cells were pelleted and virus-containing supernatants were harvested and purified by ultracentrifugation. Equal volumes of virus particles and concentrations of cell lysates were analyzed by SDS-PAGE and Western blotting using rabbit antiserum against HIV-1 Gag and an anti-actin loading control. (B) Jurkat T cells were infected with either pNL4.3 WT or pNL4.3ΔVpu, fixed and processed for TEM. Ultrathin sections of infected cells show more virions accumulating on the plasma membrane of cells infected with ΔVpu virus (lower panel) than on that of cells infected with Vpu-expressing, wild-type virus (upper panel). Virions tethered to the plasma membrane are characteristic of Vpu-defective virions and are indicated with arrows. The boxed region shows a tethered virus at a higher magnification. Scale bar, 100 nm. (C) Vpu-defective virus disseminates efficiently in culture. Jurkat T cells were infected with either pNL4.3ΔVpu (triangles) or pNL4.3 WT (squares). The amount of infectious virus in the supernatants was quantified by luciferase assay using HeLa Tzm-bl reporter cells (expressed as TCID50/ml) (upper left panel), and the percentage of infected Jurkat cells was quantified by intracellular Gag staining and flow cytometry (upper right panel). Data are from the identical experiments and are the means from three independent experiments; error bars show the SEMs. The lower left panel shows a representative example of gating on the live cell population using FACSCalibur and CellQuest 5.0, and the histogram (lower right panel) shows the intracellular staining of cells infected with either WT (gray line) or ΔVpu (black line) virus at 7 days postinfection overlaid onto stained, uninfected control cells (gray filled). The M1 marker was set to the position that excluded >99% of the uninfected cells to define the Gag-positive population. Results from a representative experiment are shown. (D) pNL4.3 WT and pNL4.3ΔVpu viruses have the same relative infectivity. Cell-free supernatants were harvested at various times postinfection and quantified for viral infectivity by luciferase assay. Data are shown as the TCID50 per ng of p24 to normalize for any differences in the viral content of supernatants. Data are from four independent experiments, and error bars show the SEMs. (E) Comparison of intracellular Gag staining with total-cell and single-cell gating. Jurkat cells infected with either WT or ΔVpu virus were fixed and stained for intracellular Gag, and data were acquired using an LSRII flow cytometer and FACSDiva software. The first gate was applied to define the total cell population (far left panel), and this population was then analyzed using FSC-A versus FSC-H for doublet discrimination (middle left panel), with a second gate applied to differentiate single cells (blue) from the total population (red) to exclude doublets. Note that there are few cells in the position expected for the doublet population (denoted with an asterisk). The histogram (middle right) shows an overlay of intracellular Gag staining of the total cell population (red line) compared to single cells (blue line). The far right panel shows an overlay of intracellular Gag staining of infected cells either left untreated (black line) or pretreated with trypsin-EDTA prior to fixing (gray line). Results from a representative experiment are shown, and similar results were seen with both WT and ΔVpu viruses.
FIG. 2.
ΔVpu virus induces VS formation more efficiently than WT Vpu-expressing virus. (A) HIV-1-infected cells were mixed with uninfected primary target T cells (asterisks) in the presence of nonblocking MAbs against Env (blue) and CD4 (green) and incubated for an hour at 37°C. Conjugates were fixed, permeabilized, stained for Gag using rabbit antiserum (red), and analyzed by LSCM. Areas of colocalization appear yellow. VSs between target cells and Jurkat-pNL4.3 WT (upper panel) or Jurkat-ΔVpu (lower panel) cells show similar copolarization of CD4 and Env/Gag to the cell-cell interface. (B) Quantification of conjugate and VS formation between HIV-1 infected T cells and target T cells. Jurkat-pNL4.3 WT (white bars; n = 183) and Jurkat-ΔVpu (black bars; n = 231) cells were mixed with target cells, stained, and scored for whether or not they had formed a conjugate with a primary T cell (defined as closely apposed pairs consisting of a single target cell and a single effector cell, excluding polysynapses) and whether the conjugate had evolved a VS (Jurkat-pNL4.3 WT, n = 65; Jurkat-ΔVpu, n = 117). Data are from three independent experiments, and error bars show the SEMs. (C) Quantification of the frequency of polysynapse formation between Jurkat-pNL4.3 WT cells (white bars; n = 45) or Jurkat-ΔVpu cells (black bars; n = 108) and target cells. Polysynapses were defined as a single effector cell engaging multiple target cells, and a representative polysynapse between a Jurkat-ΔVpu cell and a target T cell is shown. Data are from three independent experiments, and error bars show SEMs. (D) Jurkat-ΔVpu cells express more HIV-1 envelope glycoprotein (Env) at the cell surface. Jurkat-pNL4.3 WT (white bars) and Jurkat-ΔVpu (black bars) cells were surface stained for HIV-1 Env with MAb 2G12 on days 7 and 10 postinfection and analyzed by flow cytometry by gating on the live cells. Env-positive staining was defined by comparison with control stained cells (infected cells stained with secondary antibody only) and was set at the MFI that excluded >99% of the control cells (see Fig. 1); from this the MFI of the Env-positive population was determined. Data are means and SEMs from seven independent experiments. *, P = <0.05; **, P = <0.01; ***, P = <0.005; ****, P = <0.001.
FIG. 3.
Tetherin but not Vpu colocalizes with Env at the VS. (A) Jurkat-ΔVpu cells were mixed with uninfected target T cells and incubated for an hour at 37°C in the presence of MAbs against Env (blue), CD4 (green), and tetherin (red) in order to stain surface proteins. Endogenous, surface-expressed tetherin is enriched on the HIV-1-infected T cell at the VS and colocalizes with Env (n = 64). The target cell is indicated with an asterisk. (B) Tetherin is enriched at multiple contact sites on polysynapses. Env, blue; CD4, green; tetherin, red. (C) Tetherin colocalizes with HIV-1 Env on infected T cells not engaged in a VS. Jurkat-ΔVpu cells were surface stained for Env (green) and tetherin (red) in the absence of target T cells. (D) Jurkat-ΔVpu cells were mixed with uninfected target T cells and incubated for an hour at 37°C in the presence of MAbs against Env (blue) and anti-CD4 (green). Cells were fixed, permeabilized, and stained for intracellular Vpu (red). Vpu localized mainly in the perinuclear region and was not enriched at the VS (n = 26).
FIG. 4.
Quantification of cell-cell spread. (A) Flow cytometric analysis of Gag transfer. Jurkat T cells infected with pNL4.3 WT or ΔVpu were either left untreated or pretreated with 500 U/ml interferon for 24 h and mixed with dye-labeled target T cells, and the percentage of Gag+ target cells was measured by flow cytometry. Data are from two independent experiments performed in duplicate, and values show the mean percentages of Gag+ dye-labeled target cells with the SEMs. Infected cells were used when >80% of the donor cells were routinely Gag+ by flow cytometry. (B) Comparison of total-cell and single-cell gating. Jurkat T cells infected with pNL4.3 WT or ΔVpu were mixed with CellTrace dye-labeled target T cells, data were acquired using an LSRII flow cytometer with FACSDiva software, and the percentage of Gag+ dye-labeled target cells was quantified using FlowJo. The first gate was applied to define the total, live cell population (far left panel), and this population was then analyzed using FSC-A versus FSC-H for doublet discrimination and a second gate applied to differentiate single cells (blue) from the total population (red) to exclude doublets from analysis (middle panel). Note that there are few cells in the position expected for the doublet population (denoted with an asterisk). The overlay (right panel) shows a comparison of cell-cell transfer to target cells when gating on the total cell population (red) or with single cells only (blue). Values denote the percentages of Gag+ target cells identified when gating on total cells or single cells. A representative example of cell-cell spread of ΔVpu virus after 6 h of coculture with target cells is shown; similar results were obtained with WT virus. (C) Zidovudine partially reduces intracellular Gag staining in newly infected targets following cell-cell spread. Dye-labeled target cells were either left untreated or pretreated with the reverse transcriptase inhibitor zidovudine for an hour at 37°C prior to being mixed with WT-infected (white) or ΔVpu-infected (black) cells for 6 h or 24 h and subsequent intracellular Gag staining. Data show the mean fluorescence intensity of intracellular Gag staining in the newly infected Gag+ dye-labeled target cell population in the presence and absence of zidovudine following cell-cell transmission. Data were acquired using an LSRII flow cytometer as described above to exclude doublets and are means from two independent experiments with the SEMs. (D) Quantitative real-time PCR of cell-cell spread. The lower panel shows results from Jurkat T cells infected with pNL4.3 (white bars) or ΔVpu (black bars) and either left untreated or pretreated with 500 U/ml interferon for 24 h, mixed with uninfected target T cells, and incubated for 1, 3, and 6 h prior to lysis and extraction of DNA. Real-time PCR was performed using pol primers to quantify de novo HIV-1 DNA synthesis as a measure of HIV-1 cell-cell spread. Data were normalized to human serum albumin (HSA). The values obtained at t = 0 h (baseline) were subtracted from the test values, and data are shown as the relative fold enhancement over 0 h. Data are from four independent experiments, and error bars show the SEMs. Experiments were performed when >90% of the donor cells were Gag+ by flow cytometry. The upper panel shows data from experiments in which target cells were pretreated with the reverse transcriptase inhibitor zidovudine for an hour at 37°C prior to incubation with ΔVpu-infected T cells for 6 h. The values obtained at t = 0 h (baseline) were subtracted from the test values, and data are shown as the relative HIV DNA copy number normalized to human serum albumin (HSA). Error bars are the SEMs from two independent experiments. (E) Confirmation that interferon treatment reduces cell-free virus release. Jurkat-pNL4.3 WT (white bar) and Jurkat-ΔVpu (black bar) cells were either untreated or incubated with 500 U/ml interferon (gray bars) for 24 h, and cell-free supernatants were assayed for viral content by p24 ELISA. Data are relative percent p24 levels with untreated cells normalized to 100% and are means from four independent experiments with the SEMs. (F) IFN-β treatment upregulates surface tetherin but not adhesion molecule expression on T cells. Jurkat cells and primary CD4+ T cells from two different donors (A and B) were either left untreated (gray lines) or incubated for 24 h with 500 U/ml of IFN-β in the growth medium (black lines) and surface stained for tetherin (left panels), LFA-1 (middle panels), and ICAM-1 (right panels). Unstained cells are also depicted (broken lines). Representative histograms are shown. (G) VS formation is not inhibited by interferon treatment. Jurkat-pNL4.3 WT and Jurkat-ΔVpu cells were either treated with interferon or left untreated and quantified for conjugate (left panel) and VS (right panel) formation as described for Fig. 2. Data are from three independent experiments, and error bars show SEMs. Jurkat-pNL4.3 WT: untreated, white bars (n = 179); with interferon, gray bars (n = 261). Jurkat-ΔVpu: untreated, black bars (n = 162); with interferon, gray bars (n = 221). *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001.
FIG. 5.
siRNA knockdown of tetherin/Bst-2 reduces cell-cell spread. (A) Flow cytometry of tetherin knockdown. Jurkat T cells were nucleofected with control siRNA (gray line) or siRNA targeting tetherin (black line), and 48 h later surface tetherin expression was measured by flow cytometry. The gray-filled histogram shows nucleofected cells stained with the secondary antibody only. Data from a representative experiment are shown. (B) siRNA knockdown of tetherin reduces cell-cell spread of HIV-1. Jurkat T cells were nucleofected with siRNA targeting tetherin/Bst-2 (black bars) or control siRNA (white bars), and 24 h later cells were infected with ΔVpu (left panel) or WT (right panel) virus and cell-cell spread measured by real-time PCR as described above. Data are from three independent experiments and show mean relative percent cell-cell spread with the SEM. (C) Quantification of VS formation after siRNA knockdown. Jurkat-pNL4.3 WT cells and Jurkat-ΔVpu cells were nucleofected with siRNA and infected with virus as described above, and after 72 h cells were mixed with target T cells and quantified for VS (left panel) and conjugate (right panel) formation as described for Fig. 2. Data are means from three independent experiments with the SEMs. Jurkat-pNL4.3 WT control siRNA, n = 129; Jurkat-pNL4.3 WT BST-2 siRNA, n = 108; Jurkat-ΔVpu control siRNA, n = 161; Jurkat-ΔVpu BST-2 siRNA, n = 251. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001. (D) Visualization of the VS after tetherin siRNA treatment. Jurkat T cells were treated with anti-tetherin siRNA and infected with ΔVpu virus, and VS formation was examined by IF and LSCM. CD4 is green, surface tetherin is red, and Env is blue. Target cells are indicated with an asterisk. (E) Flow cytometry of surface tetherin expression on infected Jurkat T cells. Jurkat T cells were infected with pNL4.3 WT or ΔVpu, surface stained for tetherin and intracellular Gag, and analyzed by flow cytometry. Data from a representative experiment are shown.
FIG. 6.
Cell-cell spread of HIV-1 from infected primary CD4+ T cells. (A) Primary CD4+ T cells infected with pNL4.3 WT (white bars) or ΔVpu (black bars) were either left untreated or pretreated with 500 U/ml interferon for 24 h before being mixed with uninfected target T cells and incubated for 3, 6, and 12 h prior to real-time PCR analysis of cell-cell spread as described for Fig. 4. Data are from at least three independent experiments performed with three different donors, and error bars show the SEMs. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001. (B) Flow cytometry of tetherin knockdown. Primary CD4+ T cells were nucleofected with control siRNA (gray line) or siRNA targeting tetherin (black line), and 48 h later surface tetherin expression was measured by flow cytometry. The gray-filled histogram shows nucleofected cells stained with the secondary antibody only. Data from a representative experiment are shown. (C) siRNA knockdown of tetherin reduces cell-cell spread from infected primary CD4+ T cells. Primary T cells were nucleofected with siRNA targeting tetherin/Bst-2 (black bars) or control siRNA (white bars), and 24 h later cells were infected with ΔVpu HIV-1 and cell-cell spread measured by real-time PCR as described above. Data are from two independent donors and show the mean relative percentage cell-cell spread with the SEM. (D) Tetherin is enriched on primary CD4+ T cells at the VS. Primary T cells infected with ΔVpu virus were mixed with uninfected target T cells and incubated for an hour at 37°C in the presence of MAbs against Env (blue), CD4 (green), and tetherin (red). Results from a representative image from experiments performed with two different donors are shown.
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