Figure 7
Figure 7. Retargeting of Lck is critically involved in Nef-induced alterations of TCR signaling and HIV-1 replication. (A) Microscopic analysis of p-Tyr (red) distribution in Jurkat T lymphocytes expressing the indicated GFP or Nef.GFP proteins in conjugates with SEE-pulsed Raji B cells (blue) Scale bar indicates 10 μm. (B) Frequencies of cells that displayed intracellular accumulation or IS recruitment of p-Tyr. Values are the arithmetic means of at least 3 independent experiments ± SD in which more than 100 cells were counted per condition. (C) Enhanced IL-2 production by HIV-1 Nef is reversed by Unc119. Jurkat T lymphocytes were transfected with an expression plasmid for RFP or Unc119.RFP, followed by infection with WT or ΔNef HIV-1 IRES.GFP or the mock control. Cells were stimulated with SEE-pulsed Raji B cells and intracellular IL-2 was measured in infected cells positive for RFP/Unc119.RFP. CytoD refers to uninfected cells treated with the actin-disrupting drug cytochalasin D before stimulation (0.625μM for 1 hour). Depicted is the mean fold increase of IL-2 levels relative to HIV-1 ΔNef–infected cells ± SD from 3 independent experiments. (D) Representative micrographs of uninfected (mock) primary human T lymphocytes or after infection with the indicated HIV-1 IRES.GFP reporter viruses in conjugates with SEE-pulsed Raji B cells after staining for p-Erk1/2. Arrows indicate infected cells in B-cell/T-cell conjugates. Note the low level of p Erk1/2 in the HIV-1 WT IRES.Unc119.GFP-infected cell in conjugate with a B cell compared with the WT-infected cell. Scale bar indicates 10 μm. (E) Frequencies of primary human T lymphocytes with elevated p-Erk1/2 levels. (F) Total per-cell levels of p-Erk1/2 in primary human T lymphocytes infected with the indicated viruses. Values for individual cells are presented, with the arithmetic mean indicated by the black line relative to uninfected control cells in conjugates. (G) The positive effect of Nef on HIV-1 replication is antagonized by Unc119 coexpression. Replication kinetics of the indicated HIV-1 viruses in PBMC/Raji B-cell cocultures are shown. Results shown are arithmetic means ± SD from triplicate infections of p24 concentrations in the cell culture supernatants harvested at the indicated time points. (H) Depicted are the mean p24 concentrations at 8 days after infection from the experiment shown in panel G, with the SDs indicated. Statistical significance was analyzed by the Student t test. ***P ≤ .001, **P < .005, and *P < .05.

Retargeting of Lck is critically involved in Nef-induced alterations of TCR signaling and HIV-1 replication. (A) Microscopic analysis of p-Tyr (red) distribution in Jurkat T lymphocytes expressing the indicated GFP or Nef.GFP proteins in conjugates with SEE-pulsed Raji B cells (blue) Scale bar indicates 10 μm. (B) Frequencies of cells that displayed intracellular accumulation or IS recruitment of p-Tyr. Values are the arithmetic means of at least 3 independent experiments ± SD in which more than 100 cells were counted per condition. (C) Enhanced IL-2 production by HIV-1 Nef is reversed by Unc119. Jurkat T lymphocytes were transfected with an expression plasmid for RFP or Unc119.RFP, followed by infection with WT or ΔNef HIV-1 IRES.GFP or the mock control. Cells were stimulated with SEE-pulsed Raji B cells and intracellular IL-2 was measured in infected cells positive for RFP/Unc119.RFP. CytoD refers to uninfected cells treated with the actin-disrupting drug cytochalasin D before stimulation (0.625μM for 1 hour). Depicted is the mean fold increase of IL-2 levels relative to HIV-1 ΔNef–infected cells ± SD from 3 independent experiments. (D) Representative micrographs of uninfected (mock) primary human T lymphocytes or after infection with the indicated HIV-1 IRES.GFP reporter viruses in conjugates with SEE-pulsed Raji B cells after staining for p-Erk1/2. Arrows indicate infected cells in B-cell/T-cell conjugates. Note the low level of p Erk1/2 in the HIV-1 WT IRES.Unc119.GFP-infected cell in conjugate with a B cell compared with the WT-infected cell. Scale bar indicates 10 μm. (E) Frequencies of primary human T lymphocytes with elevated p-Erk1/2 levels. (F) Total per-cell levels of p-Erk1/2 in primary human T lymphocytes infected with the indicated viruses. Values for individual cells are presented, with the arithmetic mean indicated by the black line relative to uninfected control cells in conjugates. (G) The positive effect of Nef on HIV-1 replication is antagonized by Unc119 coexpression. Replication kinetics of the indicated HIV-1 viruses in PBMC/Raji B-cell cocultures are shown. Results shown are arithmetic means ± SD from triplicate infections of p24 concentrations in the cell culture supernatants harvested at the indicated time points. (H) Depicted are the mean p24 concentrations at 8 days after infection from the experiment shown in panel G, with the SDs indicated. Statistical significance was analyzed by the Student t test. ***P ≤ .001, **P < .005, and *P < .05.

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