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HIV-1 Vpr triggers natural killer cell-mediated lysis of infected cells through activation of the ATR-mediated DNA damage response.

Ward J, Davis Z, DeHart J, Zimmerman E, Bosque A, Brunetta E, Mavilio D, Planelles V, Barker E - PLoS Pathog. (2009)

Bottom Line: The presence of ULBP-1 and ULBP-2 on HIV-1 infected cells is dependent on the ability of Vpr to associate with a protein complex know as Cullin 4a (Cul4a)/damaged DNA binding protein 1 (DDB1) and Cul4a-associated factor-1(DCAF-1) E3 ubiquitin ligase (Cul4a(DCAF-1)).When T-cell blasts are infected with a vpr-deficient HIV-1, NK cells are impaired in killing the infected cells.Thus, HIV-1 Vpr actively triggers the expression of the ligands to the NK cell activation receptor.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, State University of New York, Upstate Medical University, Syracuse, New York, United States of America.

ABSTRACT
Natural killer (NK) cells are stimulated by ligands on virus-infected cells. We have recently demonstrated that NK cells respond to human immunodeficiency virus type-1 (HIV-1)-infected autologous T-cells, in part, through the recognition of ligands for the NK cell activating receptor NKG2D on the surface of the infected cells. Uninfected primary CD4(pos) T-cell blasts express little, if any, NKG2D ligands. In the present study we determined the mechanism through which ligands for NKG2D are induced on HIV-1-infected cells. Our studies reveal that expression of vpr is necessary and sufficient to elicit the expression of NKG2D ligands in the context of HIV-1 infection. Vpr specifically induces surface expression of the unique-long 16 binding proteins (ULBP)-1 and ULBP-2, but not ULBP-3, MHC class I-related chain molecules (MIC)-A or MIC-B. In these studies we also demonstrated that Vpr increases the level of ULBP-1 and ULBP-2 mRNA in primary CD4(pos) T-cell blasts. The presence of ULBP-1 and ULBP-2 on HIV-1 infected cells is dependent on the ability of Vpr to associate with a protein complex know as Cullin 4a (Cul4a)/damaged DNA binding protein 1 (DDB1) and Cul4a-associated factor-1(DCAF-1) E3 ubiquitin ligase (Cul4a(DCAF-1)). ULBP-1 and -2 expression by Vpr is also dependent on activation of the DNA damage sensor, ataxia telangiectasia and rad-3-related kinase (ATR). When T-cell blasts are infected with a vpr-deficient HIV-1, NK cells are impaired in killing the infected cells. Thus, HIV-1 Vpr actively triggers the expression of the ligands to the NK cell activation receptor.

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Expression of NKG2D ligands on CD4pos T-cells following infection with viruses unable to express Vpr, Vif, Vpu or Nef.Infected primary T-cell blasts [(A) Wild-type, (B) ΔVpr, (C) ΔVif, (D) ΔVpu and (E) ΔNef] and uninfected CD4pos T-cells were surface stained with fluorochrome-conjugated anti-CD4 Ab and a fusion protein of human NKG2D and the Fc portion of human IgG1 along with fluorochrome-conjugated goat anti-human IgG1. All cells were stained intracellularly for HIV-1 p24 Ag. Histograms were derived following acquisition on a flow cytometer of either 104 viable CD4pos cells [(for uninfected cells) blue line] or 104 viable CD4neg and HIV-1 p24 Agpos infected cells (red line). The figure is representative data from three separate experiments.
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ppat-1000613-g002: Expression of NKG2D ligands on CD4pos T-cells following infection with viruses unable to express Vpr, Vif, Vpu or Nef.Infected primary T-cell blasts [(A) Wild-type, (B) ΔVpr, (C) ΔVif, (D) ΔVpu and (E) ΔNef] and uninfected CD4pos T-cells were surface stained with fluorochrome-conjugated anti-CD4 Ab and a fusion protein of human NKG2D and the Fc portion of human IgG1 along with fluorochrome-conjugated goat anti-human IgG1. All cells were stained intracellularly for HIV-1 p24 Ag. Histograms were derived following acquisition on a flow cytometer of either 104 viable CD4pos cells [(for uninfected cells) blue line] or 104 viable CD4neg and HIV-1 p24 Agpos infected cells (red line). The figure is representative data from three separate experiments.

Mentions: We then sought to determine whether Vpr was responsible for the expression of NKG2D ligands. We generated DHIV containing a truncation in Vpr. As controls, mutants of DHIV unable to express Vif, Vpu or Nef were also generated. Figures 2 and S2 illustrate that the DHIV-ΔVpr infected cells failed to induce NKG2D ligand expression (Figure 2B; MFI = 188 for HIV-1 p24 Agpos/CD4neg infected cells and MFI = 216 for HIV-1 p24 Agneg/CD4pos infected cells). The MFI for DHIV-WT infected cells was statistically different (p<0.01) compared to the MFI for DHIV-ΔVpr infected cells. We compared expression of NKG2D ligands on CD4pos T-cells infected with DHIV-ΔVif (Figure 2C; MFI = 600 for HIV-1 p24 Agpos/CD4neg infected cells and MFI = 260 for HIV-1 p24 Agneg/CD4pos infected cells), DHIV-ΔVpu (Figure 2D; MFI = 777 for HIV-1 p24 Agpos/CD4neg infected cells and MFI = 247 for HIV-1 p24 Agneg/CD4pos infected cells), and DHIV-ΔNef (Figure 2E; MFI = 698 for HIV-1 p24 Agpos/CD4neg infected cells and MFI = 303 for HIV-1 p24 Agneg/CD4pos infected cells). Therefore, expression of NKG2D ligands induced by ΔVif, ΔVpu and ΔNef DHIV was comparable that induced by WT DHIV (Figure 2A). Thus, we conclude that Vpr is required for HIV-1-mediated up-regulation of NKG2D ligands on the surface of infected cells.


HIV-1 Vpr triggers natural killer cell-mediated lysis of infected cells through activation of the ATR-mediated DNA damage response.

Ward J, Davis Z, DeHart J, Zimmerman E, Bosque A, Brunetta E, Mavilio D, Planelles V, Barker E - PLoS Pathog. (2009)

Expression of NKG2D ligands on CD4pos T-cells following infection with viruses unable to express Vpr, Vif, Vpu or Nef.Infected primary T-cell blasts [(A) Wild-type, (B) ΔVpr, (C) ΔVif, (D) ΔVpu and (E) ΔNef] and uninfected CD4pos T-cells were surface stained with fluorochrome-conjugated anti-CD4 Ab and a fusion protein of human NKG2D and the Fc portion of human IgG1 along with fluorochrome-conjugated goat anti-human IgG1. All cells were stained intracellularly for HIV-1 p24 Ag. Histograms were derived following acquisition on a flow cytometer of either 104 viable CD4pos cells [(for uninfected cells) blue line] or 104 viable CD4neg and HIV-1 p24 Agpos infected cells (red line). The figure is representative data from three separate experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2747015&req=5

ppat-1000613-g002: Expression of NKG2D ligands on CD4pos T-cells following infection with viruses unable to express Vpr, Vif, Vpu or Nef.Infected primary T-cell blasts [(A) Wild-type, (B) ΔVpr, (C) ΔVif, (D) ΔVpu and (E) ΔNef] and uninfected CD4pos T-cells were surface stained with fluorochrome-conjugated anti-CD4 Ab and a fusion protein of human NKG2D and the Fc portion of human IgG1 along with fluorochrome-conjugated goat anti-human IgG1. All cells were stained intracellularly for HIV-1 p24 Ag. Histograms were derived following acquisition on a flow cytometer of either 104 viable CD4pos cells [(for uninfected cells) blue line] or 104 viable CD4neg and HIV-1 p24 Agpos infected cells (red line). The figure is representative data from three separate experiments.
Mentions: We then sought to determine whether Vpr was responsible for the expression of NKG2D ligands. We generated DHIV containing a truncation in Vpr. As controls, mutants of DHIV unable to express Vif, Vpu or Nef were also generated. Figures 2 and S2 illustrate that the DHIV-ΔVpr infected cells failed to induce NKG2D ligand expression (Figure 2B; MFI = 188 for HIV-1 p24 Agpos/CD4neg infected cells and MFI = 216 for HIV-1 p24 Agneg/CD4pos infected cells). The MFI for DHIV-WT infected cells was statistically different (p<0.01) compared to the MFI for DHIV-ΔVpr infected cells. We compared expression of NKG2D ligands on CD4pos T-cells infected with DHIV-ΔVif (Figure 2C; MFI = 600 for HIV-1 p24 Agpos/CD4neg infected cells and MFI = 260 for HIV-1 p24 Agneg/CD4pos infected cells), DHIV-ΔVpu (Figure 2D; MFI = 777 for HIV-1 p24 Agpos/CD4neg infected cells and MFI = 247 for HIV-1 p24 Agneg/CD4pos infected cells), and DHIV-ΔNef (Figure 2E; MFI = 698 for HIV-1 p24 Agpos/CD4neg infected cells and MFI = 303 for HIV-1 p24 Agneg/CD4pos infected cells). Therefore, expression of NKG2D ligands induced by ΔVif, ΔVpu and ΔNef DHIV was comparable that induced by WT DHIV (Figure 2A). Thus, we conclude that Vpr is required for HIV-1-mediated up-regulation of NKG2D ligands on the surface of infected cells.

Bottom Line: The presence of ULBP-1 and ULBP-2 on HIV-1 infected cells is dependent on the ability of Vpr to associate with a protein complex know as Cullin 4a (Cul4a)/damaged DNA binding protein 1 (DDB1) and Cul4a-associated factor-1(DCAF-1) E3 ubiquitin ligase (Cul4a(DCAF-1)).When T-cell blasts are infected with a vpr-deficient HIV-1, NK cells are impaired in killing the infected cells.Thus, HIV-1 Vpr actively triggers the expression of the ligands to the NK cell activation receptor.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, State University of New York, Upstate Medical University, Syracuse, New York, United States of America.

ABSTRACT
Natural killer (NK) cells are stimulated by ligands on virus-infected cells. We have recently demonstrated that NK cells respond to human immunodeficiency virus type-1 (HIV-1)-infected autologous T-cells, in part, through the recognition of ligands for the NK cell activating receptor NKG2D on the surface of the infected cells. Uninfected primary CD4(pos) T-cell blasts express little, if any, NKG2D ligands. In the present study we determined the mechanism through which ligands for NKG2D are induced on HIV-1-infected cells. Our studies reveal that expression of vpr is necessary and sufficient to elicit the expression of NKG2D ligands in the context of HIV-1 infection. Vpr specifically induces surface expression of the unique-long 16 binding proteins (ULBP)-1 and ULBP-2, but not ULBP-3, MHC class I-related chain molecules (MIC)-A or MIC-B. In these studies we also demonstrated that Vpr increases the level of ULBP-1 and ULBP-2 mRNA in primary CD4(pos) T-cell blasts. The presence of ULBP-1 and ULBP-2 on HIV-1 infected cells is dependent on the ability of Vpr to associate with a protein complex know as Cullin 4a (Cul4a)/damaged DNA binding protein 1 (DDB1) and Cul4a-associated factor-1(DCAF-1) E3 ubiquitin ligase (Cul4a(DCAF-1)). ULBP-1 and -2 expression by Vpr is also dependent on activation of the DNA damage sensor, ataxia telangiectasia and rad-3-related kinase (ATR). When T-cell blasts are infected with a vpr-deficient HIV-1, NK cells are impaired in killing the infected cells. Thus, HIV-1 Vpr actively triggers the expression of the ligands to the NK cell activation receptor.

Show MeSH
Related in: MedlinePlus