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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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Regions of Vpr that are involved in the ability of Vpr to induce arrest in the cell cycle at the G2 phase are also important in NKG2D ligand expression.Primary T-cell blasts infected with wild type HIV-1 and HIV-1 with various mutations in Vpr 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. Uninfected CD4pos T-cells were surface stained in a similar fashion. All cells were stained intracellularly for HIV-1 p24 Ag. The mean fluorescent intensity of NKG2D ligand staining was obtained from collection of 104 CD4neg p24pos cells for all infected cells, and of the 104 CD4pos p24neg cells for the uninfected control. This figure is a representative of three separate experiments.
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ppat-1000613-g006: Regions of Vpr that are involved in the ability of Vpr to induce arrest in the cell cycle at the G2 phase are also important in NKG2D ligand expression.Primary T-cell blasts infected with wild type HIV-1 and HIV-1 with various mutations in Vpr 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. Uninfected CD4pos T-cells were surface stained in a similar fashion. All cells were stained intracellularly for HIV-1 p24 Ag. The mean fluorescent intensity of NKG2D ligand staining was obtained from collection of 104 CD4neg p24pos cells for all infected cells, and of the 104 CD4pos p24neg cells for the uninfected control. This figure is a representative of three separate experiments.

Mentions: We first tested whether domains in Vpr that are involved in recruiting or activating the Cullin-4a-based E3 ubiquitin ligase were required for induction of ULBP-1 and ULBP-2. The Vpr Q65R and Vpr R80A mutations have been previously shown to abate the ability of Vpr to induce cell cycle arrest [39],[40]. Vpr Q65R is unable to bind to DCAF1; the exact defect induced by R80A mutation is unknown, and it has been proposed that R80A abates the interaction between Vpr and the ubiquitination target for the E3/Vpr complex [39],[40]. For this purpose, we generated DHIV mutants with the above substitutions in Vpr. As shown in Figure 6, expression of NKG2D ligands was diminished by either substitution, although Vpr(R80A) had a more dramatic effect than Vpr(Q65R) (18 percent of wild-type Vpr and 48 percent of wild-type Vpr, respectively). The cell cycle profiles were examined in infected cells to visualize functional deficits in these mutants. When Vpr R80A or Vpr Q65R, were expressed neither mutant virus could induce G2 arrest as effectively as WT virus (Figure S3).


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)

Regions of Vpr that are involved in the ability of Vpr to induce arrest in the cell cycle at the G2 phase are also important in NKG2D ligand expression.Primary T-cell blasts infected with wild type HIV-1 and HIV-1 with various mutations in Vpr 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. Uninfected CD4pos T-cells were surface stained in a similar fashion. All cells were stained intracellularly for HIV-1 p24 Ag. The mean fluorescent intensity of NKG2D ligand staining was obtained from collection of 104 CD4neg p24pos cells for all infected cells, and of the 104 CD4pos p24neg cells for the uninfected control. This figure is a representative of three separate experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747015&req=5

ppat-1000613-g006: Regions of Vpr that are involved in the ability of Vpr to induce arrest in the cell cycle at the G2 phase are also important in NKG2D ligand expression.Primary T-cell blasts infected with wild type HIV-1 and HIV-1 with various mutations in Vpr 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. Uninfected CD4pos T-cells were surface stained in a similar fashion. All cells were stained intracellularly for HIV-1 p24 Ag. The mean fluorescent intensity of NKG2D ligand staining was obtained from collection of 104 CD4neg p24pos cells for all infected cells, and of the 104 CD4pos p24neg cells for the uninfected control. This figure is a representative of three separate experiments.
Mentions: We first tested whether domains in Vpr that are involved in recruiting or activating the Cullin-4a-based E3 ubiquitin ligase were required for induction of ULBP-1 and ULBP-2. The Vpr Q65R and Vpr R80A mutations have been previously shown to abate the ability of Vpr to induce cell cycle arrest [39],[40]. Vpr Q65R is unable to bind to DCAF1; the exact defect induced by R80A mutation is unknown, and it has been proposed that R80A abates the interaction between Vpr and the ubiquitination target for the E3/Vpr complex [39],[40]. For this purpose, we generated DHIV mutants with the above substitutions in Vpr. As shown in Figure 6, expression of NKG2D ligands was diminished by either substitution, although Vpr(R80A) had a more dramatic effect than Vpr(Q65R) (18 percent of wild-type Vpr and 48 percent of wild-type Vpr, respectively). The cell cycle profiles were examined in infected cells to visualize functional deficits in these mutants. When Vpr R80A or Vpr Q65R, were expressed neither mutant virus could induce G2 arrest as effectively as WT virus (Figure S3).

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