HIV-1 Vpr up-regulates expression of ligands for the activating NKG2D receptor and promotes NK cell-mediated killing

Abstract

HIV up-regulates cell-surface expression of specific ligands for the activating NKG2D receptor, including ULBP-1, -2, and -3, but not MICA or MICB, in infected cells both in vitro and in vivo. However, the viral factor(s) involved in NKG2D ligand expression still remains undefined. HIV-1 Vpr activates the DNA damage/stress-sensing ATR kinase and promotes G(2) cell-cycle arrest, conditions known to up-regulate NKG2D ligands. We report here that HIV-1 selectively induces cell-surface expression of ULBP-2 in primary CD4(+) T lymphocytes by a process that is Vpr dependent. Importantly, Vpr enhanced the susceptibility of HIV-1-infected cells to NK cell-mediated killing. Strikingly, Vpr alone was sufficient to up-regulate expression of all NKG2D ligands and thus promoted efficient NKG2D-dependent NK cell-mediated killing. Delivery of virion-associated Vpr via defective HIV-1 particles induced analogous biologic effects in noninfected target cells, suggesting that Vpr may act similarly beyond infected cells. All these activities relied on Vpr ability to activate the ATR-mediated DNA damage/stress checkpoint. Overall, these results indicate that Vpr is a key determinant responsible for HIV-1-induced up-regulation of NKG2D ligands and further suggest an immunomodulatory role for Vpr that may not only contribute to HIV-1-induced CD4(+) T-lymphocyte depletion but may also take part in HIV-1-induced NK-cell dysfunction.

Figure 1. CD4⁺ T lymphocytes infected with HIV-1 express ULBP-2 in a Vpr-dependent manner

Human primary CD4⁺ T lymphocytes were mock-infected or infected with infectious CCR5-tropic HxBru.ADA.GFP or HxBru(Vpr−)ADA.GFP at an MOI of 0.5. After 5 days, mock-infected or GFP-expressing infected CD4⁺ T lymphocytes were monitored for expression of NKG2D ligands by flow cytometry using specific mAbs directed against ULBP-1, -2, and -3 and appropriate fluorochrome-conjugated secondary reagents. The histogram with the dashed line represents cells stained with the isotype control Abs; the filled histogram represents mock-infected cells, and the histograms with the bold and dotted lines represent, respectively, Vpr⁺ (HIV WT) and Vpr-defective (HIVΔVpr) HIV-infected cells, as indicated. MFI values were calculated by subtracting the corresponding isotype control values. Results shown are representative of the data obtained from 5 different donors.

Figure 2. HIV-1 Vpr enhances the killing of HIV-1–infected CD4⁺ T lymphocytes by autologous NK cells

Human primary CD4⁺ T lymphocytes were mock-infected or infected with infectious CCR5-tropic HxBru.ADA.GFP or HxBru(Vpr−)ADA.GFP at an MOI of 0.5. After 5 days, mock-infected or GFP-expressing infected primary CD4⁺ T lymphocytes were sorted and subsequently exposed to autologous primary NK cells in a 4-hour ⁵¹Cr release assay in the absence (A) or presence (B) of interfering Abs to NKG2D (α-NKG2D) or matched-IgG control Abs (IgG), as indicated. Error bars represent SEM. Results shown are representative of the data obtained with 3 different donors.

Figure 3. Up-regulation of cell-surface NKG2D ligands in cells expressing HIV-1 Vpr

(A) Human primary CD4⁺ T lymphocytes were transduced with lentiviral vectors expressing GFP alone (WPI) or coexpressing GFP and VprWT (WPI-VprWT). GFP-expressing cells were monitored for ULBP-2 cell-surface expression by flow cytometry 48 hours after transduction using specific mAbs directed against ULBP-2 and appropriate fluorochrome-conjugated secondary reagents. (B) CEM.NKR T cells were transduced with WPI-VprWT or WPI lentiviral vectors or treated with APC (4μM) as indicated. Cell-surface expression of NKG2D ligands was monitored on the GFP-expressing cells 48 hours after transduction or after a 24-hour treatment with APC, using specific mAbs directed against ULBP-1, -2, and -3, MICA, MICB, and appropriate fluorochrome-conjugated secondary reagents. MFI values were calculated by subtracting the corresponding isotype control values (dashed line). Results shown are representative of the data obtained from at least 2 independent experiments.

Figure 4. Augmentation of NKG2D ligand mRNA expression in CEM.NKR T cells expressing HIV-1 Vpr

CEM.NKR T cells were transduced with lentiviral vectors expressing GFP alone (WPI) or coexpressing GFP and VprWT (WPI-VprWT). GFP⁺ cells were sorted for analysis 48 hours after transduction. Alternatively, transduced cells were treated (or not) with 4μM APC for 24 hours. DNase-treated RNA was analyzed for NKG2D ligand expression by real-time reverse-transcriptase polymerase chain reaction. Target gene expression in Vpr-transduced and APC-treated CEM.NKR T cells was normalized for glyceraldehyde 3-phosphate dehydrogenase and hypoxanthine-guanine phosphoribosyltransferase (HPRT) expression, and the data were subsequently expressed as a fold increase relative to WPI-transduced or untreated cells, respectively. Results shown represent a mean fold increase. Error bars represent SEM. Results are representative of the data obtained from 4 independent experiments.

Figure 5. Vpr-mediated up-regulation of ULBP2 requires the recruitment of the DDB1-CUL4A (VprBP) E3 ligase complex and activation of the DNA damage/stress checkpoint arrest initiated by ATR

(A) CEM.NKR T cells were transduced with lentiviral vectors expressing GFP alone (WPI) or coexpressing GFP and VprWT (WPI-VprWT) or Vpr mutants (WPI-VprR80A or WPI-VprQ65R), as indicated. At 48 hours after transduction, GFP-expressing cells were monitored for ULBP-2 cell-surface expression by flow cytometry using specific mAbs directed against ULBP-2 and appropriate fluorochrome-conjugated secondary reagents (top panel). Expression of VprWT and Vpr mutants (VprR80A and VprQ65R) was evaluated by intracellular staining and flow cytometry using anti-Vpr mAbs and appropriate fluorochrome-conjugated secondary reagents (bottom panel). (B-C) CEM.NKR T cells were transduced with lentiviral vectors WPI or WPI-VprWT or treated with APC (4μM), in the presence or absence of caffeine (2.5mM) (B) and in the presence of DMSO or KU55933 (10μM) (C) as indicated. Cell-surface expression of ULBP-2 was monitored on GFP-expressing cells 48 hours after transduction or on the total cell population after a 24-hour treatment with APC. MFI values were calculated by subtracting the corresponding isotype control values (dashed line). Results shown are representative of the data obtained from at least 2 independent experiments. (D) HeLa cells were irradiated with γ rays (10 Gy from a Cs¹³⁷ source) in the presence of DMSO or KU55933 (10μM). Cells were then lysed and sonicated 1 hour after irradiation, and phosphorylation of Chk2 and H2AX was monitored by Western blotting using specific Abs.

Figure 6. Vpr-mediated up-regulation of NKG2D ligands in target cells promotes NK cell–mediated killing

(A) CEM.NKR T cells were transduced with lentiviral vectors WPI or WPI-VprWT and then exposed to primary NK cells in a 4-hour ⁵¹Cr release assay 48 hours after transduction, in the presence of soluble NKG2D-IgG Fc fusion proteins or matched-IgG Fc fusion molecules, as indicated. (B) CEM.NKR T cells were transduced with lentiviral vectors WPI-VprWT or WPI-VprR80A and then assessed for cell lysis by primary NK cells in a ⁵¹Cr release assay 48 hours after transduction. (C) CEM.NKR T cells were treated or not with APC (4μM) and analyzed, as in panel A, 24 hours after treatment. Primary NK cells used in panels A and C were isolated from the same donor. Error bars represent SEM. (D) Cell-surface expression of ULBP-2 was monitored on GFP⁺ and GFP⁻ CEM.NKR T cells 48 hours after transduction with lentiviral vectors expressing GFP alone (WPI) or coexpressing GFP and VprWT (WPI-VprWT) as indicated. MFI values were calculated by subtracting the corresponding isotype control values (dashed line). Results shown are representative of the data obtained from at least 2 independent experiments.

Figure 7. Virion-associated Vpr up-regulates ULBP-2 expression in noninfected target cells and triggers NK cell–mediated killing

(A) Human primary CD4⁺T lymphocytes were exposed to indinavir-treated noninfectious viral particles that were trans-packaged with VprWT or the VprQ65R mutant, as indicated, and cell-surface expression of ULBP-2 was monitored 24 hours after exposure using specific mAbs directed against ULBP-2 and appropriate fluorochrome-conjugated secondary reagents. (B) HIV-1ΔVpr and HIV-1ΔVpr LF/PS viruses (P6-mutated Gag-encoding virus that does not incorporate Vpr) trans-packaged with HA-tagged VprWT or VprQ65R were produced as described in “Production of lentiviral vectors and HIV-1 viruses.” Virion-associated HA-tagged VprWT and VprQ65R were detected by Western blotting using anti-HA mAbs. (C) Primary CD4⁺ T lymphocytes were exposed to reverse transcriptase- and integrase-defective (HIVΔVprΔRTΔIN) viral particles that were trans-packaged with VprWT or the VprQ65R mutant as indicated, and cell-surface expression of ULBP-2 was monitored 24 hours after exposure. MFI values were calculated by subtracting the corresponding isotype-control values (dashed line). (D) Primary CD4⁺ T lymphocytes exposed to indinavir-treated noninfectious viral particles containing VprWT or VprQ65R were added 24 hours after exposure to autologous primary NK cells in a 4-hour ⁵¹Cr release assay, as indicated. Error bars represent SEM. Results shown are representative of the data obtained from 2 independent donors.