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Éric A. Cohen Laboratory of Human Rétrovirology IRCM

HIV-1 Vpr-mediated ATR activation and G2 cell-cycle arrest induce up-regulation of NKG2D ligands and activate NK cell-mediated killing. Éric A. Cohen Laboratory of Human Rétrovirology IRCM 5th IAS Conference on HIV Pathogenesis, Treatment and Prevention, Cape Town July, 20, 2009.

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Éric A. Cohen Laboratory of Human Rétrovirology IRCM

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  1. HIV-1 Vpr-mediated ATR activation and G2 cell-cycle arrest induce up-regulation of NKG2D ligands and activate NK cell-mediated killing Éric A. Cohen Laboratory of Human Rétrovirology IRCM 5th IAS Conference on HIV Pathogenesis, Treatment and Prevention, Cape Town July, 20, 2009

  2. Viral Protein R (Vpr) • HIV accessory protein of 96 aa (14 kDa) • Exists in 3 different forms - Virion-associated (~275 molecules/virion) - Intracellular forms • Extracellular forms: Plasma and CSF • Displays cell transduction properties

  3. Vpr Biological Activities • Facilitates infection of nondividing cells such as terminally differentiated macrophages • Causes cell-cycle arrest at the G2 phase in CD4+ T cells via activation of the ATR-mediated DNA damage/stress signaling pathway • Conserved among HIV and SIV Vpr • Abnormal accumulation of infected cells in G2 in HIV- infected subjects

  4. How Does Vpr Induce a G2 Arrest? DNA stress/damage checkpoint G2 arrest ATR ATM P ? P Chk1 P Cdc2 Cdc2 Cyclin B Cyclin B ACTIVE INACTIVE DNA repair G2 M G2 ARREST Belzile et al., PLoS Pathogens, 2007

  5. Functional Relevance of Vpr-induced G2 Arrest Vpr-induced G2 arrest has two downstream effects: Enhances proviral transcription. Promotes apoptosis of infected cells Other Effects? Activation of the DNA damage/stress checkpoint pathway initiated by ATR or ATM kinase upregulates ligands of the activating NKG2D receptor and enhances susceptibility of target cells to NK cell killing (Gasser et al., 2005) NKG2D ATR

  6. Control of NK Cell Effector Function under control of a sophisticated and tightly regulated network of activating and inhibitory receptors NKG2D is a central activating receptor that modulates NK cell function MHC class 1-related chains A/B (MIC A/B) UL16-binding protein (ULBP-1, -2, -3, -4) NKG2D is expressed not only on NK cells, but also on gd T cells, CD8+T- cells, and a subset of CD4+ T-cells Expression of NKG2D ligands is largely confined to virus-infected, tumor and stressed cells.

  7. RATIONALE HIV-1 infection of primary CD4+ T-lymphocytes increases cell-surface expression of specific NKG2D ligands both in vitro(Cerboni et al. , 2007; Ward et al. 2007) and in vivo(Fogli et al., 2008) GOAL To investigate whether expression of Vpr could regulate cell-surface expression of NKG2D ligands and modulate NK cell cytotoxic activity

  8. HIV-1 upregulates cell-surface expression of ULBP-2 in primary CD4+ T-cells in a Vpr-dependent manner 4-5 days Cell-surface staining anti-NKG2D ligands on GFP+ cells Infection MOI= 0.5 (GFP-marked R5 virus) WT/ΔVpr- PBMCs of healthy subjects Primary CD4+ T-lymphocytes activated with PHA/IL2 Mock: 2.83 Mock: 23.91 Mock: 7.27 HIVΔVpr: 3.23 HIVΔVpr: 23.84 HIVΔVpr: 7.89 HIV WT: 2.96 HIV WT: 40.65 HIV WT: 7.38 N=5 Relative cell number ULBP-1 ULBP-2 ULBP-3 No upregulation of MICA and MICB cell-surface expression

  9. Vpr enhances susceptibility of R5 HIV-1-infected-CD4+ T-lymphocytes to NK cell-mediated killing Donor 1 Donor 2 N=3 Infected cells (GFP+) were exposed to autologous non-activated primary NK cells in a 4h-51Cr release assay • Vpr promotes NK cell-mediated killing at least in part via NKG2D

  10. Expression of Vpr alone is sufficient to upregulate ULBP2 cell-surface expression Lentiviral vectors expressing GFP WPI-Vpr WT 126.9 WPI 45.4 WPI Cell relative number WPI-Vpr WT ULBP2 Primary CD4+ T-lymphocytes N=2

  11. Vpr, in the absence of any other HIV proteins, upregulates NKG2D ligands non-selectively WPI-Vpr WT311.6 WPI-Vpr WT315.8 WPI-Vpr WT28.6 WPI-Vpr WT97.8 WPI-Vpr WT58.2 WPI 174.9 WPI 82.8 WPI 9.0 WPI 54.5 WPI 25.9 Relative cell number APC 354.9 APC 427.1 APC 20.3 APC 95.1 APC 41.6 Mock 125.2 Mock 91.7 Mock 8.7 Mock 52.1 Mock 29.4 MICA MICB ULBP-1 ULBP-2 ULBP-3 Lentiviral vector-transduced (GFP+) CEM.NKR T cells • suggest that perhaps other viral protein(s) may limit cell-surface expression of some ligands

  12. Q65R R80A Upregulation of NKG2D ligands is dependent on Vpr-mediated G2-checkpoint arrest A B WPI : 118.7 WPI-Vpr R80A : 126.2 Relative cell number WPI-Vpr Q65R : 200.1 WPI-Vpr WT : 358.1 ULBP-2 CEM.NKR T cells

  13. A B Not treated + Caffeine WPI-Vpr WT102.5 WPI-Vpr WT31.7 WPI 35.1 WPI 21.1 Relative cell number APC 327.4 APC 48.4 Mock 45.0 Mock 24.8 ULBP-2 Vpr-mediated upregulation of NKG2D ligands is inhibited by Caffeine CEM.NKR T cells

  14. Upregulation of NKG2D ligands by Vpr triggers NK cell cytotoxic responses A B Transduced cells populations were exposed to non-activated primary NK cells in a 4h-51Cr release assay CEM.NKR T cells N=2

  15. Delivery of virion-associated Vpr via defective HIV-1 particles upregulates ULBP-2 in non-infected target cells A B C Indinavir-treated HIVΔVpr HIVΔVprΔRTΔIN + Vpr WT 46.0 + Vpr WT 35.5 + Vpr Q65R 14.8 + Vpr Q65R 17.8 D Indinavir-treated HIVΔVpr Donor 1 Donor 2 N=3

  16. Conclusions HIV upregulates selectively cell-surface expression of ULBP-2 in primary CD4+ T-cells in a Vpr-dependent manner Vpr alone is sufficient to upregulate expression of NKG2D ligands, including ULBP-1, -2, -3, MICA and MICB, suggesting that HIV replication and expression of other virus proteins are not required for this activity of Vpr The absence of selective NKG2D ligand upregulation when Vpr is expressed alone suggests that another viral factor limits cell-surface expression of some ligands during HIV-1 infection Vpr upregulates expression of NKG2D ligands by a process that relies on the recruitment of the DDB1-CUL4A (DCAF1) E3 ligase and on activation of the ATR/ATM-mediated DNA damage/stress checkpoint pathway • Vpr is a key viral determinant responsible for induction of NKG2D ligands during HIV-1 infection

  17. Conclusions Vpr expressed alone or during HIV-1 infection promotes NKG2D-dependent NK cell cytotoxic responses via its ability to upregulate NKG2D ligands Delivery of virion-associated Vpr via HIV-1 defective particles induces analogous effects in non-infected target cells, suggesting that Vpr may act similarly beyond infected cells Overall, this study suggests an immunomodulatory role for Vpr that may not only contribute to HIV-1-induced CD4+ T lymphocyte depletion but may also play a part in HIV-1-induced NK cell dysfunction through sustained NKG2D receptor activation

  18. RESEARCH TEAM IN HIV PATHOGENESIS (HPAT) Acknowledgements Reagents Laboratory of Human Retrovirology • Dr. D. Trono (EPFL, Lausanne) • Dr. H. Göttlinger (U. of Mass.) • Dr. Y. Ishizaka (Tokyo) S. Sindhu J. Richard JP Belzile • Flow Cytometry • Eric Massicote (IRCM) • Martine Dupuis (IRCM) IRCM Clinic • Dr P. Larochelle • Mrs M. Gauthier All the volunteers

  19. Expression of Vpr WT and Vpr mutants (R80A and Q65R) in CEM.NKR T cells WPI : 37.9 WPI-Vpr R80A : 113.0 Relative cell number WPI-Vpr Q65R : 145.5 WPI-Vpr WT : 111.4 Vpr

  20. GFP - GFP + WPI-Vpr WT80.6 WPI-Vpr WT49.5 WPI 31.2 WPI 28.1 Relative cell number ULBP-2

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