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HIV-1 VPR causes G2 arrest and apoptosis via DNA replication stress

HIV-1 VPR causes G2 arrest and apoptosis via DNA replication stress. Erik Zimmerman Vicente Planelles Lab. Vpr functions. G2 arrest enhances proviral transcription. and translation. PIC import. G2 arrest. Abbas, Cell. and Mol. Immunology, 2003. P. Active ATR complex. P. P. Chk1. P.

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HIV-1 VPR causes G2 arrest and apoptosis via DNA replication stress

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  1. HIV-1 VPR causes G2 arrest and apoptosis via DNA replication stress Erik Zimmerman Vicente Planelles Lab

  2. Vpr functions G2 arrest enhances proviral transcription and translation PIC import G2 arrest Abbas, Cell. and Mol. Immunology, 2003

  3. P Active ATR complex P P Chk1 P Cdc25C P Cdk1/cyclin B G2  M Checkpoint activation ATR mediates G2 arrest and apoptosis Is ATR activated in the context of HIV-1 infection? What is the nature of the ATR-activating stimulus? Is transition to G2 requisite for apoptosis? Rad9-Hus1-Rad1 Rad17 ATR ? Vpr P BRCA1 GADD45a Apoptosis

  4. ATR is required for HIVNL4-3 induced G2 arrest in primary CD4+ lymphocytes

  5. HIV-infected CD4+ T cells are G2 arrested in vivo • PBMC isolated from recent seroconverters prior to initiation of HAART • Stained for CD4, p24 Gag, and DNA content

  6. Conclusions #1 • ATR is essential for HIV-1 induced G2 arrest • HIV-1 infected cells are arrested in G2 in vivo

  7. What is the ATR-activating stimulus?

  8. A model for ATR Activation DNA polymerase complex RPA 17 RPA 32-P RPA 70 RPA (ssDBP) ATR + ATRIP Rad17 complex + 9-1-1 Can we detect RPA accumulation in HIV-infected cells? Adapted from Zou and Elledge. Science, 2003

  9. HIV-1 VPR induces RPA-rich nuclear foci in primary CD4+ cells Mock HU NL4-3 NL4-3 VprX DIC P24 Gag RPA32

  10. Conclusions #2 • VPR causes DNA replication stress, accumulation of RPA-rich foci in primary CD4+ lymphocytes • Activation of ATR by VPR may require DNA replication

  11. Can we prevent the cytopathic consequences of VPR by prohibiting DNA replication/entry into G2?

  12. Cycling into G2 is required for VPR-induced apoptosis Vpr + Thym Thym 2mM Thym 48 hrs 6 hr Time post-thymidine release 12hr 18 hr 24 hr What about post-mitotic HIV-1 target cells?

  13. Activated CD4+ T cells Dividing HIV-infection is highly cytopathic ~1.6 day half-life VPR causes G2 arrest, apoptosis Macrophages Nondividing Highly resistant to HIV-induced apoptosis half-life of several days VPR enhances PIC import Two different HIV target cells: Is the ATR pathway present in nondividing cells?

  14. ATR is expressed only in dividing cells

  15. Conclusions • VPR induces an ATR-dependent G2 arrest in primary CD4+ T cells • p24+ CD4+ lymphocytes from recent seroconverters are arrested in G2 in vivo • VPR causes accumulation of RPA-rich nuclear foci, indicative of DNA replication fork stalling • VPR cannot activate ATR or induce apoptosis in non-cycling cells (G1 blocked Hela or macrophages) • Could this help explain the resistance of macrophages to HIV-1 induced apoptosis? • Can we sensitize quiescent, nondividing reservoirs to the pro-apoptotic effects of VPR?

  16. Acknowledgements Collaborators: Michael Sherman Warner Greene (Gladstone Institute) NIAID NIH Genetics Training Grant Planelles Lab: Vicente Planelles Josh Andersen Orly Ardon Jana Blackett Jason DeHart Toroti Umuerri Daniel Andrade Alberto Bosque

  17. VPR expression causes accumulation of RPA-rich nuclear foci in transduced Hela cells Mock HU pHR-GFP pHR-VPR GFP RPA-32 Do these observations apply in vivo? -primary CD4+ lymphocytes -physiological levels of VPR -presence of other viral gene products

  18. ATR is required for VPR-induced G2 arrest Mock pHR-GFP pHR-VPR G2=90% G2=88% G2=47%

  19. Vpr fails to activate ATR in primary macrophages Mock IR pHR-GFP pHR-VPR DIC -H2AX GFP

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