16th International AIDS Conference Toronto, Canada - August 13 - 18, 2006

HIV-1 VPR CAUSES G2 ARREST AND APOPTOSIS IN PRIMARY CD4+ T LYMPHOCYTES BY INDUCING DNA REPLICATION STRESS

Int Conf AIDS. 2006 Aug 13-18;16 Abstract No. TuAa0101

Zimmerman E., Andersen J., Blackett J., Planelles V.
University of Utah, Pathology, Salt Lake City, United States

BACKGROUND: HIV-1 VPR induces G2 arrest and apoptosis in infected CD4+ T lymphocytes, when expressed alone or during HIV-1 infection. We wished to investigate whether and how VPR induces the above deleterious effects in vivo. We show that G2 arrest and apoptosis are inseparable effects of VPR and stem from activation of the cellular kinase, ATR. We hypothesized that the enhanced cytopathicity of HIV-1 in T cells versus macrophages may be explained by differential abilities of VPR to activate ATR in host cells with different cell division status.

METHODS: Amaxa Nucleofection was used to perform RNAi against ATR kinase or Bax in primary CD4+ T cells. Primary CD4+ T cells were infected with the HIV-1 molecular clones NL4-3 or AD8, or VPR-deficient versions thereof. Primary human macrophages were infected with the HIV-1 molecular clone AD8 or the Vpr-deficient mutant. DNA replication was inhibited by thymidine block. Cells were stained for immunofluorescent confocal microscopy using antibodies for HIV Gag p24 and cellular RPA32 or P-H2AX, as well as apoptosis markers.

RESULTS: Using RNAi, we have determined a requirement for ATR toward VPR-induced G2 arrest and apoptosis, and Bax for apoptosis, in primary CD4+ T cells. Activation of ATR is concomitant with a hallmark of DNA replication stress, deposition of the single-stranded DNA binding protein, RPA32, in HIV-1 infected cells. We also show that cell division is necessary for VPR-mediated activation of ATR. Consequently, non-dividing cells, such as primary macrophages or G1/S-blocked T cells are refractory to VPR-induced G2 arrest and apoptosis.

CONCLUSIONS: HIV-1 VPR causes replication stress in infected T cells but not in macrophages. This replication stress activates ATR, causing G2 arrest and eventual Bax-dependent apoptosis. The differential cytopathic effects of HIV-1 in T cells and macrophages can be explained, in part, based on the dependence of VPR function on cell division status.

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2006-08-13
TuAa0101

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