Induction of Apoptosis by HIV-1 vpr

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DESCRIPTION (provided by applicant): The HIV-1 accessory gene Vpr encodes a conserved 96-amino acid protein that induces block of the cell cycle at the 62 phase. Expression of Vpr in CD4+ lymphocytes also induces apoptosis. We have identified the ATR kinase as the cellular factor that mediates Vpr-induced cell cycle arrest and apoptosis. We have also demonstrated that induction of G2 arrest and apoptosis require phosphorylation of downstream targets of ATR, such as Chk1, BRCA1 and GADD45alpha. Therefore, the ATR kinase represents a key determinant of Vpr-induced pathogenesis. The major focus of this proposal will be to elucidate the precise mechanism by which Vpr activates ATR. Macrophages infected with HIV-1 in vivo and in vitro are long-lived, when compared to infected, activated T-cells. We find that expression of Vpr in macrophages is unable to induce apoptosis, and we hypothesize that the underlying cause is that Vpr is unable to activate ATR in these cells. The differential ability of Vpr to induce apoptosis in activated T-cells versus macrophages is in complete agreement with an emerging model in which activation of ATR requires ongoing cellular DMA replication. Activation of ATR by Vpr also leads to an enhancement viral gene expression, which explains the moderate transactivation activity ascribed to Vpr in T- cells. The specific Aims of this proposal are: Specific Aim 1. To elucidate the mechanism by which Vpr activates ATR. The function of ATR is to survey cellular DNA replication and to detect stalled replication forks. We will examine whether Vpr causes activation of ATR by affecting the integrity of DNA, by hindering host cell replication, or by activating the ATR signaling complex directly. Specific Aim 2. To explore the effect of Vpr on ATR in monocyte-derived macrophages (MDM). We have observed that MDM are refractory to Vpr-induced apoptosis, but not to other pro-apoptotic stimuli. We hypothesize that Vpr is unable to activate ATR in MDM due to their non-dividing status. We also hypothesize that Vpr-induced transactivation, which is dependent on ATR activation, does not occur in MDM. Aim 3. To evaluate the contribution of ATR activation to the kinetics of replication of HIV-1. We have shown that inhibition of ATR leads to complete supression of Vpr's ability to transactivate the viral promoter. Therefore, we hypothesize that Vpr will be able to induce transactivation in dividing cells (activated T-cells) but not in non-dividing cells (MDM). We also hypothesize that ATR activation by Vpr, when allowed, will result in enhanced rate of viral replication.