Activation of the DNA-damage responsive signaling by HIV-1 Vpr, roles of ATR and Chk1 and implications for HIV-1 replication and pathogenesis

DNA damage is a universal inducer of cell cycle arrest at the G 2 phase. Invasion by certain parasitic organisms, such as the human immunodeficiency virus type 1 (HIV-1), also blocks cellular proliferation at the G2 phase. The viral accessory gene vpr encodes a conserved 96-amino acid protein that is necessary and sufficient for the HIV-1-induced G2 arrest. In the present study, we examined a recently identified DNA damage-signaling protein, the ATM- and Rad3-related protein, ATR, for its potential role in the induction of G2 arrest by Vpr. We show that inhibition of ATR by pharmacological inhibitors, by expression of the dominant negative ATR mutant, or by knockdown of ATR expression via RNAi abrogates Vpr-induced G2 arrest. Activation of ATR in response to DNA damage results in direct phosphorylation of its target, the Chk1 kinase on Ser 345. We detected phosphorylation of Chk1 in response to Vpr expression that was blocked by inhibition of ATR function. Inactivation of Chk1 reduced Vpr-induced G2 arrest. Vpr is also a mild transcriptional transactivator of the HIV-1 LTR. We have addressed a possible role of DNA damage-induced signaling on HIV-1 life cycle. We demonstrate that inhibition of ATR function reduces Vpr-induced LTR transactivation.; Cellular deoxynucleotide pools serve as substrates for the HIV-1 reverse transcriptase and for the repair of the HIV-1 integrase-induced genetic lesion. To assess a possible consequence of Vpr-induced DNA damage checkpoint on the dNTP pools of the target cells we have developed a novel sensitive assay to measure dNTP pool perturbations. Using this assay, we have demonstrated that dNTP pools increase in response to DNA damage. We hypothesize that Vpr-induced activation of the DNA-damage signaling plays a role at several stages of HIV-1 replication cycle. Activation of DNA-damage signaling increases the activity of the HIV-1 LTR, and may increase dNTP pools that are available for the HIV-1 reverse transcription and the repair of the lesion induced by the HIV-1 integration.