Session 36Poster Presentations Accessory Genes Session Day and Time: Tuesday 1:30 - 3:30 pm Room: Hall D
213 Activation of the ATR-initiated DNA Damage Signaling Pathway by HIV-1 Vpr V. Planelles1, S. Murala1, J. Walker1, M. Roshal*2 1Univ of Utah Sch of Med, Salt Lake City and 2Univ of Rochester, NY
Background: The human immunodeficiency virus type-1 (HIV-1) encodes four accessory genes (vpr, vpu, vif, and nef) that regulate various aspects of the host cell biology. Vpr encodes a 96-amino acid protein (Vpr) that causes cell cycle arrest in G2 and apoptosis in the infected lymphocytes. Several studies have indicated that the effects of Vpr on the cell are similar to the effects of genotoxic drugs and other DNA damaging agents, such as radiation. In the present study, we examine 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. ATR is a serine-threonine kinase that becomes activated in response to DNA damage. Activation of ATR typically results in direct phosphorylation of its target, the Chk1 kinase, and inactivation of Cdc-2, leading to G2 arrest.
Methods: The potential role of ATR in the induction of G2 arrest by Vpr was tested by several independent methods: phosphorylation of Chk1, a downstream target of ATR; inhibition of Vpr function with a dominant-negative ATR mutant; and knockdown of ATR via RNA interference.
Results: Expression of dominant-negative ATR or disruption of ATR using RNA interference blocked the ability of Vpr to induced G2 arrest. The activation of ATR by Vpr was concomitant with phosphorylation of it downstream target, Chk1. In addition, blocking Chk1 function with a specific Chk1 inhibitor, UCN-01, also blocked Vpr function.
Conclusions: Our observations are consistent with our hypothesis that the Vpr-induced cell cycle arrest involves a DNA damage-signaling pathway. More specifically, we demonstrate that the ATR-initiated pathway is active in Vpr-expressing cells. These observations have important ramifications in the areas of HIV pathogenesis since they provide mechanistic explanations for the cytostatic properties of HIV-1 Vpr.
