Human Immunodeficiency Virus Type 1 Vpr-Mediated G₂ Arrest Requires Rad17 and Hus1 and Induces Nuclear BRCA1 and γ-H2AX Focus Formation

Abstract

Eukaryotic cells have evolved a complex mechanism for sensing DNA damage during genome replication. Activation of this pathway prevents entry into mitosis to allow for either DNA repair or, in the event of irreparable damage, commitment to apoptosis. Under conditions of replication stress, the damage signal is initiated by the ataxia-telangiectasia-mutated and Rad3-related kinase ATR. We recently demonstrated that the human immunodeficiency virus type 1 (HIV-1) gene product viral protein R (Vpr) arrests infected cells in the G₂ phase via the activation of ATR. In the present study, we show that the activation of ATR by Vpr is analogous to activation by certain genotoxic agents, both mechanistically and in its downstream consequences. Specifically, we show a requirement for Rad17 and Hus1 to induce G₂ arrest as well as Vpr-induced phosphorylation of histone 2A variant X (H2AX) and formation of nuclear foci containing H2AX and breast cancer susceptibility protein 1. These results demonstrate that G₂ arrest mediated by the HIV-1 gene product Vpr utilizes the cellular signaling pathway whose physiological function is to recognize replication stress. These findings should contribute to a greater understanding of how HIV-1 manipulates the CD4⁺-lymphocyte cell cycle and apoptosis induction in the progressive CD4⁺-lymphocyte depletion characteristic of HIV-1 pathogenesis.

We thank Robert Weiss at Cornell University for the generous gifts of Hus1^−/− p21^−/− mouse embryonic fibroblasts and Hus1^+/+ p21^−/− mouse embryonic fibroblasts, Wayne Green at the University of Utah Flow Cytometry Core Facility for expert assistance with flow cytometry, and Chris Rodesch at the University of Utah Cell Imaging Core Facility for help with confocal microscopy. Matt Mulvey, Jamie Sundsbak, and Danelle Eto provided invaluable help with immunofluorescence techniques.

This work was supported by National Institutes of Health research grants AI49057 and AI054188 to V.P.