Abstract
BackgroundCell cycle G2 arrest induced by HIV-1 Vpr is thought to benefit viral proliferation by providing an optimized cellular environment for viral replication and by skipping host immune responses. Even though Vpr-induced G2 arrest has been studied extensively, how Vpr triggers G2 arrest remains elusive.ResultsTo examine this initiation event, we measured the Vpr effect over a single cell cycle. We found that even though Vpr stops the cell cycle at the G2/M phase, but the initiation event actually occurs in the S phase of the cell cycle. Specifically, Vpr triggers activation of Chk1 through Ser345 phosphorylation in an S phase-dependent manner. The S phase-dependent requirement of Chk1-Ser345 phosphorylation by Vpr was confirmed by siRNA gene silencing and site-directed mutagenesis. Moreover, downregulation of DNA replication licensing factors Cdt1 by siRNA significantly reduced Vpr-induced Chk1-Ser345 phosphorylation and G2 arrest. Even though hydroxyurea (HU) and ultraviolet light (UV) also induce Chk1-Ser345 phosphorylation in S phase under the same conditions, neither HU nor UV-treated cells were able to pass through S phase, whereas vpr-expressing cells completed S phase and stopped at the G2/M boundary. Furthermore, unlike HU/UV, Vpr promotes Chk1- and proteasome-mediated protein degradations of Cdc25B/C for G2 induction; in contrast, Vpr had little or no effect on Cdc25A protein degradation normally mediated by HU/UV.ConclusionsThese data suggest that Vpr induces cell cycle G2 arrest through a unique molecular mechanism that regulates host cell cycle regulation in an S-phase dependent fashion.
Highlights
Cell cycle G2 arrest induced by Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is thought to benefit viral proliferation by providing an optimized cellular environment for viral replication and by skipping host immune responses
The cell cycle G2 arrest induced by Vpr is thought to suppress human immune function by preventing T-cell clone expansion [11] and to provide an optimized cellular environment for maximal levels of viral replication [6]
HeLa cells were firstly synchronized at the G1/S boundary of the cell cycle by the double thymidine (DT) block as described previously [49]
Summary
Cell cycle G2 arrest induced by HIV-1 Vpr is thought to benefit viral proliferation by providing an optimized cellular environment for viral replication and by skipping host immune responses. Vpr displays several distinct activities in host cells. These include cytoplasm-nuclear shuttling [4,8], induction of cell cycle G2 arrest [9], and cell killing [10]. The cell cycle G2 arrest induced by Vpr is thought to suppress human immune function by preventing T-cell clone expansion [11] and to provide an optimized cellular environment for maximal levels of viral replication [6]. Further understanding of Vpr-induced cell cycle G2 arrest could provide additional insights into the molecular actions of Vpr in augmenting viral replication and modulation of host immune response
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