Cell cycle G2/M arrest through an S phase-dependent mechanism by HIV-1 viral protein R

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.

Polyploidy and Mitotic Cell Death Are Two Distinct HIV-1 Vpr-Driven Outcomes in Renal Tubule Epithelial Cells

“…A long established hallmark of Vpr expression is cell cycle arrest during G2/M [102,103,115,116,184] via activation of the ATR-Chk1 DDR signaling pathway [84,106,116,118,119,122]. This study confirms that Vpr initiates an ATR---dependent G2/M arrest in RTECs.…”

“…Strict maintenance of Vpr---mediated G2 arrest appears to be dependent on cell type [118,152]. Indeed, a previous study reported that Vpr isolates from different primate species exert cell cycle control in a species specific manner, suggesting Vpr---mediated G2 arrest has evolved to include host factors unique to particular cell types [107].…”

“…A later study found hydroxyurea and ultraviolet light (UV) induce Chk1 activation in a similar manner to Vpr. However, HU and UV---treated cells arrest in S phase, whereas vpr---expressing cells progress through S phase and arrest at the G2/M border [118]. Indeed, even the manner in which ATRi abrogates G2 arrest in Vpr+ cells suggests a mechanism fundamentally different from the DDR.…”

“…Hyper---phosphorylation of Cdk1 is also associated with G2 arrest induced by DNA damaging agents, leading to speculation that Vpr mediates cell cycle arrest via activation of the DDR [114]. In an effort to clarify the role of the DDR in Vpr---induced G2 arrest, one study expressed Vpr in cells from patients with Ataxia--- inhibition in lymphocytes and other cell lines [116,118]. Consistent with a model of DDR activation through the ATR---Chk1---Cdk1 pathway, one study found that kinase Wee1, which negatively regulates Cdk1 activity via phosphorylation, is also required for Vpr---induced G2 arrest [119].…”

“…In contrast, we find that an extended G2 is required for continue to cycle and become tetraploid following vpr expression [115]. Further, the capacity for, and magnitude of Vpr---mediated polyploidization appears to be dependent on cell type [118,152]. …”