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

Li G, Park HU, Liang D, Zhao RY - Retrovirology (2010)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, USA.

ABSTRACT

Background: 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. Even though Vpr-induced G2 arrest has been studied extensively, how Vpr triggers G2 arrest remains elusive.

Results: To 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.

Conclusions: These 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.

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Vpr induces cell cycle G2/M arrest through activation of Chk1 via Ser345 phosphorylation in S phase of the cell cycle. A. HeLa cells synchronized at the G1/S boundary by double thymine (DT) block were transduced with Adv control or Adv-Vpr (MOI 1.0) and released from the block at time 0. The cell cycle profiles measured by DNA content (a) were detected from time 0 to 11 hours after the DT release. The Cdk1-Tyr345 or Chk1-Ser345 phosphorylation status (b) were detected in the Vpr-positive or Vpr-negative cells collected at indicated time. B. HeLa cells, which were first synchronized in M phase by Nocodazole (100 ng/ml), were transduced with Adv or Adv-Vpr and detected the same way as shown in (A). Note that very weak Vpr was detected in (A-b) because Ad-Vpr was only transduced within 5 to 11 hours. The Vpr protein was clearly detected subsequently at about 15 hours after viral transduction (B-b).
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Figure 1: Vpr induces cell cycle G2/M arrest through activation of Chk1 via Ser345 phosphorylation in S phase of the cell cycle. A. HeLa cells synchronized at the G1/S boundary by double thymine (DT) block were transduced with Adv control or Adv-Vpr (MOI 1.0) and released from the block at time 0. The cell cycle profiles measured by DNA content (a) were detected from time 0 to 11 hours after the DT release. The Cdk1-Tyr345 or Chk1-Ser345 phosphorylation status (b) were detected in the Vpr-positive or Vpr-negative cells collected at indicated time. B. HeLa cells, which were first synchronized in M phase by Nocodazole (100 ng/ml), were transduced with Adv or Adv-Vpr and detected the same way as shown in (A). Note that very weak Vpr was detected in (A-b) because Ad-Vpr was only transduced within 5 to 11 hours. The Vpr protein was clearly detected subsequently at about 15 hours after viral transduction (B-b).

Mentions: To monitor the initiating event of cellular signaling for Vpr-induced G2 arrest, we adopted a single cell cycle assay to measure this event in a synchronized cell population. Specifically, HeLa cells were firstly synchronized at the G1/S boundary of the cell cycle by the double thymidine (DT) block as described previously [49]. Synchronized HeLa cells were then transduced immediately after released from the DT block with an adenoviral vector control (Adv) or a vpr-carrying adenoviral vector (Adv-Vpr) at a multiplicity of infection (MOI) of 1.0. Cells were collected at the indicated time after transduction, and cell cycle profiles were monitored by flow cytometric analysis. As shown in Figure 1A-a, >90% of cells were observed in G1 when the synchronized cells were released from the DT treatment (0 hr). Without Vpr, cells progressed to S phase by 5 hours, G2/M by 8 hours and returned back to the G1 phase by 11 hours (Figure 1A-a, left). Similar cell cycle progression was also observed in cells expressing vpr in the first 8 hours. However, cell cycling stopped at the G2/M phase by 11 hours (Figure 1A-a, right). Vpr-induced G2 arrest was further confirmed by the elevated phosphorylation of Cdk1-Tyr15 as measured by Western blot analysis (Figure 1A-b, top row). Please note that the entire cell cycle takes longer than 11 hours to complete typically around 22-24 hours. The 11 hours after release of the DT block is the shortest time within a single cell cycle that we could measure Vpr-induced G2 arrest.


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

Li G, Park HU, Liang D, Zhao RY - Retrovirology (2010)

Vpr induces cell cycle G2/M arrest through activation of Chk1 via Ser345 phosphorylation in S phase of the cell cycle. A. HeLa cells synchronized at the G1/S boundary by double thymine (DT) block were transduced with Adv control or Adv-Vpr (MOI 1.0) and released from the block at time 0. The cell cycle profiles measured by DNA content (a) were detected from time 0 to 11 hours after the DT release. The Cdk1-Tyr345 or Chk1-Ser345 phosphorylation status (b) were detected in the Vpr-positive or Vpr-negative cells collected at indicated time. B. HeLa cells, which were first synchronized in M phase by Nocodazole (100 ng/ml), were transduced with Adv or Adv-Vpr and detected the same way as shown in (A). Note that very weak Vpr was detected in (A-b) because Ad-Vpr was only transduced within 5 to 11 hours. The Vpr protein was clearly detected subsequently at about 15 hours after viral transduction (B-b).
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Figure 1: Vpr induces cell cycle G2/M arrest through activation of Chk1 via Ser345 phosphorylation in S phase of the cell cycle. A. HeLa cells synchronized at the G1/S boundary by double thymine (DT) block were transduced with Adv control or Adv-Vpr (MOI 1.0) and released from the block at time 0. The cell cycle profiles measured by DNA content (a) were detected from time 0 to 11 hours after the DT release. The Cdk1-Tyr345 or Chk1-Ser345 phosphorylation status (b) were detected in the Vpr-positive or Vpr-negative cells collected at indicated time. B. HeLa cells, which were first synchronized in M phase by Nocodazole (100 ng/ml), were transduced with Adv or Adv-Vpr and detected the same way as shown in (A). Note that very weak Vpr was detected in (A-b) because Ad-Vpr was only transduced within 5 to 11 hours. The Vpr protein was clearly detected subsequently at about 15 hours after viral transduction (B-b).
Mentions: To monitor the initiating event of cellular signaling for Vpr-induced G2 arrest, we adopted a single cell cycle assay to measure this event in a synchronized cell population. Specifically, HeLa cells were firstly synchronized at the G1/S boundary of the cell cycle by the double thymidine (DT) block as described previously [49]. Synchronized HeLa cells were then transduced immediately after released from the DT block with an adenoviral vector control (Adv) or a vpr-carrying adenoviral vector (Adv-Vpr) at a multiplicity of infection (MOI) of 1.0. Cells were collected at the indicated time after transduction, and cell cycle profiles were monitored by flow cytometric analysis. As shown in Figure 1A-a, >90% of cells were observed in G1 when the synchronized cells were released from the DT treatment (0 hr). Without Vpr, cells progressed to S phase by 5 hours, G2/M by 8 hours and returned back to the G1 phase by 11 hours (Figure 1A-a, left). Similar cell cycle progression was also observed in cells expressing vpr in the first 8 hours. However, cell cycling stopped at the G2/M phase by 11 hours (Figure 1A-a, right). Vpr-induced G2 arrest was further confirmed by the elevated phosphorylation of Cdk1-Tyr15 as measured by Western blot analysis (Figure 1A-b, top row). Please note that the entire cell cycle takes longer than 11 hours to complete typically around 22-24 hours. The 11 hours after release of the DT block is the shortest time within a single cell cycle that we could measure Vpr-induced G2 arrest.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, USA.

ABSTRACT

Background: 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. Even though Vpr-induced G2 arrest has been studied extensively, how Vpr triggers G2 arrest remains elusive.

Results: To 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.

Conclusions: These 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.

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