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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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Possible roles of Cdt1 and Cdc6 in Vpr-induced Chk1-Ser345 phosphorylation and G2 arrest in CEM-SS cells. (A) Vpr promotes accumulation of DNA polyploidy as indicated by the presence of 8N DNA. Asynchronized CEM-SS cells were grown under the normal cell culture condition, and transduced with Adv viral control or Adv-Vpr. Cells were collected at indicated time point and DNA ploidy was measured by PI staining using flow cytometric analysis. (B) Asynchronized CEM-SS cells were pretreated with Cdt1, Cdc6 or control (Ctr) siRNA, and then transduced with Adv or Adv-Vpr 24 hours after addition of siRNAs. Cells were then harvested 48 hours post-transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser345 antibody. The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as protein loading controls. (C). CEM-SS were treated the same way as described in (B). The cells were harvested 48 hours post-transduction and the cell lysates were then subjected to flow cytometric analysis.
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Figure 7: Possible roles of Cdt1 and Cdc6 in Vpr-induced Chk1-Ser345 phosphorylation and G2 arrest in CEM-SS cells. (A) Vpr promotes accumulation of DNA polyploidy as indicated by the presence of 8N DNA. Asynchronized CEM-SS cells were grown under the normal cell culture condition, and transduced with Adv viral control or Adv-Vpr. Cells were collected at indicated time point and DNA ploidy was measured by PI staining using flow cytometric analysis. (B) Asynchronized CEM-SS cells were pretreated with Cdt1, Cdc6 or control (Ctr) siRNA, and then transduced with Adv or Adv-Vpr 24 hours after addition of siRNAs. Cells were then harvested 48 hours post-transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser345 antibody. The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as protein loading controls. (C). CEM-SS were treated the same way as described in (B). The cells were harvested 48 hours post-transduction and the cell lysates were then subjected to flow cytometric analysis.

Mentions: HeLa cells are not the natural target cells of HIV-1. In addition, HeLa cells are immortalized with human papillomavirus virus 18 which encodes viral proteins that may interfere with cell cycle regulation. To see whether the same effects of Vpr on the DNA re-replication, Chk1-Ser345 phosphorylation and Cdt1 as well as Cdc6 could be observed in other cell types, we carried out the same experiments as shown in Figure 6, but we used a T-lymphocyte cell line CEM-SS, which models the natural target of HIV-1. However, instead of using synchronized cells, we used asynchronized cells that we normally grow in the laboratory. As shown in Figure 7A, CEM-SS cells transduced with the Adv control virus showed normal cell cycle profile, i.e., remained predominantly in G1 (2N) phase of the cell cycle over a period of 96 hours; however, a very strong G2 cell population (4N) was seen in the Adv-Vpr transduced cells 48 hours after viral transduction. Noticeably, a small increase of 8N DNA was also observed 72 to 96 hours after the adenoviral transduction. Very similar to what we have observed in HeLa cells, Vpr also induced relatively strong Chk1-Ser345 phosphorylation (Figure 7B, first row, lane 2 vs. 1). However, only smaller reductions of Chk1-Ser345 phosphorylation were seen in the Cdt1 or Cdc6-depleted CEM-SS cells compared to HeLa cells. This discrepancy was probably because we were only able to partially deplete Cdt1 and Cdc6 using the siRNAs in this particular T-cell line (data not shown). Consistent with the incomplete depletion of Cdt1 or Cdc6, a small but significant reduction of Vpr-induced G2 arrest (from 80.3% to 56.9%) was observed in the Cdt1-reduced cells; and a small reduction (9%) was seen in the Cdc6-reduced CEM-SS cells (Figure 7C).


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)

Possible roles of Cdt1 and Cdc6 in Vpr-induced Chk1-Ser345 phosphorylation and G2 arrest in CEM-SS cells. (A) Vpr promotes accumulation of DNA polyploidy as indicated by the presence of 8N DNA. Asynchronized CEM-SS cells were grown under the normal cell culture condition, and transduced with Adv viral control or Adv-Vpr. Cells were collected at indicated time point and DNA ploidy was measured by PI staining using flow cytometric analysis. (B) Asynchronized CEM-SS cells were pretreated with Cdt1, Cdc6 or control (Ctr) siRNA, and then transduced with Adv or Adv-Vpr 24 hours after addition of siRNAs. Cells were then harvested 48 hours post-transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser345 antibody. The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as protein loading controls. (C). CEM-SS were treated the same way as described in (B). The cells were harvested 48 hours post-transduction and the cell lysates were then subjected to flow cytometric analysis.
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Figure 7: Possible roles of Cdt1 and Cdc6 in Vpr-induced Chk1-Ser345 phosphorylation and G2 arrest in CEM-SS cells. (A) Vpr promotes accumulation of DNA polyploidy as indicated by the presence of 8N DNA. Asynchronized CEM-SS cells were grown under the normal cell culture condition, and transduced with Adv viral control or Adv-Vpr. Cells were collected at indicated time point and DNA ploidy was measured by PI staining using flow cytometric analysis. (B) Asynchronized CEM-SS cells were pretreated with Cdt1, Cdc6 or control (Ctr) siRNA, and then transduced with Adv or Adv-Vpr 24 hours after addition of siRNAs. Cells were then harvested 48 hours post-transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser345 antibody. The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as protein loading controls. (C). CEM-SS were treated the same way as described in (B). The cells were harvested 48 hours post-transduction and the cell lysates were then subjected to flow cytometric analysis.
Mentions: HeLa cells are not the natural target cells of HIV-1. In addition, HeLa cells are immortalized with human papillomavirus virus 18 which encodes viral proteins that may interfere with cell cycle regulation. To see whether the same effects of Vpr on the DNA re-replication, Chk1-Ser345 phosphorylation and Cdt1 as well as Cdc6 could be observed in other cell types, we carried out the same experiments as shown in Figure 6, but we used a T-lymphocyte cell line CEM-SS, which models the natural target of HIV-1. However, instead of using synchronized cells, we used asynchronized cells that we normally grow in the laboratory. As shown in Figure 7A, CEM-SS cells transduced with the Adv control virus showed normal cell cycle profile, i.e., remained predominantly in G1 (2N) phase of the cell cycle over a period of 96 hours; however, a very strong G2 cell population (4N) was seen in the Adv-Vpr transduced cells 48 hours after viral transduction. Noticeably, a small increase of 8N DNA was also observed 72 to 96 hours after the adenoviral transduction. Very similar to what we have observed in HeLa cells, Vpr also induced relatively strong Chk1-Ser345 phosphorylation (Figure 7B, first row, lane 2 vs. 1). However, only smaller reductions of Chk1-Ser345 phosphorylation were seen in the Cdt1 or Cdc6-depleted CEM-SS cells compared to HeLa cells. This discrepancy was probably because we were only able to partially deplete Cdt1 and Cdc6 using the siRNAs in this particular T-cell line (data not shown). Consistent with the incomplete depletion of Cdt1 or Cdc6, a small but significant reduction of Vpr-induced G2 arrest (from 80.3% to 56.9%) was observed in the Cdt1-reduced cells; and a small reduction (9%) was seen in the Cdc6-reduced CEM-SS cells (Figure 7C).

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.

Show MeSH