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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 HeLa cells. (A) a. Vpr induces cellular gross enlargement (top) with single enlarged nuclei (bottom). HeLa cells were synchronized in G1/S as described. Cells were then stained with DAPI. Images were captured 11 hours after Vpr transduction using a Leica DMR fluorescence microscope (DM4500B; Leica Microsystems) equipped with a high-performance camera (Hamamatsu) under visual light (top) and fluorescence (bottom). Scale bar: 10 μm. b. Vpr promotes the accumulation of DNA polyploidy as indicated by presence of 8N DNA. HeLa cells were synchronized in G1/S as described. DNA ploidy was measured by propidium iodide staining using flow cytometric analysis over time. (B) Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv-Vpr at time 0 and then collected at 5 hours after viral transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser345 antibody (a). The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as controls (b). (C). Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv or Adv-Vpr at time 0 and then collected at 11 hours after viral transduction for flow cytometric analysis. Ctr, control.
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Figure 6: Possible roles of Cdt1 and Cdc6 in Vpr-induced Chk1-Ser345 phosphorylation and G2 arrest in HeLa cells. (A) a. Vpr induces cellular gross enlargement (top) with single enlarged nuclei (bottom). HeLa cells were synchronized in G1/S as described. Cells were then stained with DAPI. Images were captured 11 hours after Vpr transduction using a Leica DMR fluorescence microscope (DM4500B; Leica Microsystems) equipped with a high-performance camera (Hamamatsu) under visual light (top) and fluorescence (bottom). Scale bar: 10 μm. b. Vpr promotes the accumulation of DNA polyploidy as indicated by presence of 8N DNA. HeLa cells were synchronized in G1/S as described. DNA ploidy was measured by propidium iodide staining using flow cytometric analysis over time. (B) Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv-Vpr at time 0 and then collected at 5 hours after viral transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser345 antibody (a). The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as controls (b). (C). Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv or Adv-Vpr at time 0 and then collected at 11 hours after viral transduction for flow cytometric analysis. Ctr, control.

Mentions: Since the G2-inducing signal appears to be generated in S phase of the cell cycle, one possibility is that Vpr could potentially interfere with DNA synthesis either by blocking DNA replication or by interfering with DNA replication. In eukaryotes, DNA synthesis is strictly regulated by DNA replication licensing factors Cdt1 and Cdc6 which serve to ensure that DNA replicates only once per cell cycle [56,57]. Typically, in late G1 phase, Cdt1 is activated by binding of Cdc6 to promote formation of pre-replication complexes [58]. Upon the start of DNA replication, Cdt1 is rapidly inhibited or degraded by various mechanisms to prevent re-replication (for a recent review, see [59]). However, when Cdt1 and Cdc6 are improperly elevated, DNA re-replication occurs, which causes Chk1-Ser345 phosphorylation [60]. Previous studies suggested that some HIV-infected cells increase cellular DNA ploidy [61], and Vpr promotes aneuploidy [62]. As vpr-expressing cells are obviously capable of passing through the S phase, Vpr-induced aneuploidy suggests that Vpr could either cause DNA-replication [57,63], which should occur within a single nucleus of a cell, or failed cytokinesis for which multiple nuclei should be seen in a single cell. To test these possibilities, we first compared the cellular and nuclear morphologies of HeLa cells between Adv-control and Adv-Vpr expressing cells 11 hours after adenoviral transduction. As shown in Figure 6A-a (top), the Vpr-producing cells were grossly enlarged in comparison with the Adv-control cells as described previously [64]. Nuclear staining with propidium iodide (PI) showed much larger cells with a single nucleus in each of the Vpr-producing HeLa cells when compared to control cells (Figure 6A-a, bottom). These observations suggest that Vpr may induce DNA re-replication within a single nucleus of an individual cell instead of inducing failed cytokinesis.


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 HeLa cells. (A) a. Vpr induces cellular gross enlargement (top) with single enlarged nuclei (bottom). HeLa cells were synchronized in G1/S as described. Cells were then stained with DAPI. Images were captured 11 hours after Vpr transduction using a Leica DMR fluorescence microscope (DM4500B; Leica Microsystems) equipped with a high-performance camera (Hamamatsu) under visual light (top) and fluorescence (bottom). Scale bar: 10 μm. b. Vpr promotes the accumulation of DNA polyploidy as indicated by presence of 8N DNA. HeLa cells were synchronized in G1/S as described. DNA ploidy was measured by propidium iodide staining using flow cytometric analysis over time. (B) Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv-Vpr at time 0 and then collected at 5 hours after viral transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser345 antibody (a). The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as controls (b). (C). Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv or Adv-Vpr at time 0 and then collected at 11 hours after viral transduction for flow cytometric analysis. Ctr, control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Possible roles of Cdt1 and Cdc6 in Vpr-induced Chk1-Ser345 phosphorylation and G2 arrest in HeLa cells. (A) a. Vpr induces cellular gross enlargement (top) with single enlarged nuclei (bottom). HeLa cells were synchronized in G1/S as described. Cells were then stained with DAPI. Images were captured 11 hours after Vpr transduction using a Leica DMR fluorescence microscope (DM4500B; Leica Microsystems) equipped with a high-performance camera (Hamamatsu) under visual light (top) and fluorescence (bottom). Scale bar: 10 μm. b. Vpr promotes the accumulation of DNA polyploidy as indicated by presence of 8N DNA. HeLa cells were synchronized in G1/S as described. DNA ploidy was measured by propidium iodide staining using flow cytometric analysis over time. (B) Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv-Vpr at time 0 and then collected at 5 hours after viral transduction. The cell lysates were subjected to Western blot using anti-Chk1-Ser345 antibody (a). The knockdown efficiency of Cdc6 or Cdt1 siRNA was verified by using anti-Cdc6 or anti-Cdt1 antibody with β-actin as controls (b). (C). Synchronized G1/S HeLa cells, treated with Cdc6, Cdt1 or control siRNA, were transduced with Adv or Adv-Vpr at time 0 and then collected at 11 hours after viral transduction for flow cytometric analysis. Ctr, control.
Mentions: Since the G2-inducing signal appears to be generated in S phase of the cell cycle, one possibility is that Vpr could potentially interfere with DNA synthesis either by blocking DNA replication or by interfering with DNA replication. In eukaryotes, DNA synthesis is strictly regulated by DNA replication licensing factors Cdt1 and Cdc6 which serve to ensure that DNA replicates only once per cell cycle [56,57]. Typically, in late G1 phase, Cdt1 is activated by binding of Cdc6 to promote formation of pre-replication complexes [58]. Upon the start of DNA replication, Cdt1 is rapidly inhibited or degraded by various mechanisms to prevent re-replication (for a recent review, see [59]). However, when Cdt1 and Cdc6 are improperly elevated, DNA re-replication occurs, which causes Chk1-Ser345 phosphorylation [60]. Previous studies suggested that some HIV-infected cells increase cellular DNA ploidy [61], and Vpr promotes aneuploidy [62]. As vpr-expressing cells are obviously capable of passing through the S phase, Vpr-induced aneuploidy suggests that Vpr could either cause DNA-replication [57,63], which should occur within a single nucleus of a cell, or failed cytokinesis for which multiple nuclei should be seen in a single cell. To test these possibilities, we first compared the cellular and nuclear morphologies of HeLa cells between Adv-control and Adv-Vpr expressing cells 11 hours after adenoviral transduction. As shown in Figure 6A-a (top), the Vpr-producing cells were grossly enlarged in comparison with the Adv-control cells as described previously [64]. Nuclear staining with propidium iodide (PI) showed much larger cells with a single nucleus in each of the Vpr-producing HeLa cells when compared to control cells (Figure 6A-a, bottom). These observations suggest that Vpr may induce DNA re-replication within a single nucleus of an individual cell instead of inducing failed cytokinesis.

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
Related in: MedlinePlus