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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 has little or no effect on proteasome-mediated protein degradation of Cdc25A in contrast to HU/UV. (A) Synchronized G1/S HeLa cells treated with HU, UV or transduced with Adv-Vpr were collected at the indicated time, and then subjected to Western blot analysis using anti-Cdc25A and anti-Vpr antibodies (a). β-actin was used as a loading control. The relative intensity of the Cdc25A protein levels to β-actin was determined by densitometry and the Cdc25A protein level at 0 hour was set as 1.0. (b). The results presented are the average of three independent experiments. (B) Synchronized HeLa cells were treated with 50 μm MG132 at 0 hour and collected 5 hours after treatment. The protein level of Cdc25A was detected by Western blot analysis. (C) HeLa cells were pre-treated with specific siRNA against Chk1, which were then synchronized at G1/S boundary by the DT blocks. HU- or Vpr-treated cells were collected 5 hours after the DT release. The protein level of Cdc25A was detected by Western blot analysis using the indicated antibodies.
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Figure 4: Vpr has little or no effect on proteasome-mediated protein degradation of Cdc25A in contrast to HU/UV. (A) Synchronized G1/S HeLa cells treated with HU, UV or transduced with Adv-Vpr were collected at the indicated time, and then subjected to Western blot analysis using anti-Cdc25A and anti-Vpr antibodies (a). β-actin was used as a loading control. The relative intensity of the Cdc25A protein levels to β-actin was determined by densitometry and the Cdc25A protein level at 0 hour was set as 1.0. (b). The results presented are the average of three independent experiments. (B) Synchronized HeLa cells were treated with 50 μm MG132 at 0 hour and collected 5 hours after treatment. The protein level of Cdc25A was detected by Western blot analysis. (C) HeLa cells were pre-treated with specific siRNA against Chk1, which were then synchronized at G1/S boundary by the DT blocks. HU- or Vpr-treated cells were collected 5 hours after the DT release. The protein level of Cdc25A was detected by Western blot analysis using the indicated antibodies.

Mentions: One of the downstream events driven by activated Chk1 is the inhibitory phosphorylation of Cdc25 phosphatases. Since all three Cdc25 homologues are the essential substrates of Chk1 during DNA damage/replication checkpoints, which one of the three Cdc25s is being inactivated by Chk1 could define the cell cycle outcome [17,20,21]. Previous studies suggested that Cdc25A is one of direct targets of activated Chk1, which results in the S phase arrest when cells are challenged by HU or UV [20,21]. To determine whether Cdc25A is affected by Vpr or whether it contributes to the observed differences of cell cycle profiles in cells treated with HU/UV or Vpr (Figure 3B), synchronized G1/S HeLa cells were prepared by DT block and treated with HU, UV or Adv-Vpr transduction as described above. The Cdc25A protein levels collected over time were then detected by Western blot analyses using an anti-Cdc25A antibody. As shown in Figure 4A-a, first row, and Figure 4A-b, Cdc25A protein level in a normal cell cycle rose significantly from G1/S (0 hr) to S (5 hours) and reached maximum in the G2 phase (8 hours) followed by a small decrease in G1 phase (Figure 4A-a, first row, lanes 1-4). Similar to normal cells, relatively high levels of Cdc25A, with a small dip in the G2 phase, were seen from S phase to G1 phase in the vpr-expressing cells (Figure 4A-a, first row, lanes 11-13). Since the Cdc25A protein profile in vpr-expressing cells showed similar pattern to normal cells, it suggests that Vpr has little or no effect on Chk1-mediated Cdc25A protein production or degradation. In contrast to this pattern observed in normal and vpr-expressing cells, much reduced Cdc25A proteins were observed in cells treated with HU or UV throughout the cell cycle (Figure 4A-a, first row, lanes 5-10). To test whether the low Cdc25A protein levels observed in the HU/UV-treated cells are due to prevention of protein production or promotion of proteasome-mediated protein degradation, the protein levels of Cdc25A were further compared between cells treated with the proteasome inhibitor MG132 (50 μM) and untreated control cells (Figure 4B; only cells treated with HU and collected 5 hours after the DT release are shown here as control). While the normal Cdc25A protein level was completely restored in HU-treated cells treated with MG132, only a small and non-appreciable increase of Cdc25A was noted in the vpr-expressing cells treated with MG132. These data suggest that HU/UV promotes protein degradation of Cdc25A through a proteasome-mediated mechanism. Similarly, these data further confirmed that, unlike UV or HU, Vpr has little, if any, impact on the Cdc25A protein level in these cells.


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 has little or no effect on proteasome-mediated protein degradation of Cdc25A in contrast to HU/UV. (A) Synchronized G1/S HeLa cells treated with HU, UV or transduced with Adv-Vpr were collected at the indicated time, and then subjected to Western blot analysis using anti-Cdc25A and anti-Vpr antibodies (a). β-actin was used as a loading control. The relative intensity of the Cdc25A protein levels to β-actin was determined by densitometry and the Cdc25A protein level at 0 hour was set as 1.0. (b). The results presented are the average of three independent experiments. (B) Synchronized HeLa cells were treated with 50 μm MG132 at 0 hour and collected 5 hours after treatment. The protein level of Cdc25A was detected by Western blot analysis. (C) HeLa cells were pre-treated with specific siRNA against Chk1, which were then synchronized at G1/S boundary by the DT blocks. HU- or Vpr-treated cells were collected 5 hours after the DT release. The protein level of Cdc25A was detected by Western blot analysis using the indicated antibodies.
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Related In: Results  -  Collection

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Show All Figures
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Figure 4: Vpr has little or no effect on proteasome-mediated protein degradation of Cdc25A in contrast to HU/UV. (A) Synchronized G1/S HeLa cells treated with HU, UV or transduced with Adv-Vpr were collected at the indicated time, and then subjected to Western blot analysis using anti-Cdc25A and anti-Vpr antibodies (a). β-actin was used as a loading control. The relative intensity of the Cdc25A protein levels to β-actin was determined by densitometry and the Cdc25A protein level at 0 hour was set as 1.0. (b). The results presented are the average of three independent experiments. (B) Synchronized HeLa cells were treated with 50 μm MG132 at 0 hour and collected 5 hours after treatment. The protein level of Cdc25A was detected by Western blot analysis. (C) HeLa cells were pre-treated with specific siRNA against Chk1, which were then synchronized at G1/S boundary by the DT blocks. HU- or Vpr-treated cells were collected 5 hours after the DT release. The protein level of Cdc25A was detected by Western blot analysis using the indicated antibodies.
Mentions: One of the downstream events driven by activated Chk1 is the inhibitory phosphorylation of Cdc25 phosphatases. Since all three Cdc25 homologues are the essential substrates of Chk1 during DNA damage/replication checkpoints, which one of the three Cdc25s is being inactivated by Chk1 could define the cell cycle outcome [17,20,21]. Previous studies suggested that Cdc25A is one of direct targets of activated Chk1, which results in the S phase arrest when cells are challenged by HU or UV [20,21]. To determine whether Cdc25A is affected by Vpr or whether it contributes to the observed differences of cell cycle profiles in cells treated with HU/UV or Vpr (Figure 3B), synchronized G1/S HeLa cells were prepared by DT block and treated with HU, UV or Adv-Vpr transduction as described above. The Cdc25A protein levels collected over time were then detected by Western blot analyses using an anti-Cdc25A antibody. As shown in Figure 4A-a, first row, and Figure 4A-b, Cdc25A protein level in a normal cell cycle rose significantly from G1/S (0 hr) to S (5 hours) and reached maximum in the G2 phase (8 hours) followed by a small decrease in G1 phase (Figure 4A-a, first row, lanes 1-4). Similar to normal cells, relatively high levels of Cdc25A, with a small dip in the G2 phase, were seen from S phase to G1 phase in the vpr-expressing cells (Figure 4A-a, first row, lanes 11-13). Since the Cdc25A protein profile in vpr-expressing cells showed similar pattern to normal cells, it suggests that Vpr has little or no effect on Chk1-mediated Cdc25A protein production or degradation. In contrast to this pattern observed in normal and vpr-expressing cells, much reduced Cdc25A proteins were observed in cells treated with HU or UV throughout the cell cycle (Figure 4A-a, first row, lanes 5-10). To test whether the low Cdc25A protein levels observed in the HU/UV-treated cells are due to prevention of protein production or promotion of proteasome-mediated protein degradation, the protein levels of Cdc25A were further compared between cells treated with the proteasome inhibitor MG132 (50 μM) and untreated control cells (Figure 4B; only cells treated with HU and collected 5 hours after the DT release are shown here as control). While the normal Cdc25A protein level was completely restored in HU-treated cells treated with MG132, only a small and non-appreciable increase of Cdc25A was noted in the vpr-expressing cells treated with MG132. These data suggest that HU/UV promotes protein degradation of Cdc25A through a proteasome-mediated mechanism. Similarly, these data further confirmed that, unlike UV or HU, Vpr has little, if any, impact on the Cdc25A protein level in these cells.

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