Limits...
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.

Show MeSH
Vpr promotes proteasome-mediated protein degradation of Cdc25B and Cdc25C. (A) Synchronized G1/S HeLa cells treated with HU or transduced with Adv-Vpr were collected at indicated time, and then subjected to Western blot analysis using anti-Cdc25B or anti-Cdc25C antibody, respectively (a). β-actin was used as a loading control. The relative intensity of the Cdc25B (b) or Cdc25C (c) protein levels to β-actin were determined by densitometry. The results presented are the average of three independent experiments. (B) Synchronized HeLa cells were pre-treated with specific siRNA against Chk1 or treated with 50 μm MG132 at 0 hour and collected at the indicated time. The protein level of Cdc25B was detected by Western blot analysis. (C) Synchronized HeLa cells were treated with 50 μm MG132 at 0 hour and collected 11 hours after treatment. The protein level of Cdc25C was detected by Western blot analysis (a). HeLa cells were pre-treated with specific siRNA against Chk1, which were then synchronized at G1/S boundary by DT treatment. HU or Vpr treated cells were collected 11 hours after DT release. The protein level of Cdc25C was detected by Western blot analysis using indicated antibodies (b).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2909154&req=5

Figure 5: Vpr promotes proteasome-mediated protein degradation of Cdc25B and Cdc25C. (A) Synchronized G1/S HeLa cells treated with HU or transduced with Adv-Vpr were collected at indicated time, and then subjected to Western blot analysis using anti-Cdc25B or anti-Cdc25C antibody, respectively (a). β-actin was used as a loading control. The relative intensity of the Cdc25B (b) or Cdc25C (c) protein levels to β-actin were determined by densitometry. The results presented are the average of three independent experiments. (B) Synchronized HeLa cells were pre-treated with specific siRNA against Chk1 or treated with 50 μm MG132 at 0 hour and collected at the indicated time. The protein level of Cdc25B was detected by Western blot analysis. (C) Synchronized HeLa cells were treated with 50 μm MG132 at 0 hour and collected 11 hours after treatment. The protein level of Cdc25C was detected by Western blot analysis (a). HeLa cells were pre-treated with specific siRNA against Chk1, which were then synchronized at G1/S boundary by DT treatment. HU or Vpr treated cells were collected 11 hours after DT release. The protein level of Cdc25C was detected by Western blot analysis using indicated antibodies (b).

Mentions: Since Vpr had little or no effect on Chk1-mediated Cdc25A protein degradation, we next examined whether Cdc25B/C are the substrates of Chk1 for Vpr to induce G2 arrest. Like that described above, the synchronized G1/S HeLa cells were treated with HU/UV or Adv-Vpr transduction, and the Cdc25B/C protein levels were compared by Western blot analyses using anti-Cdc25B or anti-Cdc25C antibodies. As shown in Figure 5A-a, second row, and Figure 5A-b, significant and gradual increases of Cdc25C were observed in the normal cells from 0 to 8 hours (Figure 5A-a, second row, and Figure 5A-b). HU-treatment resulted in small but perhaps insignificant decrease of Cdc25C over time; in contrast, Vpr induced a rather strong reduction of Cdc25C over time (Figure 5A-a, second row, lanes 8-10). To ascertain Vpr-mediated reduction of Cdc25C protein levels is also through proteasome-mediated proteolysis, cells were treated with the proteasome inhibitor MG132, the Chk1-specific siRNA or were untreated. Depletion of Chk1 restored the protein level of Cdc25C (Figure 5B-b, lanes 2 vs. 1). Normal protein level of Cdc25C was also seen when the same cells were treated with MG132 (Figure 5B-a). The depletion of Chk1 by siRNA was confirmed by Western blot analysis (Figure 5B-b, row 2).


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 promotes proteasome-mediated protein degradation of Cdc25B and Cdc25C. (A) Synchronized G1/S HeLa cells treated with HU or transduced with Adv-Vpr were collected at indicated time, and then subjected to Western blot analysis using anti-Cdc25B or anti-Cdc25C antibody, respectively (a). β-actin was used as a loading control. The relative intensity of the Cdc25B (b) or Cdc25C (c) protein levels to β-actin were determined by densitometry. The results presented are the average of three independent experiments. (B) Synchronized HeLa cells were pre-treated with specific siRNA against Chk1 or treated with 50 μm MG132 at 0 hour and collected at the indicated time. The protein level of Cdc25B was detected by Western blot analysis. (C) Synchronized HeLa cells were treated with 50 μm MG132 at 0 hour and collected 11 hours after treatment. The protein level of Cdc25C was detected by Western blot analysis (a). HeLa cells were pre-treated with specific siRNA against Chk1, which were then synchronized at G1/S boundary by DT treatment. HU or Vpr treated cells were collected 11 hours after DT release. The protein level of Cdc25C was detected by Western blot analysis using indicated antibodies (b).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2909154&req=5

Figure 5: Vpr promotes proteasome-mediated protein degradation of Cdc25B and Cdc25C. (A) Synchronized G1/S HeLa cells treated with HU or transduced with Adv-Vpr were collected at indicated time, and then subjected to Western blot analysis using anti-Cdc25B or anti-Cdc25C antibody, respectively (a). β-actin was used as a loading control. The relative intensity of the Cdc25B (b) or Cdc25C (c) protein levels to β-actin were determined by densitometry. The results presented are the average of three independent experiments. (B) Synchronized HeLa cells were pre-treated with specific siRNA against Chk1 or treated with 50 μm MG132 at 0 hour and collected at the indicated time. The protein level of Cdc25B was detected by Western blot analysis. (C) Synchronized HeLa cells were treated with 50 μm MG132 at 0 hour and collected 11 hours after treatment. The protein level of Cdc25C was detected by Western blot analysis (a). HeLa cells were pre-treated with specific siRNA against Chk1, which were then synchronized at G1/S boundary by DT treatment. HU or Vpr treated cells were collected 11 hours after DT release. The protein level of Cdc25C was detected by Western blot analysis using indicated antibodies (b).
Mentions: Since Vpr had little or no effect on Chk1-mediated Cdc25A protein degradation, we next examined whether Cdc25B/C are the substrates of Chk1 for Vpr to induce G2 arrest. Like that described above, the synchronized G1/S HeLa cells were treated with HU/UV or Adv-Vpr transduction, and the Cdc25B/C protein levels were compared by Western blot analyses using anti-Cdc25B or anti-Cdc25C antibodies. As shown in Figure 5A-a, second row, and Figure 5A-b, significant and gradual increases of Cdc25C were observed in the normal cells from 0 to 8 hours (Figure 5A-a, second row, and Figure 5A-b). HU-treatment resulted in small but perhaps insignificant decrease of Cdc25C over time; in contrast, Vpr induced a rather strong reduction of Cdc25C over time (Figure 5A-a, second row, lanes 8-10). To ascertain Vpr-mediated reduction of Cdc25C protein levels is also through proteasome-mediated proteolysis, cells were treated with the proteasome inhibitor MG132, the Chk1-specific siRNA or were untreated. Depletion of Chk1 restored the protein level of Cdc25C (Figure 5B-b, lanes 2 vs. 1). Normal protein level of Cdc25C was also seen when the same cells were treated with MG132 (Figure 5B-a). The depletion of Chk1 by siRNA was confirmed by Western blot analysis (Figure 5B-b, row 2).

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