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HIV-1 Vpr activates the G2 checkpoint through manipulation of the ubiquitin proteasome system.

DeHart JL, Zimmerman ES, Ardon O, Monteiro-Filho CM, ArgaƱaraz ER, Planelles V - Virol. J. (2007)

Bottom Line: We found that Vpr binds to the DCAF1 subunit of a cullin 4a/DDB1 E3 ubiquitin ligase.In contrast, the mutation Q65R, in the leucine-rich domain of Vpr that mediates DCAF1 binding, results in an inactive Vpr devoid of dominant negative behavior.Recruitment of this factor leads to its ubiquitination and degradation, resulting in failure to enter mitosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cell Biology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84112, USA. jason.dehart@path.utah.edu

ABSTRACT
HIV-1 Vpr is a viral accessory protein that activates ATR through the induction of DNA replication stress. ATR activation results in cell cycle arrest in G2 and induction of apoptosis. In the present study, we investigate the role of the ubiquitin/proteasome system (UPS) in the above activity of Vpr. We report that the general function of the UPS is required for Vpr to induce G2 checkpoint activation, as incubation of Vpr-expressing cells with proteasome inhibitors abolishes this effect. We further investigated in detail the specific E3 ubiquitin ligase subunits that Vpr manipulates. We found that Vpr binds to the DCAF1 subunit of a cullin 4a/DDB1 E3 ubiquitin ligase. The carboxy-terminal domain Vpr(R80A) mutant, which is able to bind DCAF1, is inactive in checkpoint activation and has dominant-negative character. In contrast, the mutation Q65R, in the leucine-rich domain of Vpr that mediates DCAF1 binding, results in an inactive Vpr devoid of dominant negative behavior. Thus, the interaction of Vpr with DCAF1 is required, but not sufficient, for Vpr to cause G2 arrest. We propose that Vpr recruits, through its carboxy terminal domain, an unknown cellular factor that is required for G2-to-M transition. Recruitment of this factor leads to its ubiquitination and degradation, resulting in failure to enter mitosis.

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Vpr(R80A) functions as a dominant-negative molecule. HeLa cells were infected with a constant MOI (MOI = 1) of pHR-VPR vector, and a variable MOI (1, 0.5, 0.25) of pHR-GFP, pHR- Vpr(R80A) or pHR-VPR(Q65R, R80A) as indicated. Cell cycle profiles were evaluated at 48 hours post transduction.
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Figure 4: Vpr(R80A) functions as a dominant-negative molecule. HeLa cells were infected with a constant MOI (MOI = 1) of pHR-VPR vector, and a variable MOI (1, 0.5, 0.25) of pHR-GFP, pHR- Vpr(R80A) or pHR-VPR(Q65R, R80A) as indicated. Cell cycle profiles were evaluated at 48 hours post transduction.

Mentions: In order to generate a more appropriate negative control for IP experiments, we constructed the mutation Vpr(Q65R), which disrupts a leucine-rich region required for binding to DCAF1 [15]. Vpr(Q65R) failed to associate with DDB1 (Figure 3, panel B, lane 8), DCAF1 (Figure 3, panel D, lane 6), and also failed to induce G2 arrest (Figure 4).


HIV-1 Vpr activates the G2 checkpoint through manipulation of the ubiquitin proteasome system.

DeHart JL, Zimmerman ES, Ardon O, Monteiro-Filho CM, ArgaƱaraz ER, Planelles V - Virol. J. (2007)

Vpr(R80A) functions as a dominant-negative molecule. HeLa cells were infected with a constant MOI (MOI = 1) of pHR-VPR vector, and a variable MOI (1, 0.5, 0.25) of pHR-GFP, pHR- Vpr(R80A) or pHR-VPR(Q65R, R80A) as indicated. Cell cycle profiles were evaluated at 48 hours post transduction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1904188&req=5

Figure 4: Vpr(R80A) functions as a dominant-negative molecule. HeLa cells were infected with a constant MOI (MOI = 1) of pHR-VPR vector, and a variable MOI (1, 0.5, 0.25) of pHR-GFP, pHR- Vpr(R80A) or pHR-VPR(Q65R, R80A) as indicated. Cell cycle profiles were evaluated at 48 hours post transduction.
Mentions: In order to generate a more appropriate negative control for IP experiments, we constructed the mutation Vpr(Q65R), which disrupts a leucine-rich region required for binding to DCAF1 [15]. Vpr(Q65R) failed to associate with DDB1 (Figure 3, panel B, lane 8), DCAF1 (Figure 3, panel D, lane 6), and also failed to induce G2 arrest (Figure 4).

Bottom Line: We found that Vpr binds to the DCAF1 subunit of a cullin 4a/DDB1 E3 ubiquitin ligase.In contrast, the mutation Q65R, in the leucine-rich domain of Vpr that mediates DCAF1 binding, results in an inactive Vpr devoid of dominant negative behavior.Recruitment of this factor leads to its ubiquitination and degradation, resulting in failure to enter mitosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Cell Biology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84112, USA. jason.dehart@path.utah.edu

ABSTRACT
HIV-1 Vpr is a viral accessory protein that activates ATR through the induction of DNA replication stress. ATR activation results in cell cycle arrest in G2 and induction of apoptosis. In the present study, we investigate the role of the ubiquitin/proteasome system (UPS) in the above activity of Vpr. We report that the general function of the UPS is required for Vpr to induce G2 checkpoint activation, as incubation of Vpr-expressing cells with proteasome inhibitors abolishes this effect. We further investigated in detail the specific E3 ubiquitin ligase subunits that Vpr manipulates. We found that Vpr binds to the DCAF1 subunit of a cullin 4a/DDB1 E3 ubiquitin ligase. The carboxy-terminal domain Vpr(R80A) mutant, which is able to bind DCAF1, is inactive in checkpoint activation and has dominant-negative character. In contrast, the mutation Q65R, in the leucine-rich domain of Vpr that mediates DCAF1 binding, results in an inactive Vpr devoid of dominant negative behavior. Thus, the interaction of Vpr with DCAF1 is required, but not sufficient, for Vpr to cause G2 arrest. We propose that Vpr recruits, through its carboxy terminal domain, an unknown cellular factor that is required for G2-to-M transition. Recruitment of this factor leads to its ubiquitination and degradation, resulting in failure to enter mitosis.

Show MeSH