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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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Co-immunoprecipitation studies with Vpr, DCAF1 and DDB1. A. Flag-DCAF1 was cotransfected into HeLa cells with either HA-Vpr or HA-Vpr(R80A). Immunoprecipitation was performed with either HA or Flag antibody as indicated. Immunoprecipitates were analyzed by WB for the presence of Flag-DCAF1 or HA-Vpr. B. GFP, HA-Vpr, HA-Vpr(R80A), or HA-Vpr(Q65R) constructs were transfected, and immunoprecipitations with HA antibody were performed. Immunoprecipitates were analyzed by WB with an antibody against endogenous DDB1. C. Knockdown of DCAF1 abolishes the association between Vpr and DDB1; HeLa cells were transected with HA-Vpr, in the presence of non-specific (NS) or DCAF1-specific siRNA. 48 hours later, cell extracts were immunoprecipitated with HA antibody and analyzed by WB for the presence of endogenous DDB1. D. HA-Vpr(Q65R) fails to interact with DCAF1.
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Figure 3: Co-immunoprecipitation studies with Vpr, DCAF1 and DDB1. A. Flag-DCAF1 was cotransfected into HeLa cells with either HA-Vpr or HA-Vpr(R80A). Immunoprecipitation was performed with either HA or Flag antibody as indicated. Immunoprecipitates were analyzed by WB for the presence of Flag-DCAF1 or HA-Vpr. B. GFP, HA-Vpr, HA-Vpr(R80A), or HA-Vpr(Q65R) constructs were transfected, and immunoprecipitations with HA antibody were performed. Immunoprecipitates were analyzed by WB with an antibody against endogenous DDB1. C. Knockdown of DCAF1 abolishes the association between Vpr and DDB1; HeLa cells were transected with HA-Vpr, in the presence of non-specific (NS) or DCAF1-specific siRNA. 48 hours later, cell extracts were immunoprecipitated with HA antibody and analyzed by WB for the presence of endogenous DDB1. D. HA-Vpr(Q65R) fails to interact with DCAF1.

Mentions: We transfected a Flag-DCAF1 construct along with either HA-Vpr or HA-Vpr(R80A), a Vpr mutant that is incapable of inducing G2 arrest [2,18,25] and, 48 hours later, we immunoprecipitated Flag-DCAF1 from cell extracts. When immunoprecipitates obtained with anti-HA antibody (specific for HA-Vpr) were analyzed by WB for the presence of Flag-DCAF1 (Figure 3, panel A), the presence of a reactive band of the expected molecular weight was evident both for HA-Vpr(R80A) (lane 5) and HA-Vpr (lane 6). This immunoprecipitation was reproduced when performed in the reciprocal order (lanes 8 and 9). From these experiments, we conclude that Vpr and DCAF1 physically interact.


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)

Co-immunoprecipitation studies with Vpr, DCAF1 and DDB1. A. Flag-DCAF1 was cotransfected into HeLa cells with either HA-Vpr or HA-Vpr(R80A). Immunoprecipitation was performed with either HA or Flag antibody as indicated. Immunoprecipitates were analyzed by WB for the presence of Flag-DCAF1 or HA-Vpr. B. GFP, HA-Vpr, HA-Vpr(R80A), or HA-Vpr(Q65R) constructs were transfected, and immunoprecipitations with HA antibody were performed. Immunoprecipitates were analyzed by WB with an antibody against endogenous DDB1. C. Knockdown of DCAF1 abolishes the association between Vpr and DDB1; HeLa cells were transected with HA-Vpr, in the presence of non-specific (NS) or DCAF1-specific siRNA. 48 hours later, cell extracts were immunoprecipitated with HA antibody and analyzed by WB for the presence of endogenous DDB1. D. HA-Vpr(Q65R) fails to interact with DCAF1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Co-immunoprecipitation studies with Vpr, DCAF1 and DDB1. A. Flag-DCAF1 was cotransfected into HeLa cells with either HA-Vpr or HA-Vpr(R80A). Immunoprecipitation was performed with either HA or Flag antibody as indicated. Immunoprecipitates were analyzed by WB for the presence of Flag-DCAF1 or HA-Vpr. B. GFP, HA-Vpr, HA-Vpr(R80A), or HA-Vpr(Q65R) constructs were transfected, and immunoprecipitations with HA antibody were performed. Immunoprecipitates were analyzed by WB with an antibody against endogenous DDB1. C. Knockdown of DCAF1 abolishes the association between Vpr and DDB1; HeLa cells were transected with HA-Vpr, in the presence of non-specific (NS) or DCAF1-specific siRNA. 48 hours later, cell extracts were immunoprecipitated with HA antibody and analyzed by WB for the presence of endogenous DDB1. D. HA-Vpr(Q65R) fails to interact with DCAF1.
Mentions: We transfected a Flag-DCAF1 construct along with either HA-Vpr or HA-Vpr(R80A), a Vpr mutant that is incapable of inducing G2 arrest [2,18,25] and, 48 hours later, we immunoprecipitated Flag-DCAF1 from cell extracts. When immunoprecipitates obtained with anti-HA antibody (specific for HA-Vpr) were analyzed by WB for the presence of Flag-DCAF1 (Figure 3, panel A), the presence of a reactive band of the expected molecular weight was evident both for HA-Vpr(R80A) (lane 5) and HA-Vpr (lane 6). This immunoprecipitation was reproduced when performed in the reciprocal order (lanes 8 and 9). From these experiments, we conclude that Vpr and DCAF1 physically interact.

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