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Published on: March 9, 2012
Interactions with DCAF1 and DDB1 in the CRL4 E3 ubiquitin ligase are required for Vpr-mediated G2 arrest
Yoshiyuki Hakata1, Masaaki Miyazawa, Nathaniel R Landau
1Department of Microbiology, New York University School of Medicine, 522 First Avenue, New York, NY 10016, USA. hakata@med.kindai.ac.jp.
Background:
HIV-1 Vpr-mediated G2 cell cycle arrest is dependent on the interaction of Vpr with an E3 ubiquitin ligase that contains damage-specific DNA binding protein 1 (DDB1), Cullin 4A (Cul4A), DDB1 and Cul4-associated factor 1 (DCAF1), and Rbx1. Vpr is thought to associate directly with DCAF1 in the E3 ubiquitin ligase complex although the exact interaction pattern of the proteins in the complex is not completely defined. The Vpr of SIVagm induces G2 arrest of cognate African Green Monkey (AGM) cells but not human cells. The molecular mechanism by which SIVagm Vpr exhibits its species-specific function remained unknown.
Methods:
Physical interaction of proteins in the E3 ubiquitin ligase complex was assessed by co-immunoprecipitation followed by western blotting. In addition, co-localization of the proteins in cells was investigated by confocal microscopy. The cell cycle was analyzed by propidium iodide staining and flow cytometry. DNA damage response elicited by Vpr was evaluated by detecting phosphorylation of H2AX, a marker for DNA damage response.
Results:
We show that RNAi knock-down of DCAF1 prevented the co-immunoprecipitation of DDB1 with HIV-1 Vpr while DDB1 knock-down did not influence the binding of Vpr to DCAF1. HIV-1 Vpr mutants with a L64P or a R90K mutation maintained the ability to associate with DCAF1 but did not appear to be in a complex with DDB1. SIVagm Vpr associated with AGM DCAF1 and DDB1 while, in human cells, it binds to human DCAF1 but hardly binds to human DDB1, resulting in the reduced activation of H2AX.
Conclusions:
The identification of Vpr mutants which associate with DCAF1 but only poorly with DDB1 suggests that DCAF1 is necessary but the simple binding of Vpr to DCAF1 is not sufficient for the Vpr association with DDB1-containing E3 ligase complex. Vpr may interact both with DCAF1 and DDB1 in the E3 ligase complex. Alternatively, the interaction of Vpr and DCAF1 may induce a conformational change in DCAF1 or Vpr that promotes the interaction with DDB1. The ability of SIVagm Vpr to associate with DDB1, but not DCAF1, can explain the species-specificity of SIVagm Vpr-mediated G2 arrest.
Insights
HIV-1 Vpr protein interacts with DCAF1 and DDB1 to cause cell cycle arrest. SIVagm Vpr
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- HIV-1 Vpr protein mediates G2 cell cycle arrest through interaction with an E3 ubiquitin ligase complex.
- This complex includes DDB1, Cul4A, DCAF1, and Rbx1, with Vpr potentially binding directly to DCAF1.
- The Vpr protein from SIVagm induces G2 arrest in African Green Monkey cells but not human cells, with the mechanism of this species-specificity being unclear.
Purpose of the Study:
- To elucidate the precise interaction pattern of HIV-1 Vpr within the DDB1-containing E3 ubiquitin ligase complex.
- To investigate the molecular mechanism underlying the species-specific G2 cell cycle arrest induced by SIVagm Vpr.
Main Methods:
- Co-immunoprecipitation and western blotting to assess physical protein interactions.
- Confocal microscopy to evaluate protein co-localization within cells.
- Flow cytometry with propidium iodide staining for cell cycle analysis.
- Detection of phosphorylated H2AX to evaluate DNA damage response.
Main Results:
- RNAi knockdown of DCAF1 disrupted the co-immunoprecipitation of DDB1 with HIV-1 Vpr.
- HIV-1 Vpr mutants (L64P, R90K) bound DCAF1 but showed reduced association with DDB1.
- SIVagm Vpr associated with both AGM DCAF1 and DDB1, but primarily with human DCAF1, with minimal binding to human DDB1, leading to reduced H2AX phosphorylation.
Conclusions:
- DCAF1 is essential for Vpr's association with the DDB1-containing E3 ligase complex, but binding to DCAF1 alone is insufficient.
- Vpr may interact with both DCAF1 and DDB1, or its interaction with DCAF1 may induce conformational changes facilitating DDB1 binding.
- The differential binding of SIVagm Vpr to DDB1 explains its species-specific G2 arrest activity.
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