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Lentiviral Vpr usurps Cul4-DDB1[VprBP] E3 ubiquitin ligase to modulate cell cycle.

ABSTRACT: The replication of viruses depends on the cell cycle status of the infected cells. Viruses have evolved functions that alleviate restrictions imposed on their replication by the host. Vpr, an accessory factor of primate lentiviruses, arrests cells at the DNA damage checkpoint in G2 phase of the cell cycle, but the mechanism underlying this effect has remained elusive. Here we report that Vpr proteins of both the human (HIV-1) and the distantly related simian (SIVmac) immunodeficiency viruses specifically associate with a protein complex comprising subunits of E3 ubiquitin ligase assembled on Cullin-4 scaffold (Cul4-DDB1[VprBP]). We show that Vpr binding to Cul4-DDB1[VprBP] leads to increased neddylation and elevated intrinsic ubiquitin ligase activity of this E3. This effect is mediated through the VprBP subunit of the complex, which recently has been suggested to function as a substrate receptor for Cul4. We also demonstrate that VprBP regulates G1 phase and is essential for the completion of DNA replication in S phase. Furthermore, the ability of Vpr to arrest cells in G2 phase correlates with its ability to interact with Cul4-DDB1[VprBP] E3 complex. Our studies identify the Cul4-DDB1[VprBP] E3 ubiquitin ligase complex as the downstream effector of lentiviral Vpr for the induction of cell cycle arrest in G2 phase and suggest that Vpr may use this complex to perturb other aspects of the cell cycle and DNA metabolism in infected cells.

SUBMITTER: Hrecka K

PROVIDER: S-EPMC1906728 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundHIV-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.MethodsPhysical 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.ResultsWe 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.ConclusionsThe 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.

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Lentiviral Vpr usurps Cul4-DDB1[VprBP] E3 ubiquitin ligase to modulate cell cycle.

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