Lentiviral Vpr usurps Cul4-DDB1[VprBP] E3 ubiquitin ligase to modulate cell cycle - PubMed (original) (raw)

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

Kasia Hrecka et al. Proc Natl Acad Sci U S A. 2007.

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Vpr binds DDA1–DDB1–VprBP complex. (A) Biochemical interactions among Vpr, VprBP, DDB1, and DDA1. Detergent extracts from HEK293T cells transiently expressing FLAG-tagged HIV-1 Vpr (lane 2), VprBP (lane 3), DDB1 (lane 4), or DDA1 (lane 5) subunits were immunoprecipitated (IP) with α-FLAG beads, and immunoprecipitates were analyzed by immunoblotting for VprBP, DDB1, DDA1, Vpr, and COP9 signalosome subunits CSN3 and CSN8. (B) Vpr binds the DDA1–DDB1–VprBP complex. FLAG-VprBP (Upper) and FLAG-Vpr (Lower) containing complexes were sedimented through 10–40% glycerol gradients. Aliquots of fractions collected from the tops of the gradients were immunoblotted for the indicated proteins.

Fig. 2.

Fig. 2.

Vpr elevates neddylation and intrinsic ubiquitin-ligase activity of the Cul4–DDB1[VprBP] E3 complex. (A) Vpr binds to and specifically elevates neddylation of Cul4A associated with VprBP. Myc-tagged Cullin 4A (_m_-Cul4) was coexpressed with Vpr, VprBP, and DDB2, in various combinations, in HEK293T cells. FLAG-tagged (f) versions of these proteins were used in some experiments to facilitate their immunoprecipitation. _m_-Cul4A (arrow) and its neddylated form (∗) were detected in detergent extracts (Extr) and α-FLAG immune complexes (IP) by immunoblotting with α-myc and α-Nedd8 antibodies, respectively. (B) Vpr stimulates intrinsic ubiquitin ligase activity of the Cul4–DDB1[VprBP] E3 complex. _m_-Cul4–DDB1[VprBP] complexes were assembled with or without Vpr in HEK293T cells, purified via their FLAG-VprBP subunits, and incubated with E1, ubiquitin, and/or E2 as indicated. Cul4A and its ubiquitinated forms were detected by immunoblotting for Cul4.

Fig. 3.

Fig. 3.

VprBP depletion leads to G1 and G2 phase arrests. (A) The effect of RNAi to VprBP on the ability of Vpr to arrest cells in G2. U2OS cells expressing shRNAs to VprBP (Lower) or DOCK2 (Upper) were transduced with a lentiviral TEIG vector expressing HIV-1 Vpr (Vpr) or a control empty vector (Vector). Three days later, the cells were stained with PI, and the DNA content was analyzed by flow cytometry. (B) VprBP-depleted cells arrest in G1 and G2. VprBP-depleted (Lower) and control cell populations (Upper) were labeled with BrdU for 1 h. BrdU incorporation and DNA content were analyzed by flow cytometry either immediately after BrdU labeling or after 6 and 12 h chase. Bivariant distributions of BrdU incorporation and DNA content are shown. The percent fractions of cells in the G1 (Lower Left), S (Upper), and G2 (Lower Right) phases are indicated. (C) Histone γ-H2A.X and cyclin A expression in VprBP-depleted (VprBP) and control cells (vector) were revealed by indirect fluorescence. Cells were counterstained with DAPI, and fluorescent signals were imaged with an iCys laser scanning cytometer. Histograms of DNA content and bivariate distributions of γ-H2A.X or cyclin A fluorescence versus DNA content are shown. (D) Levels of cyclins, checkpoint, and replication licensing proteins in lysates of VprBP-depleted and control cells were analyzed by immunoblotting with antibodies to the indicated proteins. Splicing factor 2 (SF2) was used as a loading control. The asterisk indicates a band reacting nonspecifically with the α-VprBP IgG. Lanes 1 and 3 contain 3-fold dilutions of the amounts loaded in lanes 2 and 4, respectively.

Fig. 4.

Fig. 4.

Effects of mutations on Vpr abilities to arrest cells in the G2 phase, bind VprBP and DDB1, and elevate Cul4 neddylation. (A) Mutations disrupt the ability of Vpr to arrest cells in G2. U2OS cells were transduced with retroviral vectors expressing wild type (HIV-1 Vpr), the indicated mutant forms of HIV-1 Vpr, or a control empty vector (mock). Two days later, cells were labeled with BrdU, and their cell cycle profiles were analyzed by flow cytometry. (Left) Quantification of cells in the G1, S, and G2/M phases is shown. Cells with >4N DNA content are boxed. (Right) DNA content histograms also are shown. (B) Vpr mutations disrupt binding to DDB1 and VprBP. FLAG-tagged wild-type and mutant HIV-1 Vpr proteins were immunoprecipitated from transiently transfected HEK293T cells, and immune complexes were analyzed by immunoblotting for VprBP, DDB1, and Vpr. (C) Vpr mutants unable to arrest cells in G2 do not increase Cul4 neddylation. Wild-type and mutant HIV-1 Vpr proteins were transiently coexpressed with myc-Cul4A and VprBP in HEK293T cells, as described in the legend for Fig. 2_A_. Detergent extracts were immunoblotted for Cul4A and its neddylated form with α-myc antibody.

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