Interactions with DCAF1 and DDB1 in the CRL4 E3 ubiquitin ligase are required for Vpr-mediated G2 arrest

Abstract

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.

Figure 1

DCAF1 is required for the association of Vpr with DDB1. (A) A model of the Vpr-CRL4-DCAF1 E3 ubiquitin ligase complex in which Vpr directly contacts DCAF1. (B) HeLa cells were transfected with siRNAs targeting DCAF1 or DDB1. A control siRNA is included and two siRNAs were used against DCAF1. A day later, the cells were transfected again with HA-Vpr expression vector. After another two days, HA-Vpr complexes were immunoprecipitated with anti-HA antibody. Co-immunoprecipitated DCAF1 and DDB1 were detected on an immunoblot. A nonspecific band is indicated with an asterisk.

Figure 2

Vpr-DCAF interaction is not sufficient for Vpr association with DDB1. (A) 293 T cells were cotransfected with HA-Vpr or HA-VprL64P and Flag-DCAF1 expression vectors. Vpr was immunoprecipitated with anti-HA antibody and the immunoprecipitates were subjected to immunoblot analysis with anti-Flag MAb, anti-DDB1 antibody, and anti-HA antibody. (B) HA-Vpr, HA-VprR90K, or HA-VprR90D were expressed with Flag-DCAF1 and were immunoprecipitated with anti-HA antibody. Coimmunoprecipitated DDB1 and Flag-DCAF1 were detected on the immunoblot. (C) 293 T cells were transfected with increasing amounts (0.05 μg, 0.1 μg, and 0.2 μg) of HA-Vpr, HA-VprR90K, or HA-VprR90D expression vector together with a constant amount of HA-UNG2 and Flag-DCAF1 expression vectors. Two days later, the cells were lysed and Vpr and UNG2 were detected by immunoblot analysis with anti-HA antibody. The βtubulin was detected as a loading control. (D) UNG2 band intensities obtained in (C) were quantified and normalized to the UNG2 signal of the third lane from the left in (C). The results are representative data of three independent experiments.

Figure 3

The species-specificity of SIV_agm Vpr is correlated with an inability to associate with DDB1 in human cells. (A) 293 T cells were transfected with SIV_mac Vpr or SIV_agm Vpr, Flag-DCAF1, and EGFP expression vectors. After two days, the cells were fixed, stained with propidium iodide and analyzed by flow cytometry. The G₂:G₁ ratio was calculated after gating for the GFP⁺ cells. The results are representative of three independent experiments. (B) COS cells were transfected and the cell cycle profiles were analyzed as in (A). (C) 293 T cells were cotransfected with myc-Vpr of SIV_mac or SIV_agm and Flag-DCAF1 expression vectors. Myc-Vpr was immunoprecipitated with anti-myc MAb and the immunoprecipitated complexes were analyzed on an immunoblot probed with anti-Flag MAb, anti-DDB1 antibody, and anti-myc MAb. (D) COS cells were transfected with myc-Vpr of SIV_mac or SIV_agm. Myc-Vpr was immunoprecipitated with anti-myc MAb and coimmunoprecipitated endogenous AGM DCAF1 and DDB1 were detected with anti-DCAF1 and anti-DDB1 antibodies.

Figure 4

In human cells, SIV_agm Vpr does associates less efficiently with the CRL4 E3 ubiquitin ligase. (A) 293 T cells were cotransfected with HA-Vpr, HA-VprR90K, or SIV_agm HA-Vpr and Flag-DCAF1 expression vectors. Vpr was immunoprecipitated with anti-HA antibody and the immunoprecipitates were analyzed on an immunoblot probed with anti-Flag MAb, anti-DDB1 antibody, and anti-HA antibody. (B) HeLa cells were transfected with HA-Vpr or SIV_agm HA-Vpr expression vector. The transfected cells were permeabilized, fixed, and then incubated with anti-DDB1 and anti-HA antibodies followed by Alexa Fluor 594-anti-rabbit IgG and Alexa Fluor 488-anti-rat IgG. Representative images from an HA-Vpr or SIV_agm HA-Vpr expressing sample are shown. Arrows indicate Vpr foci and DDB1 dots which colocalize. (C) The percentage of Vpr foci colocalized with DDB1 among total Vpr foci was calculated. More than 170 Vpr foci were evaluated for each sample and three independent experiments were done. The data are the mean values with standard deviations. P values were calculated by the Student’s t-test with P < 0.05 considered significant.

Figure 5

SIV_agm Vpr induces less DNA damage than HIV-1 Vpr. (A) HeLa cells were transfected with HA-Vpr or SIV_agm HA-Vpr expression vector. After two days, the cells were fixed, permeabilized and incubated with anti-γH2AX and anti-HA antibodies. Subsequently, the cells were stained with Alexa Fluor 594-anti-rabbit IgG and Alexa Fluor 488-anti-rat IgG. (B) The number of γH2AX dots in Vpr expressing cells was counted. More than 40 Vpr + cells were evaluated in each experiment. Control sample was transfected with pcDNA3.1 instead of Vpr expression vector. The data represent means with standard deviations from three independent experiments. P values were calculated by Student’s t-test with P < 0.05 considered as significant.