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Defining the interactions and role of DCAF1/VPRBP in the DDB1-cullin4A E3 ubiquitin ligase complex engaged by HIV-1 Vpr to induce a G2 cell cycle arrest.

Gérard FC, Yang R, Romani B, Poisson A, Belzile JP, Rougeau N, Cohen ÉA - PLoS ONE (2014)

Bottom Line: Herein, we show that the minimal domain of DCAF1 that retained the ability to bind Vpr and DDB1 was mapped to residues 1041 to 1393 (DCAF1 WD).Mutagenic analyses identified an α-helical H-box motif and F/YxxF/Y motifs located in the N-terminal domain of DCAF1 WD that are involved in exclusive binding to DDB1.Importantly, we provide evidence that binding of Vpr to DCAF1 appears to modulate the formation of a DDB1/DCAF1 complex.

View Article: PubMed Central - PubMed

Affiliation: Institut de Recherches Cliniques de Montréal (IRCM), Montréal, Québec, Canada.

ABSTRACT
HIV viral protein R (Vpr) induces a cell cycle arrest at the G2/M phase by activating the ATR DNA damage/replication stress signalling pathway through engagement of the DDB1-CUL4A-DCAF1 E3 ubiquitin ligase via a direct binding to the substrate specificity receptor DCAF1. Since no high resolution structures of the DDB1-DCAF1-Vpr substrate recognition module currently exist, we used a mutagenesis approach to better define motifs in DCAF1 that are crucial for Vpr and DDB1 binding. Herein, we show that the minimal domain of DCAF1 that retained the ability to bind Vpr and DDB1 was mapped to residues 1041 to 1393 (DCAF1 WD). Mutagenic analyses identified an α-helical H-box motif and F/YxxF/Y motifs located in the N-terminal domain of DCAF1 WD that are involved in exclusive binding to DDB1. While we could not identify elements specifically involved in Vpr binding, overall, the mutagenesis data suggest that the predicted β-propeller conformation of DCAF1 is likely to be critical for Vpr association. Importantly, we provide evidence that binding of Vpr to DCAF1 appears to modulate the formation of a DDB1/DCAF1 complex. Lastly, we show that expression of DCAF1 WD in the absence of endogenous DCAF1 was not sufficient to enable Vpr-mediated G2 arrest activity. Overall, our results reveal that Vpr and DDB1 binding on DCAF1 can be genetically separated and further suggest that DCAF1 contains determinants in addition to the Vpr and DDB1 minimal binding domain, which are required for Vpr to enable the induction of a G2 arrest.

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Mutations in the putative H-box motif of DCAF1 abrogate DDB1 binding without affecting Vpr interaction.A. Sequence alignment of the H-box motifs of indicated cellular DCAF’s and viral proteins. HBX and WHX indicate viral protein X from Hepatitis B virus and Woodchuck Hepatitis virus, respectively, while SV5-V represents viral protein V encoded by paramyxovirus simian virus 5. Residues of the H-box motif previously reported to contact DDB1 are boxed. Alignment adapted from Li & al.[21]. The asterisks indicate the position of substitution mutation (L1054 and R1057). B. HEK293T cells were mock-transfected (lanes 1 and 2) or transfected with Myc-DCAF1 WD (lanes 3 and 4), Myc-DCAF1 WD L1054P (lanes 5 and 6) or with Myc-DCAF1 WD R1057E (lanes 7 and 8) -encoding plasmids in the presence of empty vector (lanes 1, 3, 5 and 7) or HA-tagged Vpr-expressing plasmid (lanes 2, 4, 6, and 8). Total amounts of DNA were adjusted with empty vector so that similar quantities of plasmids were transfected in each sample. Immunoprecipitations were performed on cell extracts using anti-Myc antibodies. The levels of HA-Vpr, endogenous DDB1, Myc-DCAF1 WD (WT and mutants) and actin were monitored in cell extracts as well as, when applicable, in immunoprecipitated fractions by Western Blot using specific antibodies. * denotes the light chain of the IgG used for immunoprecipitation. C. Quantitation of DDB1 and HA-Vpr binding to Myc-DCAF1 WD. The percentage of DDB1 or HA-Vpr bound to Myc-DCAF1 WD was determined by evaluating the ratio of DDB1 or HA-Vpr band signal over that of Myc-DCAF1 WD WT or mutant in the immunoprecipitated fractions. Ratios obtained with Myc-DCAF1 WD WT were assigned a value of 100%. Error bars indicate the SEM from the quantitative analysis of at least 3 independent experiments. Statistical analysis was performed as described in the Experimental Procedures (p<0.05; ns: non significant).
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pone-0089195-g002: Mutations in the putative H-box motif of DCAF1 abrogate DDB1 binding without affecting Vpr interaction.A. Sequence alignment of the H-box motifs of indicated cellular DCAF’s and viral proteins. HBX and WHX indicate viral protein X from Hepatitis B virus and Woodchuck Hepatitis virus, respectively, while SV5-V represents viral protein V encoded by paramyxovirus simian virus 5. Residues of the H-box motif previously reported to contact DDB1 are boxed. Alignment adapted from Li & al.[21]. The asterisks indicate the position of substitution mutation (L1054 and R1057). B. HEK293T cells were mock-transfected (lanes 1 and 2) or transfected with Myc-DCAF1 WD (lanes 3 and 4), Myc-DCAF1 WD L1054P (lanes 5 and 6) or with Myc-DCAF1 WD R1057E (lanes 7 and 8) -encoding plasmids in the presence of empty vector (lanes 1, 3, 5 and 7) or HA-tagged Vpr-expressing plasmid (lanes 2, 4, 6, and 8). Total amounts of DNA were adjusted with empty vector so that similar quantities of plasmids were transfected in each sample. Immunoprecipitations were performed on cell extracts using anti-Myc antibodies. The levels of HA-Vpr, endogenous DDB1, Myc-DCAF1 WD (WT and mutants) and actin were monitored in cell extracts as well as, when applicable, in immunoprecipitated fractions by Western Blot using specific antibodies. * denotes the light chain of the IgG used for immunoprecipitation. C. Quantitation of DDB1 and HA-Vpr binding to Myc-DCAF1 WD. The percentage of DDB1 or HA-Vpr bound to Myc-DCAF1 WD was determined by evaluating the ratio of DDB1 or HA-Vpr band signal over that of Myc-DCAF1 WD WT or mutant in the immunoprecipitated fractions. Ratios obtained with Myc-DCAF1 WD WT were assigned a value of 100%. Error bars indicate the SEM from the quantitative analysis of at least 3 independent experiments. Statistical analysis was performed as described in the Experimental Procedures (p<0.05; ns: non significant).

Mentions: In order to assess the critical motifs involved in Vpr and/or DDB1 binding, we performed a mutagenic analysis of the different motifs present in DCAF1 WD, and characterized the complexes formed between endogenous DDB1, Vpr and the resulting DCAF1 mutant proteins. Alignment of the DCAF1 putative H-box motif with those previously found in several DCAF’s and viral proteins, such as HBX and SV5-V, revealed a moderate degree of amino-acid conservation at specific positions (Fig. 2A). Conserved leucine and arginine residues at position 1054 and 1057, respectively, were selected for single-point mutation. Leucine 1054 was mutated to proline to completely disrupt the predicted α-helix, while arginine 1057 was substituted for glutamic acid since changing the charge at that position in the HBX H-box motif impaired the recruitment of the DDB1-CRL4A E3 ubiquitin ligase [21]. The data of figure 2 reveals that DCAF1 WD L1054P and DCAF1 WD R1057E completely lost their ability to recruit DDB1 (Fig 2B, lanes 5 and 7 and Fig. 2C), yet they retained the capacity to interact with Vpr (Fig. 2B, compare lanes 4, 6 and 8) almost as efficiently as DCAF WD WT (Fig. 2C). Consistent with the data shown in figure 1, Vpr did not appear to modify the amounts of DDB1 bound to DCAF1 WD (Fig. 2B, lanes 3 and 4, and Fig. 2C) and the presence of Vpr in the mutant DCAF1 WD-containing complexes did not restore any binding to endogenous DDB1 (lanes 6 and 8), thus indicating that DCAF1 acts as a bridge between DDB1 and Vpr within the ternary complex. Taken together, these results suggest that the region spanning residues 1049 to 1061 (NFTSRLNRRASSFP) in DCAF1 is likely to contain the H-box motif required for DDB1 binding and further reveal that the domains of DCAF1 responsible for DDB1 and Vpr binding can be genetically separated.


Defining the interactions and role of DCAF1/VPRBP in the DDB1-cullin4A E3 ubiquitin ligase complex engaged by HIV-1 Vpr to induce a G2 cell cycle arrest.

Gérard FC, Yang R, Romani B, Poisson A, Belzile JP, Rougeau N, Cohen ÉA - PLoS ONE (2014)

Mutations in the putative H-box motif of DCAF1 abrogate DDB1 binding without affecting Vpr interaction.A. Sequence alignment of the H-box motifs of indicated cellular DCAF’s and viral proteins. HBX and WHX indicate viral protein X from Hepatitis B virus and Woodchuck Hepatitis virus, respectively, while SV5-V represents viral protein V encoded by paramyxovirus simian virus 5. Residues of the H-box motif previously reported to contact DDB1 are boxed. Alignment adapted from Li & al.[21]. The asterisks indicate the position of substitution mutation (L1054 and R1057). B. HEK293T cells were mock-transfected (lanes 1 and 2) or transfected with Myc-DCAF1 WD (lanes 3 and 4), Myc-DCAF1 WD L1054P (lanes 5 and 6) or with Myc-DCAF1 WD R1057E (lanes 7 and 8) -encoding plasmids in the presence of empty vector (lanes 1, 3, 5 and 7) or HA-tagged Vpr-expressing plasmid (lanes 2, 4, 6, and 8). Total amounts of DNA were adjusted with empty vector so that similar quantities of plasmids were transfected in each sample. Immunoprecipitations were performed on cell extracts using anti-Myc antibodies. The levels of HA-Vpr, endogenous DDB1, Myc-DCAF1 WD (WT and mutants) and actin were monitored in cell extracts as well as, when applicable, in immunoprecipitated fractions by Western Blot using specific antibodies. * denotes the light chain of the IgG used for immunoprecipitation. C. Quantitation of DDB1 and HA-Vpr binding to Myc-DCAF1 WD. The percentage of DDB1 or HA-Vpr bound to Myc-DCAF1 WD was determined by evaluating the ratio of DDB1 or HA-Vpr band signal over that of Myc-DCAF1 WD WT or mutant in the immunoprecipitated fractions. Ratios obtained with Myc-DCAF1 WD WT were assigned a value of 100%. Error bars indicate the SEM from the quantitative analysis of at least 3 independent experiments. Statistical analysis was performed as described in the Experimental Procedures (p<0.05; ns: non significant).
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pone-0089195-g002: Mutations in the putative H-box motif of DCAF1 abrogate DDB1 binding without affecting Vpr interaction.A. Sequence alignment of the H-box motifs of indicated cellular DCAF’s and viral proteins. HBX and WHX indicate viral protein X from Hepatitis B virus and Woodchuck Hepatitis virus, respectively, while SV5-V represents viral protein V encoded by paramyxovirus simian virus 5. Residues of the H-box motif previously reported to contact DDB1 are boxed. Alignment adapted from Li & al.[21]. The asterisks indicate the position of substitution mutation (L1054 and R1057). B. HEK293T cells were mock-transfected (lanes 1 and 2) or transfected with Myc-DCAF1 WD (lanes 3 and 4), Myc-DCAF1 WD L1054P (lanes 5 and 6) or with Myc-DCAF1 WD R1057E (lanes 7 and 8) -encoding plasmids in the presence of empty vector (lanes 1, 3, 5 and 7) or HA-tagged Vpr-expressing plasmid (lanes 2, 4, 6, and 8). Total amounts of DNA were adjusted with empty vector so that similar quantities of plasmids were transfected in each sample. Immunoprecipitations were performed on cell extracts using anti-Myc antibodies. The levels of HA-Vpr, endogenous DDB1, Myc-DCAF1 WD (WT and mutants) and actin were monitored in cell extracts as well as, when applicable, in immunoprecipitated fractions by Western Blot using specific antibodies. * denotes the light chain of the IgG used for immunoprecipitation. C. Quantitation of DDB1 and HA-Vpr binding to Myc-DCAF1 WD. The percentage of DDB1 or HA-Vpr bound to Myc-DCAF1 WD was determined by evaluating the ratio of DDB1 or HA-Vpr band signal over that of Myc-DCAF1 WD WT or mutant in the immunoprecipitated fractions. Ratios obtained with Myc-DCAF1 WD WT were assigned a value of 100%. Error bars indicate the SEM from the quantitative analysis of at least 3 independent experiments. Statistical analysis was performed as described in the Experimental Procedures (p<0.05; ns: non significant).
Mentions: In order to assess the critical motifs involved in Vpr and/or DDB1 binding, we performed a mutagenic analysis of the different motifs present in DCAF1 WD, and characterized the complexes formed between endogenous DDB1, Vpr and the resulting DCAF1 mutant proteins. Alignment of the DCAF1 putative H-box motif with those previously found in several DCAF’s and viral proteins, such as HBX and SV5-V, revealed a moderate degree of amino-acid conservation at specific positions (Fig. 2A). Conserved leucine and arginine residues at position 1054 and 1057, respectively, were selected for single-point mutation. Leucine 1054 was mutated to proline to completely disrupt the predicted α-helix, while arginine 1057 was substituted for glutamic acid since changing the charge at that position in the HBX H-box motif impaired the recruitment of the DDB1-CRL4A E3 ubiquitin ligase [21]. The data of figure 2 reveals that DCAF1 WD L1054P and DCAF1 WD R1057E completely lost their ability to recruit DDB1 (Fig 2B, lanes 5 and 7 and Fig. 2C), yet they retained the capacity to interact with Vpr (Fig. 2B, compare lanes 4, 6 and 8) almost as efficiently as DCAF WD WT (Fig. 2C). Consistent with the data shown in figure 1, Vpr did not appear to modify the amounts of DDB1 bound to DCAF1 WD (Fig. 2B, lanes 3 and 4, and Fig. 2C) and the presence of Vpr in the mutant DCAF1 WD-containing complexes did not restore any binding to endogenous DDB1 (lanes 6 and 8), thus indicating that DCAF1 acts as a bridge between DDB1 and Vpr within the ternary complex. Taken together, these results suggest that the region spanning residues 1049 to 1061 (NFTSRLNRRASSFP) in DCAF1 is likely to contain the H-box motif required for DDB1 binding and further reveal that the domains of DCAF1 responsible for DDB1 and Vpr binding can be genetically separated.

Bottom Line: Herein, we show that the minimal domain of DCAF1 that retained the ability to bind Vpr and DDB1 was mapped to residues 1041 to 1393 (DCAF1 WD).Mutagenic analyses identified an α-helical H-box motif and F/YxxF/Y motifs located in the N-terminal domain of DCAF1 WD that are involved in exclusive binding to DDB1.Importantly, we provide evidence that binding of Vpr to DCAF1 appears to modulate the formation of a DDB1/DCAF1 complex.

View Article: PubMed Central - PubMed

Affiliation: Institut de Recherches Cliniques de Montréal (IRCM), Montréal, Québec, Canada.

ABSTRACT
HIV viral protein R (Vpr) induces a cell cycle arrest at the G2/M phase by activating the ATR DNA damage/replication stress signalling pathway through engagement of the DDB1-CUL4A-DCAF1 E3 ubiquitin ligase via a direct binding to the substrate specificity receptor DCAF1. Since no high resolution structures of the DDB1-DCAF1-Vpr substrate recognition module currently exist, we used a mutagenesis approach to better define motifs in DCAF1 that are crucial for Vpr and DDB1 binding. Herein, we show that the minimal domain of DCAF1 that retained the ability to bind Vpr and DDB1 was mapped to residues 1041 to 1393 (DCAF1 WD). Mutagenic analyses identified an α-helical H-box motif and F/YxxF/Y motifs located in the N-terminal domain of DCAF1 WD that are involved in exclusive binding to DDB1. While we could not identify elements specifically involved in Vpr binding, overall, the mutagenesis data suggest that the predicted β-propeller conformation of DCAF1 is likely to be critical for Vpr association. Importantly, we provide evidence that binding of Vpr to DCAF1 appears to modulate the formation of a DDB1/DCAF1 complex. Lastly, we show that expression of DCAF1 WD in the absence of endogenous DCAF1 was not sufficient to enable Vpr-mediated G2 arrest activity. Overall, our results reveal that Vpr and DDB1 binding on DCAF1 can be genetically separated and further suggest that DCAF1 contains determinants in addition to the Vpr and DDB1 minimal binding domain, which are required for Vpr to enable the induction of a G2 arrest.

Show MeSH
Related in: MedlinePlus