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. 2012 Apr 6;287(15):12550-8.
doi: 10.1074/jbc.M112.340711. Epub 2012 Feb 23.

HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1

Jinwoo Ahn  1 , Caili Hao, Junpeng Yan, Maria DeLucia, Jennifer Mehrens, Chuanping Wang, Angela M Gronenborn, Jacek Skowronski
Affiliations

HIV/simian immunodeficiency virus (SIV) accessory virulence factor Vpx loads the host cell restriction factor SAMHD1 onto the E3 ubiquitin ligase complex CRL4DCAF1

Jinwoo Ahn et al. J Biol Chem. .

Abstract

The sterile alpha motif and HD domain-containing protein-1 (SAMHD1) inhibits infection of myeloid cells by human and related primate immunodeficiency viruses (HIV and SIV). This potent inhibition is counteracted by the Vpx accessory virulence factor of HIV-2/SIVsm viruses, which targets SAMHD1 for proteasome-dependent degradation, by reprogramming cellular CRL4(DCAF1) E3 ubiquitin ligase. However, the precise mechanism of Vpx-dependent recruitment of human SAMHD1 onto the ligase, and the molecular interfaces on the respective molecules have not been defined. Here, we show that human SAMHD1 is recruited to the CRL4(DCAF1-Vpx) E3 ubiquitin ligase complex by interacting with the DCAF1 substrate receptor subunit in a Vpx-dependent manner. No stable association is detectable with DCAF1 alone. The SAMHD1 determinant for the interaction is a short peptide located distal to the SAMHD1 catalytic domain and requires the presence of Vpx for stable engagement. This peptide is sufficient to confer Vpx-dependent recruitment to CRL4(DCAF1) and ubiquitination when fused to heterologous proteins. The precise amino acid sequence of the peptide diverges among SAMHD1 proteins from different vertebrate species, explaining selective down-regulation of human SAMHD1 levels by Vpx. Critical amino acid residues of SAMHD1 and Vpx involved in the DCAF1-Vpx-SAMDH1 interaction were identified by mutagenesis. Our findings show that the N terminus of Vpx, bound to DCAF1, recruits SAMHD1 via its C terminus to CRL4, in a species-specific manner for proteasomal degradation.

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Figures

FIGURE 1.
FIGURE 1.
C terminus of human SAMHD1 mediates Vpx-directed down-regulation of SAMHD1 levels. A, schematic representation of SAMHD1. The location of the SAM and HD/COG1078 phosphohydrolase domains in the 626 amino acid residues long human SAMHD1 protein is shown. B, U937 cells stably expressing wild type or truncated FLAG-tagged SAMHD1 proteins were either not infected (−), or infected with increasing amounts of VLP(Vpx). Detergent extracts prepared after 2 days from the infected cells were separated by SDS-PAGE and SAMHD1 levels were determined by immunoblotting with anti-FLAG antibody. C, alignment of the amino acid sequences of SAMHD1 proteins from different species was generated with ClustalW and Jalview (43). Amino acid residues are assigned Clustal color code. D, FLAG-tagged human (Hs), mouse (Mm), zebrafish (Dr) SAMHD1 proteins, or chimeric proteins comprising the C-terminal region (residue 585–626) of human SAMHD1 (Mm·Hs, Dr·Hs) were expressed stably in U937 cells, and then tested for Vpx sensitivity, as described for B, above.
FIGURE 2.
FIGURE 2.
Vpx recruits SAMHD1 to the DCAF1-DDB1 complex via the SAMHD1 C-terminal region. Human (Hs), mouse (Mm), and zebrafish (Dr) FLAG-tagged SAMHD1 proteins, or chimeric SAMHD1 proteins in which mouse (Mm·Hs) and zebrafish (Dr·Hs) C-terminal domains were exchanged for that of human SAMHD1 were transiently co-expressed with Vpx in HEK 293T cells. SAMHD1, and its associated proteins, were precipitated from detergent extracts via SAMHD1 FLAG-tag. Extracts and immune complexes were separated by SDS-PAGE and immunoblotted for DCAF1, DDB1, SAMHD1, and Vpx. (*) indicates endogenous proteins that cross-react with SAMHD1 and DCAF1 antibodies.
FIGURE 3.
FIGURE 3.
The DCAF1-Vpx complex binds the C terminus of SAMHD1. A and B, SAM-domain deleted SAMHD1(115–626) (A) and SAM-domain and the C terminus-deleted SAMHD1(115–585) (B) were separated on an analytical size exclusion column at a flow rate of 0.8 ml/min. C and D, mixtures of NusA·Vpx(1–102), DCAF1c, and SAMHD1(115–626) (C) or SAMHD1(115–585) (D) were analyzed as described for panel A. Peak elution volumes are indicated above the peaks. Numbers and positions of peak fractions are indicated below each peak. E, proteins in peak fractions were concentrated 10-fold, resolved by SDS-PAGE, and stained with Coomassie Brilliant Blue. The SDS-PAGE gel lane numbers correspond to the peak fractions numbers indicated in panels A–D.
FIGURE 4.
FIGURE 4.
Mutational analyses of the human SAMHD1 C terminus identify a Vpx-dependent degradation signal. A, U937 cells stably expressing wild type or mutant FLAG-SAMHD1 proteins with the alanine substitutions at the indicated positions in their C-terminal domains were either not infected (−), or infected (triangle) with increasing amounts of VLP(Vpx). SAMHD1 levels were determined 2 days later by immunoblotting with anti-FLAG antibody. B, preassembled DDB1-DCAF1c-Vpx(1–102) complex was incubated in vitro with recombinant fusion proteins comprising wild type or mutated SAMHD1 C-terminal peptide residues 575–626 fused to thioredoxin (Trx·SAMHD1c). The ability of mutant SAMHD1c peptides to bind DDB1-DCAF1-Vpx(1–102) was analyzed by analytical gel filtration chromatography as described in the legend to Fig. 3, and results are summarized with + (binding) or − (no binding). A schematic representation of the Trx·SAMHD1c fusion protein is shown at the top of the panel and the SAMHD1c region mediating binding to DDB1-DCAF1c-Vpx(1–102) complex is indicated by a bold line. Mutant SAMHD1c moieties are aligned with the wild type SAMHD1c amino acid sequence to visualize the location of the deleted regions. C, panel of Trx·SAMHD1c fusion proteins with single alanine substitution at the indicated positions within the SAMHD1c peptide (residues 575–626) was tested for binding to preassembled DDB1-DCAF1-Vpx(1–102), as described in the Fig. 3 legend, and results are summarized with + (binding) or − (no binding). The substituted residues are underlined and listed in bold. D, in vitro ubiquitination assays of wild type (lanes 1, 2, 17, 18) and single alanine-substituted Trx·SAMHD1c fusion proteins (lanes 3–14). Trx·SAMHD1c proteins were incubated with DDB1-DCAF1c-Vpx(1–102) in the presence of E1 (UBA1), E2 (UbcH5b), CUL4A-RBX1, and FLAG-tagged ubiquitin at 37 °C. A reaction assembled and incubated without Trx·SAMHD1c substrate (−) (lanes 15 and 16) provided a negative control to identify bands corresponding to ubiquitinated Trx·SAMHD1c, indicated on the right side of the panel (Trx·SAMHD1c Ubn). The ubiquitinated species seen in the negative control reaction probably reflect ubiquitination of the CRL4 subunits and other reaction components. The reactions were terminated after 10 min (odd numbered lanes) and 30 min (even numbered lanes) by the addition of Laemmli buffer and incubation at 95 °C for 5 min. The reaction mixtures were separated by 10–20% SDS-PAGE, transferred to PVDF, and ubiquitin was revealed with anti-FLAG antibody.
FIGURE 5.
FIGURE 5.
The N terminus of Vpx mediates DCAF1-dependent down-regulation of SAMHD1 levels. HEK 293T cells were transiently co-transfected with plasmids expressing DCAF1, wild type, or mutant Vpx (N12A, E15A, E16A, and T17A), and/or human SAMHD1, as indicated. Each transfection was performed in duplicate. Extracts prepared from the transfected cells were separated on 10–20% SDS-PAGE and Vpx, SAMHD1, and DCAF1 were revealed by immunoblotting. A nonspecific protein band (*) cross-reacting with the DCAF1 antibody (*) is shown as a loading control.
FIGURE 6.
FIGURE 6.
Model for SAMHD1 recruitment to CRL4DCAF1-Vpx. The N terminus of Vpx interacts with the C-terminal region of SAMHD1 and recruits SAMHD1 to CRL4DCAF1-Vpx E3 ubiquitin ligase for ubiquitination. The relative positioning of the individual subunits in the CRL4DCAF-Vpx complex is based on the known architecture of CRL4 E3 ubiquitin ligases (44).
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