DCAF1 interacts with UNG2 via Vpr, but not Vpx. A–C, 50 μl of ΔN-UNG2 (residues 99–313, 12 μm), NusA-Vpr-ΔC (residues 1–79, 12 μm), or DCAF1C (residues 1057–1396, 12 μm) was injected into an analytical Superdex 200 gel filtration column at a flow rate of 0.8 ml/min. Absorbance at 280 nm was recorded. Elution peaks were collected in fractions of 0.5 ml. The peak elution volume (ml) is labeled in each panel. Proteins in each peak were concentrated, separated by SDS-PAGE, and visualized by Coomassie Brilliant Blue staining (M and N). The numbers under each peak refer to the lane number in M and N. D–F, NusA-Vpr-ΔC (12 μm), DCAF1C (12 μm), and ΔN-UNG2 (24 μm) were mixed in pairs and incubated at 4 °C for 30 min. Protein mixtures were injected into the gel filtration column, and eluted proteins were analyzed as described in A–C. The components in each mixture are indicated. G, a mixture of ΔN-UNG2 (24 μm), NusA-Vpr-ΔC (12 μm), and DCAF1C (12 μm) was injected into the column, and the eluted proteins were analyzed as described in A–C. H, a mixture of the same three proteins at equal molar concentrations (12 μm) was subjected to tobacco etch virus (TEV) protease digestion at 4 °C for 1 h followed by column fractionation and analyzed as described above. I and J, 50 μl of NusA (12 μm) or NusA-Vpx-ΔC (residues 1–102, 12 μm) was injected into the column. K and L, ΔN-UNG2 (24 μm) was mixed with either NusA or NusA-Vpx-ΔC (12 μm) and injected into the gel filtration column.

Vpr increases ubiquitination of UNG2 by CRL4^DCAF1 E3 ubiquitin ligase. A, E1 (UBA1), E2 (UbcH5b), and preassembled CRL4^DCAF1B E3 ubiquitin ligase were incubated with full-length UNG2 and N-terminally His₆- and FLAG-tagged ubiquitin (Ub) in various combinations as indicated. NusA or NusA-Vpr-ΔC was also added as indicated. The reaction mixtures were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-FLAG (upper panel) or anti-UNG antibodies (lower panel). Ubiquitinated species are indicated. B, protein mixtures of DDB1-DCAF1B, CUL4A-RBX1, and NusA-Vpr-ΔC were incubated on ice. Tobacco etch virus protease was added to the reaction mixtures to cleave NusA from the E3 ligase complex. The CRL4^{DCAF1B-Vpr-ΔC} was purified over a Mono Q column and analyzed by SDS-PAGE with Coomassie Brilliant Blue staining (2, 4, and 8 μg, respectively in lanes 2–4). C, the CRL4^{DCAF1B-Vpr-ΔC} E3 ubiquitin ligase (E3-Vpr-ΔC) was incubated with E1, E2, N-terminally His₆- and FLAG-tagged Ub, and UNG2 in the ubiquitination buffer as indicated. The reaction mixtures were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-FLAG (left panel) and anti-UNG (right panel) antibody.

Comparison between the E3-ligase-substrate complexes of the CRL4^DCAF1-Vpr-UNG2 and CRL4^SV5-STAT2/STAT1. HIV-1 Vpr recruits UNG2 to the substrate acceptor DCAF1, with DCAF1 binding to DDB1 of CRL4. Other viral proteins, such as the SV5 V protein, use an alternate mode, namely directly interacting with DDB1 and thereby loading STAT1-STAT2 onto CRL4.

Characterization of CRL4^DCAF1 E3 ubiquitin ligase. A, SDS-PAGE analysis of CUL4A-RBX1 (lane 2), DDB1-DCAF1B (lane 3), and CRL4^DCAF1B E3 ubiquitin ligase (lane 4) complexes. 1.0 μg of each complex was separated by SDS-PAGE and stained with Coomassie Brilliant Blue. B, SDS-PAGE analysis of CRL4^DCAF1B E3 ubiquitin ligase complexes. 1.0–3.0 μg of the complex was analyzed as described in A. C, multi-angle light scattering of DDB1-DCAF1, CUL4A-RBX1, and their mixtures. 10 μm DDB1-DCAF1B or CUL4A-RBX1 was injected into an analytical Superdex 200 gel filtration column, respectively, at a flow rate of 0.5 ml/min. Molecular masses of DDB1-DCAF1B (absorbance at 280 nm in blue filled squares, and molecular mass in light blue filled triangles) and CUL4A-RBX1 (absorbance at 280 nm in pink inverted triangles, and molecular mass in light pink filled diamonds) were estimated to be 180 and 120 kDa, respectively. The theoretical molecular mass of each complex is 180 and 101 kDa, respectively. 1.5-fold molar excess of DDB1-DCAF1B was incubated with CUL4-RBX1. The mixture was injected into the analytical gel filtration column. The molecular mass of CRL4^DCAF1B (absorbance at 280 nm in black filled diamonds, and molecular mass in gray filled circles) was estimated to be 300 kDa. Abs, absorbance; arb. Unit, arbitrary unit.

Vpr down-regulates UNG2 in a Vpr- and DCAF1-dependent manner in vivo. A, HEK293 cells were transfected with pCDNA3.1 plasmids expressing Myc-tagged full-length Vpr (Vpr-FL, 0.6 μg), Myc-tagged UNG2 (1.2 μg), and FLAG-tagged DCAF1B (residues 987–1396, 2.2 μg) in 6-well plates as indicated. The parent plasmid pCDNA3.1 was used to balance the total amount of DNA (4 μg) in each transfection when needed. After 42 h of transient transfection, cells were treated with either MG132 (15 μm, lanes 1–4) or vehicle only (dimethyl sulfoxide (DMSO), lanes 5–8). After 6 h of treatment, cells were harvested, and the cell lysates were analyzed by immunoblotting with anti-Myc, anti-FLAG, and anti-actin antibodies as indicated. The average of relative intensity of Myc-UNG2 from three independent experiments was determined using a densitometer and is indicated in each lane. B, HEK293 cells, plated in 10-cm plates, were transfected with Myc-tagged Vpr-FL (3.6 μg), Myc-tagged UNG2 (3.6 μg), and FLAG-tagged DCAF1B (16.8 μg) and an appropriate amount of the parent pCDNA3.1 plasmid. After 42 h of transfection, cells were treated with 10 μm MG132 for 6 h. The cell lysates were immunoprecipitated (IP) with anti-FLAG antibodies. The pulled down mixtures were separated by SDS-PAGE and analyzed by immunoblotting with anti-Myc and anti-FLAG antibodies as indicated. C, HEK293 cells were transfected with HA-tagged Vpr-ΔC (3.6 μg), Myc-tagged UNG2 (3.6 μg), FLAG-tagged DCAF1B (16.8 μg), and the appropriate amount of the parent plasmids in 10-cm plates. N-terminally HA-tagged WT, W54R (defective in UNG2 binding), or H71R (defective in DCAF1 binding) of Vpr-ΔCs (residues 1–79) was used for transfection. Cells were harvested 48 h after transfection. Cell lysates were immunoprecipitated with anti-FLAG antibodies, and the proteins bound to antibodies were separated by SDS-PAGE and analyzed by immunoblotting with anti-Myc, anti-HA, anti-actin, and anti-FLAG antibodies as indicated. D, HEK293 cells were co-transfected with constructs expressing HA-tagged, full-length DCAF1 (DCAF1-FL), DDB1, CUL4A-RBX1, and Vpr (5, 5, 4, and 2 μg, respectively) as indicated. The cells were also co-transfected with Myc-tagged UNG2 and His₆-tagged ubiquitin (4 μg each). At 40 h after transfection, cells were harvested and lysed. His₆-Ub-conjugated proteins were pulled down with Ni-NTA agarose beads. The proteins that bound to Ni-NTA beads were eluted and separated by SDS-PAGE and analyzed by immunoblotting with anti-Myc antibody.