HIV-1 Vpr induces the K48-linked polyubiquitination and proteasomal degradation of target cellular proteins to activate ATR and promote G2 arrest

Abstract

HIV-1 viral protein R (Vpr) induces cell cycle arrest at the G(2)/M phase by a mechanism involving the activation of the DNA damage sensor ATR. We and others recently showed that Vpr performs this function by subverting the activity of the DDB1-CUL4A (VPRBP) E3 ubiquitin ligase. Vpr could thus act as a connector between the E3 ligase and an unknown cellular factor whose ubiquitination would induce G(2) arrest. While attractive, this model is based solely on the indirect observation that some mutants of Vpr retain their interaction with the E3 ligase but fail to induce G(2) arrest. Using a tandem affinity purification approach, we observed that Vpr interacts with ubiquitinated cellular proteins and that this association requires the recruitment of an active E3 ligase given that the depletion of VPRBP by RNA interference or the overexpression of a dominant negative mutant of CUL4A decreased this association. Importantly, G(2)-arrest-defective mutants of Vpr in the C-terminal putative substrate-interacting domain displayed a decreased association with ubiquitinated proteins. We also found that the inhibition of proteasomal activity increased this association and that the ubiquitin chains were at least in part constituted of classical K48 linkages. Interestingly, the inhibition of K48 polyubiquitination specifically impaired the Vpr-induced phosphorylation of H2AX, an early target of ATR, but did not affect UV-induced H2AX phosphorylation. Overall, our results provide direct evidence that the association of Vpr with the DDB1-CUL4A (VPRBP) E3 ubiquitin ligase induces the K48-linked polyubiquitination of as-yet-unknown cellular proteins, resulting in their proteasomal degradation and ultimately leading to the activation of ATR and G(2) arrest.

FIG. 1.

Vpr interacts with VPRBP and DDB1 during infection of primary CD4⁺ T lymphocytes. Ten million activated primary CD4⁺ T lymphocytes were mock infected or infected with viruses defective for Vpr [HxBru(Vpr⁻)] or encoding HA-tagged Vpr [HxBru(HA-Vpr)] at a multiplicity of infection of 0.01. Five days after infection cells were harvested in Triton lysis buffer, and immunoprecipitation (IP) against HA was performed as described in Materials and Methods. Coimmunoprecipitated endogenous VPRBP and DDB1 were detected by Western blotting by using specific rabbit polyclonal antibodies. HA-tagged Vpr was detected by using a monoclonal anti-HA antibody.

FIG. 2.

Vpr interacts with unknown cellular ubiquitinated proteins. (A) HEK293T cells were transfected with plasmids encoding TAP (lanes 1 and 3) or TAP-Vpr (lanes 2 and 4). Cells were cotransfected with either an empty plasmid (lanes 1 and 2) or a Myc-Ub(K48R)-encoding plasmid (lanes 3 and 4). Two days later, tandem affinity purification was performed on cell lysates as described in Materials and Methods. The levels of ubiquitinated proteins were determined by Western blotting of crude lysates and pulled-down fractions by using a monoclonal anti-Myc antibody. (B) Cells were cotransfected with plasmids expressing TAP-Vpr and Myc-Ub(K48R) (lanes 3 and 4) or with plasmids expressing TAP and Myc-Ub(K48R) as a control (lanes 1 and 2). Forty-eight hours posttransfection, cells were lysed in Triton lysis buffer (lanes 1 and 3) or were heat denatured following lysis in RIPA buffer (lanes 2 and 4). Cell extracts were subjected to IgG pulldowns with precoupled beads, and complexes were eluted following cleavage with tobacco etch virus (TEV) protease. The levels of ubiquitinated proteins were determined by using a monoclonal anti-Myc antibody, and Vpr was detected by using a polyclonal antibody.

FIG. 3.

Association of Vpr with ubiquitinated proteins involves the recruitment of an active E3 ubiquitin ligase complex. HEK293T cells were cotransfected with a plasmid encoding Myc-Ub(K48R) and with either TAP-encoding (lanes 1 and 2) or TAP-Vpr-encoding (lanes 3 and 4) plasmids. Cells were transcomplemented with plasmids expressing scrambled shRNA or shRNA targeting VPRBP (A) and with an empty plasmid or a plasmid encoding a dominant negative mutant form of CUL4A (Cul4A DN) (B), as indicated. Two days after transfection, cell extracts were subjected to tandem affinity purification as described in Materials and Methods. The levels of ubiquitinated proteins were determined by Western blotting of crude lysates and pulled-down fractions by using a monoclonal anti-Myc antibody. Vpr and VPRBP were detected by using polyclonal antibodies. An asterisk denotes a nonspecific band detected by the anti-VPRBP antibody used as a loading control.

FIG. 4.

Analysis of the association of Vpr mutants with ubiquitinated proteins. (A) HEK293T monoclonal cell lines stably expressing a control shRNA (shControl) or an shRNA against VPRBP (shVprBP) were cotransfected with a plasmid encoding Myc-Ub(K48R) and plasmids expressing TAP, TAP-Vpr(WT), and TAP-Vpr(Q65R), as indicated. Two days after transfection, cell extracts were subjected to tandem affinity purification as described in Materials and Methods. (B) HEK293T cells were cotransfected with a plasmid encoding Myc-Ub(K48R) and plasmids expressing TAP, TAP-Vpr(WT), and TAP-Vpr(1-78), as indicated. Cell extracts were processed as described above for A. (C) HEK293T cells were cotransfected with a plasmid encoding Myc-Ub(K48R) and plasmids expressing TAP, TAP-Vpr(WT), TAP-Vpr(R87A,R88A), TAP-Vpr(1-84), TAP-Vpr(1-86), and TAP-Vpr(1-90), as indicated. Two days after transfection, cell extracts were subjected to tandem affinity purification as described in Materials and Methods. Pulldown eluates (lanes 7, 8, 10, 12, 14, and 16) and 2-fold dilutions of pulldown eluates (lanes 9, 11, 13, 15, and 17) were resolved by SDS-PAGE for analysis. For all panels, the levels of ubiquitinated proteins were determined by Western blotting of crude lysates and pulled-down fractions by using a monoclonal anti-Myc antibody. Vpr and VPRBP were detected by using polyclonal antibodies. An asterisk denotes nonspecific bands used as loading controls.

FIG. 5.

Vpr-associated ubiquitinated proteins are degraded by the proteasome. (A) HEK293T cells were cotransfected with a plasmid encoding HA-Ub(WT) and with either TAP-encoding (lanes 1 and 2) or TAP-Vpr-encoding (lanes 3 and 4) plasmids. Twenty-four hours after transfection, cells were treated (lanes 2 and 4) or not (lanes 1 and 3) with 5 μM MG132 for 16 h. (B) Cells were cotransfected with a plasmid encoding HA-Ub(WT) and with either TAP-encoding (lanes 1 and 2) or TAP-Vpr-encoding (lanes 3 and 4) plasmids. Cells were transcomplemented with plasmids expressing scrambled shRNA or shRNA targeting VPRBP, as indicated. Twenty-four hours after transfection, cells were treated with 5 μM MG132 for 16 h. For both panels, cell extracts were subjected to tandem affinity purification. Ubiquitinated proteins were detected by using a monoclonal anti-HA antibody. Vpr and VPRBP were detected by using polyclonal antibodies. An asterisk denotes a nonspecific band detected by the anti-VPRBP antibody used as a loading control.

FIG. 6.

Vpr induces the K48-linked polyubiquitination of unknown cellular substrates. HEK293T cells were transfected with a plasmid encoding either TAP (lanes 1 to 4) or TAP-Vpr (lanes 5 to 8). Cells were transcomplemented with plasmids expressing HA-Ub(WT) (lanes 2 and 6), HA-Ub(K0) (lanes 3 and 7), and HA-Ub(K48R) (lanes 4 and 8) or with an empty plasmid as a negative control (lanes 1 and 5). Twenty-four hours after transfection, cells were treated with 5 μM MG132 for 16 h, and cell extracts were subjected to tandem affinity purification. The levels of ubiquitinated proteins were determined by Western blotting of crude lysates and pulled-down fractions by using a monoclonal anti-HA antibody. Vpr was detected by using a polyclonal antibody.

FIG. 7.

K48-linked polyubiquitination is required for Vpr-induced H2AX phosphorylation. (A) HeLa cells were transiently transfected with an empty plasmid or plasmids expressing HA-Ub(K48R) or HA-Ub(K63R). Twenty-four hours after transfection, cells were transduced with a lentiviral vector expressing GFP alone (WPI) or coexpressing Vpr and GFP (Vpr). Two days later, cells were fixed, permeabilized, and stained with antibodies against GFP (green), γ-H2AX (red), and HA (blue). Cells with more than 10 γ-H2AX foci were considered to be positive for H2AX phosphorylation. (B) Results depicted in the graphs are the means of data from three independent experiments. Error bars represent standard deviations. Statistical significance was determined with the Student t test with 95% confidence (P < 0.05).