HIV-1 Vpr Induces the K48-Linked Polyubiquitination and Proteasomal Degradation of Target Cellular Proteins To Activate ATR and Promote G ₂ 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.

G 2 /M Cell Cycle Arrest Correlates with Primate Lentiviral Vpr Interaction with the SLX4 Complex

“…However, we also note a further caveat that should be borne in mind. Most of the data published on the mechanism of Vpr-mediated cell cycle arrest, as well as the data presented here, have been obtained by transient transfection or ectopic expression of Vpr (3,4,11,13,14,27,28). The levels of HIV-1 Vpr incorporated into incoming particles have been shown to be sufficient to induce some G 2 /M arrest (27), and of course de novo expression in the infected cells does this robustly.…”

“…Although several putative Vpr targets have been described, the identity of that which triggers this event has until recently remained elusive. The requirement for the ubiquitin-proteasome system and, in particular, K48-ubiquitin linkages supported a notion that the Vpr target is degraded (14). However, Laguette and colleagues recently found that Vpr interacts with the SLX4 complex, members of the Fanconi anemia DNA repair pathway (15).…”

Rapamycin activates autophagy in Hutchinson-Gilford progeria syndrome

“…Specifically, different effects have been described for different types of polyubiquitin chains; for example, the K48 polyubiquitin (polyUb) chain is recognized by, and promotes degradation of proteins through, the proteasome, whereas the K63 polyUb chain promotes autophagic clearance of oligomeric and aggregated proteins. [23][24][25] We found that progerin is preferentially ubiquitinated by K63-linked polyUb chains, suggesting that it is a substrate for autophagy. Additionally, progerin co-immunoprecipitates with the autophagic adaptor protein p62, further demonstrating that the clearance of progerin is mediated by autophagy.…”

SLX4-SLX1 Protein-independent Down-regulation of MUS81-EME1 Protein by HIV-1 Viral Protein R (Vpr)

“…However, the detailed mechanism of Vpr-induced G 2 /M arrest is not yet clear. Several lines of evidence suggest that G 2 arrest requires interaction of Vpr with the CRL4 E3 ubiquitin ligase complex, comprising DDB1, CUL4, and RBX1, via its cellular partner DCAF1 (22)(23)(24)(25)(26)(27)(28)(29)(30). DCAF1 is a substrate receptor component of the CRL4 (31)(32)(33).…”