HIV-1 Vpr induces the K48-linked polyubiquitination and proteasomal degradation of target cellular proteins to activate ATR and promote G2 arrest.
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.
Belzile
Jean-Philippe
JP
Laboratory of Human Retrovirology, Institut de Recherches Cliniques de Montréal, 110, Avenue des Pins Ouest, Montreal, Quebec H2W 1R7, Canada.
Richard
Jonathan
J
Rougeau
Nicole
N
Xiao
Yong
Y
Cohen
Eric A
EA
D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000502:physiology*; D002460:Cell Line; D044842:Cullin Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D016195:G2 Phase; D006657:Histones / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D025821:Polyubiquitin / Q000737:chemistry*; D046988:Proteasome Endopeptidase Complex / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D025801:Ubiquitin / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000502:physiology; D054875:Ubiquitination; D054325:vpr Gene Products, Human Immunodeficiency Virus / Q000502:physiology*
C114234:CUL4A protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; C091435:DDB1 protein, human; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D025801:Ubiquitin; D054325:vpr Gene Products, Human Immunodeficiency Virus; C514912:vpr protein, Human immunodeficiency virus 1; D025821:Polyubiquitin; D044767:Ubiquitin-Protein Ligases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C530533:DCAF1 protein, human; D017346:Protein Serine-Threonine Kinases; D046988:Proteasome Endopeptidase Complex