Formation of mobile chromatin-associated nuclear foci containing HIV-1 Vpr and VPRBP is critical for the induction of G2 cell cycle arrest.
HIV-1 Viral protein R (Vpr) induces a cell cycle arrest at the G2/M phase by activating the ATR DNA damage/stress checkpoint. Recently, we and several other groups showed that Vpr performs this activity by recruiting the DDB1-CUL4A (VPRBP) E3 ubiquitin ligase. While recruitment of this E3 ubiquitin ligase complex has been shown to be required for G2 arrest, the subcellular compartment where this complex forms and functionally acts is unknown. Herein, using immunofluorescence and confocal microscopy, we show that Vpr forms nuclear foci in several cell types including HeLa cells and primary CD4+ T-lymphocytes. These nuclear foci contain VPRBP and partially overlap with DNA repair foci components such as gamma-H2AX, 53BP1 and RPA32. While treatment with the non-specific ATR inhibitor caffeine or depletion of VPRBP by siRNA did not inhibit formation of Vpr nuclear foci, mutations in the C-terminal domain of Vpr and cytoplasmic sequestration of Vpr by overexpression of Gag-Pol resulted in impaired formation of these nuclear structures and defective G2 arrest. Consistently, we observed that G2 arrest-competent sooty mangabey Vpr could form these foci but not its G2 arrest-defective paralog Vpx, suggesting that formation of Vpr nuclear foci represents a critical early event in the induction of G2 arrest. Indeed, we found that Vpr could associate to chromatin via its C-terminal domain and that it could form a complex with VPRBP on chromatin. Finally, analysis of Vpr nuclear foci by time-lapse microscopy showed that they were highly mobile and stable structures. Overall, our results suggest that Vpr recruits the DDB1-CUL4A (VPRBP) E3 ligase to these nuclear foci and uses these mobile structures to target a chromatin-bound cellular substrate for ubiquitination in order to induce DNA damage/replication stress, ultimately leading to ATR activation and G2 cell cycle arrest.
Belzile
Jean-Philippe
JP
Laboratory of Human Retrovirology, Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec, Canada.
Abrahamyan
Levon G
LG
Gérard
Francine C A
FC
Rougeau
Nicole
N
Cohen
Eric A
EA
D000328:Adult; D064007:Ataxia Telangiectasia Mutated Proteins; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000502:physiology*; D002843:Chromatin / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D016195:G2 Phase / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D046988:Proteasome Endopeptidase Complex / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination; D014779:Virus Replication; D054325:vpr Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002843:Chromatin; D054325:vpr Gene Products, Human Immunodeficiency Virus; C514912:vpr protein, Human immunodeficiency virus 1; 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
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