HIV-1 Vpr protein directly loads helicase-like transcription factor (HLTF) onto the CRL4-DCAF1 E3 ubiquitin ligase

J Biol Chem. 2017 Dec 22;292(51):21117-21127. doi: 10.1074/jbc.M117.798801. Epub 2017 Oct 27.

Abstract

The viral protein R (Vpr) is an accessory virulence factor of HIV-1 that facilitates infection in immune cells. Cellular functions of Vpr are tied to its interaction with DCAF1, a substrate receptor component of the CRL4 E3 ubiquitin ligase. Recent proteomic approaches suggested that Vpr degrades helicase-like transcription factor (HLTF) DNA helicase in a proteasome-dependent manner by redirecting the CRL4-DCAF1 E3 ligase. However, the precise molecular mechanism of Vpr-dependent HLTF depletion is not known. Here, using in vitro reconstitution assays, we show that Vpr mediates polyubiquitination of HLTF, by directly loading it onto the C-terminal WD40 domain of DCAF1 in complex with the CRL4 E3 ubiquitin ligase. Mutational analyses suggest that Vpr interacts with DNA-binding residues in the N-terminal HIRAN domain of HLTF in a manner similar to the recruitment of another target, uracil DNA glycosylase (UNG2), to the CRL4-DCAF1 E3 by Vpr. Strikingly, Vpr also engages a second, adjacent region, which connects the HIRAN and ATPase/helicase domains. Thus, our findings reveal that Vpr utilizes common as well as distinctive interfaces to recruit multiple postreplication DNA repair proteins to the CRL4-DCAF1 E3 ligase for ubiquitin-dependent proteasomal degradation.

Keywords: Vpr; human immunodeficiency virus (HIV); polyubiquitin chain; protein degradation; ubiquitin; ubiquitin ligase; virulence factor.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Binding Sites
  • Carrier Proteins / antagonists & inhibitors
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cell Line, Tumor
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Dimerization
  • Gene Deletion
  • HEK293 Cells
  • Humans
  • Models, Molecular*
  • Oligopeptides / chemistry
  • Oligopeptides / genetics
  • Oligopeptides / metabolism
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Point Mutation
  • Proteasome Endopeptidase Complex / metabolism*
  • Protein Interaction Domains and Motifs
  • Protein Multimerization
  • Protein Serine-Threonine Kinases
  • RNA Interference
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Transcription Factors / chemistry
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Ubiquitin-Protein Ligases / antagonists & inhibitors
  • Ubiquitin-Protein Ligases / chemistry
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism*
  • Ubiquitination
  • WD40 Repeats
  • vpr Gene Products, Human Immunodeficiency Virus / chemistry
  • vpr Gene Products, Human Immunodeficiency Virus / genetics
  • vpr Gene Products, Human Immunodeficiency Virus / metabolism*

Substances

  • Carrier Proteins
  • DNA-Binding Proteins
  • HLTF protein, human
  • IL17RB protein, human
  • Oligopeptides
  • Peptide Fragments
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Transcription Factors
  • vpr Gene Products, Human Immunodeficiency Virus
  • vpr protein, Human immunodeficiency virus 1
  • FLAG peptide
  • Ubiquitin-Protein Ligases
  • DCAF1 protein, human
  • Protein Serine-Threonine Kinases
  • Proteasome Endopeptidase Complex

Associated data

  • PDB/5JK7
  • PDB/2HYE
  • PDB/4XZG