biostudies-literatureZhou XNIAID NIH HHSNIGMS NIH HHS21117-21127https://www.ebi.ac.uk/biostudies/studies/S-EPMC5743084biostudies-literatureUnknown292(51)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.The Journal of biological chemistryHIV-1 Vpr protein directly loads helicase-like transcription factor (HLTF) onto the CRL4-DCAF1 E3 ubiquitin ligase.PMC5743084R01 GM116642R01 AI100673P50 GM082251R01 GM123973Hrecka KMonnie CAhn JSkowronski JDeLucia MZhou XHao CfalseHIV-1 Vpr protein directly loads helicase-like transcription factor (HLTF) onto the CRL4-DCAF1 E3 ubiquitin ligase.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.2017-01-01T00:00:00Z2017 Dec2020-11-19T08:26:02Z2019-03-27T00:13:00ZS-EPMC57430842907957510.1074/jbc.m117.79880110.1074/jbc.M117.798801