HIV-1 Vpr Loads Uracil DNA Glycosylase-2 onto DCAF1, a Substrate Recognition Subunit of a Cullin 4A-RING E3 Ubiquitin Ligase for Proteasome-dependent Degradation

Jinwoo Ahn,Thomas Vu,Zach Novince,Jennifer Guerrero-Santoro,Vesna Rapic-Otrin,Angela M Gronenborn

doi:10.1074/jbc.m110.133181

Jinwoo Ahn, Thomas Vu + Show 4 more

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https://doi.org/10.1074/jbc.m110.133181

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Abstract

The human immunodeficiency virus type 1 (HIV-1) accessory protein, Vpr, interacts with several host cellular proteins including uracil DNA glycosylase-2 (UNG2) and a cullin-RING E3 ubiquitin ligase assembly (CRL4(DCAF1)). The ligase is composed of cullin 4A (CUL4A), RING H2 finger protein (RBX1), DNA damage-binding protein 1 (DDB1), and a substrate recognition subunit, DDB1- and CUL4-associated factor 1 (DCAF1). Here we show that recombinant UNG2 specifically interacts with Vpr, but not with Vpx of simian immunodeficiency virus, forming a heterotrimeric complex with DCAF1 and Vpr in vitro as well as in vivo. Using reconstituted CRL4(DCAF1) and CRL4(DCAF1-Vpr) E3 ubiquitin ligases in vitro reveals that UNG2 ubiquitination (ubiquitylation) is facilitated by Vpr. Co-expression of DCAF1 and Vpr causes down-regulation of UNG2 in a proteasome-dependent manner, with Vpr mutants that are defective in UNG2 or DCAF1 binding abrogating this effect. Taken together, our results show that the CRL4(DCAF1) E3 ubiquitin ligase can be subverted by Vpr to target UNG2 for degradation.

Highlights

DCAF1 cifically, viral protein R (Vpr) interacts with the CRL4 E3 ubiquitin ligase, assembled with cullin 4A (CUL4A), RBX1, DDB1 (DNA damage-binding protein 1), and DCAF1 (DDB1- and CUL4-associated factor 1) [1, 21]
Certain aspects of the interplay between human immunodeficiency virus type 1 (HIV-1) Vpr and CRL4DCAF1 E3 ubiquitin ligase in cell cycle arrest and viral infectivity have been described recently [23,24,25,26,27,28,29,30,31], many gaps are still present in our understanding of Vpr activity in proteasomal-dependent degradation of host cell proteins
We demonstrate for the first time that HIV-1 Vpr mediates uracil-DNA glycosylase-2 (UNG2) binding to the substrate recognition subunit, DCAF1 (Fig. 1), and that a stable heterotrimeric complex is formed

Summary

Introduction

In Vitro Ubiquitination Assays—Typically, E1 (UBA1, 0.2 ␮M), E2 (UbcH5b, 2.5 ␮M), and appropriate E3 complexes (RBX1-CUL4A-DDB1-DCAF1B or RBX1-CUL4A-DDB1DCAF1B-Vpr-⌬C, 0.2 ␮M) were incubated with 1 ␮M UNG2 and 2.5 ␮M His6-FLAG-tagged ubiquitin in a buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5% glycerol, 20 units/ml pyrophosphatase, 2 mM DTT, and 5 mM ATP at 37 °C for 2 h. A peak eluting at 12.3 ml (Fig. 1G) was found to contain all three proteins (Fig. 1M, lane 10), indicating that a triple complex of DCAF1C-NusA-Vpr-⌬C-⌬N-UNG2 was formed. The NusA-Vpr-⌬C-⌬N-UNG2 peak eluting at 11 ml (Fig. 1F) was not observed in this experiment, suggesting that DCAF1C formed protein complexes with all of NusA-Vpr⌬C-⌬N-UNG2.

Results

Conclusion