Human Immunodeficiency Virus Type 1 Vpr-Binding Protein VprBP, a WD40 Protein Associated with the DDB1-CUL4 E3 Ubiquitin Ligase, Is Essential for DNA Replication and Embryonic Development

Abstract

Damaged DNA binding protein 1, DDB1, bridges an estimated 90 or more WD40 repeats (DDB1-binding WD40, or DWD proteins) to the CUL4-ROC1 catalytic core to constitute a potentially large number of E3 ligase complexes. Among these DWD proteins is the human immunodeficiency virus type 1 (HIV-1) Vpr-binding protein VprBP, whose cellular function has yet to be characterized but has recently been found to mediate Vpr-induced G₂ cell cycle arrest. We demonstrate here that VprBP binds stoichiometrically with DDB1 through its WD40 domain and through DDB1 to CUL4A, subunits of the COP9/signalsome, and DDA1. The steady-state level of VprBP remains constant during interphase and decreases during mitosis. VprBP binds to chromatin in a DDB1-independent and cell cycle-dependent manner, increasing from early S through G₂ before decreasing to undetectable levels in mitotic and G₁ cells. Silencing VprBP reduced the rate of DNA replication, blocked cells from progressing through the S phase, and inhibited proliferation. VprBP ablation in mice results in early embryonic lethality. Conditional deletion of the VprBP gene in mouse embryonic fibroblasts results in severely defective progression through S phase and subsequent apoptosis. Our studies identify a previously unknown function of VprBP in S-phase progression and suggest the possibility that HIV-1 Vpr may divert an ongoing chromosomal replication activity to facilitate viral replication.

ACKNOWLEDGMENTS

We thank members of the Cook and Xiong labs for discussion throughout this work, in particular Xinhai Pei for his aid with flow cytometric analysis and mouse studies. We thank Ling-Jun Zhao (St. Louis University) for providing a VprBP cDNA expression plasmid and an antibody to VprBP and Keiichi Nakayama (Kyushu University, Fukuoka, Japan) for providing the pMX and pMX-Cre retrovirus vectors.

C.M.M. is supported by a DOD Breast Cancer Predoctoral Fellowship and S.C.J. is supported in part by a Development Biology Training Grant to the University of North Carolina. This study is supported by NIH grant K01-CA094907 and by an American Cancer Society grant (GMC-111880) to J.G.C. and an NIH grant (GM067113) to Y.X.