HIV-1 Vpr-Mediated G2 Arrest Involves the DDB1-CUL4AVPRBP E3 Ubiquitin Ligase

Jean-Philippe Belzile,Ghislaine Duisit,Éric A Cohen,Johanne Mercier,Nicole Rougeau,Andrés Finzi,Klaus Früh

doi:10.1371/journal.ppat.0030085

Jean-Philippe Belzile, Ghislaine Duisit + Show 5 more

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https://doi.org/10.1371/journal.ppat.0030085

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Abstract

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has been shown to cause G2 cell cycle arrest in human cells by inducing ATR-mediated inactivation of p34cdc2, but factors directly engaged in this process remain unknown. We used tandem affinity purification to isolate native Vpr complexes. We found that damaged DNA binding protein 1 (DDB1), viral protein R binding protein (VPRBP), and cullin 4A (CUL4A)—components of a CUL4A E3 ubiquitin ligase complex, DDB1-CUL4AVPRBP —were able to associate with Vpr. Depletion of VPRBP by small interfering RNA impaired Vpr-mediated induction of G2 arrest. Importantly, VPRBP knockdown alone did not affect normal cell cycle progression or activation of ATR checkpoints, suggesting that the involvement of VPRBP in G2 arrest was specific to Vpr. Moreover, leucine/isoleucine-rich domain Vpr mutants impaired in their ability to interact with VPRBP and DDB1 also produced strongly attenuated G2 arrest. In contrast, G2 arrest–defective C-terminal Vpr mutants were found to maintain their ability to associate with these proteins, suggesting that the interaction of Vpr with the DDB1-VPRBP complex is necessary but not sufficient to block cell cycle progression. Overall, these results point toward a model in which Vpr could act as a connector between the DDB1-CUL4AVPRBP E3 ubiquitin ligase complex and an unknown cellular factor whose proteolysis or modulation of activity through ubiquitination would activate ATR-mediated checkpoint signaling and induce G2 arrest.

Highlights

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) accessory protein is a small 96Àamino acid protein that plays several roles during virus infection
We demonstrated that association of Vpr with this ubiquitinating complex might be responsible for the defect in cell growth
Vpr was fused to a tandem affinity purification (TAP) tag containing two immunoglobulin-binding domains of protein A from Staphylococcus aureus, a cleavage site for the tobacco etch virus (TEV) protease, and the calmodulin-binding peptide (CBP)

Summary

Introduction

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) accessory protein is a small 96Àamino acid protein that plays several roles during virus infection (reviewed in [1,2]). Accumulating evidence indicates that Vpr-mediated cell cycle arrest depends on DNA damage response, but precise mechanisms of its induction remain obscure. Depending on the type of stress, ATR- or ATMmediated checkpoints are fully activated by the coordinated activity of Rad9-Rad1-Hus (9-1-1), Rad17-RFC, breast cancerÀassociated protein (BRCA1), and p53 binding protein (53BP) (reviewed in [11,12,13]). ATR- or ATM-dependent phosphorylation of the histone variant H2AFX (H2AX) triggers the formation of c-H2AFX/BRCA1 or 53BP foci. These foci are presumed to help in the recruitment and/or retention of DNA repair machinery and checkpoint effectors at the damaged DNA sites, promoting checkpoint signal amplification [12]. Inactivation of cdc2/cyclin by hyperphosphorylation and cytoplasmic retention prevents entry into mitosis before the completion of DNA repair [10,12]

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