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

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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. Human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS, encodes several proteins termed accessory, which play a critical role in viral pathogenesis. Collectively, HIV-1 accessory proteins Vif, Vpr, Vpu, and Nef have been shown to manipulate host cell biology to promote viral replication, immune evasion, and AIDS progression. In this context, Vpr has been found to block normal cell growth. This interruption of cell division by Vpr is thought to increase viral replication and induce programmed cell death. However, how Vpr is able to block cell division remains unknown. To understand this mechanism, we have studied the interaction between Vpr and novel cellular proteins. We identified a novel complex comprising Vpr and cellular proteins involved in a process called ubiquitination. Ubiquitination is a mechanism by which a small protein, ubiquitin, is conjugated to cellular proteins to modulate their activity or induce their degradation. We demonstrated that association of Vpr with this ubiquitinating complex might be responsible for the defect in cell growth. Further characterization of this protein complex and the elucidation of the mechanism by which it affects cell growth might lead to the development of new anti-HIV drugs and an improved understanding of the basic cellular processes controlling cell division.