Cytoplasmic retention of HIV-1 regulatory protein Vpr by protein-protein interaction with a novel human cytoplasmic protein VprBP

Vpr is an HIV-1 auxiliary regulatory protein packaged in the virion. It has been shown to enhance the nuclear transport of the HIV-1 preintegration co mplex, activate transcription of cellular and viral promoters, and arrest t he cell cycle at the G2/M check-point. We previously identified a cellular protein of 180 kDa (RIP) that interacted with HIV-1 Vpr specifically. We no w rename this cellular protein as Vpr-binding protein, or VprBP. In this re port, we describe the cloning of the VprBP cDNA that encodes 1507 aa residu es and is identical to the previously cloned cDNA KIAA0800. We demonstrate that Vpr specifically interacts with recombinantly expressed VprBP in vitro as well as in vivo. Furthermore, Vpr interacts with the cellular endogenou s VprBP in the context of the HIV-1 life cycle. Mutational analysis of VprB P suggests that the Vpr binding domain is located within the C-terminal hal f of VprBP, which has a Pro-rich domain and several Phe-x-x-Phe repeats. Su bcellular fractionation studies show that both the endogenous VprBP and the adenovirus-expressed VprBP are distributed predominantly in the cytoplasmi c fraction. Consistent with previous reports, the adenovirus-expressed Vpr is distributed in both the cytoplasmic and the nuclear fractions. However, when VprBP and Vpr are expressed together, Vpr is found almost exclusively in the cytoplasm. Expression of VprBP does not affect the nuclear transport of the adenoviral nuclear protein, pTP. VprBP expressed in insect cells al so blocks the nuclear transport of a Vpr-GFP fusion protein, and VprBP muta nts incapable of interacting with Vpr fail to block Vpr-GFP nuclear transpo rt. We hypothesize that Vpr interaction with VprBP may cause changes in the host cell cytoplasm that affect HIV-1 pathogenesis as well as HIV-1 replic ation. (C) 2001 Elsevier Science B.V. All rights reserved.