Abstract
HIV-1 Vpr is a viral accessory protein that activates ATR through the induction of DNA replication stress. ATR activation results in cell cycle arrest in G2 and induction of apoptosis. In the present study, we investigate the role of the ubiquitin/proteasome system (UPS) in the above activity of Vpr. We report that the general function of the UPS is required for Vpr to induce G2 checkpoint activation, as incubation of Vpr-expressing cells with proteasome inhibitors abolishes this effect. We further investigated in detail the specific E3 ubiquitin ligase subunits that Vpr manipulates. We found that Vpr binds to the DCAF1 subunit of a cullin 4a/DDB1 E3 ubiquitin ligase. The carboxy-terminal domain Vpr(R80A) mutant, which is able to bind DCAF1, is inactive in checkpoint activation and has dominant-negative character. In contrast, the mutation Q65R, in the leucine-rich domain of Vpr that mediates DCAF1 binding, results in an inactive Vpr devoid of dominant negative behavior. Thus, the interaction of Vpr with DCAF1 is required, but not sufficient, for Vpr to cause G2 arrest. We propose that Vpr recruits, through its carboxy terminal domain, an unknown cellular factor that is required for G2-to-M transition. Recruitment of this factor leads to its ubiquitination and degradation, resulting in failure to enter mitosis.
Highlights
The HIV-1 encoded viral protein R induces cell cycle arrest and apoptosis through activation of the serine/threonine kinase known as the ataxia telangiectasia-mutated and Rad3-related (ATR) protein [1,2]
A protein known as RIP, that was discovered as an interaction partner of viral protein R (Vpr) [15], was recently shown to be part of a family of WDrepeat proteins that are found in association with cullin 4a/damaged DNA-binding protein 1 (DDB1) E3 ubiquitin ligases [9]
(page number not for citation purposes) http://www.virologyj.com/content/4/1/57 aphidicolin both activate ataxia telangiectasia-mutated and Rad3-related protein (ATR), our results suggest, do not demonstrate, the possibility that the presence of DCAF1 may be normally required for ATR activation
Summary
The HIV-1 encoded viral protein R induces cell cycle arrest and apoptosis through activation of the serine/threonine kinase known as the ataxia telangiectasia-mutated and Rad3-related (ATR) protein [1,2]. Destruction of cell cycle regulators is typically mediated by the proteasome and involves polyubiquitination by E3 ubiquitin ligases. Certain viral proteins are known to bind to the substrate specificity subunits of E3 ligases to redirect specificity to non-cognate targets. Examples of these viral proteins include hepatitis B protein X [7], human papilloma virus E6 [8], simian virus 5 V protein [9,10], HIV-1 Vif [11,12,13], and HIV-1 Vpu [14]. We examined in detail the potential role of the UPS in the ability of HIV-1 Vpr to induce G2 arrest
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