DeHart Jason L, Zimmerman Erik S, Ardon Orly, Monteiro-Filho Carlos M R, Argañaraz Enrique R, Planelles Vicente
Division of Cell Biology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT 84112, USA.
Virol J. 2007 Jun 8;4:57. doi: 10.1186/1743-422X-4-57.
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.
HIV-1病毒蛋白R（Vpr）是一种病毒辅助蛋白，可通过诱导DNA复制应激来激活共济失调毛细血管扩张症突变基因（ATR）。ATR激活导致细胞周期停滞于G2期并诱导细胞凋亡。在本研究中，我们探究了泛素/蛋白酶体系统（UPS）在Vpr上述活性中的作用。我们报告称，UPS的一般功能是Vpr诱导G2检查点激活所必需的，因为用蛋白酶体抑制剂处理表达Vpr的细胞可消除这种效应。我们进一步详细研究了Vpr操纵的特定E3泛素连接酶亚基。我们发现Vpr与cullin 4a/DDB1 E3泛素连接酶的DCAF1亚基结合。能够结合DCAF1的羧基末端结构域Vpr（R80A）突变体在检查点激活中无活性，并具有显性负性特征。相比之下，Vpr富含亮氨酸结构域中介导DCAF1结合的Q65R突变导致Vpr无活性且不具有显性负性作用。因此，Vpr与DCAF1的相互作用是Vpr导致G2期停滞所必需的，但并不充分。我们提出，Vpr通过其羧基末端结构域招募一种未知的细胞因子，该因子是G2期向M期转换所必需的。招募这种因子会导致其泛素化和降解，从而导致无法进入有丝分裂。
