Department of Microbiology, New York University School of Medicine, 522 First Avenue, New York, NY 10016, USA.
Virol J. 2014 Jun 9;11:108. doi: 10.1186/1743-422X-11-108.
HIV-1 Vpr-mediated G2 cell cycle arrest is dependent on the interaction of Vpr with an E3 ubiquitin ligase that contains damage-specific DNA binding protein 1 (DDB1), Cullin 4A (Cul4A), DDB1 and Cul4-associated factor 1 (DCAF1), and Rbx1. Vpr is thought to associate directly with DCAF1 in the E3 ubiquitin ligase complex although the exact interaction pattern of the proteins in the complex is not completely defined. The Vpr of SIVagm induces G2 arrest of cognate African Green Monkey (AGM) cells but not human cells. The molecular mechanism by which SIVagm Vpr exhibits its species-specific function remained unknown.
Physical interaction of proteins in the E3 ubiquitin ligase complex was assessed by co-immunoprecipitation followed by western blotting. In addition, co-localization of the proteins in cells was investigated by confocal microscopy. The cell cycle was analyzed by propidium iodide staining and flow cytometry. DNA damage response elicited by Vpr was evaluated by detecting phosphorylation of H2AX, a marker for DNA damage response.
We show that RNAi knock-down of DCAF1 prevented the co-immunoprecipitation of DDB1 with HIV-1 Vpr while DDB1 knock-down did not influence the binding of Vpr to DCAF1. HIV-1 Vpr mutants with a L64P or a R90K mutation maintained the ability to associate with DCAF1 but did not appear to be in a complex with DDB1. SIVagm Vpr associated with AGM DCAF1 and DDB1 while, in human cells, it binds to human DCAF1 but hardly binds to human DDB1, resulting in the reduced activation of H2AX.
The identification of Vpr mutants which associate with DCAF1 but only poorly with DDB1 suggests that DCAF1 is necessary but the simple binding of Vpr to DCAF1 is not sufficient for the Vpr association with DDB1-containing E3 ligase complex. Vpr may interact both with DCAF1 and DDB1 in the E3 ligase complex. Alternatively, the interaction of Vpr and DCAF1 may induce a conformational change in DCAF1 or Vpr that promotes the interaction with DDB1. The ability of SIVagm Vpr to associate with DDB1, but not DCAF1, can explain the species-specificity of SIVagm Vpr-mediated G2 arrest.
HIV-1 Vpr 介导的 G2 细胞周期停滞依赖于 Vpr 与 E3 泛素连接酶的相互作用，该酶包含损伤特异性 DNA 结合蛋白 1（DDB1）、Cullin 4A（Cul4A）、DDB1 和 Cul4 相关因子 1（DCAF1）以及 Rbx1。尽管复合物中蛋白质的确切相互作用模式尚未完全确定，但人们认为 Vpr 直接与 E3 泛素连接酶复合物中的 DCAF1 相关。然而，SIVagm 的 Vpr 诱导其同源的非洲绿猴（AGM）细胞而不是人类细胞的 G2 停滞。SIVagm Vpr 表现出其种属特异性功能的分子机制仍然未知。
通过共免疫沉淀和 Western blot 评估 E3 泛素连接酶复合物中蛋白质的物理相互作用。此外，通过共聚焦显微镜研究细胞中蛋白质的共定位。通过碘化丙啶染色和流式细胞术分析细胞周期。通过检测 DNA 损伤反应标志物 H2AX 的磷酸化来评估 Vpr 引起的 DNA 损伤反应。
我们表明，DCAF1 的 RNAi 敲低阻止了 DDB1 与 HIV-1 Vpr 的共免疫沉淀，而 DDB1 的敲低并不影响 Vpr 与 DCAF1 的结合。具有 L64P 或 R90K 突变的 HIV-1 Vpr 突变体仍然能够与 DCAF1 结合，但似乎不能与 DDB1 形成复合物。SIVagm Vpr 与 AGM DCAF1 和 DDB1 结合，而在人类细胞中，它与人类 DCAF1 结合，但几乎不与人类 DDB1 结合，导致 H2AX 的激活减少。
鉴定出与 DCAF1 结合但与 DDB1 结合不良的 Vpr 突变体表明，DCAF1 是必需的，但 Vpr 与 DCAF1 的简单结合不足以与包含 DDB1 的 E3 连接酶复合物结合。Vpr 可能在 E3 连接酶复合物中与 DCAF1 和 DDB1 相互作用。或者，Vpr 和 DCAF1 的相互作用可能会诱导 DCAF1 或 Vpr 的构象变化，从而促进与 DDB1 的相互作用。SIVagm Vpr 与 DDB1 结合而不是 DCAF1 的能力可以解释 SIVagm Vpr 介导的 G2 停滞的种属特异性。
