Department of Structural Biology and Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Biomedical Science Tower 3, RM 1055, 3501 Fifth Ave., Pittsburgh, PA, 15260, USA.
Virol J. 2021 Mar 1;18(1):48. doi: 10.1186/s12985-021-01514-2.
Vpr is a virion-associated protein that is encoded by lentiviruses and serves to counteract intrinsic immunity factors that restrict infection. HIV-1 Vpr mediates proteasome-dependent degradation of several DNA repair/modification proteins. Mechanistically, Vpr directly recruits cellular targets onto DCAF1, a substrate receptor of Cullin 4 RING E3 ubiquitin ligase (CRL4) for poly-ubiquitination. Further, Vpr can mediate poly-ubiquitination of DCAF1-interacting proteins by the CRL4. Because Vpr-mediated degradation of its known targets can not explain the primary cell-cycle arrest phenotype that Vpr expression induces, we surveyed the literature for DNA-repair-associated proteins that interact with the CRL4-DCAF1. One such protein is SIRT7, a deacetylase of histone 3 that belongs to the Sirtuin family and regulates a wide range of cellular processes. We wondered whether Vpr can mediate degradation of SIRT7 via the CRL4-DCAF1.
HEK293T cells were transfected with cocktails of plasmids expressing DCAF1, DDB1, SIRT7 and Vpr. Ectopic and endogeneous levels of SIRT7 were monitered by immunoblotting and protein-protein interactions were assessed by immunoprecipitation. For in vitro reconstitution assays, recombinant CRL4-DCAF1-Vpr complexes and SIRT7 were prepared and poly-ubiqutination of SIRT7 was monitored with immunoblotting.
We demonstrate SIRT7 polyubiquitination and degradation upon Vpr expression. Specifically, SIRT7 is shown to interact with the CRL4-DCAF1 complex, and expression of Vpr in HEK293T cells results in SIRT7 degradation, which is partially rescued by CRL inhibitor MNL4924 and proteasome inhibitor MG132. Further, in vitro reconstitution assays show that Vpr induces poly-ubiquitination of SIRT7 by the CRL4-DCAF1. Importantly, we find that Vpr from several different HIV-1 strains, but not HIV-2 strains, mediates SIRT7 poly-ubiquitination in the reconstitution assay and degradation in cells. Finally, we show that SIRT7 degradation by Vpr is independent of the known, distinctive phenotype of Vpr-induced cell cycle arrest at the G2 phase, CONCLUSIONS: Targeting histone deacetylase SIRT7 for degradation is a conserved feature of HIV-1 Vpr. Altogether, our findings reveal that HIV-1 Vpr mediates down-regulation of SIRT7 by a mechanism that does not involve novel target recruitment to the CRL4-DCAF1 but instead involves regulation of the E3 ligase activity.
Vpr 是一种病毒相关蛋白，由慢病毒编码，用于对抗限制感染的固有免疫因子。HIV-1 Vpr 介导几种 DNA 修复/修饰蛋白的蛋白酶体依赖性降解。在机制上，Vpr 直接将细胞靶标募集到 DCAF1 上，DCAF1 是 Cullin 4 RING E3 泛素连接酶（CRL4）用于多泛素化的底物受体。此外，Vpr 可以通过 CRL4 介导 DCAF1 相互作用蛋白的多泛素化。由于 Vpr 介导的其已知靶标的降解不能解释 Vpr 表达诱导的主要细胞周期停滞表型，因此我们查阅文献以寻找与 CRL4-DCAF1 相互作用的与 DNA 修复相关的蛋白质。其中一种蛋白质是 SIRT7，它是组蛋白 3 的去乙酰化酶，属于 Sirtuin 家族，调节广泛的细胞过程。我们想知道 Vpr 是否可以通过 CRL4-DCAF1 介导 SIRT7 的降解。
用表达 DCAF1、DDB1、SIRT7 和 Vpr 的质粒混合物转染 HEK293T 细胞。通过免疫印迹监测外源性和内源性 SIRT7 水平，并通过免疫沉淀评估蛋白质-蛋白质相互作用。对于体外重组测定，制备重组 CRL4-DCAF1-Vpr 复合物和 SIRT7，并通过免疫印迹监测 SIRT7 的多泛素化。
我们证明了 Vpr 表达后 SIRT7 的多泛素化和降解。具体而言，SIRT7 被证明与 CRL4-DCAF1 复合物相互作用，并且在 HEK293T 细胞中表达 Vpr 导致 SIRT7 降解，这部分被 CRL 抑制剂 MNL4924 和蛋白酶体抑制剂 MG132 挽救。此外，体外重组测定表明 Vpr 诱导 CRL4-DCAF1 介导的 SIRT7 多泛素化。重要的是，我们发现来自几种不同 HIV-1 株的 Vpr 而不是 HIV-2 株在重组测定和细胞中介导 SIRT7 的多泛素化和降解。最后，我们发现 Vpr 对 SIRT7 的降解不依赖于 Vpr 诱导的 G2 期细胞周期停滞的已知独特表型。
将组蛋白去乙酰化酶 SIRT7 作为靶标进行降解是 HIV-1 Vpr 的保守特征。总之，我们的研究结果表明，HIV-1 Vpr 通过一种不涉及将新靶标募集到 CRL4-DCAF1 而不涉及调节 E3 连接酶活性的机制来介导 SIRT7 的下调。
