Vpr Revisited

描述(申请人提供)：HIV-1 VPR是一种由所有慢病毒编码的核、病毒颗粒包装的辅助蛋白。此外，SIV/HIV-2还编码VPX，它保持了密切的序列相似性。VPR的特点是诱导细胞周期停滞于G2/M期。了解VPR/VPX在病毒复制和致病中的作用的关键是鉴定它们与之相互作用的细胞蛋白。包括我们在内的几个小组最近报告了一种细胞复合体的鉴定，VPR和VPX可能与之相互作用，并可能通过它发挥作用。该复合体是由DDB1/DCAF1/CUL4A/ROC1组成的E3泛素连接酶。该复合体通过介导DNA复制许可因子CDT1、STAT1和STAT2等细胞蛋白质的破坏来调节基因组DNA复制和DNA损伤修复，这些底物尚未确定。与E3泛素连接酶相互作用的鉴定为VPR如何介导其作用提供了一个统一的解释，并为阐明VPR在HIV-1致病中所起的作用提供了一个框架。其他病毒编码针对这种复合体的蛋白质，利用它作为一种手段来诱导STAT蛋白的降解，以抑制I型干扰素反应。我们将确定VPR是否通过与该复合体的相互作用来阻止G2期细胞，诱导细胞凋亡，并激活DNA损伤信号通路。此外，还将鉴定和表征与VPX和DCAF1结合的细胞蛋白。第三，该项目将检验他们的假设，即VPR/VPX在抑制对病毒的先天免疫反应方面发挥作用。在过去的几年里，先天免疫已经成为一个非常有趣的话题。该项目将确定VPR/VPX是否可构成另一种此类机制。与公共卫生相关的细胞具有抵抗病毒感染的机制。这些机制引起了人们的极大兴趣，因为它们为抗病毒药物的开发提供了新的途径。病毒已经进化出逃避这些所谓的先天免疫机制的方法。例如，艾滋病毒会产生一种名为病毒感染性因子(VIF)的蛋白质，它会使血细胞产生的一种抗病毒蛋白质失活。在这个项目中，我们将研究一种名为病毒蛋白R(VPR)的艾滋病毒蛋白质，它可能是病毒避开先天免疫机制的另一种方式。当一个细胞感染艾滋病毒时，VPR会产生，正如我们最近发现的那样，它与一种名为受损DNA结合蛋白1(DDB1)的细胞蛋白结合，并与另一种名为DCAF1的蛋白结合。DCAF1/DDB1是细胞的一种分子机器，可以破坏不需要的蛋白质。我们的发现表明，VPR导致DDB1降解某些细胞蛋白，我们假设其中至少有一种是病毒需要破坏才能复制的抗病毒蛋白。该项目将确定DDB1的靶标，并了解这些蛋白质如何对抗病毒。此外，VPR似乎影响DCAF1/DDB1复合体的正常功能，从而扰乱细胞内的功能，如DNA修复和DNA复制。该项目将确定这是如何发生的，以及它如何影响艾滋病毒的复制。

DESCRIPTION (provided by applicant): HIV-1 Vpr is a nuclear, virion packaged accessory protein encoded by all lentiviruses. SIV/HIV-2 encodes in addition, Vpx which maintains close sequence similarity. The hallmark of Vpr is that it induces G2/M cell cycle arrest. A key to understanding the role of Vpr/Vpx in virus replication and pathogenesis is the identification of the cellular proteins with which they interact. Several groups, including ours, recently reported the identification of a cellular complex with which Vpr and perhaps Vpx interact and through which they may function. The complex is an E3 ubiquitin ligase that consists of DDB1/DCAF1/CUL4A/ROC1. This complex regulates genomic DNA replication and damaged DNA repair by mediating the destruction of cellular proteins including the DNA replication-licensing factor CDT1, STAT1 and STAT2 and other, yet to be identified substrates. The identification of the interaction with the E3 ubiquitin ligase presents a unified explanation of how Vpr may mediate its effects and provides a framework to elucidate the role that Vpr plays in HIV-1 pathogenesis. Other viruses encode proteins that target this complex, using it as a means to induce degradation of STAT proteins to inhibit type-I interferon responses. We will determine whether Vpr acts through its interaction with the complex to arrest cells in G2, induce apoptosis and activate DNA damage signaling pathways. In addition, cellular proteins that bind to Vpx and to DCAF1 will be identified and characterized. Thirdly, the project will test they hypothesis that Vpr/Vpx play a role in dampening innate immune responses to the virus. Innate immunity has over the past several years become a topic of great interest. The project will determine whether Vpr/Vpx may constitute another such mechanism. PUBLIC HEALTH RELEVANCE Cells have mechanism by which they resist infection by viruses. These mechanism are of great interest because they provide new avenues for the development of antiviral drugs. Viruses have evolved means of escaping these so called innate immune mechanisms. HIV, for example produces a protein called virion infectivity factor( VIF) that inactivates one of the antiviral proteins that are produced by blood cells. In this project we will study an HIV protein called viral protein R (VPR) that may serve as yet another means by which the virus avoids the innate immune mechanisms. When a cell is infected with HIV, Vpr is produced and as we recently discovered, binds to a cellular protein called damaged DNA binding protein 1 (DDB1) in association with another protein called DCAF1. DCAF1/DDB1 is a molecular machine of the cell that destroys unwanted proteins. Our findings have shown that Vpr causes DDB1 to degrade certain cellular proteins and we hypothesize that at least one of these is an antiviral protein that the virus needs to destroy in order to replicate. The project will identify the targets of DDB1 and understand how these proteins work against the virus. In addition, Vpr seems to affect the normal function of the DCAF1/DDB1 complex and thereby disrupt functions in the cell such as DNA repair and DNA replication. The project will determine how this happens and how it affects replication of HIV.