Regulation of SAMHD1 antiviral activity

项目摘要/摘要 新近发现的人类限制因子SAMHD1的表达与感染有关 原代巨噬细胞、树突状细胞和巨噬细胞对慢病毒如HIV-1、HIV-2和SIVmac的阻断 静息的CD4T细胞。SAMHD1通过阻止反转录的发生来阻止慢病毒的感染。 SAMHD1具有降解脱氧核苷酸的脱氧核苷酸三磷酸水解酶(DNTPase)活性 三磷酸(DNTPs)转化为核苷酸和三磷酸盐，这一活性是限制HIV-1所必需的。 然而，更多的研究表明，SAMHD1的dNTPase活性不足以应对HIV-1 限制。因此，限制HIV-1需要SAMHD1的额外属性。SAMHD1与 核酸在体外，但这种相互作用在体内对HIV-1限制的贡献仍然是 下定决心。我们使用SAMHD1突变体的初步数据表明，核酸结合对 HIV-1限制。我们确定了SAMHD1与寡脱氧核苷酸结合的结构并利用 这种结构作为结构-功能研究的工具。几种氨基酸构成了SAMHD1之间的界面 这些残基的突变导致SAMHD1蛋白不能 限制HIV-1。这些结果表明，SAMHD1与核酸相互作用的能力对 HIV-1限制。除其抗病毒活性外，人类SAMHD1基因的突变可导致艾卡迪- 古提埃尔综合征(AGS)。AGS患者表现出I型干扰素(干扰素)水平升高，据信 由先天免疫感受器识别内源核酸所致。因此，它一直是 提示SAMHD1在体内的作用是通过以下途径阻止天然免疫反应的激活 内源性核酸，SAMHD1的核酸结合活性被认为是 对此功能很重要。我们的初步数据表明SAMHD1基因敲除(KO)小鼠对 单纯疱疹病毒2型(HSV-2)可导致肢体瘫痪和死亡。这些结果表明SAMHD1 阻止先天免疫传感器识别HSV-2，可能是通过与HSV-2病毒相互作用 并保护它不被发现。这种在体内的强烈表型将被用来揭示SAMHD1在 先天免疫力。基于这些公布的和初步的结果，这一提议的中心假设 SAMHD1与核酸的相互作用对其抗病毒和先天免疫是重要的 功能。我们的理论基础是确定SAMHD1调节病毒感染的机制 先天免疫反应将使新的抗病毒和AGS疗法的开发成为可能。测试我们的 中心假设，我们将追求以下具体目标：1)确定SAMHD1的机制 抑制HIV-1感染，2)评估SAMHD1乙酰化在SAMHD1介导的病毒限制中的作用，以及 3)研究SAMHD1在先天免疫中的作用。

Project Summary/Abstract Expression of the recently discovered human restriction factor SAMHD1 is responsible for the infection block imposed to lentiviruses such as HIV-1, HIV-2 and SIVmac by primary macrophages, dendritic cells and resting CD4+ T-cells. SAMHD1 blocks lentiviral infection by preventing the occurrence of reverse transcription. SAMHD1 has deoxynucleotide triphosphohydrolase (dNTPase) activity, which degrades deoxynucleotide triphosphates (dNTPs) into nucleotides and triphosphates, and this activity is required for HIV-1 restriction. However, additional studies have revealed that the dNTPase activity of SAMHD1 is not sufficient for HIV-1 restriction. Thus, an additional property of SAMHD1 is required for HIV-1 restriction. SAMHD1 interacts with nucleic acids in vitro, but the contribution of this interaction to HIV-1 restriction in vivo remains to be determined. Our preliminary data using SAMHD1 mutants indicate that nucleic acid binding is important for HIV-1 restriction. We determined the structure of SAMHD1 bound to an oligodeoxynucleotide and leveraged this structure as a tool for structure-function studies. Several amino acids form the interface between SAMHD1 and the oligodeoxynucleotide, and mutation of these residues resulted in SAMHD1 proteins that are unable to restrict HIV-1. These results suggest that the ability of SAMHD1 to interact with nucleic acids is important for HIV-1 restriction. Separate from its antiviral activity, mutations in the human SAMHD1 gene cause Aicardi- Goutières syndrome (AGS). AGS patients exhibit increased levels of type I interferon (IFN) that are believed to result from the recognition of endogenous nucleic acids by innate immune sensors. Thus, it has been suggested that the in vivo role of SAMHD1 is to prevent activation of the innate immune response by endogenous nucleic acids, and the nucleic acid binding activity of SAMHD1 has been suggested to be important for this function. Our preliminary data indicate that SAMHD1 knockout (KO) mice are resistant to herpes simplex virus 2 (HSV-2)-induced limb paralysis and death. These results suggest that SAMHD1 prevents the recognition of HSV-2 by innate immune sensors, potentially by interacting with the HSV-2 viral DNA and shielding it from detection. This strong phenotype in vivo will be used to unveil the role of SAMHD1 in innate immunity. Based on these published and preliminary results, the central hypothesis of this proposal is that the interaction of SAMHD1 with nucleic acids is important for its antiviral and innate immune functions. Our rationale is that identification of the mechanisms by which SAMHD1 modulates viral infection and innate immune responses will enable the development of novel antiviral and AGS therapies. To test our central hypothesis, we will pursue the following specific aims: 1) Determine the mechanism by which SAMHD1 inhibits HIV-1 infection, 2) Evaluate the role of SAMHD1 acetylation in SAMHD1-mediated viral restriction, and 3) Characterize the role of SAMHD1 in innate immunity.