Regulation of SAMHD1 antiviral activity

项目总结/摘要 最近发现的人类限制性因子SAMHD 1的表达是感染的原因 阻断由原代巨噬细胞、树突细胞和巨噬细胞对慢病毒如HIV-1、HIV-2和SIVmac的作用。 静息CD 4 + T细胞。SAMHD 1通过阻止逆转录的发生来阻断慢病毒感染。 SAMHD 1具有脱氧核苷酸三磷酸水解酶（dNTH）活性，其降解脱氧核苷酸 三磷酸（dNTPs）转化为核苷酸和三磷酸，并且这种活性是HIV-1限制所需的。 然而，另外的研究表明，SAMHD 1的dNTR活性对于HIV-1来说是不够的。 限制.因此，SAMHD 1的额外性质是HIV-1限制所必需的。SAMHD 1与 核酸在体外，但这种相互作用的贡献，HIV-1的限制在体内仍然是 测定我们使用SAMHD 1突变体的初步数据表明，核酸结合对于 HIV-1限制。我们确定了与寡脱氧核苷酸结合的SAMHD 1的结构，并利用 这种结构作为结构-功能研究的工具。几个氨基酸形成SAMHD 1之间的界面 和寡脱氧核苷酸，这些残基的突变导致SAMHD 1蛋白不能 限制HIV-1。这些结果表明，SAMHD 1与核酸相互作用的能力对于 HIV-1限制。除了其抗病毒活性，人类SAMHD 1基因的突变会导致Abladii- Goutières综合征（AGS）。AGS患者表现出I型干扰素（IFN）水平升高，据信I型干扰素（IFN） 由先天免疫传感器识别内源性核酸引起。因此， 提示SAMHD 1的体内作用是通过以下途径阻止先天免疫应答的激活： 内源性核酸，并且已经提出SAMHD 1的核酸结合活性是 这一功能很重要。我们的初步数据表明，SAMHD 1敲除（KO）小鼠对 单纯疱疹病毒2型（HSV-2）引起肢体瘫痪和死亡。这些结果表明，SAMHD 1 可能通过与HSV-2病毒相互作用，阻止先天免疫传感器识别HSV-2 DNA并屏蔽它不被发现。这种强的体内表型将被用来揭示SAMHD 1在细胞凋亡中的作用。 先天免疫根据这些已发表的初步结果，本提案的中心假设 SAMHD 1与核酸的相互作用对于其抗病毒和天然免疫是重要的 功能协调发展的我们的基本原理是SAMHD 1调节病毒感染的机制的鉴定 和先天免疫反应将使新的抗病毒和AGS疗法的发展成为可能。来测试我们 中心假设，我们将追求以下具体目标：1）确定SAMHD 1 抑制HIV-1感染，2）评估SAMHD 1乙酰化在SAMHD 1介导的病毒限制中的作用，和 3)SAMHD 1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.