Defining a common mechanism of SAMHD1 function in cancer, HIV, and AGS

项目摘要 截至2019年，全球有3800万艾滋病毒携带者。这个数字每年都在继续上升。 尽管在艾滋病毒治疗方面取得了进展。开发治疗艾滋病毒的方法的一个重大挑战仍然是 潜伏感染细胞的蓄水池。目前的抗逆转录病毒药物不能针对这一子集的未分裂细胞 转录不活跃的前病毒。众所周知，艾滋病毒在非分裂细胞中的感染与 在细胞分裂方面比艾滋病毒感染具有更多的特性。例如，未分裂细胞的dNTP浓度低于 逆转录酶的KM显著减慢病毒复制的动力学。SAM和HD域 含有蛋白1(SAMHD1)是负责这一限制的宿主dNTPase。透彻了解 SAMHD1在未分裂的水库细胞中感染HIV-1的独特环境中的贡献 提供新的治疗方法来靶向这个储蓄者的可能性。为此，我建议将一部小说描述为 一系列与几种癌症有关的SAMHD1突变，并利用这些突变作为工具 进一步探讨SAMHD1在HIV-1限制中的作用。我发现了一种白血病和结肠癌 SAMHD1突变体，R366C/H，保持蛋白质稳定性，但失去dNTPase活性。与细胞周期的相互作用 SAMHD1的蛋白质和参与双链DNA断裂修复的dNTPase不依赖的功能是 保存在这个突变体中。有趣的是，导致dNTPase活性受损的SAMHD1突变是第一个 Aicardi Gtières综合征(AGS)是一种罕见的以异常类型为特征的遗传性脑病 1干扰素的产生。这些ags突变体中的许多不能限制巨噬细胞中HIV-1的感染 因为它们无法耗尽细胞内的dNTP。鉴于癌细胞中的dNTP浓度要高出6-11倍 SAMHD1基因敲除导致癌细胞表型，如增殖增加 SAMHD1作为dNTPase的作用可能还涉及该酶在癌症中的作用。 因此，我推测R366C/H SAMHD1癌症突变体的dNTPase缺陷对 对癌细胞表型和取消HIV-1在非分裂细胞中的限制和天然免疫 响应抑制。在目标1中，我将完成R366C/H癌症突变体的功能描述 并利用生化和细胞化学方法确定这些功能缺陷对癌细胞表型的影响 生物学技术。随后，在目标2中，我将使用病毒学、细胞生物学和免疫学方法来 分析R366C/H癌突变对HIV-1限制性内切酶和天然免疫抑制的影响，从而 使用这种癌症突变来探测更多传统上研究的SAMHD1疾病表型。总而言之，这些 免费的AIMS将突出SAMHD1 dNTPase活性在癌症中的作用，并将 SAMHD1参与癌症、HIV-1限制和AGS。

Project Summary As of 2019 there were 38.0 million individuals living with HIV globally. This number continues to rise yearly despite advances in HIV treatment. A significant challenge to developing a cure for HIV continues to be the reservoir of latently infected cells. Current antiretrovirals cannot target this subset of non-dividing cells that harbor transcriptionally inactive provirus. It is well established that HIV infection in non-dividing cells has different properties than HIV infection in dividing cells. For example, non-dividing cells harbor dNTP concentrations below the Km of reverse transcriptase which significantly slows the kinetics of viral replication. SAM and HD domain containing protein 1 (SAMHD1) is the host dNTPase responsible for this restriction. A thorough understanding of the contribution of SAMHD1 to the unique environment of HIV-1 infection in non-dividing reservoir cells has the potential to inform novel therapeutics to target this reservoir. To this effect, I propose to characterize a novel series of SAMHD1 mutations that have been implicated in several cancers and use these mutants as tools to further probe the role of SAMHD1 in HIV-1 restriction. I have identified a leukemia and colon cancer associated SAMHD1 mutant, R366C/H, that retains protein stability but loses dNTPase activity. Interactions with cell cycle proteins and involvement in double strand DNA break repair, dNTPase independent functions of SAMHD1, are preserved in this mutant. Interestingly, SAMHD1 mutations that result in impaired dNTPase activity were first reported in Aicardi Goutières Syndrome (AGS), a rare inherited encephalopathy characterized by aberrant type 1 interferon production. Many of these AGS mutants are unable to restrict HIV-1 infection in macrophages due to their inability to deplete cellular dNTPs. Given that the dNTP concentration in cancer cells is 6-11 fold higher than in normal cells and that SAMHD1 knock-out results in cancer cell phenotypes such as increased proliferation and reduced apoptosis, it is likely that the role of SAMHD1 as a dNTPase also involves the enzyme in cancer. Therefore, I hypothesize that the dNTPase deficiency of the R366C/H SAMHD1 cancer mutant contributes to cancer cell phenotypes and abrogates both HIV-1 restriction in non-dividing cells and innate immune response suppression. In Aim 1, I will complete my functional characterization of the R366C/H cancer mutant and determine the impact of these functional deficits on cancer cell phenotypes using biochemical and cell biology techniques. Subsequently in Aim 2, I will use virology, cell biology, and immunology approaches to analyze the effect of the R366C/H cancer mutation on HIV-1 restriction and innate immune suppression, thus using this cancer mutation to probe more traditionally studied SAMHD1 disease phenotypes . Collectively, these complimentary aims will highlight the role of SAMHD1 dNTPase activity in cancer and mechanistically link the involvement of SAMHD1 in cancer, HIV-1 restriction, and AGS.