Reconstitution of a protective antiviral RNAi response in somatic human cells

描述(申请人提供)：以前的工作已经证明，几个哺乳动物胚胎细胞系能够从转基因的长dsRNAs中产生功能性siRNAs，最近的一份报告表明，胚胎干细胞也可以安装保护性的抗病毒RNAi反应。相反，终末分化的哺乳动物体细胞不能从转基因的长dsRNA或从病毒感染期间产生的dsRNA产生功能上显著水平的siRNAs。我们假设这些胚胎细胞表达一种从dsRNAs中高效产生siRNA所需的因子，到目前为止，我们一直专注于单个哺乳动物的DICER蛋白，部分原因是以前的生化工作证明了N-末端结构域的存在，该结构域抑制了人类DICER在体外处理长dsRNAs的能力，但不能抑制前miRNAs。值得注意的是，我们的初步数据确实确定了一种N末端截短的DICER形式，它在胚胎畸胎癌细胞中表达，但在人类体细胞中不表达。这一DICER异构体的特性和功能分析是这项赠款申请的主要初始目标。一旦确定了这种新的DICER异构体的特性，我们将在实验室产生的一种独特的人类细胞系NoDice中表达该蛋白，以及人工合成的人类DICER缺失突变体和果蝇Dcr1/LOCS-PB和Dcr2/LOCS-PD蛋白伙伴，该细胞系缺乏任何功能的内源性DICER酶。然后，我们将确定是否可以挽救microRNA的加工，并在这些细胞中重建siRNA生产，在后一种情况下，在与RNA病毒挑战之后。因此，本研究的主要目的是确定为什么胚胎哺乳动物细胞能够在病毒感染时启动保护性RNAi反应，而体细胞则缺乏这种能力，并在哺乳动物细胞中重建RNAi。然后我们将分析RNAi是否可以保护细胞免受病毒的攻击，以期稍后将这项研究扩展到病毒致病的体内模型。总体而言，该项目不仅有可能揭示为什么RNAi在哺乳动物体细胞中不起作用，而且还可以有效地重建这种潜在的高度保护性的抗病毒先天免疫反应，最初是在培养细胞中，后来也可能在体内。

DESCRIPTION (provided by applicant): Previous work has demonstrated that several mammalian embryonic cell lines are able to generate functional siRNAs from transfected long dsRNAs and a recent report has demonstrated that embryonic stem cells can also mount a protective antiviral RNAi response. In contrast, terminally differentiated mammalian somatic cells are unable to generate a functionally significant level of siRNAs from either transfected long dsRNAs or from dsRNAs generated during viral infection. We hypothesize that these embryonic cells express a factor required for efficient siRNA production from dsRNAs and we have so far focused on the single mammalian Dicer protein, in part because previous biochemical work has documented the presence of an N- terminal domain that inhibits the ability of human Dicer to process long dsRNAs, but not pre-miRNAs, in vitro. Remarkably, our preliminary data have indeed identified an N-terminally truncated form of Dicer that is expressed in embryonic teratocarcinoma cells but not in human somatic cells. The characterization and functional analysis of this Dicer isoform are key initial goals of this grant application. Once th identity of this novel Dicer isoform is defined, we will express this protein, as well as artificia N-terminal human Dicer deletion mutants and the Drosophila Dcr1/locs-PB and Dcr2/locs-PD protein partners, in a unique human cell line generated in the laboratory, called NoDice, that lacks any functional endogenous Dicer enzyme. We will then determine if we can rescue microRNA processing and reconstitute siRNA production in these cells, in the latter case after challenge with RNA viruses. The main goal of this research is therefore to determine why embryonic mammalian cells are able to mount a protective RNAi response upon viral infection, while somatic cells lack this ability, and to reconstitute RNAi in mammalian cells. We will then analyze whether RNAi can protect cells against viral challenge with a view to later extending this research into in vivo models of viral pathogenesis. Overall, this project has the potential to not only reveal why RNAi is non- functional in mammalian somatic cells but also to allow the effective reconstitution of this potentially highly protective antiviral innate immune response, initially in cultured cells but potentially later also in vivo.