Reconstitution of a protective antiviral RNAi response in somatic human cells

重建人体细胞中保护性抗病毒 RNAi 反应

基本信息

批准号：
8849841
负责人：
BRYAN R. CULLEN
金额：
$ 19.85万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-01 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8849841
关键词：
Animals Antiviral Agents Antiviral Response Applications Grants Biochemical Biogenesis Cell Line Cells Cleaved cell Cloning Complementary DNA Cultured Cells Data Dicer Enzyme Drosophila genus Drosophila melanogaster Embryo Excision Genes Genome engineering Germ Cells Goals Human Human Cell Line Immune response In Vitro Infection Invertebrates Laboratories Length Malignant Epithelial Cell Mammalian Cell Mammals Mediating MicroRNAs Mutate N-terminal Plants Process Production Protein Isoforms Proteins Publishing RNA Interference RNA Viruses Reporting Research Small Interfering RNA Somatic Cell Teratocarcinoma Testing Variant Vesicular stomatitis Indiana virus Viral Viral Pathogenesis Virus Virus Diseases Virus Replication Work base blastomere structure cell type embryonic stem cell helicase human DICER1 protein in vivo in vivo Model mutant novel pathogen prevent protective effect public health relevance reconstitution research study response selective expression

Animals Antiviral Agents Antiviral Response Applications Grants Biochemical Biogenesis Cell Line Cells Cleaved cell Cloning Complementary DNA Cultured Cells Data Dicer Enzyme Drosophila genus Drosophila melanogaster Embryo Excision Genes Genome engineering Germ Cells Goals Human Human Cell Line Immune response In Vitro Infection Invertebrates Laboratories Length Malignant Epithelial Cell Mammalian Cell Mammals Mediating MicroRNAs Mutate N-terminal Plants Process Production Protein Isoforms Proteins Publishing RNA Interference RNA Viruses Reporting Research Small Interfering RNA Somatic Cell Teratocarcinoma Testing Variant Vesicular stomatitis Indiana virus Viral Viral Pathogenesis Virus Virus Diseases Virus Replication Work base blastomere structure cell type embryonic stem cell helicase human DICER1 protein in vivo in vivo Model mutant novel pathogen prevent protective effect public health relevance reconstitution research study response selective expression

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项目摘要

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.

描述（由申请人提供）：以前的工作表明，几种哺乳动物的胚胎细胞系能够从转染的长DSRNA中产生功能性siRNA，并且最近的一份报告表明，胚胎干细胞还可以安装保护性抗病毒药RNAI响应。相比之下，末端分化的哺乳动物体细胞无法从转染的长DSRNA或病毒感染过程中产生的DSRNA产生功能上显着的siRNA。我们假设这些胚胎细胞表达了从DSRNA高效产生siRNA所需的因素，到目前为止，我们专注于单个哺乳动物丁香蛋白，部分原因是先前的生物化学作品已经证明存在N末端结构域的存在，这些n-终端结构域抑制了人dicer可以处理长dsrnas的能力，但不是Pre-Mirnas，而不是Vitro。值得注意的是，我们的初步数据确实已经确定了在胚胎畸胎瘤细胞中表达的DICER的N末端截断形式，但在人类体细胞细胞中却没有。此迪切尔同工型的表征和功能分析是该赠款应用程序的关键初始目标。一旦定义了这种新颖的迪切尔同工型的身份，我们将表达该蛋白质，以及n末端的人dicer删除突变体和果蝇DCR1/locs-pb和dcr2/locs-pd蛋白质伴侣，在实验室中产生的独特人细胞系，称为Nodice endecem，这是任何功能性的dicer dicer dicer dicer dicer dice dice dice dice dice dice dice dice eNZ中的独特人细胞系中。然后，我们将确定在挑战RNA病毒之后，在后一种情况下，我们是否可以在这些细胞中挽救MicroRNA加工并重建这些细胞中的siRNA产生。因此，这项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在保护性RNAi反应上，而体细胞缺乏这种能力，并在哺乳动物细胞中重新构建RNAi。然后，我们将分析RNAi是否可以保护细胞免受病毒挑战的影响，以期将这项研究扩展到病毒发病机理的体内模型中。总体而言，该项目不仅有可能揭示RNAi在哺乳动物的体细胞中不起作用，而且还可以有效地重构这种潜在的高度保护性的抗病毒先天免疫反应，最初是在培养的细胞中，但后来有可能在体内。