Interferon-Stimulated Gene Inhibition of Rotavirus Replication and Viral Antagonism

干扰素刺激的轮状病毒复制基因抑制和病毒拮抗作用

• 批准号: 10117185
• 负责人: Siyuan Ding
• 金额: $ 39.38万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-03 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10117185
• 关键词: Adsorption; Antiviral Agents; Antiviral Response; Binding; Binding Proteins; Biochemistry; Biological Assay; Biological Models; Biology; CEB1 Gene; Cells; Cessation of life; Child; Chronic diarrhea; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Data; Data Set; Diarrhea; Disease; Early Endosome; Ectopic Expression; Elongation Factor; Endocytosis; Enteral; Epithelial; Epithelial Cells; Evolution; Foundations; Gastroenteritis; Generations; Genes; Genetic Transcription; Goals; House mice; Human; Immune Evasion; Immunoprecipitation; In Vitro; Individual; Infant; Infection; Integration Host Factors; Interferons; Intestines; Knock-out; Knowledge; Label; Letters; Libraries; Mass Spectrum Analysis; Mediating; Medical; Messenger RNA; Modeling; Modification; Molecular; Morbidity - disease rate; Mucous Membrane; Mutagenesis; Mutation; Neonatal; Nonstructural Protein; Organoids; Outcome; Patients; Physiological; Poxviridae; Predisposition; Process; Production; Proteins; Proteomics; Protocols documentation; RNA; Rattus; Role; Rotavirus; Rotavirus Infections; Rotavirus Vaccines; SAM Domain; Severities; Side; Signal Transduction; Site-Directed Mutagenesis; Small Interfering RNA; Small Intestines; Structure; Surface; Symptoms; Syndrome; System; Testing; Therapeutic Intervention; Viral; Viral Antigens; Viral Pathogenesis; Virus; Virus Diseases; Virus Replication; Vomiting; attenuation; base; burden of illness; cell type; crosslink; design; enteric virus infection; fluorophore; gain of function; human pathogen; improved; in vivo; intestinal epithelium; major vault protein; mortality; mutant; novel; overexpression; prevent; response; reverse genetics; screening; suckling; therapeutic development; ubiquitin-protein ligase; vaccine candidate; viral RNA

Project Summary Rotaviruses (RVs) are a medically important human pathogen and the predominant cause of severe gastroenteritis, vomiting, and diarrhea in infants and young children worldwide. RVs are also a great model to interrogate the antiviral responses at the host mucosal surfaces. Our overall objectives are to better understand RV-host interactions and to use that information to develop improved RV vaccines and therapeutic interventions, thereby preventing and treating enteric virus infections. The host interferon (IFN) signaling underlies the basis of RV host range restriction and suppresses the replication of RVs not native to that species in vivo. However, the specific IFN-mediated antiviral effectors are not known and the associated molecular mechanisms remain unclear. To bridge this gap in knowledge, we sought to define the most highly induced IFN-stimulated genes (ISGs) in primary human intestinal epithelial cells (IECs). Using an IEC-specific ISG gain-of-function screening approach, we identified several novel host factors that restrict RV replication, including sterile alpha motif domain-containing 9 (encoded by SAMD9). Intracellular viral RNA levels and virus progeny production were significantly enhanced in SAMD9 CRISPR knockout cells. In parallel, we also made the exciting discovery that RV encodes non-structural protein 1 (NSP1) to target SAMD9 for proteasomal degradation. In this R01 application, using a set of novel, powerful, and tractable model systems, we will test the hypotheses that SAMD9 confines early RV replication in an epithelial cell-specific manner and that RV NSP1 functions to overcome SAMD9 restriction to promote viral replication and pathogenesis in vivo. In Aim 1, we will examine the mechanistic basis underlying SAMD9 inhibition of RV replication in vitro using several newly available fluorophore-labeled RVs and a recently developed RV reverse genetics system. We will test these findings in a physiologically relevant human small intestinal organoid culture derived from healthy individuals and SAMD9-mutation patients. In Aim 2, we will examine how RV NSP1 binds to SAMD9 via a novel recognition motif and induces its degradation. We will determine whether NSP1 degrades SAMD9 in IECs in vivo and if this process contributes to successful RV intestinal replication using a novel neonatal rat model. Collectively, we expect these studies on SAMD9-RV interactions to have a substantial impact on elucidating the basic biology of ISG mode of action, identifying new viral innate immune evasion mechanisms, and laying the scientific foundation for the rational design of new RV vaccine candidates based on targeted NSP1 attenuation.

项目摘要 轮状病毒（RV）是一种医学上重要的人类病原体，并且是导致严重呼吸道感染的主要原因。 肠胃炎，呕吐和腹泻的婴儿和幼儿在世界各地。房车也是一个伟大的模式， 询问宿主粘膜表面的抗病毒反应。我们的总体目标是 了解RV与宿主的相互作用，并利用这些信息开发改进的RV疫苗和治疗药物， 因此，预防和治疗肠道病毒感染。宿主干扰素（IFN）信号传导 是RV宿主范围限制的基础，并抑制非RV宿主的RV复制。 体内的物种然而，特异性干扰素介导的抗病毒效应物尚不清楚，相关的抗病毒效应物也不清楚。 分子机制仍不清楚。为了弥合这一知识差距，我们试图定义最高的 在原代人肠上皮细胞（IEC）中诱导IFN刺激基因（ISG）。使用IEC专用 ISG功能获得性筛选方法，我们确定了几个限制RV复制的新宿主因子， 包括含有无菌α基序结构域的9（由SAMD 9编码）。细胞内病毒RNA水平和病毒 在SAMD 9 CRISPR敲除细胞中，子代产生显著增强。与此同时，我们还 令人兴奋的发现是RV编码非结构蛋白1（NSP 1），靶向SAMD 9蛋白酶体， 降解在这个R 01应用程序中，我们将使用一组新颖、强大且易于处理的模型系统来测试 假设SAMD 9以上皮细胞特异性方式限制早期RV复制，并且RV NSP 1的功能是克服SAMD 9的限制，以促进病毒在体内的复制和致病。在目标1中， 我们将研究SAMD 9在体外抑制RV复制的机制基础， 新可用的荧光团标记的RV和最近开发的RV反向遗传学系统。我们将测试 这些发现在来自健康人的生理学相关的人小肠类器官培养中， 个体和SAMD 9突变患者。在目标2中，我们将研究RV NSP 1如何通过一个结合位点与SAMD 9结合。 新的识别基序，并诱导其降解。我们将确定NSP 1是否在细胞中降解SAMD 9。 IEC在体内，如果这一过程有助于成功的RV肠道复制使用新的新生大鼠 模型总的来说，我们期望这些关于SAMD 9-RV相互作用的研究对以下方面产生重大影响： 阐明ISG作用模式的基础生物学，鉴定新的病毒先天免疫逃避机制， 为基于靶向的RV候选疫苗的合理设计奠定科学基础， NSP 1衰减。