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: 10756871
• 负责人: Siyuan Ding
• 金额: $ 7.78万
• 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/10756871
• 关键词: Accountability; Adsorption; Amino Acids; Antiviral Response; Binding; Binding Proteins; Biochemical; Biochemistry; Biological Assay; Biological Models; Biology; CEB1 Gene; Cells; Cellular biology; Cessation of life; Child; Chronic diarrhea; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cryoelectron Microscopy; Data; Data Set; Development; Diarrhea; Disease; Early Endosome; Ectopic Expression; Elongation Factor; Endocytosis; Enteral; Epithelial Cells; Epithelium; Expression Library; Foundations; Fractionation; Gastroenteritis; Generations; Genes; Genetic Transcription; Goals; House mice; Human; Immune Evasion; Immunoprecipitation; In Vitro; Individual; Infant; Integration Host Factors; Interferons; Intestines; Knock-out; Knowledge; Label; Letters; Maps; Mass Spectrum Analysis; Mediating; Medical; Messenger RNA; Modeling; Modification; Molecular; Morbidity - disease rate; Mucous Membrane; Mutagenesis; Mutate; Mutation; Neonatal; Nonstructural Protein; Nucleic Acids; Organoids; Patients; Penetration; Persons; 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; Therapeutic Intervention; Viral; Viral Antigens; Viral Pathogenesis; Virus; Virus Replication; Vomiting; antagonist; attenuation; burden of illness; cell type; cofactor; crosslink; enteric virus infection; fluorophore; gain of function; human pathogen; improved; in vivo; intestinal epithelium; major vault protein; mortality; mutant; novel; nucleoside triphosphatase; overexpression; parent grant; prevent; rational design; response; reverse genetics; screening; suckling; superresolution microscopy; ubiquitin-protein ligase; vaccine candidate; viral RNA

Project Summary From the parent grant: 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， 是全球婴幼儿严重胃肠炎、呕吐和腹泻的主要原因。 RV也是询问宿主粘膜表面的抗病毒反应的一个很好的模型。我们的整体 目标是更好地了解RV-宿主相互作用，并利用这些信息开发改进的RV 疫苗和治疗干预，从而预防和治疗肠道病毒感染。主机 干扰素（IFN）信号转导是RV宿主范围限制的基础，并抑制 RV不是该物种在体内的原生动物。然而，特异性IFN介导的抗病毒效应物是未知的， 相关的分子机制仍不清楚。为了弥补这一知识差距，我们试图定义 在原代人肠上皮细胞（IEC）中最高度诱导的IFN刺激基因（ISG）。使用 一种IEC特异性ISG功能获得性筛选方法，我们确定了几种新的宿主因子， RV复制，包括含无菌α基序结构域9（由SAMD 9编码）。细胞内病毒RNA 在SAMD 9 CRISPR敲除细胞中，CRISPR水平和病毒子代产生显著增强。同时， 我们还取得了令人兴奋的发现，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，使用一组新颖、强大且易于处理的模型系统， 我们将检验SAMD 9以上皮细胞特异性方式限制早期RV复制的假设， 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。 在目的1中，我们将使用以下方法研究SAMD 9体外抑制RV复制的机制基础： 几个新的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 将在生理学相关的人类小肠类器官培养物中测试这些发现， 健康个体和SAMD 9突变患者。在目标2中，我们将研究RV NSP 1如何与SAMD 9结合 通过一种新的识别基序，并诱导其降解。我们将确定NSP 1是否降解SAMD 9 在体内的IEC中，如果该过程有助于使用新的新生大鼠成功进行RV肠道复制， 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。