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: 10581528
• 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/10581528
• 关键词: Accountability; Adsorption; 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; Development; Diarrhea; Disease; Early Endosome; Ectopic Expression; Elongation Factor; Endocytosis; Enteral; Epithelial Cells; Epithelium; Expression Library; Foundations; 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; Mutation; Neonatal; Nonstructural Protein; Organoids; Outcome; 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 Diseases; 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; overexpression; prevent; rational design; response; reverse genetics; screening; suckling; 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（RVS）是医学上重要的人类病原体，是严重的主要原因 全世界婴儿和幼儿的胃肠炎，呕吐和腹泻。 RVS也是一个很好的模型 询问宿主粘膜表面的抗病毒反应。我们的整体目标是更好 了解RV-host互动并使用该信息来开发改进的RV疫苗和治疗性 干预措施，从而预防和治疗肠道病毒感染。主机干扰素（IFN）信号传导 基础RV主机范围限制的基础并抑制了不本地的RV的复制 体内物种。但是，尚不清楚特定的IFN介导的抗病毒效应子 分子机制尚不清楚。为了弥合知识的差距，我们试图定义最高的 原代人肠上皮细胞（IEC）诱导的IFN刺激基因（ISG）。使用特定于IEC的 ISG功能筛查方法，我们确定了几种限制RV复制的新型宿主因素， 包括无菌α基序域9（由SAMD9编码）。细胞内病毒RNA水平和病毒 SAMD9 CRISPR敲除细胞中的后代产生显着增强。同时，我们也做了 RV编码非结构蛋白1（NSP1）的令人兴奋的发现以靶向蛋白酶体 降解。在此R01应用程序中，使用一组新颖，功能强大且可操作的模型系统，我们将测试 SAMD9以上皮细胞特异性方式限制了早期RV复制的假设 NSP1的功能以克服SAMD9限制以促进体内病毒复制和发病机理。在AIM 1中， 我们将使用几个 新近可用的荧光团标签的RV和最近开发的RV反向遗传学系统。我们将测试 这些发现在生理相关的人类小肠癌培养 个体和SAMD9妇女的患者。在AIM 2中，我们将检查RV NSP1如何通过A结合SAMD9 新颖的识别基序并引起其降解。我们将确定NSP1是否降低了SAMD9 IEC在体内，如果此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，我们希望这些关于SAMD9-RV相互作用的研究对 阐明ISG作用方式的基本生物学，确定新的病毒先天免疫逃避机制， 并为基于有针对性的新RV疫苗的合理设计奠定了科学基础 NSP1衰减。