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

基本信息

批准号：
10355504
负责人：
Siyuan Ding
金额：
$ 39.38万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
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/10355504
关键词：
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 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 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 Intervention Viral Viral Antigens Viral Pathogenesis Virus Virus Diseases Virus Replication Vomiting antagonist attenuation base burden of illness cell type 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 therapeutic development ubiquitin-protein ligase vaccine candidate viral RNA

项目摘要

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.

项目概要轮状病毒 (RV) 是医学上重要的人类病原体，也是导致严重疾病的主要原因全世界婴儿和幼儿的胃肠炎、呕吐和腹泻。房车也是一个很好的模型询问宿主粘膜表面的抗病毒反应。我们的总体目标是更好了解 RV 与宿主的相互作用，并利用该信息开发改进的 RV 疫苗和治疗方法干预措施，从而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，并抑制非本地 RV 的复制体内的物种。然而，具体的 IFN 介导的抗病毒效应物尚不清楚，相关的分子机制仍不清楚。为了弥合这一知识差距，我们试图定义最高在原代人肠上皮细胞（IEC）中诱导IFN刺激基因（ISG）。使用 IEC 特定的 ISG 功能获得筛选方法，我们确定了几种限制 RV 复制的新宿主因素，包括含有9个不育α基序结构域（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量显着增强。与此同时，我们还做了 RV 编码非结构蛋白 1 (NSP1) 以靶向 SAMD9 进行蛋白酶体的激动人心的发现降解。在这个 R01 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 SAMD9 以上皮细胞特异性方式限制早期 RV 复制，并且 RV NSP1 的功能是克服 SAMD9 限制，促进体内病毒复制和发病机制。在目标 1 中，我们将使用几种方法在体外检查 SAMD9 抑制 RV 复制的机制基础新推出的荧光团标记的 RV 和最近开发的 RV 反向遗传学系统。我们将测试这些发现来自于健康的生理相关的人类小肠类器官培养物个体和 SAMD9 突变患者。在目标 2 中，我们将研究 RV NSP1 如何通过新的识别基序并诱导其降解。我们将确定 NSP1 是否会降解 SAMD9 体内 IEC 以及该过程是否有助于使用新型新生大鼠成功进行 RV 肠道复制模型。总的来说，我们预计这些关于 SAMD9-RV 相互作用的研究将对阐明 ISG 作用模式的基本生物学，确定新的病毒先天免疫逃避机制，为基于针对性的合理设计新型RV候选疫苗奠定科学基础 NSP1 衰减。