Interferon-Stimulated Gene Inhibition of Rotavirus Replication and Viral Antagonism

项目摘要 来自父母的资助：轮状病毒(RV)是一种重要的人类病原体， 全球婴幼儿严重胃肠炎、呕吐和腹泻的主要原因。 RVS也是询问宿主粘膜表面抗病毒反应的一个很好的模型。我们的整体 目标是更好地了解房车与宿主的相互作用，并利用这些信息来开发改进的房车 疫苗和治疗干预，从而预防和治疗肠道病毒感染。东道主 干扰素信号是限制RV宿主范围的基础，并抑制RV的复制 房车不是该物种在活体内原生的。然而，特定的干扰素介导的抗病毒效应尚不清楚， 相关的分子机制仍不清楚。为了弥合这一知识鸿沟，我们试图定义 在原代人类肠上皮细胞(IECS)中诱导最高水平的干扰素刺激基因(ISGs)。vbl.使用 一种IEC特异性的ISG功能增益筛选方法，我们确定了几种新的宿主因素，它们限制了 RV复制，包括无菌α基序结构域9(由SAMD9编码)。细胞内病毒RNA 在SAMD9 CRISPR基因敲除细胞中，病毒水平和病毒后代产量显著增加。同时， 我们还发现，轮状病毒编码非结构蛋白1(NSP1)，以SAMD9为靶点 蛋白酶体降解。在这个R01应用程序中，使用了一组新颖、强大和易处理的模型系统， 我们将测试SAMD9以上皮细胞特异性的方式限制早期RV复制的假设，并 RV NSP1的功能是克服SAMD9限制，促进病毒在体内的复制和致病。 在目标1中，我们将研究SAMD9在体外抑制RV复制的机制基础 几个新近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 将在一种与生理相关的人类小肠器官培养中测试这些发现 健康人和SAMD9突变患者。在目标2中，我们将研究RV NSP1如何与SAMD9绑定 通过一种新的识别基序并诱导其降解。我们将确定NSP1是否会降低SAMD9 在活体IECS中，以及这一过程是否有助于使用新生大鼠成功复制RV肠道 模特。总体而言，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，识别新的病毒先天免疫逃避机制， 为基于靶向的新型RV候选疫苗的合理设计奠定了科学基础 NSP1衰减。

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.