Interferon-Stimulated Gene Inhibition of Rotavirus Replication and Viral Antagonism

项目摘要 轮状病毒(RV)是一种重要的医学上的人类病原体，也是引起严重 全世界婴幼儿的胃肠炎、呕吐和腹泻。房车也是一款很好的车型 询问宿主粘膜表面的抗病毒反应。我们的总体目标是更好地 了解轮状病毒与宿主的相互作用，并利用这些信息开发改进的轮状病毒疫苗和治疗方法 干预措施，从而预防和治疗肠道病毒感染。宿主干扰素信号转导 奠定了RV主机范围限制的基础，并抑制了非本机RV的复制 活体内的物种。然而，特异性干扰素介导的抗病毒效应尚不清楚，相关的 分子机制尚不清楚。为了弥合这一知识鸿沟，我们试图定义最高 在原代培养的人肠上皮细胞中诱导干扰素刺激基因(ISGs)。使用特定于IEC的 ISG功能增益筛选方法，我们发现了几种限制RV复制的新宿主因素， 包括无菌α基序结构域9(由SAMD9编码)。细胞内病毒RNA水平与病毒 SAMD9 CRISPR基因敲除细胞的后代产量显著提高。同时，我们还制作了 RV编码非结构蛋白1(NSP1)靶向蛋白酶体SAMD9的令人兴奋的发现 退化。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 SAMD9以上皮细胞特异性方式限制早期轮状病毒复制的假说 NSP1的功能是克服SAMD9的限制，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 最新可用的荧光标记房车和最近开发的房车反向遗传学系统。我们将测试 这些发现来自健康人的生理相关的人类小肠器官培养 个体和SAMD9突变患者。在目标2中，我们将研究RV NSP1如何通过一个 新的识别基序，并导致其退化。我们将确定NSP1是否会降低SAMD9在 体内的IECS以及这一过程是否有助于使用新的新生大鼠成功复制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 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.