Role of CD300 Family in Norovirus Tropism, Persistence, and Immunity

CD300 家族在诺如病毒趋向性、持久性和免疫中的作用

• 批准号: 9913436
• 负责人: Megan T Baldridge
• 金额: $ 69.99万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-22 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9913436
• 关键词: Amino Acids; Animals; Bile Acids; Binding; Biological Models; Biology; CRISPR screen; CRISPR/Cas technology; Calicivirus; Cell surface; Cells; Code; Color; Crystallization; Data; Data Pooling; Dependence; Detection; Enteral; Family; Family member; Family suidae; Gastroenteritis; Genes; Growth; Hela Cells; Human; Immune system; Immunity; In Vitro; Infection; Integration Host Factors; Intestines; Knowledge; Laboratories; Letters; Ligand Binding; Link; Maps; Medical; Microglia; Molecular; Mus; Norovirus; Pathogenesis; Plaque Assay; Play; Predisposition; Production; RNA Viruses; Reporting; Role; Science; Seminal; Structure; Subfamily lentivirinae; Testing; Time; Tissues; Tropism; Vaccination; Viral; Virion; Virus; Work; base; cell type; cofactor; enteric infection; experimental study; genetic manipulation; genome-wide; in vivo; receptor; receptor function; transmission process; vaccine response; vector

PROJECT SUMMARY Human noroviruses (HNoVs) are medically important non-enveloped positive-sense RNA viruses that cause gastroenteritis. This revised proposal leverages the recent discovery of CD300lf as a receptor for murine NoV (MNoV, Science 2016 353:933) to define mechanisms of NoV pathogenesis, persistence and intestinal immunity. HNoVs do not robustly replicate in small animals and so murine MNoV has emerged as a model system to discover mechanisms of NoV biology and principles of intestinal immunity that would be difficult to identify in human studies or using animals that are not easily manipulated genetically. Studies utilizing MNoV have delineated important aspects of the interplay between virus and host in the intestine. While our proposal was initially restricted to MNoV, seminal work from the Estes lab demonstrates for the first time robust replication of HNoVs in human enteric enteroids and a role for bile acids in HNoV growth (see letter, Science 2016 353:1387) allowing us to collaborate with the Estes laboratory to perform HNoV studies. New data provided in Aim 1 show that specific bile acids function as the soluble 'co-factor' that we reported as essential for the function of CD300lf as an MNoV receptor1. These data suggest a remarkable conservation of dependence on a host factor present in the intestine for both HNoV and MNoV. Based on these advances we propose new studies herein and believe that these studies will enhance the value of our proposed work. NoV species and cell tropism is determined at the level of cell binding and entry. Thus, host factors including receptor(s) required for infection and pathogenesis are incompletely understood for this viral genus, creating key knowledge gaps for understanding NoV spread, pathogenesis, vaccination, species tropism and persistence. To identify host molecules required for MNoV infection we performed a CRISPR-Cas9-based screen, and discovered that CD300lf is a receptor for MNoV in primary cells and BV2 microglial cells and that it is required for infection in vivo. CD300lf confers the ability of MNoV to bind to cells at 4oC, and is sufficient to render human HeLa cells fully permissive for MNoV replication. The crystal structure of the CD300lf ectodomain revealed an Ig-domain with a ligand binding pocket; amino acids comprising this binding pocket are critical for receptor function for MNoV infection. The soluble ectodomain (sCD300lf) neutralized viral infectivity in vitro and in vivo and we now show that it directly binds to the virion. A soluble cofactor was required for CD300lf receptor function, and new experiments show that specific bile acids function as soluble cofactors. Given these data we propose to test the hypothesis that CD300lf confers tissue and cell tropism responsible for the capacity of MNoV to establish persistent intestinal infection and to elicit immunity through the following Aims: Aim 1: Determine the molecular basis of the role of CD300lf in NoV infection. Aim 2: Determine the role of CD300lf in MNoV tropism, persistence and immunity.

项目摘要 人类北病毒（HNOVS）是医学上重要的非发育的阳性RNA病毒， 引起胃肠炎。这项修订的建议利用了最近发现CD300LF作为鼠的受体的发现 NOV（MNOV，Science 2016 353：933），以定义Nov Nov发病机理，持久性和肠道的机制 免疫。小动物在小动物中不会重复强烈复制，因此鼠Mnov已成为模型 发现Nov Biology的机制和肠道免疫原理的系统 在人类研究中识别或使用不容易通过遗传操纵的动物。利用MNOV的研究 已经描绘了肠道病毒和宿主之间相互作用的重要方面。而我们的建议 最初仅限于MNOV，Estes Lab的开创性工作首次证明 在人类肠肠域中复制HNOV，并在HNOV生长中起作用胆汁酸的作用 2016 353：1387）允许我们与ESTES实验室合作进行HNOV研究。新数据 在AIM 1中提供的表明，特定的胆汁酸作为可溶的“ co因子”，我们报告为必不可少 对于CD300LF作为MNOV受体的功能。这些数据表明对 HNOV和MNOV的肠中存在的宿主因子的依赖性。根据这些进步，我们 在此提出了新的研究，并认为这些研究将增强我们提议的工作的价值。 在细胞结合和进入的水平上确定了nov物种和细胞向量。因此，宿主因素 包括感染和发病机理所需的受体不完全了解这种病毒属， 建立关键知识差距，以理解Nov传播，发病机理，疫苗接种，物种端主和 持久性。为了识别MNOV感染所需的宿主分子，我们进行了基于CRISPR-CAS9的宿主分子 屏幕，发现CD300LF是原代细胞和BV2小胶质细胞中MNOV的受体，并且 是体内感染所必需的。 CD300LF赋予MNOV在4OC时与细胞结合的能力，并且足以使 使人类HeLa细胞完全允许进行MNOV复制。 CD300LF的晶体结构 外生域揭示了一个带有配体结合口袋的Ig域。包括这个结合口袋的氨基酸 对于MNOV感染的受体功能至关重要。可溶性外生域（SCD300LF）中和病毒 体外和体内感染性，现在我们表明它直接与病毒座结合。可溶性辅因子是 CD300LF受体功能所需的需要，新实验表明特定的胆汁酸起作用 辅因子。鉴于这些数据，我们建议测试CD300LF赋予组织和细胞向量的假设 负责MNOV的能力建立持续性肠道感染并通过 以下目的： AIM 1：确定CD300LF在NOV感染中作用的分子基础。 AIM 2：确定CD300LF在MNOV的持久性和免疫力中的作用。