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.

项目概要 人类诺如病毒 (HNoV) 是医学上重要的无包膜正义 RNA 病毒， 引起胃肠炎。该修订后的提案利用了最近发现的 CD300lf 作为小鼠受体 NoV (MNoV, Science 2016 353:933) 定义 NoV 发病机制、持续性和肠道机制 免疫。 HNoV 无法在小动物体内进行稳健复制，因此鼠类 MNoV 已成为一种模型 系统来发现NoV生物学机制和肠道免疫原理，这是很难的 在人类研究中或使用不容易进行基因操纵的动物来识别。利用 MNoV 的研究 描述了病毒与肠道宿主之间相互作用的重要方面。虽然我们的建议 最初仅限于 MNoV，Estes 实验室的开创性工作首次证明了稳健性 HNoV 在人肠类肠中的复制以及胆汁酸在 HNoV 生长中的作用（参见《科学》杂志上的信件） 2016 353:1387）使我们能够与 Estes 实验室合作进行 HNoV 研究。新数据 目标 1 中提供的结果表明，特定的胆汁酸充当我们报告为必需的可溶性“辅助因子” CD300lf 作为 MNoV 受体的功能。这些数据表明显着的保护 HNoV 和 MNoV 都依赖于肠道中存在的宿主因子。基于这些进步，我们 在此提出新的研究，并相信这些研究将提高我们提出的工作的价值。 NoV 种类和细胞向性是在细胞结合和进入水平上确定的。因此，宿主因素 包括感染所需的受体和发病机制尚未完全了解该病毒属， 造成了解新病毒传播、发病机制、疫苗接种、物种向性和 坚持。为了鉴定 MNoV 感染所需的宿主分子，我们进行了基于 CRISPR-Cas9 的研究 筛选，发现 CD300lf 是原代细胞和 BV2 小胶质细胞中 MNoV 的受体，并且它 是体内感染所必需的。 CD300lf 赋予 MNoV 在 4oC 下与细胞结合的能力，并且足以 使人类 HeLa 细胞完全允许 ​​MNoV 复制。 CD300lf的晶体结构 胞外域显示出带有配体结合袋的 Ig 结构域；包含该结合口袋的氨基酸 对于 MNoV 感染的受体功能至关重要。可溶性胞外域 (sCD300lf) 中和病毒 体外和体内的感染性，我们现在证明它直接与病毒体结合。可溶性辅助因子是 CD300lf 受体功能所需的，新的实验表明，特定的胆汁酸具有可溶性胆汁酸的功能 辅助因子。鉴于这些数据，我们建议检验 CD300lf 赋予组织和细胞趋向性的假设 负责 MNoV 建立持续性肠道感染并通过以下方式引发免疫力的能力： 以下目标： 目标 1：确定 CD300lf 在 NoV 感染中作用的分子基础。 目标 2：确定 CD300lf 在 MNoV 趋向性、持久性和免疫中的作用。