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

项目摘要 人诺如病毒（HNoV）是医学上重要的无包膜正义RNA病毒， 引起肠胃炎。这项修订的建议利用了最近发现的CD 300 lf作为小鼠的受体， NoV（MNoV，Science 2016 353：933），以定义NoV发病机制、持久性和肠毒性的机制。 免疫力HNoV不能在小动物中稳健地复制，因此鼠MNoV已经作为模型出现 系统，以发现新的生物学机制和肠道免疫的原则，将难以 在人类研究中识别或使用不易遗传操纵的动物。使用MNoV的研究 描述了肠道中病毒和宿主之间相互作用的重要方面。虽然我们的提议 最初仅限于MNoV，Estes实验室的开创性工作首次证明了其强大性 HNoV在人肠类肠中的复制和胆汁酸在HNoV生长中的作用（参见letter，Science 2016 353：1387），使我们能够与Estes实验室合作进行HNoV研究。新数据 目的1中提供的结果表明，特定的胆汁酸作为可溶性“辅因子”发挥作用，我们报告为必需的 对于CD 300 lf作为MNoV受体的功能1。这些数据表明， HNoV和MNoV均依赖于肠中存在的宿主因子。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拟议工作的价值。 在细胞结合和进入的水平上确定NoV种类和细胞向性。因此，宿主因素 包括感染和发病机理所需的受体对于该病毒属还不完全了解， 创造关键的知识差距，以了解新冠病毒的传播，发病机制，疫苗接种，物种嗜性和 持久性。为了鉴定MNoV感染所需的宿主分子，我们进行了基于CRISPR-Cas9的免疫组化。 筛选，发现CD 300 lf是原代细胞和BV 2小胶质细胞中MNoV的受体， 是体内感染所必需的。CD 300 lf赋予MNoV在4 ° C下结合细胞的能力，并且足以 使人HeLa细胞完全允许MNoV复制。CD 300 lf的晶体结构 胞外域显示具有配体结合口袋的Ig结构域;包含该结合口袋的氨基酸 对MNoV感染的受体功能至关重要。可溶性胞外域（sCD 300 lf）中和病毒 在体外和体内的感染性，我们现在表明它直接结合到病毒粒子。可溶性辅因子是 CD 300 lf受体功能所需，新的实验表明，特定的胆汁酸作为可溶性 辅因子鉴于这些数据，我们建议测试CD 300 lf赋予组织和细胞向性的假设 负责MNoV建立持续性肠道感染并通过以下途径引起免疫的能力： 以下目标： 目的1：探讨CD 300 lf在NoV感染中作用的分子基础。 目的2：探讨CD 300 lf在MNoV嗜性、持久性和免疫中的作用。

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.