Cellular receptor for enterovirus D68

 描述（由申请方提供）：小核糖核酸病毒肠道病毒D-68（EV-D 68）是一种二十面体无包膜病毒，是2014年美国呼吸道疾病爆发的原因。在这些爆发中，据报道，类似于脊髓灰质炎病毒引起的急性弛缓性麻痹与幼儿感染EV-D 68有关，这表明该病毒可能侵入中枢神经系统。因此，EV-D 68是一种新兴的具有医学意义的人类病原体。像大多数其他病毒一样，EV-D 68进入细胞需要受体分子。据报道，两种不同的细胞受体，唾液酸和衰变加速因子（CD 55）介导EV-D 68进入。在HeLa和A549细胞中，EV-D 68的NY 68和Fermon株的复制不受神经氨酸酶处理或针对ESTA 1的SCR 1或SCR 3区域的抗体的存在的影响。然而，两种细胞系中的秘鲁68菌株的感染对用神经氨酸酶处理细胞敏感。这些数据表明，EV-D 68进入需要一种以上的细胞特异性分子，并且该分子取决于病毒基因型。该提案的目标是对编码参与EV-D 68细胞进入的分子的基因进行遗传筛选。为了鉴定EV-D 68的细胞受体，我们建议在感染EV-D 68之前使用CRISPR-Cas9方法遗传改变细胞群体，并鉴定不再支持病毒复制的细胞。从这些候选基因中，我们将确定哪些分子在病毒复制的早期步骤中起作用：细胞附着， 使用各种工具，包括先前发表的工作和生物信息学，进行细胞进入和病毒脱壳。通过将野生型基因重新引入相应的改变的细胞中，对确定具有编码跨膜蛋白和具有已知特征的蛋白质的基因的细胞进行补充，所述蛋白质相对于其他小核糖核酸病毒受体蛋白具有已知特征。将通过病毒复制评估细胞结合和对多种EV-D 68基因型感染的易感性。如果鉴定出一种以上的细胞蛋白介导EV-D 68感染，我们将通过研究不同基因型之间衣壳编码区交换的嵌合病毒来重新理解病毒-受体相互作用。

 DESCRIPTION (provided by applicant): The picornavirus enterovirus D-68 (EV-D68) is an icosahedral non-enveloped virus that was responsible for an outbreak of respiratory disease in the United States during 2014. During these outbreaks, acute flaccid paralysis similar to that caused by poliovirus was reported to correlate with EV-D68 infection in young children, suggesting that the virus might invade the central nervous system. EV-D68 is therefore an emerging human pathogen of medical significance. Like most other viruses, EV-D68 entry into the cell requires a receptor molecule. Two different cell receptors, sialic acid and decay-accelerating factor (DAF/CD55) have been reported to mediate EV-D68 entry. Replication of the NY68 and Fermon strains of EV-D68 in HeLa and A549 cells was unaffected by either neuraminidase treatment or the presence of antibodies against the SCR1 or SCR3 regions of DAF. However, infection by the Peru 68 strain in both cell lines was sensitive to treatment of cells with neuraminidase. These data suggest that more than one cell specific molecule is required for EV-D68 entry and that the molecule depends on the virus genotype. The goal of this proposal is to carry out a genetic screen for genes encoding molecules that participate in cell entry of EV-D68. To identify the cell receptor for EV-D68 we propose to genetically alter a population of cells using CRISPR-Cas9 methodology prior to infection with EV-D68, and identify those cells that no longer support viral replication. From this list of candidate genes, we will determine which molecule(s) functions during the early steps of viral replication: cell attachment, cell entry and viral uncoating using various tools including previous published work and bioinformatics. Cells determined to have disruptions in genes encoding transmembrane proteins and proteins with known characteristics to other picornavirus receptor proteins will be complemented by re-introducing the wild type gene into the corresponding altered cell. Cell binding and susceptibility to infection with multiple EV- D68 genotypes will be assessed by viral replication. If more than one cell protein is identified to mediate EV- D68 infection, we will refie our understanding of the virus-receptor interaction, by studying chimeric viruses in which the capsid coding region has been exchanged between the different genotypes.