Receptor utilization for influenza virus entry in vivo

描述（由申请人提供）：流感病毒进入宿主细胞是病毒生命周期的一个基本部分。流感病毒与细胞表面唾液酸部分的初始结合和内体内低ph依赖性的膜融合通常具有很好的特征；然而，细胞内化和运输事件之间的结合和融合仍然不完全了解。我们的初步数据显示唾液酸结合本身并不足以使功能性流感病毒进入体内。使用Lec 1细胞（CHO的突变细胞系，其n -连接糖蛋白上缺乏末端唾液酸），我们发现流感进入和感染受到严重抑制。相反，在lec1细胞中，病毒与细胞表面的结合是正常的。我们假设Lec 1细胞缺乏流感病毒内化和/或内吞分选通常所需的功能性受体。在这个项目中，我们的目标是使用Lec 1细胞在体内识别流感病毒感染的功能性次级或“co”受体。具体来说，我们将；1)利用多种已充分表征的结合、内化和融合试验表征Lec 1细胞的病毒进入缺陷，2)通过分离和筛选单克隆抗体鉴定缺失受体，这些单克隆抗体可区分识别CHO/Lec 1细胞表面表位并阻止病毒进入。我们的初步数据清楚地显示了病毒进入突变细胞的阻断，然而，确定一个拟议的“共受体”和病毒进入突变细胞的分子特征需要一定程度的探索性实验。流感病毒在体内内化和/或内噬运输的受体需求的鉴定，将对我们了解这种高致病性和潜在致命的病毒具有重要意义，这种病毒被列为NIAID生物防御和新发传染病研究的C类优先病原体。

DESCRIPTION (provided by applicant): The entry of influenza virus into its host cell is a fundamental part of the virus life cycle. Initial binding to cell surface sialic acid moieties and low pH-dependent envelope fusion within the endosome have generally been well characterized for influenza virus; however, the cellular internalization and trafficking events between binding and fusion remain incompletely understood. Our preliminary data show that sialic acid binding per se is not sufficient for functional influenza virus entry in vivo. Using Lec 1 cells (a mutant cell line of CHO that lacks terminal sialic acid on its N-linked glycoproteins), we show that influenza entry and infection is severely inhibited. In contrast, virus binding to the cell surface is normal in Lec 1 cells. We hypothesize that Lec 1 cells lack a functional receptor normally required for influenza virus internalization and/or endocytic sorting. Our goals in this project are to use Lec 1 cells to identify a functional secondary, or "co"-receptor for influenza virus infection in vivo. Specifically, we will; 1) characterize the defect in virus entry for Lec 1 cells using a variety of well-characterized binding, internalization and fusion assays, and 2) identify the missing receptor by isolation and screening of monoclonal antibodies that differentially recognize CHO/Lec 1 cell surface epitopes and that will prevent virus entry. Our preliminary data clearly show a block in virus entry in mutant cells, however the identification of a proposed "co-receptor" and molecular characterization of virus entry in the mutant cells requires some degree of experimentation of an exploratory nature. The characterization of the entry block in Lec 1 cells and the identification of receptor requirements for internalization and/or endocytic trafficking of influenza virus in vivo will be of fundamental importance to our understanding of this highly pathogenic and potentially deadly virus, which is classified as an NIAID category C priority pathogen for biodefense and emerging infectious disease research.