Receptor utilization for influenza virus entry in vivo

描述（由申请方提供）：流感病毒进入其宿主细胞是病毒生命周期的基本组成部分。流感病毒与细胞表面唾液酸部分的初始结合和内体内的低pH依赖性包膜融合通常已得到很好的表征;然而，结合和融合之间的细胞内化和运输事件仍不完全清楚。我们的初步数据显示唾液酸结合本身不足以使流感病毒进入体内发挥功能。使用Lec 1细胞（CHO的一种突变细胞系，其N-连接糖蛋白上缺乏末端唾液酸），我们表明流感病毒进入和感染受到严重抑制。相反，在Lec 1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/或内吞分选所需的功能性受体。我们在这个项目中的目标是使用Lec 1细胞，以确定一个功能性的第二，或“共”受体的流感病毒感染在体内。具体来说，我们将1）使用各种充分表征的结合、内化和融合测定来表征病毒进入Lec 1细胞的缺陷，和2）通过分离和筛选差异识别CHO/Lec 1细胞表面表位并将阻止病毒进入的单克隆抗体来鉴定缺失的受体。我们的初步数据清楚地显示了突变细胞中病毒进入的阻断，然而，所提出的“共受体”的鉴定和突变细胞中病毒进入的分子表征需要一定程度的探索性实验。Lec 1细胞中的进入阻断的表征和流感病毒在体内内化和/或内吞运输的受体要求的鉴定对于我们理解这种高致病性和潜在致命性的病毒具有根本重要性，该病毒被归类为NIAID C类优先病原体用于生物防御和新兴传染病研究。

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.