Receptor utilization for influenza virus entry in vivo

说明(申请人提供)：流感病毒进入宿主细胞是病毒生命周期的基本部分。流感病毒与细胞表面唾液酸部分的初始结合和内体内的低pH依赖的包膜融合已被很好地描述；然而，结合和融合之间的细胞内化和转运事件仍然不完全清楚。我们的初步数据显示，唾液酸结合本身不足以使功能性流感病毒进入体内。使用Lec-1细胞(一种突变的CHO细胞系，其N-连接的糖蛋白上缺乏末端唾液酸)，我们发现流感病毒的进入和感染被严重抑制。相反，病毒与细胞表面的结合在Lec-1细胞中是正常的。我们假设Lec-1细胞缺乏正常情况下流感病毒内化和/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细胞来鉴定体内流感病毒感染的功能性次级受体或“共”受体。具体地说，我们将：1)使用各种具有良好特性的结合、内化和融合分析来表征病毒进入Lec 1细胞的缺陷，以及2)通过分离和筛选区别识别CHO/Lec 1细胞表面表位并将阻止病毒进入的单抗来识别缺失的受体。我们的初步数据清楚地表明，病毒进入突变细胞是被阻断的，然而，鉴定所提议的“共同受体”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。确定Lec-1细胞进入阻断的特性和确定流感病毒在体内内化和/或内吞转运的受体要求，将对我们理解这种高致病性和潜在致命的病毒至关重要。这种病毒被归类为NIAID C类优先病原体，用于生物防御和新发传染病的研究。

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.