Entry of Coronaviruses into Host Cells

描述（由申请人提供）：冠状病毒进入宿主细胞是病毒学研究的一个未开发领域。在本申请中，我们建议使用鸡传染性支气管炎病毒（IBV）作为模型，将直接适用于高致病性人类冠状病毒，如SARS冠状病毒。本申请的主要目标是定义关于IBV的融合机制和内化途径的基本信息。在我们对冠状病毒进入的理解中仍然存在某些重要的差距：具体来说，1）到目前为止，还没有使用病毒-细胞融合试验来研究冠状病毒的进入，2）尽管冠状病毒通常被认为在中性pH值下进行细胞-细胞融合，但以前的数据也表明了通过内体的pH依赖性途径。为了解决这些问题，我们有两个具体的目标：1 -开发冠状病毒的病毒-细胞融合检测。本实验室利用流感病毒-细胞融合的方法，主要目的是建立一种基于荧光的病毒-细胞融合检测方法。2 -确定IBV进入宿主细胞的内化途径。我们以前的特点是使用分子，药理学和形态学方法相结合的其他病毒的进入。使用类似的技术，第二个主要目标是确定IBV进入过程中内吞作用的作用。我们的工作不仅旨在阐明IBV的基本进入机制，而且还可以作为一般冠状病毒的模型-特别是高致病性人类冠状病毒，如SARS-CoV。IBV将作为了解冠状病毒进入和融合的极好模型系统，并促进未来抗病毒药物的开发。

DESCRIPTION (provided by applicant): Entry of coronaviruses into host cells is an under-explored area of virology research. In this application, we propose to use infectious bronchitis virus (IBV) of chickens as a model that would be directly applicable to highly pathogenic human coronaviruses, such as SARS-CoV. The major goal of this application is to define basic information regarding the fusion mechanism and route of internalization of IBV. Certain important gaps remain in our understanding of coronavirus entry: specifically 1) the fact that, to date, no virus--cell fusion assays have been employed to study coronavirus entry, and 2) although coronaviruses are generally considered to undergo cell--cell fusion at neutral pH, previous data also suggest a pH-dependent route of entry through endosomes. To address these issues, we have two specific aims: 1 - To develop a virus-cell fusion assay for coronaviruses. Using protocols in our laboratory to study influenza virus--cell fusion, a principal goal is to develop a fluorescence-based virus-cell fusion assay for the coronavirus IBV. 2 - To determine the internalization route of IBV into host cells. We have previously characterized the entry of other viruses using a combination of molecular, pharmacological and morphological approaches. Using similar techniques, a second principal goal is to define the role of endocytosis during IBV entry. Our work is designed not only to elucidate the basic entry mechanism of IBV, but also to serve as a model for coronaviruses in general--especially highly pathogenic human coronaviruses, such as SARS-CoV. IBV would serve as an excellent model system to understand coronavirus entry and fusion, and facilitate the development of future anti-viral drugs.