Cell Entry of Human Metapneumovirus

描述（由申请方提供）：本提案的主要目的是确定人偏肺病毒（hMPV）的进入机制，hMPV是一种最近描述的副粘病毒，是全球婴儿和儿童下呼吸道感染的主要原因。了解导致hMPV对接和内化的病毒-细胞相互作用对于阐明发病机制至关重要。宿主细胞受体是病毒嗜性的重要决定因素，并且可以引发导致病毒内化或引发细胞进行病毒复制的信号传导途径。此外，对病毒-受体相互作用的理解可能会导致靶向抗病毒化合物的开发，例如Pleconaril，它可以阻断小RNA病毒与其受体的结合。几种致病性人病毒利用结合RGD的整联蛋白作为受体或共受体，包括腺病毒、柯萨奇病毒、双埃柯病毒、轮状病毒和人疱疹病毒-8。我们的初步数据表明，结合RGD的整联蛋白作为hMPV的受体，融合蛋白（F）介导这种相互作用。在目标1中，我们将定义整合素介导hMPV细胞进入的机制。hMPV感染性需要1V 21整合素，这种相互作用由hMPV F蛋白介导。我们假设hMPV F-整联蛋白相互作用不是简单的结合事件，而是整联蛋白连接触发融合。将在流式细胞术和表面等离子体共振测定中检测野生型和突变体重组F蛋白结合1V 21整联蛋白的能力。将通过反向遗传学产生整联蛋白结合基序发生改变的突变病毒，并检测其结合1V 21整联蛋白和感染细胞的能力。整合素结合对膜融合的贡献将使用融合试验来确定，该试验基于表达野生型或RGD突变型hMPV F的稳定转染细胞系。这些实验将确定1V 21整联蛋白在hMPV细胞附着、融合和进入中的作用。在目标2中，我们将确定hMPV F是否与整合素结合触发细胞内信号传导。整合素介导由外向内和由内向外的信号传导。这些实验将确定hMPV融合和细胞进入是否需要整联蛋白活化，以及下游信号传导是否由hMPV F-整联蛋白结合介导。我们将使用促进或阻碍整合素活化的实验条件来确定整合素活化是否是F结合、融合和细胞进入所必需的。将使用重组F蛋白结合流式细胞术试验、基于细胞的融合试验和hMPV病毒感染性试验确定hMPV进入的这三个步骤。将进行单独的实验以确定F结合对下游信号传导的影响，例如粘着斑激酶（FAK）磷酸化和第二介体向1V 21整联蛋白的募集。下游整合素信号传导抑制剂、免疫沉淀和siRNA技术将用于确定整合素介导的信号传导是否是hMPV进入或复制所需的。这些实验将有助于广泛了解整合素信号特性是如何利用致病性病毒。人类偏肺病毒（Human metapneumovirus，hMPV）是一种在2001年发现的新型病毒。hMPV是全球呼吸道疾病的主要原因，并导致许多婴儿和儿童住院。这项提议的目的是确定hMPV附着在人类细胞上的蛋白质（或“受体”），以便进入并感染细胞。这些研究将增加对hMPV如何导致人类疾病的理解，并可能有助于开发抗病毒药物和疫苗的靶点。

DESCRIPTION (provided by applicant): The central objective of this proposal is to determine mechanisms of entry for human metapneumovirus (hMPV), a recently described paramyxovirus that is a major cause of lower respiratory tract infection in infants and children worldwide. Understanding the virus-cell interactions that lead to docking and internalization of hMPV is critical to elucidating mechanisms of pathogenesis. Host cell receptors are important determinants of virus tropism and can evoke signaling pathways that lead to virus internalization or prime the cell for viral replication. Furthermore, an understanding of virus-receptor interactions may lead to development of targeted antiviral compounds, such as pleconaril, which blocks picornavirus binding to its receptor. Several pathogenic human viruses utilize RGD-binding integrins as receptors or co-receptors, including adenovirus, Coxsackievirus, parechovirus, rotavirus and human herpes virus-8. Our preliminary data suggest that RGD- binding integrins serve as receptors for hMPV and that the fusion (F) protein mediates this interaction. In Aim 1, we will define mechanisms by which integrins mediate hMPV cell entry. hMPV infectivity requires 1V21 integrin, and this interaction is mediated by hMPV F protein. We hypothesize that hMPV F-integrin interaction is not a simple binding event, but that integrin ligation triggers fusion. Wild type and mutant recombinant F protein will be tested for the capacity to bind 1V21 integrin in flow cytometry and surface plasmon resonance assays. Mutant viruses with alterations in integrin-binding motifs will be generated by reverse genetics and tested for the capacity to bind 1V21 integrin and infect cells. The contribution of integrin binding to membrane fusion will be determined using a fusion assay based on a stably transfected cell line expressing wild type or RGD-mutant hMPV F. These experiments will determine the role of 1V21 integrin in hMPV cell attachment, fusion and entry. In Aim 2, we will determine whether integrin engagement by hMPV F triggers intracellular signaling. Integrins mediate both outside-in and inside-out signaling. These experiments will determine whether integrin activation is required for hMPV fusion and cell entry and whether downstream signaling is mediated by hMPV F-integrin binding. We will use experimental conditions that promote or hinder integrin activation to determine whether integrin activation is required for F binding, for fusion and for cell entry. These three steps in hMPV entry will be determined using a recombinant F protein binding flow cytometry assay, a cell-based fusion assay, and hMPV virus infectivity assay. Separate experiments will be performed to determine the effect of F binding on downstream signaling such as focal adhesion kinase (FAK) phosphorylation and the recruitment of second mediators to 1V21 integrins. Inhibitors of downstream integrin signaling, immunoprecipitation and siRNA techniques will be used to determine whether integrin-mediated signaling is required for hMPV entry or replication. These experiments will contribute broadly to an understanding of how integrin signaling properties are utilized by pathogenic viruses. PUBLIC HEALTH RELEVANCE: Human metapneumovirus (hMPV) is an emerging virus that was discovered in 2001. hMPV is a major cause of respiratory tract disease worldwide, and leads to many hospitalizations of infants and children. The purpose of this proposal is to determine the protein (or "receptor") on human cells that hMPV attaches to in order to enter and infect the cell. These studies will increase understanding of how hMPV causes disease in humans, and may help develop targets for antiviral medicines and vaccines.