Structural Controls of Functional Receptor and Antibody Binding to Viral Capsids.

动物病毒的细胞感染是由它们的结构蛋白和这些结构的变异控制的。病毒蛋白与宿主细胞受体相互作用，导致结合、摄取和细胞内运输，同时与宿主抗体相互作用可能以不同的效率中和感染。病毒结构蛋白的许多功能仍未完全了解，包括受体或抗体结合在控制感染中的结构作用。在这里，我们将以细小病毒衣壳为模型继续研究这些过程。正在研究的病毒包括犬细小病毒（CPV），由于其与宿主转铁蛋白受体1型（TfR）相互作用的变化而成为一种新的大流行病原体。细小病毒衣壳也将抗体结合到几个表位上，产生的中和作用有显著差异。在过去的一段时间里，我们对衣壳的结构和功能、TfR变异在感染中的作用以及抗体的结合产生了重要的新信息。开发的工具使我们能够操纵每个组件，以便了解它们如何控制病毒感染。这些研究提出了三个互补的目的：目的1。解释受体在感染过程中的结构作用，以及宿主特异性结构变异的影响。定义TfR顶端结构域突变体和变异体与互补病毒衣壳结构的相互作用，并解释它们如何控制细胞感染和病毒宿主趋向性。展示受体结合如何改变衣壳使其为感染做好准备。利用其他配体结合衣壳导致内吞作用，并研究其对感染的影响。目标2。定义衣壳介导的过程和细胞感染所需的结构变异。我们已经确定了不同衣壳结构中的单个变化，这些变化可以防止感染，其中一些似乎控制了成功细胞感染所需的关键变异。我们将定义这些结构如何控制衣壳功能，特别是特定衣壳内切割的来源。我们将使用冷冻电镜方法确定具有改变感染特性的衣壳的高分辨率结构。这些位点是不对称的，因为它们只出现在约10%的衣壳蛋白中。因此，我们将使用各种结构和生化方法来研究这些结构，并解释它们在细胞感染过程中的功能。目标3。分析抗体与衣壳的相互作用，解释结合、中和和抗体逃逸的机制。使用衣壳- fab复合物的高分辨率结构来解释抗体结合特异性，并了解这些相互作用如何在某些情况下导致中和而不是其他情况。利用抗体和衣壳突变来改变抗体的结合、中和和交叉反应性，以确定衣壳、抗体和受体之间的功能相互作用。

Cell infection by animal viruses is controlled by their structural proteins and by variation in those structures. Viral proteins interact with host cell receptors, leading to binding, uptake and intracellular trafficking, while interactions with host antibodies may neutralize infection with varying efficiencies. Many functions of viral structural proteins are still not fully understood, including the structural effects of receptor or antibody binding in controlling infection. Here we will continue to investigate those processes using parvovirus capsids as a model. The viruses being studied include canine parvovirus (CPV), which arose as a new pandemic pathogen due to changes in its interaction with the host transferrin receptor type-1 (TfR). The parvovirus capsids also bind antibodies to several epitopes with significant variation in the resulting neutralization. In the previous period we have generated important new information about the structures and functions of capsids, the roles of TfR variants in infection, and the binding of antibodies. The tools developed allow us to manipulate each component in order to understand how they control virus infection. The studies are presented as three complementary Aims: Aim 1. Explain the structural role of the receptor in the infectious process, and the effects of host- specific structural variation. Define the interactions of TfR apical domain mutants and variants with the complementary viral capsid structures, and explain how those control cell infection and viral host tropisms. Show how the receptor binding alters the capsid to prepare it for infection. Use other ligands to bind the capsid to result in endocytosis, and investigate the effects on infection. Aim 2. Define the capsid-mediated processes and structural variation required for cell infection. We have identified single changes in different capsid structures that prevent infection, and some of those appear to control key variation required for successful cell infection. We will define how those structures control the capsid functions, in particular the sources of specific intra-capsid cleavages. We will determine the high resolution structures of capsids with altered infectious properties using cryo-EM approaches. Those sites are asymmetric, as they occur in only ~10% of the capsid proteins. We will therefore use a variety of structural and biochemical approaches to study those structures and to explain their functions in the processes of cell infection. Aim 3. Analyze the interactions of antibodies with the capsid to explain the mechanisms of binding, neutralization, and antibody escape. Use high-resolution structures of capsid-Fab complexes to explain antibody-binding specificity, and also to understand how those interactions result in neutralization in some cases but not others. Use antibody and capsid mutations to alter the binding, neutralization, and cross- reactivity of antibodies, to define the functional interactions between capsids, antibodies, and receptors.