Structural controls of functional receptor and antibody binding to viral capsids

描述（由申请人提供）：在此，我们试图了解细小病毒衣壳的结构灵活性和变异如何控制其结合受体的能力，从而导致细胞感染以及宿主范围的变异，以及衣壳结构如何控制抗体结合和中和。这些研究领域意义重大，因为它们是所有动物和人类病毒的特征。虽然细小病毒衣壳结构简单，但它们显然是复杂的生物分子机器，使用单个衣壳蛋白的变体执行许多功能，并且控制许多功能的特征现在已经映射到特定的突变和衣壳结构，提供了一个机会，以获得对病毒-宿主相互作用如何发生的详细了解。细小病毒包括B19病毒、人类博卡病毒和Parv4，所有这些病毒都会引起人类疾病。在这里，我们使用猫和犬细小病毒作为模型，建立在我们以前的研究表明，细胞感染和动物宿主范围控制的特定相互作用的衣壳与转铁蛋白受体1型（TfR）的不同主机。根据结合位点和附着角度，抗体结合的病毒感染也有不同的结果。在这个项目中要调查的三个重叠领域是：1。定义细小病毒衣壳的变异和柔性结构的功能效应。在病毒衣壳中存在灵活性和结构变异，我们将进一步详细表征。其中一些变异是不对称的，仅发生在一小部分衣壳亚基中，而其他变异则发生在大多数病毒位点中。已知这些位点中的许多会影响病毒功能。2.表征细小病毒衣壳与TfR或其他受体之间的不同相互作用如何控制感染。衣壳与不同TfR的结合受蛋白质结构和柔性控制，并且可能受蛋白酶切割控制。为了定义功能性TfR与衣壳的结合，我们将使用多种方法，包括cryoEM分析和影响结合的衣壳突变体的分析。为了定义TfR相互作用，我们将使用含有氨基酸变化或在相互作用位点内添加聚糖的受体。3.利用抗体与衣壳的结合来定义其结构，并解释抗体中和的机制。我们有许多抗体可以结合衣壳结构。有些人作为Fab而中和，有些人则不是。我们将使用这些抗体作为衣壳结构变异的探针。抗体及其结构域将用于竞争测定以进一步确定不同TfR与衣壳的结合，并检测切割的亚基或其他衣壳修饰。将测试具有改变的结合亲和力、不同附着位点或不同附着角度的抗体对参与细胞感染的病毒功能的影响。 公共卫生相关性：该项目解决了对所有感染人类和其他动物的病毒的成功至关重要的几个问题。其中包括：病毒蛋白在其生命周期中的结构如何变化，与不同受体结合的机制，病毒结构蛋白的差异如何控制其宿主范围，细胞感染的过程，以及抗体结合如何在某些情况下中和病毒，而不是其他情况。这些研究将展示受体结合的改变如何导致新的流行病或大流行病毒的出现，并将揭示制造更有效疫苗或治疗方法的新方法。正在研究的特定模型病毒感染动物，但它们与人类的各种重要病毒相似。细小病毒的小尺寸和简单的遗传和衣壳结构使它们成为定义病毒-宿主相互作用的最基本方面的极好模型，并且结果和广泛的结论将与更好地理解人类的许多不同病毒直接相关。

DESCRIPTION (provided by applicant): Here we seek to understand how structural flexibility and variation in parvoviral capsids control their ability to bind receptors leading to cell infection and also to variation in host range, and also how capsid structures control antibody binding and neutralization. Those areas of study are significant because they are features of all animal and human viruses. While parvovirus capsids appear structurally simple, they are clearly sophisticated biomolecular machines that carry out many functions using variants of a single capsid protein, and the features controlling many functions have now been mapped to specific mutations and capsid structures, presenting an opportunity to gain a complete understanding of how virus-host interactions occur in fine detail. Parvoviruses include the B19 virus, human bocavirus, and Parv4, all of which cause disease in humans. Here we use feline and canine parvoviruses as models to build on our previous studies showing that cell infection and animal host ranges are controlled by specific interactions of the capsids with the transferrin receptors type-1 (TfR) of different hosts. There are also distinct outcomes for viral infection of antibody binding, depending on the binding site and angle of attachment. The three overlapping areas to be investigated in this project are: 1. Define the functional effects of variant and flexible structures of the parvovirus capsids. There is both flexibility and structural variation in the viral capsids which we will further characterize in detail. Some of that variation is asymmetric, only occurring in a small proportion of the capsid subunits, while other variation occurs in most viral sites. Many of those sites are known to affect viral functions. 2. Characterize how different interactions between parvovirus capsids and TfR or other receptors control infection. Capsid binding to different TfRs is controlled by the protein structure, and flexibility, and potentially by protease cleavages. To define the functional TfR binding to the capsids we will use a variety of approaches, including cryoEM analysis and analysis of capsid mutants affecting binding. To define the TfR interaction we will use receptors containing amino acid changes or added glycans within the interaction sites. 3. Use antibody binding to capsids to define their structures, and also to explain the mechanisms of antibody neutralization. We have many antibodies that bind the capsid structures. Some neutralize as Fabs, and others do not. We will use those antibodies as probes for structural variation in the capsids. The antibodies and their domains will be used in competition assays to further define the binding of different TfRs to the capsids, and to detect cleaved subunits or other capsid modifications. Antibodies with altered binding affinities, different attachment sites, or different angles of attachment would be tested for effects on the virus functions involved in cell infection. PUBLIC HEALTH RELEVANCE: This project addresses several issues of central importance to the success of all viruses infecting humans and other animals. Those include: how viral proteins vary in structure over their life cycles, the mechanisms of binding to different receptors, how differences in viral structural proteins can control their host ranges, the processes of cell infection, and how antibody binding can neutralize the virus in some cases but not others. These studies will show how altered receptor binding can lead to the emergence of new epidemic or pandemic viruses, and will also reveal new ways to make more effective vaccines or therapeutics. The specific model viruses being examined infect animals, but they are similar to a variety of important viruses of humans. The small sizes and simple genetic and capsid structures of the parvoviruses make them excellent models for defining the most basic aspects of the viral-host interaction, and the results and broad conclusions will be directly relevant to the better understanding of many different viruses of humans.