Structural controls of functional receptor and antibody binding to viral capsids

描述(申请人提供)：在这里，我们试图了解细小病毒衣壳结构的灵活性和变异如何控制它们与受体结合的能力，从而导致细胞感染和宿主范围的变异，以及衣壳结构如何控制抗体的结合和中和。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。虽然细小病毒衣壳看起来结构简单，但它们显然是复杂的生物分子机器，使用单一衣壳蛋白的变体执行许多功能，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，这为全面了解病毒-宿主相互作用是如何发生的细节提供了机会。细小病毒包括B19病毒、人类博卡病毒和Parv4病毒，所有这些病毒都会导致人类疾病。在这里，我们使用猫和犬细小病毒作为模型，以我们之前的研究为基础，表明细胞感染和动物宿主范围由衣壳与不同宿主的转铁蛋白受体1型(TFR)的特定相互作用控制。抗体结合的病毒感染也有不同的结果，这取决于结合的位置和附着的角度。本项目要研究的三个重叠领域是：1.确定细小病毒衣壳变异和柔性结构的功能效应。病毒衣壳既有灵活性，又有结构变异，我们将进一步详细描述这一点。其中一些变异是不对称的，只出现在一小部分衣壳亚基中，而另一些变异发生在大多数病毒部位。众所周知，这些站点中的许多都会影响病毒功能。2.描述细小病毒衣壳与TFR或其他受体之间的不同相互作用如何控制感染。衣壳与不同的TFR结合受蛋白质结构和柔韧性的控制，也可能受蛋白酶的裂解控制。为了定义功能性TFR与衣壳的结合，我们将使用各种方法，包括低温EM分析和影响结合的衣壳突变体的分析。为了定义TFR的相互作用，我们将使用相互作用部位中含有氨基酸变化或添加的多糖的受体。3.利用抗体与衣壳的结合来确定其结构，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。一些人以Fabs的身份中和，另一些人则不是。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分析，以进一步确定不同TFR与衣壳的结合，并检测裂解的亚基或其他衣壳修饰。结合亲和力改变、不同附着位置或不同附着角度的抗体将被测试对细胞感染所涉及的病毒功能的影响。 公共卫生相关性：该项目涉及对感染人类和其他动物的所有病毒的成功至关重要的几个问题。这些包括：病毒蛋白在其生命周期中结构如何变化，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，细胞感染过程，以及抗体结合如何在某些情况下中和病毒，而不是在其他情况下。这些研究将展示受体结合的改变如何导致新的流行或大流行病毒的出现，并将揭示制造更有效的疫苗或疗法的新方法。正在检测的特定模型病毒会感染动物，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与宿主相互作用的最基本方面的极佳模型，其结果和广泛的结论将直接与更好地理解人类的许多不同病毒有关。

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.