Engineering altered receptors and antibodies to study viral functions

描述（由申请方提供）：所有感染脊椎动物的病毒在细胞感染过程中与受体相互作用，并且还受到宿主中预先存在或感染后不久产生的抗体的攻击。这两种相互作用对病毒感染的成功至关重要，但作用方向相反。因此，拟议工作的长期目标是研究这些配体与病毒衣壳的相互作用，以了解结合的差异如何影响感染。该项目围绕两个具体目标建立：1）确定受体结合特性对细胞感染的影响。假设：受体与衣壳相互作用的差异影响感染过程。构建犬细小病毒（CPV）亲和力改变的转铁蛋白受体，并确定其对衣壳结合和细胞感染性的影响。2)选择抗衣壳抗体的变体，并检查它们对病毒感染的影响。假设：结合亲和力的增加或附着位置的增加对感染有不同的影响。我将通过定向进化选择具有改变的结合特性的抗病毒抗体，并测试它们对病毒受体附着和细胞感染的影响。为了完成所提出的目标，我将使用分子诱变技术与蛋白质工程方法的组合沿着。定向进化研究提出，使用突变的产品，将显示在酵母细胞的表面上的库，允许选择的克隆与改变亲和力的衣壳。公共卫生相关性：病毒受体结合和感染之间的关系，以及抗体中和和感染之间的关系，是理解病毒感染，适应性和宿主范围的核心。这两种相互作用都很复杂，许多细节仍然没有得到很好的理解。通过使用各种方法来设计具有改变的亲和力和特异性的受体和抗体，我将能够在一个很好理解的模型系统中具体地研究这些问题。

DESCRIPTION (provided by applicant): All viruses infecting vertebrates interact with receptors during the process of cell infection, and also are challenged by antibodies that are either pre-existing in the host or generated shortly after infection. Both these interactions are critical to the success of the viral infection, but work in opposite directions. The long-term objectives of the proposed work are therefore to examine the interactions of those ligands with viral capsids in order to understand how differences in binding influences infection. This project is built around two specific aims: 1) Determine the effects of receptor binding properties on the infection of cells. Hypothesis: That differences in receptor interactions with the capsid influence the process of infection. Construct canine parvovirus (CPV) affinity-altered transferrin receptors and determine their effect on capsid binding and cell infectivity. 2) Select for variants of anti-capsid antibodies and examine their effects on viral infection. Hypothesis: That increased affinity of binding or the position of attachment will have different effects on infection. I will select antiviral antibodies with altered binding properties by directed evolution, and test their effects on viral receptor attachment and on cell infection. To complete the proposed aims, I will use a combination of molecular mutagenesis techniques along with protein engineering methods. Directed evolution studies are proposed, using libraries of mutated products which will be displayed on the surface of yeast cells, allowing selection of clones with altered affinities to capsids. PUBLIC HEALTH RELEVANCE: Both the relationships between viral receptor binding and infection, and between antibody neutralization and infection, are central to understanding viral infection, fitness and host range. Both interactions are complex and many details are still not well understood. By using a variety of approaches to engineer receptors and antibodies with altered affinities and specificities, I will be able to specifically examine these questions in a well understood model system.