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.