: Parvoviruses cause significant disease in humans and other animals, and canine parvovirus (CPV) emerged as a new virus of dogs through a natural change in the host range of a feline parvovirus (feline panleukopenia virus - FPV). In these studies we seek to understand the receptors and pathways that the viruses use during cell infection, and to define molecular mechanisms of host range control. The 3 related specific aims will define the roles for receptor binding, endocytic trafficking, and cytoplasmic and nuclear transport in cell infection and host range of these viruses. Specific Aim 1. We will define viral interactions with the transferrin receptor (TfR) and the role of TfR binding in cell infection and host range. CPV and FPV bind to the feline and human TfRs, and that leads to rapid endocytosis and sorting of the virus capsids through specific endosomal pathways. We hypothesize that TfR binding is species specific, and that the new host range of CPV (infection of dogs) was due to the viruses acquiring the ability to bind the canine TfR, or perhaps some other receptor. We will clone and express the canine TfR and determine that receptor's ability to bind and allow infection by CPV and FPV, or by defined mutant viruses. Chimeric receptors or cryo-EM analysis and image reconstruction would be used to reveal the interactions between the TfR and the viral capsids, so that we can define the molecular relationships between the TfR binding site and host range-determining sites of the capsid. Specific Aim 2. Define the endosomal pathways used for infection of the cells, and compare the ability of virus in alternative pathways to successfully infect cells. TfRs with altered cytoplasmic tails will be prepared to cause slow endosomal uptake, or to divert the virus into alternative pathways in the cell. Endocytosis would also be changed using treatments involving wild type or mutant Rab proteins and other mediators of endosomal functions. Specific Aim 3. Define the role of cytoplasmic and nuclear transport in cell infection. We will follow the transport of capsids within the cytoplasm, will use specific antibodies to bind capsid structures in the cytoplasm of the cell, and will also use drug treatments to examine for effects on infection. Nuclear transport will be examined using viruses added to the outside of cells, by microinjecting capsids, or with inhibitors of nuclear transport. Irnmuno-electron microscopy will be used to locate the capsids within the cell, around the nuclear pore, and within the nucleus.