These studies seek to define the mechanisms by which parvovirus capsid protein genes control host range. Parvoviruses are the etiological agents of many diseases of humans and animals - human parvovirus (B19) causes the childhood fifth disease (erythema infectiosum), and also transient aplastic crisis or fetal infections. Canine parvovirus (CPV) is a new virus infecting dogs, and is an extended host range variant of the long- recognized feline panleukopenia virus (FPV). Two or three changes in the capsid protein of CPV determine the canine host range, and atomic structures of CPV and FPV show that those differences are on the capsid surface, near regions of contact between protein molecules. Restriction of FPV infection in dog cells occurs after viral entry into the cell, but before DNA replication in the nucleus. The objectives of these studies are: 1) Define virus structures and functions involved in infection. The roles of the virus capsid and changes in the structure in infection and host range determination will be examined using a variety of techniques. Some capsids with mutations or treated in various ways will be examined for changes using methods such as X ray crystallography and by reaction with antibodies against internal capsid protein sequences. Altered or treated capsid genes will be tested by infection of canine cells, or will be used in a genetic transduction assay to give a rapid testing of mutant capsid proteins by examining their ability to deliver a reporter gene to the nucleus. 2) Define and compare the cellular uptake and infection pathways of the viruses. The mechanisms of normal virus entry into the various cells will be defined. A variety of approaches will be used to follow the uptake of virus into cellular vesicles, and then to test for virus disassembly, release of viral nucleic acid, and transport to the cell nucleus. The uptake pathways of the capsids and those used for infection will be defined by treatments of the cells during virus infection - e.g. by preventing endosomal acidification or transport, or with protease inhibitors. We will compare the uptake and infection by mutant viruses with 1 or 2 changes in their capsids which prevent infection of canine cells, to define any differences between the permissive and restricted uptake and infection pathways. In other studies we will introduce native or treated virus directly into the cytoplasm and then examine for infection. 3) Compare CPV susceptible or resistant cells. Various cells will be compared in an attempt to define differences which affect host range. We will determine whether the restriction occurs in cis or trans by testing hybrid cells, test for differences in pH of endocytosis or in binding properties. FPV susceptible canine cells in the thymus or bone marrow will also be identified by examination of surface markers and by in vitro culture.
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