These studies will combine the efforts of three laboratories - at Cornell, Purdue and Warwick Universities - in continued studies of the structures and functions of parvovirus capsids. Parvoviruses are the cause of disease in many animals, including humans. The B19 human parvovirus causes the childhood fifth disease, as well as rarer but more severe disease of adults and fetuses. Canine parvovirus (CPV), the primary subject of these studies, is an example of an emerging virus that gained its new canine host range through mutation of the capsid protein. The parvovirus capsid is very simple, being comprised of 60 copies of essentially one protein yet it performs a wide variety of structural and biological functions. The objectives of these studies are: 1) Virus Purification. Continue to produce purified capsids for use in crystallographic and biochemical studies in the three cooperating laboratories. 2) Assembly of the Capsid in vivo and in vitro. Define the assembly and disassembly processes of the virus capsid using in vitro translation, as well as in different in vivo expression systems. The applicants will define the kinetics and concentration-dependence of the assembly of empty and full capsids, and will use mutants blocked in specific assembly interactions to identify the pathways involved. 3) Define Antibody Neutralization mechanisms. Antibodies are the primary protective immunity against parvoviruses. They will examine in detail the binding of two neutralizing antibodies which recognize different antigenic sites on the capsid. They will produce single chain antibodies, and examine their interactions with the capsid using co-crystallization, and by mutagenisis of the interacting sites on the virus and the antibody. 4) Structural flexibility in the Parvovirus Capsid. Examine for transient changes in the structure of the CPV capsid under altered environmental conditions, including treatment at low pH, dialysis with EDTA to remove predicted Ca++ ions, and reduction of disulfide bonds. In this work they would use crystallography of the capsid, as well as structure-specific antibodies and fluorescent chemical probes. 5) Cell Surface and other Receptors required for Binding Infection. Purify and identify the 42 kDa and 116 kDa proteins which bind to CPV in virus-overlay blots of cell membrane proteins. Genetic transfer methods will also be used to screen cDNA libraries or genome DNA for the presence of genes that can render cells susceptible to infection - some of which may encode virus receptors or possibly co-receptors.
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