The role that cellular receptors serve as determinants of coxsackievirus tropism in the pathogenesis of human and animal diseases continues to be the long range goal of this program. The HeLa cell receptor (HR1) for the coxsackieviruses of group B (CB) has been purified in a detergent-stable complex with virions. A second receptor (HR2) for binding the CB-RD variant viruses to RD cells and to HeLA cells has been discovered. Purification methods other than that used for HR1 must be developed due to the labile nature of the CB-RD variants. Assays with monoclonal antibodies, reveals that the HeLa cell RD-virus receptor (HR2) is not functional (does not bring the virus into the infectious pathway), while the receptor (HR2) on RD cells is fully active. We plan to look for structural differences between the HR2 receptors on RD and HeLa cells which may be required for biological activity. "Probes" are needed for characterization, isolation and quantitation of receptors, and for cDNA cloning. Additional receptor-specific monoclonal antibodies are being produced. Adenovirus fiber protein which also specifically binds to both human and mouse receptors for the group B coxsackieviruses will serve as another probe. We have begun to use these probes in attempts to clone the HeLa cell HR1 receptor in a lambda gt11 expression vector. The amino acid sequence of the coxsackievirus receptors will be predicted from the cDNA sequences. We will also attempt to use human DNA probes for identification of mouse cDNA clones. Expression of functional receptors in either bacterial or eucaryotic systems may provide larger amounts of the receptor proteins for purifiction and use in biochemical study of virus-receptor interaction, and also for preparation of specific high titer antibody. One of the goals is the characterization of the virus-receptor complex by X-ray crystallography (collaboration with M. Rossman at Purdue). The contribution of the receptor to the process of viral uncoating will also be investigated. In addition, the relationship between the distribution and activity of host cell receptors in inbred mice in the development of viral pathogenesis will be investigated. DNA probes for receptor mRNA and antibodies to receptors will allow mapping of receptor distribution in organs and cells. Sites of virus multiplication will be determined by titration of infectivity, by immunohistochemistry, and by in situ hybridization using DNA or RNA probes for viral RNA. These results will be correlated with the known patterns of coxsackievirus pathogenesis.