9604680 Fane The main objectives of this study are to elucidate the critical interactions during viral assembly, and to define the structural domains of the responsible macromolecules, the scaffolding proteins, in Microviridae viruses. A combination of genetic, biochemical and structural approaches including X-ray crystallography will be employed. The results of these analyses may provide further insights into the assembly of viruses and the design of recombinant proteins. The Microviridae internal scaffolding proteins, gpB, share many properties with molecular chaperones. Prior results indicate that the internal scaffolding proteins either possess inherent flexibility or interact with their substrates in nonspecific manners, perhaps via interfaces. Determining the atomic structures of hybrid procapsids, containing foreign scaffolding proteins, will directly address this question. For those instances in which foreign scaffolding proteins do not cross function, genetic analyses will be employed to define domains which may confer substrate specificity. Gain of function mutations in viral coat proteins, allowing for the utilization of foreign scaffolding proteins, have been isolated and will be examined. The atomic structure of the external scaffolding proteins, gpD, of Microviridae suggests that they also share many features with molecular chaperones. They bind other proteins in many different ways using various surfaces, the specificity for a particular viral coat protein may thus reside in specific domains. Unlike the internal scaffolding proteins which exhibit a great deal of divergence in primary structure, the external scaffolding proteins share 75% sequence identity. The inability for Phi X174 protein to productively direct the assembly of other Microviridae virions is therefore attributable to the divergent residues localized its NH2-termini, which forms a large alpha-helical structure. This hypothesis will be tested with chimeric polypeptides, and the plasmid-based cross complementation system will be used. *** All viruses must assemble themselves by means of multiple macromolecular interactions, the assembly is often dependent on proteins known as scaffolding proteins. Analogous to scaffoldings used in the construction of buildings, scaffolding proteins are found in virus assembly intermediates but not in the mature viruses. Two different scaffolding proteins, external and internal, are required for the assembly of the Microviridae family of viruses. With these viruses it is possible to purify viral intermediates which still include the external scaffolding protein. By examining the atomic structure of the external scaffolding protein and performing genetic analyses with both the internal and external proteins, it has been determined that different regions of the scaffolding proteins may have specific and identifiable functions. The main objectives of this research are to refine the atomic structures of these proteins and to test hypotheses regarding their various functions by constructing hybrid Microviridae scaffolding proteins. The results of these analyses will provide further insights into viral assembly.
