MOLECULAR ANALYSIS OF VIRUS ASSEMBLY AND UNCOATING

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Coat proteins of non-enveloped, icosahedral animal viruses perform a multitude of functions during the course of viral replication, including subunit assembly to form the viral capsid, specific encapsidation of the viral genome, proper maturation cleavage, binding to a cellular receptor and disassociation. Each of these activities is a potential target for antiviral therapy, but the rational design of agents to control viral disease requires detailed knowledge of the chemical interactions and reaction involved at each step. To date, structure function relationships of viral coat proteins and intact virions have been studied in detail only for the enveloped orthomyxoviridae (primarily influenza virus) and the non-enveloped picornaviridae. Much of the known mechanistic details of virus assembly, stability, maturation and disassembly have been derived from these viruses and the basic concepts are commonly applied to other virus systems. In this application we propose to expand this data base by extending our studies of the remarkably simple and accessible animal nodaviruses. Members of this family display many properties of structurally similar but more complex viruses and are relevant as prototypes to guide investigation medically important virus pathogens. We have previously used X-ray crystallography and cryo-electron microscopy to characterize three nodaviruses at high resolution. These studies have led to specific proposals for molecular mechanisms of particle assembly, maturation, and uncoating. We will test these proposals in detail through the methods of molecular genetics, biochemistry and biophysics. Specifically, (1) the importance of selected nodaviral coat protein regions in virion assembly, stability and maturation will be tested by characterizing the properties of a series of constructed mutants. Regions in the high resolution structure of nodavirus FHV have been identified where mutations may produce predictable, biologically relevant phenotypes. (2) Molecular processes associated with FHV uncoating will be investigated. These will include an analysis of release of RNA from heat-treated particles with particular emphasis on the specificity with which the encapsidated genome is liberated. Further , the role of cleavage product gamma in the uncoating process will be determined. This will be done primarily by studying the effect of gamma chain mutations on viral binding, internalization and RNA release into the cytosol. The results of our studies will contribute to a better understanding of the general principles underlying structure-function relationships in icosahedral virus.