The goal of this investigation is to define the regulatory effects exerted by sequential proteolytic processing on cell-free synthesis and assembly of picornaviral proteins. The experiments outlined in this proposal are expected to answer important questions about picornaviral replication and morphogenesis, and may set a precedent for the examination of other eukaroytic virusesin view of potential proteolytic involvement. In addition, development of a cell-free system capable of synthesizing active viral proteins should have valuable applications in many other experimental programs. The rabbit reticulocyte translation system will be used to study in vitro synthesis and assembly of encephalomyocarditis (EMC) viral capsid intermediate structures. Proteolytic interconversion of viral protein and its effects on viral morphogenesis will be determined by following radiolabeled capsid precursors through various assembly steps. We will attempt to couple cell-free translation, transcription and assembly reactions to examine the parameters required for viral RNA encapsidation. The role of cellular protease(s) in polyprotein cleavage events will be characterized by monitering the effects of various chemical inhibitors. If possible, a specific assay will be developed, so this enzyme can be isolated and characterized. Another experimental priority of the proposed research is to complete the nucleotide sequencing of the EMC genome. Selective radiolabeling of the viral proteins, combined with carboxy and amino termini determinations will test deduced polyprotein sequence for correct orientation and placement of proteolytic cleavage products, as well as establish the primary sequence of the proteolytic cleavage sites.
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