The assembly of spherical viruses is not well understood. The objective of this proposal is to examine the initial events in the assembly of a small icosahedral virus, cowpea chlorotic mottle virus (CCMV). The specific objectives of this proposal are to: (i) define and map the viral RNA and coat protein sequences responsible for initiating viral assembly; (ii) characterize the biochemical properties of the initiation complex for viral assembly; (iii) demonstrate both in vitro and in vivo that RNA packaging signals are essential for virus particle assembly and (iv) determine the role protein-protein and protein-RNA interactions contribute to the formation of the assembly initiation complex. The long-term goal of this research is to determine the precise mechanisms and regulation of viral assembly. An understanding of virion assembly may reveal ways to interfere with assembly, providing novel approaches to the control of viral diseases. CCMV provides an attractive system to unravel the assembly pathway because assembly questions can be addressed using the tools of molecular genetics, biochemistry, and structural biology. The assembly initiation complex will be characterized by three complementary approaches: (i) a biochemical approach which seeks to isolate the minimum ribonucleoprotein complex required to initiate viral assembly, (ii) a structural approach which takes advantage of the high resolution virus structure determined by X-ray crystallography and (iii) a molecular genetic approach in which mutations in the viral RNA are isolated which prevent assembly. Both in vitro and in vivo assembly assays will be used to characterize the initiation complex. Both the viral RNA sequences and the protein(s) constituting the origin of assembly will be examined.
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