Studies on the assembly of cowpea chlorotic mottle virus
Abstract
Viral assembly is the process by which the viral genome becomes encapsidated by viral structural protein(s) to produce progeny virions. The detailed chemical mechanisms of the protein-protein and protein-RNA interactions that dictate the assembly process are not well understood. Understanding such chemical interactions not only holds the potential to develop novel anti-viral strategies for interfering with viral assembly but also serves as a model for studying the chemical basis for macromolecular assembly in general. Cowpea chlorotic mottle virus (CCMV) was used as a model to examine how changes in the coat protein alter specific protein-protein and protein-RNA interactions. An in vitro viral assembly system, which uses CCMV coat protein expressed and purified from E. coli and viral RNA in vitro transcribed, was developed. Cryo-electron microscopy and image reconstruction determined that the in vitro assembled virions were identical to virions purified from infected plants. Two classes of mutants have been analyzed using the in vitro assembly system: (i) mutants based on the atomic resolution structure of CCMV, and (ii) mutants with altered assembly phenotypes induced by nitrous acid mutagenesis. The C-terminus is involved in non-covalent dimer formation, while the N-terminus plays a role in binding vRNA and stabilizing the hexamers. Metal binding stabilizes the quasi three-fold axis of the virion. The chemical basis of a salt-stable assembly mutant was determined to result from a K42R mutation. The combination of structural biology, genetics, and molecular biology allowed for examination of the critical structure-function relationships that influence virion assembly. A model for CCMV assembly was developed based on the results of these studies.
Degree
Ph.D.
Advisors
Kuhn, Purdue University.
Subject Area
Molecular biology|Microbiology|Plant pathology
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