Nodaviral morphogenesis involves formation of labile precursor particles, called provirions, which mature by autocatalytic cleavage of the 407-residue coat precursor protein between asparagine residue 363 and alanine residue 364. It has previously been demonstrated that maturation results in increased physicochemical stability of the virion. We show here that cleavage of coat protein in purified provirions of Flock House virus was accompanied by a five- to eightfold increase in specific infectivity. Cleavage-negative provirions, produced by site-directed mutagenesis of asparagine residue 363 to aspartate, threonine, or alanine, displayed no infectivity above revertant frequencies as measured by plaque assay. All viable revertants (nine of nine) restored asparagine to the mutated position, suggesting high specificity for asparagine at the cleavage site.