The effect of large-scale synonymous substitutions in a small icosahedral， single-stranded RNA viral genome on virulence， viral titer， and protein evolution were analyzed. The coat protein (CP) gene of the Fny stain of (CMV) was modified. We created four CP mutants in which all the codons of nine amino acids in the 5'- or 3'-half of the CP were replaced by either the most frequently or the least frequently used synonymous codons in monocot plants. When the dicot host () was inoculated with these four CP mutants， viral RNA titers in uninoculated symptomatic leaves decreased， while all mutants eventually showed mosaic symptoms similar to wildtype. The codon adaptation index of these four CP mutants against dicots genes was similar to those of the wild type CP gene， indicating that the reduction of viral RNA titer was due to deleterious changes of the RNA secondary structure of RNA 3 and 4. When two 5'-mutants were serial-passaged in ， viral RNA titers were rapidly restored but competitive fitness remained decreased. Although no nucleic acid changes were observed in the passaged wildtype CMV， one to three amino acid changes were observed in the synonymously-mutated CP of each passaged virus， which were involved in recovery of viral RNA titer of 5'-mutants. Thus， we demonstrated that deleterious effects of the large-scale synonymous substitutions in the RNA viral genome facilitated the rapid amino acid mutation(s) in the CP to restore the viral RNA titer. Recently， it has been known that the synonymous substitutions in RNA virus genes affect viral pathogenicity and competitive fitness by alteration of global or local RNA secondary structure of the viral genome. We confirmed that the large-scale synonymous substitutions in the CP gene of CMV resulted in decreased viral RNA titer. Importantly， when viral evolution was stimulated by serial-passage inoculation， viral RNA titer was rapidly restored concurrent with a few amino acid changes in the CP. This novel finding indicates that the deleterious effects of large-scale nucleic acid mutations on viral RNA secondary structure is readily tolerated by structural changes in the CP， demonstrating a novel part of the adaptive evolution of an RNA viral genome. In addition， our serial inoculation experimental system of large-scale synonymous mutants could uncover the role of new amino acid residues in the viral protein that have not been observed in the wildtype virus strains.