The infectivity of flavivirus particles depends on a maturation process that is 
triggered by the proteolytic cleavage of the precursor of the M protein (prM). 
This activation cleavage is naturally performed by ubiquitous cellular proteases 
of the furin family, which typically recognize the multibasic sequence motif 
R-X-R/K-R. Previously, we demonstrated that a tick-borne encephalitis virus 
(TBEV) mutant with an altered cleavage motif, R-X-R, produced immature, 
noninfectious particles that could be activated by exogenous trypsin, which 
cleaves after single basic residues. Here, we report the adaptation of this 
mutant to chymotrypsin, a protease specific for large, hydrophobic amino acid 
residues. Using selection pressure in cell culture, two different mutations 
conferring a chymotrypsin-dependent phenotype were identified. Surprisingly, one 
of these mutations (Ser85Phe) occurred three positions upstream of the natural 
cleavage site. The other mutation (Arg89His) arose at the natural cleavage 
position but involved a His residue, which is not a typical chymotrypsin 
cleavage site. Efficient cleavage of protein prM and activation by the 
heterologous protease were confirmed using various recombinant TBEV mutants. 
Mutants with only the originally selected mutations exhibited unimpaired export 
kinetics and were genotypically stable during at least six cell culture 
passages. However, in contrast to the wild-type virus or trypsin-dependent 
mutants, chymotrypsin-dependent mutants were not neurovirulent in suckling mice. 
Our results demonstrate that flaviviruses with altered protease specificities 
can be generated and suggest that this approach can be used for the construction 
of viral mutants or vectors that can be activated on demand and have restricted 
tissue tropism and virulence.