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Journal of Virology, September 2008, p. 8272-8282, Vol. 82, No. 17
0022-538X/08/$08.00+0     doi:10.1128/JVI.00587-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Changing the Protease Specificity for Activation of a Flavivirus, Tick-Borne Encephalitis Virus{triangledown}

Wolfgang Fischl,{dagger} Sigrid Elshuber,{ddagger} Sabrina Schrauf, and Christian W. Mandl*

Clinical Institute of Virology, Medical University of Vienna, Vienna, Austria

Received 16 March 2008/ Accepted 5 June 2008

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.

* Corresponding author. Mailing address: Clinical Institute of Virology, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria. Phone: 43 1 40490, ext. 79502. Fax: 43 1 40490 9795. E-mail: christian.mandl{at}meduniwien.ac.at

{triangledown} Published ahead of print on 18 June 2008.

{dagger} Present address: Department of Molecular Virology, University of Heidelberg, Heidelberg, Germany.

{ddagger} Present address: Intercell AG, Campus Vienna Biocenter, Vienna, Austria.

Journal of Virology, September 2008, p. 8272-8282, Vol. 82, No. 17
0022-538X/08/$08.00+0     doi:10.1128/JVI.00587-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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