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Changing the protease specificity for activation of a flavivirus, tick-borne encephalitis virus
Wolfgang Fischl1, Sigrid Elshuber, Sabrina Schrauf
1Clinical Institute of Virology, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria.
Abstract:
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.
Insights
Flavivirus infectivity relies on M protein cleavage. Researchers engineered a tick-borne encephalitis virus (TBEV) mutant activated by chymotrypsin, a protease not usually involved, demonstrating potential for creating on-demand activated viral vectors.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Flavivirus infectivity is critically dependent on the proteolytic cleavage of the precursor M protein (prM) by cellular furin proteases.
- This cleavage typically occurs at multibasic motifs (R-X-R/K-R), activating the virus.
- Previous work showed a tick-borne encephalitis virus (TBEV) mutant with an altered motif (R-X-R) could be activated by trypsin.
Purpose of the Study:
- To adapt a TBEV mutant to chymotrypsin, a protease with different substrate specificity.
- To investigate the genetic basis and biological consequences of altered protease specificity in flaviviruses.
- To explore the potential for creating engineered flaviviruses with conditional activation and reduced virulence.
Main Methods:
- Selection pressure in cell culture to isolate TBEV mutants with chymotrypsin-dependent phenotypes.
- Site-directed mutagenesis to confirm the role of identified mutations (Ser85Phe, Arg89His) in chymotrypsin activation.
- Analysis of prM cleavage, virus export kinetics, genotypic stability, and neurovirulence in suckling mice.
Main Results:
- Two mutations (Ser85Phe and Arg89His) were identified, conferring chymotrypsin-dependent activation of TBEV.
- These mutations allowed efficient prM cleavage and virus activation by chymotrypsin, despite unusual cleavage site characteristics.
- Chymotrypsin-dependent TBEV mutants showed reduced neurovirulence in mice compared to wild-type or trypsin-dependent viruses.
Conclusions:
- Flaviviruses can be engineered to utilize heterologous proteases like chymotrypsin for activation, altering their protease specificity.
- This approach yields genetically stable viral mutants with unimpaired export kinetics.
- The generation of virulence-attenuated flaviviruses with on-demand activation is feasible, suggesting applications in creating novel viral vectors or attenuated vaccine candidates.
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