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Recombinant measles virus requiring an exogenous protease for activation of infectivity
A Maisner1, B Mrkic, G Herrler
1Institut für Virologie, Philipps-Universität Marburg, Robert-Koch-Str. 17, 35037 Marburg, Germany. maisner@mailer.uni-marburg.de
Abstract:
Proteolytic cleavage of the fusion protein (F) is an important control mechanism of the biological activity of paramyxoviruses. The sequence R-R-H-K-R(112) at the cleavage site of the F protein of measles virus (MV) was altered by site-directed mutagenesis to R-N-H-N-R(112), which is not recognized by the ubiquitous cellular protease furin. When transiently expressed in cell cultures standard F protein was cleaved, whereas the mutant remained in the uncleaved form. Syncytium formation by the mutant that was analysed after coexpression with haemagglutinin protein depended on the presence of trypsin. Recombinant MV containing the mutation required trypsin activation for fusion and infectivity in cell culture. Intranasal infection of transgenic mice susceptible to MV infection (Ifnar(tm)-CD46Ge) resulted in a moderately productive infection and inflammation of the lung. In contrast to parental virus, intracerebral inoculation did not induce neural disease. The possible effects of the change in cleavage activation on tissue tropism and pathogenicity are discussed.
Insights
Altering the measles virus fusion protein cleavage site prevents furin recognition, requiring trypsin for viral activation and reducing neurovirulence in mice.
Area of Science:
- Virology
- Molecular Biology
Background:
- Proteolytic cleavage of paramyxovirus fusion (F) proteins regulates viral activity.
- The measles virus (MV) F protein cleavage site sequence is crucial for its biological function.
Purpose of the Study:
- To investigate the impact of altering the MV F protein cleavage site on viral activation and pathogenicity.
- To determine if modifying the furin cleavage site affects MV infectivity and tissue tropism.
Main Methods:
- Site-directed mutagenesis was used to change the MV F protein cleavage site from R-R-H-K-R(112) to R-N-H-N-R(112).
- Transient expression in cell cultures assessed F protein cleavage and syncytium formation.
- Recombinant MV with the mutation was generated and tested for trypsin-dependent infectivity.
- Transgenic mice (Ifnar(tm)-CD46Ge) were infected intranasally and intracerebrally to evaluate pathogenicity.
Main Results:
- The R-N-H-N-R(112) mutant F protein remained uncleaved by furin in cell culture.
- Syncytium formation and viral infectivity required exogenous trypsin for the mutant.
- Intranasal infection in mice led to moderate lung infection and inflammation.
- Intracerebral inoculation of the mutant virus did not cause neural disease in susceptible mice.
Conclusions:
- The F protein cleavage site mutation renders MV dependent on exogenous proteases for activation.
- Altering the cleavage site significantly impacts MV tissue tropism and reduces neurovirulence.
- This study provides insights into the role of F protein processing in paramyxovirus pathogenesis.