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Changes in specific cleavability of the Sendai virus fusion protein: implications for pathogenicity in mice
M Tashiro1, J T Seto, S Choosakul
1Department of Virology, Jichi Medical School, Tochigi, Japan.
Abstract:
Sendai virus mutants, KDe-21 and KDe-62, which had undergone multiple cycles of replication in Madin Darby canine kidney (MDCK) cells in the absence of exogenous proteases were isolated. The fusion (F) protein of the mutants regained proteolytic cleavability in MDCK cells and chick embryos, but the F protein remained non-cleavable in other cell lines. Unlike the F protein of wild-type (wt) virus, the mutant F was resistant to trypsin but was sensitive to elastase and, to a lesser extent, to chymotrypsin. Sequence analyses of the F gene and the F protein revealed an amino acid substitution at the cleavage site, Arg(116) to Ile, which conferred trypsin resistance and enhanced cleavability at Ile(116) by elastase and host proteases present in MDCK cells and in chicken embryos. In contrast to the pneumopathogenicity in mice of wt Sendai virus, the KDe mutants were non-pathogenic; cleavage activation of the F protein did not occur in the lungs and thereby infection was terminated after an initial cycle of replication.
Insights
Sendai virus mutants with altered fusion (F) proteins were isolated. These mutants showed protease sensitivity changes, impacting pathogenicity and viral replication in vivo.
Area of Science:
- Virology
- Molecular Biology
- Protein Biochemistry
Background:
- Sendai virus requires fusion (F) protein cleavage for infectivity.
- Proteolytic activation of the F protein is crucial for viral spread and pathogenesis.
- Previous studies focused on wild-type virus F protein cleavage mechanisms.
Purpose of the Study:
- To isolate and characterize Sendai virus mutants with altered F protein cleavage properties.
- To investigate the impact of F protein mutations on protease sensitivity and pathogenicity.
- To elucidate the molecular basis for altered F protein activation in specific cell types.
Main Methods:
- Isolation of Sendai virus mutants (KDe-21, KDe-62) after serial replication in Madin Darby canine kidney (MDCK) cells.
- Protease sensitivity assays using trypsin, elastase, and chymotrypsin.
- Sequence analysis of the F gene and F protein.
- Assessment of pneumopathogenicity in mice.
Main Results:
- Mutant F proteins regained proteolytic cleavability in MDCK cells and chick embryos, but not in other cell lines.
- Mutant F protein was resistant to trypsin but sensitive to elastase and chymotrypsin.
- An Arg(116) to Ile substitution at the F protein cleavage site conferred trypsin resistance and enhanced elastase cleavage.
- Mutants were non-pathogenic in mice due to lack of F protein cleavage activation in the lungs.
Conclusions:
- The Arg(116) to Ile substitution in the Sendai virus F protein alters protease susceptibility.
- This mutation leads to reduced pathogenicity by preventing efficient F protein activation in the host.
- MDCK cells and chick embryos possess proteases capable of activating the modified F protein, unlike other tested cell lines.