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Mutational analysis of the murine coronavirus spike protein: effect on cell-to-cell fusion
E C Bos1, L Heijnen, W Luytjes
1Department of Virology, Faculty of Medicine, Leiden University, The Netherlands.
Abstract:
The spike (S) protein of murine coronavirus strain A59 (MHV-A59) is a type I membrane protein that induces membrane fusion. In this study we have analyzed the role of two domains in the S protein on fusion. The 180-kDa mature S protein is partially cleaved into two 90-kDa subunits during transport to the plasma membrane. We have identified several amino acids that are important for cleavage of S, and we show that cleavage is not strictly required for fusion. However, the level of cleavage seems to influence the fusion kinetics. After introduction of an arginine at position P2 to mimick the MHV-JHM cleavage site, full cleavage of the spike protein was obtained. Further, we analyzed the effect of mutations in the transmembrane (TM) domain of the S protein. Maturation and cell surface expression of the mutant proteins were not affected, and all proteins became acylated. The mutant in which the predicted transmembrane domain was shortened did not induce syncytia. From a group of mutants in which several conserved cysteines in the TM domain had been replaced by serines, one was unable to induce syncytia, another showed delayed syncytia formation, and the third mutant induced syncytia as did the wild-type protein. The potential role of the transmembrane domain in fusion is discussed.
Insights
Murine coronavirus spike (S) protein cleavage influences fusion kinetics but is not essential. Mutations in the transmembrane domain can inhibit or delay syncytia formation, highlighting its role in viral fusion.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- The spike (S) protein of murine coronavirus strain A59 (MHV-A59) mediates membrane fusion.
- The mature S protein undergoes partial cleavage into two subunits during transport.
Purpose of the Study:
- To investigate the role of S protein domains in membrane fusion.
- To identify amino acids critical for S protein cleavage and analyze its necessity for fusion.
- To examine the impact of mutations in the transmembrane (TM) domain on fusion.
Main Methods:
- Site-directed mutagenesis to alter S protein cleavage sites and TM domain.
- Analysis of S protein cleavage, maturation, cell surface expression, and acylation.
- Assessment of syncytia formation (cell-cell fusion) induced by wild-type and mutant S proteins.
Main Results:
- Cleavage of S protein is not strictly required for fusion but affects fusion kinetics.
- Modifying the cleavage site to mimic MHV-JHM resulted in full S protein cleavage.
- A mutant with a shortened TM domain failed to induce syncytia.
- Mutations in conserved cysteines within the TM domain differentially affected syncytia formation.
Conclusions:
- S protein cleavage influences the rate of viral-induced membrane fusion.
- The transmembrane domain of the S protein plays a critical role in mediating coronavirus-induced cell fusion.