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A Sendai virus vector leading to the efficient expression of mutant M proteins interfering with virus particle
G Mottet1, A Mühlemann, C Tapparel
1Department of Genetics and Microbiology, University of Geneva Medical School, Switzerland.
Abstract:
A Sendai virus expression vector in the form of a transcribing copy-back defective interfering RNA was constructed and shown to efficiently express a tagged matrix protein in the only context of a Sendai virus infection. In an attempt to identify relevant M protein domains involved in viral assembly and budding, a series of deletion mutants were tested for their ability to bind to cellular membrane fractions. The deletion of a region spanning amino acids 105-137 significantly decreased this binding when the protein was expressed in a system driven by the T7 RNA polymerase away from any other viral proteins. Plus or minus charges were introduced in the hydrophobic portion of a predicted amphiphilic helix in this region, and M proteins with altered membrane binding properties were produced. The genes encoding these mutant M proteins were then inserted in the Sendai virus vector and shown to be expressed at levels similar to that of the endogenous wild-type M protein. The presence of a negative charge in the hydrophobic region of the putative amphiphilic helix prevented the incorporation of the mutant protein into virus particles and appeared to decrease the efficiency of virus particle budding. In contrast, the introduction of a positive charge appeared to increase the M mutant uptake into virions. The use a Sendai virus vector has therefore been shown instrumental in the identification of mutant M proteins interfering with the viral assembly-budding process.
Insights
Researchers engineered a Sendai virus vector to study matrix (M) protein functions. This revealed specific M protein regions crucial for viral assembly and budding, impacting virus particle formation.
Area of Science:
- Virology
- Molecular Biology
- Protein Biochemistry
Background:
- Sendai virus matrix (M) protein is essential for viral assembly and budding.
- Understanding M protein domains involved in membrane binding is key to elucidating its role in viral morphogenesis.
Purpose of the Study:
- To identify specific domains of the Sendai virus M protein critical for viral assembly and budding.
- To investigate the role of charged residues within a predicted amphipathic helix in M protein membrane association and viral particle formation.
Main Methods:
- Construction of a Sendai virus expression vector using defective interfering RNA.
- Generation and characterization of M protein deletion and point mutants.
- Assessment of M protein binding to cellular membrane fractions.
- Analysis of mutant M protein expression, incorporation into virions, and effect on virus budding using the Sendai virus vector.
Main Results:
- Deletion of amino acids 105-137 in the M protein significantly reduced membrane binding.
- Introduction of negative charges in a predicted amphipathic helix impaired M protein incorporation into virus particles and budding.
- Introduction of positive charges enhanced M protein uptake into virions.
Conclusions:
- A specific region (amino acids 105-137) of the Sendai virus M protein is vital for membrane binding and subsequent viral assembly and budding.
- Charged residues within this region modulate M protein's interaction with viral components and membranes, influencing virion formation.
- The Sendai virus vector is a powerful tool for identifying M protein mutants that disrupt the viral assembly-budding process.