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Characterization of Sendai virus M protein mutants that can partially interfere with virus particle production
Geneviève Mottet1, Virginie Müller1, Laurent Roux1
1Department of Genetics and Microbiology, University of Geneva Medical School, CMU, 9 avenue de Champel, 1211 Geneva 4, Switzerland 1.
Abstract:
Substitution of Val(113) in Sendai virus (SeV) M protein generates non-functional polypeptides, characterized by their exclusion from virus particles and by their ability to interfere with virus particle production. These phenotypic traits correlate with a single-band PAGE migration profile, in contrast to wild-type M (M(wt )), which separates into two species, one of which is a phosphorylated form. The single-band migration is likely to result from a conformational change, as evidenced by the lack of maturation of a native epitope and by a particular tryptic digestion profile, and not from the phosphorylation of all M molecules, an assumption consistent with the PAGE migration feature. One of the M mutants (HA-M(30 ), an M protein carrying Thr(112)Met and Val(113) Glu substitutions tagged with an influenza virus haemagglutinin epitope) was characterized further in the context of SeV infection, i.e. under conditions of co-expression with M(wt). HA-M (30) is shown (i) to bind mainly to membrane fractions, (ii) not to co-precipitate M(wt), as HA-M(wt) does, (iii) to interfere with the binding of nucleocapsids to membranes and (iv) to accumulate in perinuclear regions, in contrast to HA-M(wt ), which is also found at the cell periphery. Such mutants constitute potential tools for the identification of critical steps in paramyxovirus assembly and budding.
Insights
Mutating the Sendai virus M protein at Val(113) creates non-functional versions that block virus production. These M protein mutants offer new tools to study paramyxovirus assembly and budding processes.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- The M protein of Sendai virus (SeV) is crucial for viral particle assembly and budding.
- Wild-type M protein (M(wt)) exists in two species, including a phosphorylated form, and separates into two bands on PAGE.
- Mutations in the M protein can lead to non-functional polypeptides that interfere with virus production.
Purpose of the Study:
- To investigate the functional consequences of substituting Val(113) in the SeV M protein.
- To characterize the properties of M protein mutants and their impact on virus assembly.
- To explore the potential of these mutants as tools for studying paramyxovirus replication.
Main Methods:
- Site-directed mutagenesis of the SeV M protein (Val(113) substitution).
- PAGE analysis to assess protein migration and phosphorylation status.
- Characterization of M protein mutants (e.g., HA-M(30)) in SeV-infected cells using co-precipitation and subcellular localization studies.
Main Results:
- Substitution of Val(113) resulted in non-functional M proteins that were excluded from virus particles.
- Mutant M proteins exhibited a single-band PAGE profile, suggesting a conformational change rather than uniform phosphorylation.
- The HA-M(30) mutant interfered with nucleocapsid-membrane binding and accumulated in perinuclear regions, unlike M(wt).
Conclusions:
- Val(113) substitution in SeV M protein generates non-functional polypeptides that disrupt virus assembly.
- These mutants display altered cellular localization and impaired interactions with viral components.
- SeV M protein mutants serve as valuable tools for dissecting critical steps in paramyxovirus assembly and budding.