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Phosphorylation of the Sendai virus M protein is not essential for virus replication either in vitro or in vivo
T Sakaguchi1, K Kiyotani, A Kato
1Department of Bacteriology, Hiroshima University School of Medicine, 1-2-3 Kasumi, Hiroshima, 734, Japan. takemasa@mcai.med.hiroshima-u.ac.jp
Abstract:
A large proportion of intracellular Sendai virus (SeV) M proteins is phosphorylated, but in mature virions the M protein is not phosphorylated or dephosphorylated. Phosphorylated M protein in cells is bound to the cytoskeletal components more firmly than unphosphorylated M protein. Thus it has been hypothesized that M protein phosphorylation plays an important role in the virus life cycle, especially in the step of maturation. Here, a transient expression-mutation experiment of the M gene demonstrated that a change of the Ser residue at the 70th position from the N-terminus to Ala (S70A) totally abolished M protein phosphorylation, strongly suggesting that this residue is phosphorylated. The mutated M gene was then placed in the corresponding region in the cDNA plasmid which generates a full-length antigenome SeV RNA, and a mutant SeV M-S70A was successfully recovered from the cDNA. This mutant virus was indeed defective in M protein phosphorylation but did not differ at all from the wild-type SeV recovered from the parental cDNA either in the replication kinetics and plaque morphology in cultured cells or in in vivo replication and pathogenicity for mice. We thus concluded that no phosphorylation of the M protein was required for SeV replication either in vitro or in vivo.
Insights
Sendai virus (SeV) M protein phosphorylation is not essential for viral replication or pathogenicity. A mutant SeV lacking M protein phosphorylation showed no differences in replication or disease in mice, challenging previous hypotheses.
Area of Science:
- Virology
- Molecular Biology
- Protein Phosphorylation
Background:
- Intracellular Sendai virus (SeV) M protein is heavily phosphorylated, but M protein in mature virions is not.
- Phosphorylated M protein binds more strongly to cytoskeletal components than unphosphorylated M protein.
- M protein phosphorylation was hypothesized to be crucial for SeV life cycle, particularly maturation.
Purpose of the Study:
- To investigate the role of M protein phosphorylation in the Sendai virus life cycle.
- To determine if M protein phosphorylation is required for viral replication and pathogenicity in vitro and in vivo.
Main Methods:
- Site-directed mutagenesis of the SeV M gene, specifically changing Ser70 to Ala (S70A), to abolish phosphorylation.
- Recovery of a mutant SeV (M-S70A) expressing the mutated M gene from a cDNA plasmid.
- Comparative analysis of the replication kinetics, plaque morphology, in vivo replication, and pathogenicity of the mutant SeV M-S70A and wild-type SeV in cultured cells and mice.
Main Results:
- Mutation of Ser70 to Ala (S70A) completely abolished M protein phosphorylation.
- The recovered mutant SeV M-S70A exhibited defective M protein phosphorylation.
- No significant differences were observed between mutant SeV M-S70A and wild-type SeV in replication kinetics, plaque morphology, in vivo replication, or pathogenicity.
Conclusions:
- M protein phosphorylation is not required for Sendai virus replication in cultured cells.
- M protein phosphorylation is dispensable for Sendai virus replication and pathogenicity in vivo.
- The hypothesis that M protein phosphorylation plays a critical role in SeV maturation or other life cycle steps is not supported by these findings.