nsP3 is one of the four RNA replicase subunits encoded by alphaviruses. The specific essential functions of nsP3 remain unknown, but it is known to be phosphorylated on serine and threonine residues. Here we have completed mapping of the individual phosphorylation sites on Semliki Forest virus nsP3 (482 amino acids) by point mutational analysis of threonine residues. This showed that threonines 344 and 345 represented the major threonine phosphorylation sites in nsP3. Experiments with deletion variants suggested that nsP3 itself had no kinase activity; instead, it was likely to be phosphorylated by multiple cellular kinases. Phosphorylation was not necessary for the peripheral membrane association of nsP3, which was mediated by the N-terminal region preceding the phosphorylation sites. Two deletion variants of nsP3 with either reduced or undetectable phosphorylation were studied in the context of virus infection. Cells infected with mutant viruses produced close to wild type levels of infectious virions; however, the rate of viral RNA synthesis was significantly reduced in the mutants. A virus totally defective in nsP3 phosphorylation and exhibiting a decreased rate of RNA synthesis also exhibited greatly reduced pathogenicity in mice.
Elimination of Phosphorylation Sites of Semliki Forest Virus Replicase Protein nsP3*
H. Vihinen,T. Ahola,Minna T. Tuittila,A. Merits,L. Kääriäinen
Published 2001 in Journal of Biological Chemistry
ABSTRACT
PUBLICATION RECORD
- Publication year
2001
- Venue
Journal of Biological Chemistry
- Publication date
2001-02-23
- Fields of study
Biology, Medicine
- Identifiers
- External record
- Source metadata
Semantic Scholar, PubMed
CITATION MAP
EXTRACTION MAP
CLAIMS
CONCEPTS
- cellular kinases
Host-cell kinases proposed here to phosphorylate nsP3 rather than an intrinsic kinase activity of nsP3 itself.
Aliases: host kinases
- mutant viruses
Engineered virus variants carrying nsP3 phosphorylation-site deletions or loss-of-phosphorylation mutations used in infection experiments.
Aliases: mutant virus variants
- pathogenicity in mice
The disease-causing ability of virus infection in mice, used here to evaluate the impact of nsP3 phosphorylation loss.
Aliases: mouse pathogenicity, virulence in mice
- peripheral membrane association
The membrane-associated localization pattern of nsP3 at the cell periphery that was assessed in relation to phosphorylation.
Aliases: peripheral membrane binding
- phosphorylation
The addition of phosphate groups to nsP3 serine and threonine residues examined as a protein modification in this paper.
Aliases: nsP3 phosphorylation
- semliki forest virus
An alphavirus used here as the source of the nsP3 replicase protein studied in the paper.
Aliases: SFV
- semliki forest virus nsp3
A 482-amino-acid nonstructural replicase protein of Semliki Forest virus analyzed for phosphorylation sites and infection phenotypes in this paper.
Aliases: nsP3, SFV nsP3, replicase protein nsP3
- threonine phosphorylation sites
The threonine residues in nsP3 that can carry phosphate groups and were mapped by point mutational analysis in this study.
Aliases: phosphorylation sites on threonine residues
- viral rna synthesis
The production of viral RNA during infection, measured here as an outcome of nsP3 phosphorylation mutations.
Aliases: RNA synthesis
REFERENCES
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