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Elimination of phosphorylation sites of Semliki Forest virus replicase protein nsP3.




TekijätVihinen H, Ahola T, Tuittila M, Merits A, Kääriäinen L

Julkaisuvuosi2001

JournalJournal of Biological Chemistry

Tietokannassa oleva lehden nimiThe Journal of biological chemistry

Lehden akronyymiJ Biol Chem

Vuosikerta276

Numero8

Aloitussivu5745

Lopetussivu52

Sivujen määrä8

ISSN0021-9258

DOIhttps://doi.org/10.1074/jbc.M006077200


Tiivistelmä
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.



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