Elimination of Phosphorylation Sites of Semliki Forest Virus Replicase Protein nsP3

Leevi Kääriäinen,Tero Ahola,Andres Merits,Helena Vihinen,Minna Tuittila

doi:10.1074/jbc.m006077200

Leevi Kääriäinen, Tero Ahola

Open Access

https://doi.org/10.1074/jbc.m006077200

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Abstract

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.

Highlights

Phosphorylation and dephosphorylation have been recognized as major processes by which protein function is regulated
NsP1 is an enzyme responsible for methylation and capping of viral mRNAs [7, 8]. It mediates membrane association of the RNA replication complex [9] and its targeting onto the cytoplasmic surface of endosomes and lysosomes [10]. nsP2 is an RNA helicase [11], RNA triphosphatase [12], and an autoprotease responsible for the cleavage of the nonstructural polyprotein [3]. nsP4 is the catalytic subunit of this RNA-dependent RNA polymerase [13]
Phosphoamino acid analysis had revealed that the wild type nsP3 was phosphorylated on serine and threonine, as described for the protein synthesized during Semliki Forest virus (SFV) infection

Summary

Introduction

Phosphorylation and dephosphorylation have been recognized as major processes by which protein function is regulated. Phosphorylation of Wild Type nsP3 and Deletion Mutant N-nsP3—To study phosphorylation of SFV nsP3, we synthesized the protein in relatively large amounts in eukaryotic cells by transfection. Phosphoamino acid analysis had revealed that the wild type nsP3 was phosphorylated on serine and threonine, as described for the protein synthesized during SFV infection (data not shown; Ref. 19).

Results

Conclusion