Elimination of phosphorylation sites of semliki forest virus replicase protein nsP3

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 ma pping 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 phosphor ylation sites in nsP3. Experiments with deletion variants suggested that ns P3 itself had no kinase activity; instead, it was likely to be phosphorylat ed by multiple cellular kinases. Phosphorylation was not necessary for the peripheral membrane association of nsP3, which was mediated by the N-termin al region preceding the phosphorylation sites. Two deletion variants of nsP 3 with either reduced or undetectable phosphorylation were studied in the c ontext of virus infection. Cells infected with mutant viruses produced clos e to wild type levels of infectious virions; however, the rate of viral RNA synthesis was significantly reduced in the mutants. A virus totally defect ive in nsP3 phosphorylation and exhibiting a decreased rate of RNA synthesi s also exhibited greatly reduced pathogenicity in mice.