Phosphorylation of measles virus nucleoprotein affects viral growth by changing gene expression and genomic RNA stability.

The measles virus (MV) nucleoprotein associates with the viral RNA genome to form the N-RNA complex, providing a template for viral RNA synthesis. In our previous study, major phosphorylation sites of the nucleoprotein were identified as S479 and S510. However, the functions of these phosphorylation sites have not been clarified. In this study, we rescued recombinant MVs (rMVs) whose phosphorylation sites in the nucleoprotein were substituted (rMV-S479A, rMV-S510A, and rMV-S479A/S510A) by reverse genetics and used them in subsequent analyses. In a one-step growth experiment, rMVs showed rapid growth kinetics compared with wild-type MV, although the peak titer of the wild-type MV was the same as or slightly higher than those of the rMVs. Time course analysis of nucleoprotein accumulation also revealed that viral gene expression of rMV was enhanced during the early phase of infection. These findings suggest that nucleoprotein phosphorylation has an important role in controlling viral growth rate through the regulation of viral gene expression. Conversely, multistep growth curves revealed that nucleoprotein-phosphorylation intensity inversely correlated with viral titer at the plateau phase. Additionally, the phosphorylation intensity of the wild-type nucleoprotein in infected cells was significantly reduced through nucleoprotein-phosphoprotein binding. Excessive nucleoprotein-phosphorylation resulted in lower stability against RNase and faster turnover of viral genomic RNA. These results suggest that nucleoprotein-phosphorylation is also involved in viral genomic RNA stability.