The roles of phosphorylation in mumps virus rna synthesis and replication
Zengel, James Robert
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Mumps virus (MuV) is a paramyxovirus with a negative-sense non-segmented, negative-strand RNA-genome. MuV infects humans causing acute infection with hallmark enlargement of the parotid gland . Although in incidence of mumps has greatly decreased since introduction of vaccines in the late 1960’s, there have been large outbreaks in highly vaccinated population [2–6]. To better control these outbreaks, we need to better understand the replication of MuV and the role of MuV proteins in replication and pathogenesis. It has been known for over 30 years that many of the MuV proteins are phosphorylated, but until recently the role of phosphorylation of these proteins had not been studied . In the follow body of work, we aim to better understand the role of phosphorylation in MuV RNA synthesis and replication and apply these finding to enhance the yield of a candidate vaccine in cell culture. We determined the major phosphorylation site in the nucleoprotein (NP) to be at position S439, and show that phosphorylation at this site has a negative effect on RNA synthesis and peak viral titers. We also build on previous research on phosphorylation of the phosphoprotein (P)  by determine the kinase involved in the phosphorylation of the MuV P protein to be PLK1 and showing the NP is important for P phosphorylation. The binding site for P was determined to be at residue T147, and mutation of this residue to prevent PLK1 binding and phosphorylation of other residues in P resulted in increased MuV growth in cell culture. We used what was learned in the basic research into the phosphorylation of MuV proteins to enhance the growth of a novel MuV vaccine candidate previously developed in our lab . By introducing an alanine substation mutation at position T147 and rescuing a vaccine virus containing the T147A mutation, we increased vaccine yield 10-fold in vero cells. This work allows for a better understanding of how phosphorylation effects MuV RNA replication and growth and the findings advanced the development of a next generation MuV vaccine candidate.