The role of phosphorylation of the mumps virus phosphoprotein in viral pathogenesis
Pickar, Adrian Kathryn
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Mumps virus (MuV) is a human pathogen that causes acute parotitis and is highly neurotropic. Even though the mumps vaccine has dramatically reduced disease incidence, large outbreaks have recently occurred in vaccinated populations. There is no antiviral drug for MuV infection. MuV is a paramyxovirus containing a negative-sense nonsegmented RNA genome. The RNA-dependent RNA polymerase of MuV minimally consists of the phosphoprotein (P) and the large protein (L). While P does not have intrinsic enzymatic activity, it is an essential cofactor for the viral polymerase, and has a variety of functions, including enabling binding of the P-L complex to the nucleocapsid template and preventing self-assembly of nascent nucleoprotein (NP). The P protein is heavily phosphorylated. The importance of P phosphorylation in paramyxovirus transcription and replication has been studied in numerous viruses, and it has been shown to play a role in regulating viral mRNA synthesis. We hypothesized that phosphorylation of MuV P plays roles in viral transcription and replication. Understanding functions of viral proteins will aid development of antiviral strategies. In this study, a strain of MuV from a recent outbreak in Iowa in 2006, MuVIowa/US/06, was used to examine the role of phosphorylation of MuV P in viral RNA synthesis. Using mutational analysis and a minigenome system, the roles of the MuV P domains was investigated. We found that the functions of the N-terminal and C-terminal domains can be trans-complemented and that this complementation requires the oligomerization domain. To investigate the roles of serine (S) and threonine (T) residues of P in viral RNA synthesis, P was subjected to mass spectrometry and mutational analysis. We found ten critical S/T residues for minigenome activity, and analysis of a recombinant virus containing a P-T101A mutation suggests that phosphorylation of MuV-P-T101 plays a negative role in viral RNA synthesis. We also identified a host kinase, PLK1, that phosphorylates MuV P and this phosphorylation negatively regulates viral transcription. Together, these results show a role for phosphorylation of MuV P in regulating viral RNA synthesis and provide insight into potential targets for development of antiviral strategies and vaccines.