Roles of Serine and Threonine Residues of Mumps Virus P Protein in Viral Transcription and Replication

Abstract

Mumps virus (MuV), a paramyxovirus containing a negative-sense nonsegmented RNA genome, is a human pathogen that causes an acute infection with symptoms ranging from parotitis to mild meningitis and severe encephalitis. Vaccination against mumps virus has been effective in reducing mumps cases. However, recently large outbreaks have occurred in vaccinated populations. There is no anti-MuV drug. Understanding replication of MuV may lead to novel antiviral strategies. MuV RNA-dependent RNA polymerase minimally consists of the phosphoprotein (P) and the large protein (L). The P protein is heavily phosphorylated. To investigate the roles of serine (S) and threonine (T) residues of P in viral RNA transcription and replication, P was subjected to mass spectrometry and mutational analysis. P, a 392-amino acid residue protein, has 64 S and T residues. We have found that mutating nine S/T residues significantly reduced and mutating residue T at 101 to A (T101A) significantly enhanced activity in a minigenome system. A recombinant virus containing the P-T101A mutation (rMuV-P-T101A) was recovered and analyzed. rMuV-P-T101A grew to higher titers and had increased protein expression at early time points. Together, these results suggest that phosphorylation of MuV-P-T101 plays a negative role in viral RNA synthesis. This is the first time that the P protein of a paramyxovirus has been systematically analyzed for S/T residues that are critical for viral RNA synthesis. IMPORTANCE Mumps virus (MuV) is a reemerging paramyxovirus that caused large outbreaks in the UnitedStates, where vaccination coverage is very high. There is no anti-MuV drug. In this work, we have systematically analyzed roles of Ser/Thr residues of MuV P in viral RNA synthesis. We have identified S/T residues of P critical for MuV RNA synthesis and phosphorylation sites that are important for viral RNA synthesis. This work leads to a better understanding of viral RNA synthesis as well as to potential novel strategies to control mumps. Mumps virus (MuV) is a human pathogen that causes acute parotitis and is highly neurotropic (1). Invasion of the central nervous system is evident in almost half of all clinical cases, with asceptic meningitis occurring in approximately 10% of cases and encephalitis in less than 1% (1). Even though the mumps vaccine has dramatically reduced disease incidence, large outbreaks have recently occurred in vaccinated populations (2, 3). Over 5,700 mumps cases were reported in a 2006 outbreak that originated at a university in Iowa and spread to 10 other states (2). A mumps outbreak occurred in New York and New Jersey in 2009 to 2010 where 88% of the patients had one dose of mumps vaccine and 75% of patients had two doses (3). There is no antiviral drug for MuV infection. 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 (4), MuV Iowa/US/06 (referred to here as MuV), was used to examine the role of phosphorylation of the M...

Mumps Virus Nucleoprotein Enhances Phosphorylation of the Phosphoprotein by Polo-Like Kinase 1

“…For virus infections, cells were inoculated with viruses at a multiplicity of infection (MOI) of 0.1 or 3 in DMEM plus 1% bovine serum albumin (BSA) and incubated at 37°C with 5% CO 2 for 1 to 2 h. The inocula were then replaced with DMEM supplemented with 2% FBS and 1% P/S. rMuV-P-T147A, rMuV-P-T204A, rMuV-P-S292A/S294A, rMuV-P-T147A/T204A, and rMuV-P-T147A/S292A/S294A were rescued from the plasmids containing their respective full-length genome as described before (7). A plasmid containing the full-length genome (5 g), along with plasmids pCAGGS-L (2 g), pCAGGS-NP (100 ng), and pCAGGS-P (320 ng), was transfected into BSR-T7 cells.…”

“…The MuV minigenome system used in this study was described previously (7). BSR-T7 cells in 24-well plates were transfected with pCAGGS-P (80 ng), pCAGGS-NP (25 ng), pCAGGS-L (500 ng), pMG-RLuc (100 ng), pFFLuc (1 ng), and various amounts of Flag-PLK1 or Flag-PLK1 (K82M) (0, 16, 32, or 64 ng).…”

“…Plasmids containing the full-length genome for rMuV-P-T147A, rMuV-P-S292A/S294A, and rMuV-P-T147A/ S292A/S294A viruses were made similarly to that of rMuV-P-T101A as described before (7). The MuV minigenome plasmid (BH526/pMGRLuc), containing Renilla luciferase, and a plasmid containing firefly luciferase (pFF-Luc) were described previously (7). Construction details and sequence files of the plasmids are available upon request.…”

“…A systematic mutational analysis of MuV P has identified residue T101 of P as important in viral RNA synthesis. Analysis of a recombinant MuV containing this mutation (rMuV-P-T101A) indicates that phosphorylation at P-T101 plays a negative role in viral transcription (7). Host kinases that are important for MuV phosphorylation have not been identified.…”

“…MuV infection causes acute parotitis, and it is a neurotropic agent with symptoms ranging from mild meningitis to severe encephalitis (16). Phosphorylation of MuV P plays a role in viral RNA synthesis (7). A systematic mutational analysis of MuV P has identified residue T101 of P as important in viral RNA synthesis.…”