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 United States, 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.

FIG 1

Mass spectrometry analysis of MuV P protein products. (A) Phosphorylation of P in infected cells. Vero cells were infected with MuV and then labeled with [³⁵S]Met or [³³P]phosphate. The cells were lysed and immunoprecipitated with monoclonal anti-MuV-P antibody. The immunoprecipitated products were resolved in SDS-PAGE. (B) Purification of P from MuV-infected cells. Vero cells were infected with MuV. The cells were lysed and then immunoprecipitated with monoclonal anti-MuV-P antibody. The products were resolved in SDS-PAGE and stained with Commassie blue. Both the major (P₁) and minor (P₂) phosphorylated P bands were excised and processed for mass spectrometry analysis. The excised proteins were digested with trypsin, enriched using TiO₂, and analyzed by LC-MS/MS. (C) Schematic of LC-MS/MS coverage of the major phosphorylated MuV P protein (P₁). All serine and threonine residues are boxed, and underlined residues represent the detected peptides. MS achieved 73.9% P coverage. S/T residues highlighted in black represent detected phosphorylated residues, which included T147, T165, S188, and T265. (D) Schematic of LC-MS/MS coverage of the minor phosphorylated MuV P protein (P₂). MS achieved 88.5% P coverage. S/T residues highlighted in black represent detected phosphorylated residues, which included T10, T16, T39, S69, T91, T150, T165, S188, T250, S257, T258, T282, S374, and T375.

FIG 2

Mutation of phosphorylated residues identified by MS resulted in similar levels of minigenome activity. (A) Minigenome activity of P-T165A. Increasing amounts of P or P-T165A were transfected together with other plasmids as described in Materials and Methods. The negative control (NC) contained no transfected P. Renilla luciferase was the reporter gene in the minigenome, and Firefly luciferase expression was used as a transfection control. The minigenome activity was measured and normalized as the ratio of Renilla luciferase activity to firefly luciferase activity (relative luciferase activity). (B) Minigenome activity of P-S69A. P values were calculated using Student's t test. Error bars represent the standard errors of the means (SEM) of data from 6 replicates. *, P < 0.001. (C) Immunoblotting was performed to detect the expression levels of NP and P, P-T165A, or P-S69A.

FIG 3

Identification of the S/T residues that were critical for RNA synthesis using the MuV minigenome system. Minigenome activity of P-T10A (A) and P-T101A (B) is shown. P values were calculated using Student's t test. Error bars represent the SEM of data from 6 replicates. *, P < 0.001. Western blotting was performed to detect the expression levels of NP and P, P-T10A, and P-T101A.

FIG 4

Reduced phosphorylation of P in rMuV-P-T101A-infected cells. (A) MuV P protein band patterns of transfected and infected cells. HeLa cells were transfected with pCAGGS-P or pCAGGs-P mutants or infected with MuV and labeled with ³⁵S or ³³P. Cell lysates were immunoprecipitated with monoclonal anti-MuV-P antibody and resolved on a 10% SDS-PAGE gel. (B) Sequence confirmation of rMuV-P-T101A. The genome of rMuV-P-T101A was sequenced, and the region that contained the mutation is shown. (C) Phosphorylation of P in rMuV-P-T101A-infected cells. Mock-, MuV-, and rMuV-P-T101A-infected HeLa cells were labeled with ³⁵S or ³³P. Cell lysates were immunoprecipitated with monoclonal anti-MuV-P and resolved on a 10% SDS-PAGE gel. (D) Relative phosphorylation level of P in infected cells. The relative level was calculated as the ratio of phosphorylated protein (³³P-labeled P) to total protein (³⁵S-labeled P) and standardized to that of MuV. P values were calculated using Student's t test. Error bars represent the SEM of data from 4 individual experiments.

FIG 5

Growth rates of rMuV-P-T101A in cells. (A) Growth rates of MuV and rMuV-P-T101A in Vero cells at an MOI of 0.01. (B) Growth rates of MuV-IA and rMuv-P-T101A in HeLa cells at an MOI of 0.01. P values were calculated using Student's t test. *, P < 0.05; **, P < 0.01. Error bars represent the SEM of data from 3 replicates.

FIG 6

Viral protein expression was initially enhanced in rMuV-P-T101A-infected cells. (A) Detection of viral protein expression using immunoblotting. Vero cells were mock, MuV, or rMuV-P-T101A infected at an MOI of 0.01. Cell lysates were collected at 12, 24, 48, and 72 hpi, and monoclonal anti-MuV-NP antibody was used for NP protein expression. β-Actin expression is provided as a control to show Vero cell growth. (B) Detection of viral protein expression using flow cytometry. MuV- or rMuV-P-T101A-infected Vero cells (MOI of 0.1) were collected at 12, 24, 48, and 72 hpi and processed for flow cytometry. The mean fluorescent intensity of infected cells (for MuV NP protein) was used to show viral protein expression levels. Error bars represent the SEM from 4 replicates.

FIG 7

Viral RNA synthesis in rMuV-P-T101A-infected cells. (A) Viral mRNA levels. Vero cells were infected with MuV or rMuV-P-T101A at an MOI of 0.01. Total RNA was extracted at 0, 2, 4, 8, 12, 16, and 20 hpi. To measure viral mRNA levels, oligo(dT) primers and a MuV-F-specific 6-carboxyfluorescein (FAM)-tagged probe were used for real-time PCR. Genome levels of MuV or rMuV-P-T101A at 0 hpi were used as the baseline for normalization. (B) Genome replication of MuV and rMuV-P-T101A in Vero cells at an MOI of 0.01. To measure genomic RNA levels, gene-specific primers flanking the negative-sense genomic MuV-F gene and a MuV-F-specific FAM-tagged probe were used for real-time PCR. Genome levels at 0 hpi for each virus were used as a baseline for normalization. (C) Relative viral mRNA (vmRNA) levels per genome at each time point were calculated for MuV- or rMuV-P-T101A-infected Vero cells at an MOI of 0.01. P values were calculated using Student's t test. *, P < 0.05; **, P < 0.01. Error bars represent the SEM of data from 3 replicates.

FIG 8

Interactions of P with itself, NP, and L. (A) Oligomerization of P-T101A. Mock-, MuV-, or rMuV-P-T101A-infected Vero cells were labeled with ³⁵S. DSP (DMSO as a negative control) was used for cross-linking. The cross-linked products were immunoprecipitated using monoclonal anti-MuV-P antibody. Half of the products were mixed with the loading buffer without DTT, and the other half were mixed with the loading buffer with DTT. (B) Interaction between P-T101A and NP and L. Vero cells were mock infected or infected with MuV or rMuV-P-T101A and labeled with [³⁵S]Met. Cell lysates were immunoprecipitated with monoclonal anti-MuV-NP.