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The roles of phosphorylation of the nucleocapsid protein of mumps virus in 1
regulating viral RNA transcription and replication 2
3
James Zengel, Adrian Pickar, Pei Xu §, Alita Lin¶, and Biao He* 4
5
Department of Infectious Disease, University of Georgia College of Veterinary Medicine, 6
Athens, GA 7
§ Current Address: Microbiology Department, University of Chicago, Chicago, IL 8 ¶ Current Address: Simon Fraser University, Burnaby, BC V5A 1S6 Canada 9 10 *Corresponding author: 11 Department of Infectious Diseases 12 College of Veterinary Medicine 13 University of Georgia 14 501 D.W. Brooks Dr. 15 Athens, GA 30602-7388 16 Tel: 706 542 2855 17 Email: [email protected] 18
19
JVI Accepted Manuscript Posted Online 6 May 2015J. Virol. doi:10.1128/JVI.00686-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Abstract 20
Mumps virus (MuV) is a paramyxovirus with a negative sense non-segmented RNA 21
genome. The viral RNA genome is encapsidated by the nucleocapsid protein (NP) to 22
form the ribonucleoprotein (RNP), which serves as a template for transcription and 23
replication. In this study, we investigated the roles of phosphorylation sites of NP in MuV 24
RNA synthesis. Using radioactive labeling, we first demonstrated that NP was 25
phosphorylated in MuV-infected cells. Using both liquid chromatography-mass 26
spectrometry (LC-MS) and in silico modeling, we identified nine putative phosphorylated 27
residues within NP. We mutated these nine residues to alanine. Mutation of the serine 28
residue at position 439 to alanine (S439A) was found to reduce the phosphorylation of 29
NP in transfected cells by over 90%. The effects of these mutations on the MuV mini-30
genome system were examined. S439A was found to have higher activity, four mutants 31
had lower activity and four mutants had similar activity compared to wild-type NP. MuV 32
containing the S439A mutation had reduced phosphorylation of NP by 90% and 33
enhanced viral RNA synthesis and viral protein expression at early time point after 34
infection, indicating that S439 is the major phosphorylation site of NP and its 35
phosphorylation plays an important role in down-regulating viral RNA synthesis. 36
37
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Significance 38
Mumps virus (MuV), a paramyxovirus, is an important human pathogen that is re-39
emerging in human populations. Nucelocapsid protein (NP) of MuV is essential for viral 40
RNA synthesis. We have identified the major phosphorylation site of NP. We have 41
found that phosphorylation of NP plays a critical role in regulating viral RNA synthesis. 42
The work will lead to a better understanding of viral RNA synthesis and possible novel 43
targets for anti-viral drug development. 44
45
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Introduction 46
Mumps virus (MuV) infects humans, causing acute infection with hallmark enlargement 47
of the parotid gland (1). Before widespread vaccination in the late 1960s, mumps was 48
the leading cause of aseptic meningitis and caused deafness in children (2). Although 49
vaccination has greatly reduced the number of infections, large outbreaks have 50
occurred recently in vaccinated populations. The largest recent outbreak in the United 51
States originated at a university in Iowa in 2006, where over 5000 cases were reported, 52
compared to approximately 250 cases per year in the preceding years (3). In 2014, 53
there were over 1100 cases of mumps reported, mainly centered around universities 54
(4). At least 90% of the individual infected received the Measles, Mumps, and Rubella 55
(MMR) vaccine, and the majority of people received two doses (3). New strategies to 56
control these outbreaks are needed. Understanding the roles of each MuV protein in 57
virus replication and pathogenesis will aid development of countermeasures for MuV. 58
59
Mumps virus (MuV) is a member of the family Paramyxoviridae in the genus 60
Rubulavirus (1). It has a negative sense, non-segmented, RNA genome of 15,384 61
nucleotides. The genome is comprised of seven transcriptional units that encode nine 62
viral proteins in the following order: 3’-NP-V/I/P-M-F-SH-HN-L-5’ with RNA synthesis 63
initiating at a single site at the 3’ end. The RNA genome associates with NP to form the 64
helical ribonucleoprotein (RNP), which protects the genome from degradation, and 65
serves as the template for RNA synthesis. The NP also associates with the 66
phosphoprotein (P) and indirectly with the large protein (L), in which P and L forms the 67
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viral RNA-dependent RNA polymerase (vRdRp) (5). The vRdRp uses NP-encapsidated 68
RNA as a template for both replication of the vRNA genome and production of mRNA 69
(2). The vRdRp transcribes the NP-encapsidated RNA into 5’ capped and 3’ 70
polyadenylated mRNAs in cytoplasm (6). Although the exact details of mRNA 71
production are not known, the process is currently believed to involve termination and 72
reinitiation (stop and start) at each gene junction. The vRdRp also replicates viral RNA 73
genome (7-10). It is thought that vRdRp transcribes vRNA first and replicates vRNA at a 74
later stage after entry into host cells. The regulation of the switch from transcription to 75
replication by vRdRp is not clear. It is thought that phosphorylation state of the P protein 76
plays a critical role. Interestingly, P interacts with NP in RNP as well as free NP (11-14). 77
Mumps NP is also involved in virus budding. It interacts with the matrix (M) protein, 78
which is critical for virus egress (15). 79
80
For all negative-sense non-segmented RNA viruses, their genomes are encapsidated 81
with nucleoprotein to form RNP. It has been shown that phosphorylation of NP plays a 82
role in transcription, replication, and genome stability. In measles virus, phosphorylation 83
of NP has been shown to up-regulate transcriptional activity in a minigenome assay 84
(16). A similar phenotype has also been seen in rabies virus (17) and Nipah virus (18). 85
In measles virus, phosphorylation of NP is involved in genome stability and 86
phosphorylation of NP affects genome stability (19). In Marburg virus, only 87
phosphorylated NP is incorporated into nucleocapsid complexes (20). Similarly in 88
measles virus, phosphorylated NP is preferentially incorporated into the nucleocapsid 89
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(21). Although it has been shown that NP is phosphorylated in MuV-infected chicken 90
cells, the role of phosphorylation is unclear (22). 91
92
In this study, we used in silico modeling and mass spectrometry to determine 93
phosphorylation sites in the NP of MuV. We studied the function of NP in RNA 94
transcription and replication through the use of a minigenome system (23) and a 95
reverse genetics system (24). 96
97
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Materials and Methods 98
Plasmids and cells 99
All plasmids were constructed using standard molecular cloning techniques. Plasmid 100
sequences were based on the sequence of a mumps virus isolated during an outbreak 101
in Iowa from 2006 (GenBank: JN012242.1). MuV NP, P, and L were previously cloned 102
into the pCAGGS expression vector (24, 25). Firefly-luciferase (pFF-Luc) and a MuV 103
mini-genome plasmid expressing Renilla luciferase flanked by MuV-IA trailer and leader 104
sequences and under a T7 promoter (pT7-MG-RLuc) were also previously produced 105
(23). Mutations were introduced into pCAGGS-NP as previously described for 106
introduction of pCAGGS-P mutations (23). Plasmids encoding the full-length genome of 107
MuV-IA previously used to rescue virus were mutated as necessary. Plasmids and 108
sequences are available upon request. 109
110
HEK293T cells were maintained in Dulbecco’s modified Eagle medium (DMEM) 111
supplemented with 5% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S). 112
BSR-T7 cells were maintained in DMEM supplemented with 10% FBS, 1% P/S, 10% 113
tryptose phosphate broth (TPB), and 400 µg/ml G418 to maintain T7 RNA polymerase 114
(RNAP) expression. Vero and HeLa cells were maintained in DMEM with 10% FBS and 115
1% P/S. All cells were cultured at 37°C and 5% CO2. Cells were passaged the day 116
before to achieve about 85-95% confluence for infection and 60-80% confluence for 117
transfection. 118
119
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Minigenome 120
BSR-T7 cells (1 day, 60-80% confluent, 24-well plate) were transfected with pCAGGS-P 121
(80 ng), pCAGGS-L (500 ng), pT7-MG-RLuc (100 ng), pFF-Luc (1 ng), and varying 122
amounts of pCAGGS-NP (wt or mutant at 0, 12.5, 25, 50, or 100 ng) using jetPRIME 123
(Polyplus Transfection, France) following manufacture’s protocol. Empty pCAGGS 124
vector was used to maintain a constant amount of total plasmid transfected per well. 125
After 48 hr, media was removed and 100 µl of passive lysis buffer (Promega, Madison, 126
WI) was added to each well, followed by shaking on an orbital shaker for 15 min. 40 µl 127
of lysate was transferred to a white 96-well plate and a dual luciferase assay (Promega) 128
was performed according to the manufacturer’s protocol. Luminescence was detected 129
using a GloMax 96 Microplate Luminometer (Promega). The ratio of Renilla to firefly 130
luminescence was determined for each well, and the average of 3-6 biological replicates 131
was calculated. The peak activity for each NP plasmid was determined and each 132
experimental data set was normalized to wt NP. The data reported is the combined 133
data for a least 3 experimental replicates. 134
135
Virus Rescue and sequencing 136
BSR-T7 cells (1 day, 60-80% confluent, 6-well plate) were transfected with pCAGGS-137
NP (100 ng), pCAGGS-P (160 ng), pCAGGS-L (2000 ng), and full-length genome (2500 138
ng) using jetPRIME. After 48-72hr, transfected BSR-T7 cells were trypsinized and co-139
cultured with Vero cells at a ratio of 1:5 in a 10-cm dish. When CPE was observed (2-7 140
days), the media, likely containing virus, was collected and a plaque assay was 141
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performed using Vero cells. Single plaques were isolated 6-7 days later and cultured in 142
fresh Vero cells in 6-well plates to produce passage 1 (P1). After titer determination, P1 143
was passaged again in T75 or T150 flasks at an MOI of 0.01 to produce P2. After 144
72hrs, virus was collected, BSA was added to 1% final concentration, and aliquots were 145
stored at -80°C. Titer was determined by plaque assay. Viral RNA was isolated using 146
QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA) followed by synthesis of DNA 147
templates using SuperScript III One-Step RT-PCR System with Platinum Taq (Life 148
Technologies, Grand Island, NY) and 5 sets of primers were used to amplify the entire 149
genome. Fragments were sent to Genewiz (South Plainfield, NJ) for sequencing using 150
6-10 primers per fragment. Only viruses matching the full-length plasmid sequence 151
were used for further experiments. Primer sequences are available upon request. 152
153
Immunoprecipitation 154
Cells were lysed with whole cell extraction buffer (WCEB) (50mM Tris-HCl [pH 8], 155
280mM NaCl, 0.5% NP-40, 0.2mM EDTA, 2mM EGTA, and 10% glycerol) 156
supplemented with protease inhibitors (1x protease inhibitor, 0.1mM 157
phenylmethylsulfonyl fluoride OR 1x protease/phosphatase inhibitor cocktail for 158
radioactive labeling experiments). Insoluble material was pelleted at 14000xg for 2 159
minutes and the supernatant was transferred to a new tube. Rec-Protein G-Sepharose 160
4B beads and anti-P or anti-NP mAb was added to each tube and nutated overnight at 161
4°C. The next day, tubes were spun at 600xg for 2 min and supernatant was aspirated. 162
Three washes were performed with 1 ml of WCEB using the same process. The bead 163
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pellet was resuspended in 50-200 µl of 2x Laemmli Sample Buffer (Bio-Rad, Hercules, 164
CA) + 5% β-Mercaptoethanol followed by heating at 95°C for 5 min. 165
166
Mass spectrometry 167
Vero cells in a 10-cm plate were infected with MuV-IA at an MOI of 0.5. After 24 hr, 168
immunoprecipitation was performed as described above with an anti-MuV-P mAb. After 169
overnight incubation, the sample was spun to pellet the beads and the supernatant was 170
collected. The washes were continued and loading buffer was added as above. This 171
produced the “anti-P” sample. The supernatant collected prior to the first wash was 172
used for a second immunoprecipitation with anti-MuV-NP mAb. This produced the “anti-173
NP” sample. Both samples were resolved on a 10% acrylamide gel by SDS-PAGE. 174
The gel was stained with Coomassie Blue G250 in 10% acetic acid and 45% methanol 175
for 4 hours, followed by destaining with destain buffer (10% acetic acid, 40% methanol, 176
50% water). Bands were excised from the gel and sent to the MS & Proteomics WM 177
Keck Foundation Biotechnology Resource Laboratory (Yale University, New Haven, CT) 178
for further processing and mass spectrometry (MS). Briefly, the protein was digested 179
with trypsin and enriched for phosphoproteins on a TiO2 column (2x). Peptides were 180
separated on a nanoACQUITY (Waters, Milford, MA) (75µm x 250mm eluted at 181
300nl/min, 80 minute run) with MS analysis on an Orbitrap Elite mass spectrometer 182
(Thermo Scientific). Both the fraction enriched by the column and the flow-through were 183
analyzed by LC-MS/MS, and peak lists were combined prior to a Mascot search against 184
the NCBInr database with taxonomy restricted to viruses. Phosphorylated peptides 185
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were considered significant with a random probability score of less than 5%. For 186
peptides with more than one possible phosphorylation site, the Mascot Delta Score and 187
PhosphoRS score were used to determine which site was phosphorylated. 188
189
Radioactive labeling for phosphorylation analysis. 190
In order to examine phosphorylation of NP expressed from transfected plasmid, 1 µg of 191
pCAGGS-NP (wt or mutant) was transfected into HEK293T cells in a 6-well plate using 192
JetPrime in duplicate. After 24 hr, cells were starved in 1 ml DMEM lacking methionine 193
and cysteine for 30 min followed by labeling with about 50 µCi/ml 35S-EasyTag 194
Express35S Protein Labeling Mix (PerkinElmer, Waltham, MA) for 6 hr. Alternatively, 195
the cells were starved with 1 ml DMEM lacking sodium phosphate followed by labeling 196
with about 100 µCi 33P-Orthophosphoric acid (Perkin Elmer) for 6 hr. The cells were 197
then lysed and immunoprecipitation was performed with anti-NP mAb as described 198
above. The samples were resolved on a 10% acrylamide gel by SDS-PAGE and gels 199
were dried. Radioactivity was detected by exposing the gel to a Storage Phosphor 200
Screen BAS-IP MS (Fuji) overnight. The screen was read on a Typhoon FLA 7000 (GE 201
Healthcare Life Sciences, Pittsburgh, PA) and the densitometry analysis was performed 202
using ImageQuant TL software (GE Healthcare). The ratio of 33P/35S was calculated 203
and reported. 204
205
In order to determine NP phosphorylation in infected cells, Vero cells in a 6-well plate 206
were infected with MuV (wt, S439A, S520A, or 542A) at an MOI of 0.1 for 1 hr. Media 207
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was replaced with DMEM containing 2% FBS and 1% P/S and incubated for 24 hr. 208
After 24 hr, the cells were lysed, labeled, and immunoprecipitation and quantification 209
were performed as above. 210
211
Growth Curves. 212
Vero or HeLa cells in a 10-cm dish were infected with MuV (wt, S439A, S520A, or 213
542A) at an MOI of 0.01 or 5 in 5 ml of DMEM+2% FBS+1% P/S for 1 hr in triplicate. 214
Cells were washed four times with PBS and 10 ml of DMEM+2% FBS+1% P/S was 215
added to the cells. One sample was taken immediately after the DMEM was added and 216
labeled as 0 hpi (hours post infection). For MOI of 5, samples were collected at 0, 6, 217
12, 24, 48, and 72 hpi. For MOI of 0.01, samples were collected at 0, 24, 48, 72, 96, 218
and 120 hpi. All samples were supplemented with 1% BSA after collection and stored 219
at -80°C. Virus titers were determined by plaque assay on Vero cells. Results were 220
confirmed in a second experiment. Significance was determined by two-way ANOVA 221
using the Holm-Sidak method to correct for multiple comparisons. 222
223
Real-time PCR. 224
Vero cells in a 6-well plate were infected with MuV (wt, S439A, S520A, or S542A) at an 225
MOI of 0.1 for 1 hr, washed three times with PBS, and 2 ml of DMEM+2%FBS+1%P/S 226
was added to each well. At 0, 6, 12, and 18 hpi, total RNA was collected using the 227
RNeasy Plus Mini Kit with QIAshredder homogenization (Qiagen) according to the 228
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manufacturer’s instruction. cDNA was generated using SuperScript III Reverse 229
Transcriptase (Life Technologies) using 5 µl of RNA according to the manufacturer’s 230
directions. Oligo(dT)15 (Promega) were used for mRNA cDNA synthesis and a primer 231
specific for the negative sense genome (TGAACTAGCGAGGCCTATCCCCAAG) was 232
used for genomic cDNA synthesis. 5µl of cDNA was used for real-time PCR using a 233
MuV-F specific, FAM-tagged probe (Life Technologies) and TaqMan Gene Expression 234
Master Mix (Life Technologies) according to the manufacturer’s instructions. Real-time 235
PCR was run on a StepOnePlus Real Time PCR System (Life Technologies). 236
Biological triplicate samples were run for each sample. Ct values were normalized to 237
genomic RNA at 0 hpi. Significance was determined by two-way ANOVA using the 238
Holm-Sidak method to correct for multiple comparisons. 239
240
Protein quantification. 241
Cells were infected at an MOI of 0.1 for 6 hr or MOI of 5 for 24 hr. Cells were washed 242
once with PBS and trypsinized. Cells were collected into a 1.5 ml tube and pelleted (all 243
spins at 600 g), washed twice with DMEM+2% FBS, and fixed and permeabilized using 244
Cytofix/Cytoperm solution (BD Biosciences, San Jose, CA) overnight at 4°C. The mAbs 245
were conjugated using Zenon Alexa 488 (A488) or Allophycocyanin (APC) Mouse IgG1 246
Labeling Kits (Life Technologies) according to the manufactures specifications. Cells 247
were then washed twice with Perm/Wash buffer (BD Biosiences) followed by staining 248
with anti-NP(A488) or anti-P(APC). After staining for 20 min at 4°C, samples were 249
washed twice with Perm/Wash buffer and once with PBS+1%BSA. Cells were then 250
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resuspended in 500 µl of PBS+1% BSA. Flow cytometry was performed using the 251
LSRII Flow Cytometer (BD) and data was collected and analyzed using FACSDiva 252
(BD). The mean fluorescence intensity was calculated for the stained population. 253
254
Total protein was also measured by infecting cells at an MOI of 5 as above, lysing with 255
2x Laemmli Sample Buffer (Bio-Rad), and heating at 95°C for 5 min. Samples were 256
then resolved on a 10% acrylamide gel by SDS-PAGE and transferred to Amersham 257
Hybond LFP PVDF membranes (GE Healthcare Life Sciences). Immunoblotting was 258
performed by incubating the membranes with anti-NP and anti-P mAb and anti-GAPDH 259
[GT239] (Genetex, Irvine, CA) in 5% milk+PBS+0.1% Tween 20 (PBST) overnight at 260
4°C, followed by three washes with PBST, followed by incubation with Cy3 conjugated 261
goat anti-mouse IgG diluted 1:2500 (Jackson ImmunoResearch,West Grove, PA) in 5% 262
milk+PBST for 1hr at room temperature. After the incubation, the membrane was 263
washed four times with PBST and dried. The blot was visualized on the Typhoon FLA 264
7000 (GE Healthcare Life Sciences) and the densitometry analysis was performed 265
using ImageQuant TL software (GE Healthcare). 266
267
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Results 268
269
MuV NP is phosphorylated. 270
Previously, it was shown that MuV NP was phosphorylated when mumps virus was 271
grown in chicken embryo cells. In order to determine if NP is phosphorylated in 272
mammalian cells, Vero cells were infected with the recombinant mumps virus, 273
rMuV(Iowa/US/06) (referred to as MuV), at an MOI of 0.1 for 24 hr and labeled with 35S-274
Met/Cys or 33P- Orthophosphoric acid. Immunoprecipitation was performed using anti-275
NP mAb (24) and samples were resolved by SDS-PAGE. NP was detected in the 33P 276
labeling, indicating that NP was phosphorylated in infected cells (Figure 1A, left panel). 277
To determine if NP phosphorylation was dependent on other viral proteins, cells were 278
transfected with a plasmid encoding MuV NP, labeled with radioactive reagents and 279
immunoprecipitated as above (Figure 1A, right panel). NP was detected in NP-280
transfected cells labeled with 33P, indicating that NP was phosphorylated without any 281
other viral proteins present. 282
283
Phosphorylation sites in NP were determined by in silico modeling and mass 284
spectrometry. 285
Potential phosphorylation sites within NP were first identified using NetPhos 2.0 286
(http://www.cbs.dtu.dk/services/NetPhos/), a sequence-based prediction (26). Using 287
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this program, 12 phosphorylation sites were predicted above the cutoff value of 0.5. 288
These sites are summarized in Table 1. 289
290
To identify the phosphorylated resides in MuV NP in infected cells, Vero cells were 291
infected with MuV at an MOI of 0.1 for 48hr. In order to determine if there were 292
differences in the phosphorylation state of NP interacting with P, two sequential 293
immunoprecipitation were performed. Cells were first lysed and immunoprecipitated 294
with a mAb specific for MuV-P, which pulled down all of the P protein in the sample, as 295
well as NP that was associated with P (Figure 1B, Sample 1). The unbound protein 296
from the first immunoprecipitation was then immunoprecipitated again using a mAb 297
specific for MuV-NP, which pulled down all of the non-P associated NP (Figure 1B, 298
Sample 2). These samples were resolved by SDS-PAGE and stained with Coomassie 299
Blue. The labeled NP bands were excised from the gel. The samples were subjected 300
to tryptic digestion, phosphopeptide enrichment, and analyzed by LC-MS/MS. The 301
coverage was between 93-94% for each sample (Figure 1C and 1D), with residues 302
T387 and S439 found to be phosphorylated in both samples, and residues S25 and 303
S542 phosphorylated in only in sample 1. The detected sites along with other sites that 304
were below the defined cutoff for confirmed phosphorylation are summarized in Table 1. 305
306
The S439 residue in MuV NP was found to be the major phosphorylation site in 307
transfected cells. 308
309
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To assess which serine and threonine residues contributed to NP phosphorylation, we 310
chose to examine seven residues (S25, S94, T183, S298, T387, S439, and S542) 311
based on identification by mass spectrometry and two residues (S67 and S520) based 312
on high in silico prediction scores (0.990 and 0.979). Plasmids encoding NP were made 313
with mutations to convert the serine or threonine residues to alanine in the encoded 314
protein. Effects of the mutations on phosphorylation were determined by transfection of 315
cells with plasmids expressing wt NP or the NP with alanine substitutions in duplicate. 316
After 24 hours, one replicate was labeled with 35S and the other was labeled with 33P. 317
After 6 hours of labeling, the cells were lysed and immunoprecipitation with anti-NP 318
antibody was performed. The samples were resolved by SDS-PAGE (Figure 2A) and 319
the ratio of 33P to 35S was calculated (Figure 2B). NP-S439A had little or no 320
phosphorylation, while there was no significant difference between wt NP and the other 321
mutants. The addition of P and L in the transfection had no effect on the 322
phosphorylation of wt NP during transfection (data not shown). 323
324
The role of NP residues was assessed with a MuV minigenome system. 325
The role of NP in transcription and replication was studied using the MuV minigenome 326
system previously developed in our lab (23). The minigenome system consists of 327
plasmids required for transcription and replication of viral RNA (NP, P, and L), as wells 328
as a plasmid that encodes a viral negative sense minigenome under a T7 promoter. 329
When transfected into T7 RNAP-expressing BSR-T7 cells, the minigenome plasmid 330
produces negative-sense RNA containing the sequence for Renilla luciferase flanked by 331
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the MuV leader and trailer. The MuV replication machinery (NP, P, and L) replicates 332
this RNA through a positive sense intermediate and produces Renilla luciferase mRNA, 333
which is translated by host machinery. Changes in Renilla luciferase activity are due to 334
changes in the replicative and transcriptional activity of the MuV replication system. The 335
plasmids encoding NP mutants were tested at four concentrations and the peak activity 336
was reported for each (Figure 3A). An example minigenome titration is shown 337
comparing wt NP and the S439A mutant (Figure 3B). Western blots were performed for 338
each minigenome set to examine NP expression levels (Figure 3C). Two mutants and 339
wt NP are shown, but all mutants had similar protein amounts when the same amount of 340
plasmid was transfected, with some slight variation. The same plasmids showed no 341
difference in protein levels when using radioactive labeling. The use of multiple 342
concentrations of plasmid in the minigenome system also controls for any differences in 343
expression. We found that the S439A mutant had a higher level of minigenome activity. 344
Four of the substitutions (S25A, S94A, T183A, S298A) had lower activity and there was 345
no change with the other four substitutions (S67A, T387A, S520A, S542A). 346
347
S439 was the major phosphorylation site in NP in virus. 348
We have constructed plasmids containing full-length MuV genome with alanine 349
substitutions in NP and produced seven plasmids (S25A, S94A, S183A, T387A, S439A, 350
S520A, S542A). The reverse genetics system previously developed in our lab was 351
used to successfully rescue three viruses (rMuV-NP-S439A, S520A, S542A) (24, 25). 352
Complete genome sequences were confirmed as outlined in the methods. At least 353
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three rescue attempts were made to rescue the other viruses, without success, while 354
wild-type viruses were consistently rescued. To examine the phosphorylation states of 355
NP in these viruses, Vero cells were infected with wt MuV and the three mutant viruses. 356
Radioactive labeling of infected cells and immunoprecipitation of cell lysates were 357
performed, and phosphorylation was determined as in the previous experiment (Figure 358
4A). After calculating the ratio of 33P to 35S (Figure 4B), we found that there was a 359
significant decrease in phosphorylation for MuV-NP-S439A, while there was no change 360
in the other two mutant viruses, indicating that S439 is the major phosphorylation site of 361
NP. This is consistent with the results obtained using transfected NP. 362
363
rMuV(wt), rMuV-NP-S439A, S520A, and S542A had changed growth rates in cell 364
culture. 365
To determine the effect of the NP mutations on virus growth in cell culture, single-cycle 366
and multi-cycle growth curves were performed in Vero and HeLa cells. In a single-cycle 367
growth curve, cells were infected with an MOI of 5 and supernatant was collected at 0, 368
6, 12, 24, 48, and 72 hours post infection (hpi). In a multi-cycle growth curve, cells were 369
infected at an MOI of 0.01 and supernatant was collected every 24 hours until 144 hpi. 370
During single-cycle replication in Vero cells (Figure 5A), there was lower virus titer for 371
rMuV-NP-S439A at 6 hpi when compared to rMuV(wt), but an increased titer at 12, 24, 372
48, and 72 hpi. rMuV-NP-S542A had decreased titers at 12, 24, 48, and 72 hpi and 373
rMuV-NP-S520A had even lower titers at each of those time-points. During multi-cycle 374
replication in Vero cells (Figure 5B), rMuV-NP-S439A had increased titers at 48, 96, and 375
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120 hpi, while both rMuV-NP-S520A and S542A had reduced titers at 72 and 120 hpi 376
when compared to rMuV(wt). 377
378
In HeLa cells, the growth characteristics of the viruses were similar to those in Vero 379
cells for the single-cycle growth, but there were differences between HeLa and Vero for 380
the multi-cycle growth. There still was a lag in single-cycle growth for MuV-NP-S439A 381
(Figure 5C), but the virus was able to reach a significantly higher titer than rMuV(wt) by 382
48hpi. During multi-cycle growth in HeLa cells (Figure 5D), MuV-NP-S439A had lower 383
titers from 72 to 144 hpi. MuV-NP-S542A has higher titers compared to rMuV(wt) at 48 384
and 96hpi with slightly lower titers at 144hpi. MuV-NP-S520A had decreased titers 385
compared to rMuV(wt) after 72hpi, similar to growth in Vero cells. 386
387
rMuV-NP-S439A had increased protein present at 6 and 24 hours post infection. 388
To determine if there were differences in protein production for these viruses, the 389
amount of protein produced during viral infection was examined by western blotting first 390
(Figure 6A). rMuV-NP-S439A had increased viral protein in cells (Figure 6B). rMuV-391
NP-S542A also had a small increase in viral protein levels by western blot, although the 392
increase was not significant. To determine if this difference was due to protein 393
production on a per cell basis, flow cytometry was used to stain for viral protein 394
expression after infection. In order to determine early protein production, cells were 395
collected 6 hours after infection (MOI of 0.1) and stained with antibodies specific to MuV 396
NP or P. The mean fluorescence intensity was determined for each of the stained 397
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populations (Figure 6C). rMuV-NP-S439A produced more protein at 6 hpi, as seen by 398
staining for NP, while amounts of P were not detectable at this time. Furthermore, 399
rMuV-NP-S439A had an increase in both the amount of NP and P produced on a per 400
cell basis using high MOI (MOI of 5) infection at 24 hpi (Figure 6D). rMuV-NP-S542A 401
had a trend toward higher protein levels by flow cytometry, but the difference was not 402
significantly different from rMuV (wt) (p=0.12 to 0.4). 403
404
rMuv-NP-S439A had increased genome replication and mRNA production. 405
To understand the difference in protein production and viral titer, the amount of genome 406
RNA and mRNA were measured by quantitative real-time, reverse transcription PCR 407
(qRT-PCR) in infected Vero cells (MOI of 0.1). The cDNA was generated using a 408
genome specific primer to quantify genome RNA and oligo dT to quantify mRNA. The 409
probe used for all samples was specific to MuV F or HN. Data using the MuV F specific 410
probe is reported. It was found that rMuV-NP-S439A had increased genomic RNA 411
production at 6, 12, and 18 hpi, while rMuV-NP-S520A and S542A had decreased 412
genomic RNA production at 12 and 18 hpi when compared to rMuV(wt) (Figure 7A). 413
rMuV-NP-S439A also had increased mRNA production at all timepoints, while rMuV-414
NP-S520A and S542A had decreased levels at 6, 12, and 18 hpi when compared to 415
MuV(wt) (Figure 7B). When comparing the ratio of mRNA to genomic RNA, rMuV-NP-416
S439A had increased relative mRNA levels at 0 and 6 hpi, rMuV-NP-S542A had 417
reduced relative levels at 6 and 12 hpi, and rMuV-NP-S520A had no significant 418
differences when compared to MuV(wt) (Figure 7C). 419
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420
Mutations in NP affect NP-P interaction during infection but not transfection. 421
To investigate the mechanism of the phosphorylation of NP in regulating viral RNA 422
synthesis, NP and P interaction was examined. Cells were transfected with plasmids 423
encoding NP and P and immunoprecipitation was performed using either anti-NP 424
(Figure 8A) or anti-P (Figure 8B) mAbs. After co-immunoprecipitation of NP and P 425
when using plasmids encoding any of the mutant NPs, no difference was detected. To 426
assess differences in NP and P association in infected cells, Vero cells were infected 427
with MuV-wt, S439A, S520A, and S542A. Co-immunoprecipitation and total protein 428
visualization was performed (Figure 8C). The ratio of NP to P was calculated (Figure 429
8D) and MuV-S439A had decreased amounts of NP co-immunoprecipitated with P 430
during the anti-P pull down. While there was a difference in NP-P interaction during 431
infection, there was no difference in the amount of NP or P in sucrose gradient purified 432
virus from infected cells (data not shown). 433
434
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Discussion 435
436
In this study, we identified and confirmed multiple phosphorylated residues in MuV NP 437
by mass spectrometry and directed mutagenesis. We showed that S439 was the major 438
site of phosphorylation. Mutating this residue to alanine caused an increase in 439
minigenome activity and higher levels of viral RNA and protein expression in rMuV-NP-440
S439A-infected cells than wild type virus-infected cells at early time points after 441
infection. This is in contrast to previous work on Measles, Rabies, and Nipah viruses, 442
which have decreased activity in their respective minigenome systems when NP 443
phosphorylation is reduced (16-18). To the best of our knowledge this is the only case in 444
which decreased phosphorylation of the nucleoprotein of a virus resulted in increased 445
activity, indicating that phosphorylation of NP down-regulate viral RNA synthesis. We 446
hypothesize that phosphorylation can both up- and down-regulate activity and that these 447
differences may depend on the site that is being phosphorylated. 448
449
The mechanism of down-regulation of viral RNA synthesis by S439 of NP is not clear. 450
We assessed the RNA binding of S439A along with all other alanine substitution 451
mutants, but no differences were found compared to any of the mutants and wt NP 452
(data not shown). One interesting difference between wt NP and NP-S439A is their 453
interaction with P. While we were not able to show any differences in interactions 454
between NP and P or NP and M in transfected cells, we found that there was much less 455
NP pulled down in cells infected with rMuV-NP-S439A by an anti-P mAb in co-456
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immunoprecipitation. This result suggests that phosphorylation at S439 increases NP 457
association with P in infected cells, although phosphorylation at S439 was found in both 458
P-associated NP and free NP (Figure 1b, Table 1). This is rather surprising because 459
the domain of NP interacting with P is located at N-terminal 400 amino acid residues of 460
NP and mutation at residue 439 should not have affected binding with P (13, 14). The 461
difference in expression levels of NP, P and L in transfection and infection may 462
contribute to the difference observed in the NP-P binding. It is possible that a previous 463
un-detected region of NP (C-terminal tail domain) can interact with P, in the presence of 464
other viral protein such as L and M. 465
466
The fact that rMuV-NP-S439A caused a slight lag in virion production even though there 467
was more RNA and protein at that time point when compared to the other viruses 468
suggests that there may be some defect in packaging of RNA for production and 469
release of progeny virus. The lag in virion production may be detrimental to virus 470
growth in vivo, which could explain why this position is highly conserved among all MuV 471
strains (data not shown). The lower titer of rMuV-NP-S439A in HeLa cells, a type I 472
interferon (IFN) producing cell, compared to wt MuV is consistent with this residue being 473
critical for MuV growth in vivo. It is possible that the rMuV-NP-S439A virus was more 474
sensitive to type I IFN during HeLa infection, which is not observed in Vero cells, an IFN 475
defective cell. 476
477
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While mutations at other amino acid residues did not produce a significant decrease in 478
phosphorylation in transfected cells, they did play critical roles. There was a small 479
decrease in phosphorylation of the S542A mutant. There was also a small, but 480
significant decrease in both genomic RNA and mRNA, although there was a slight 481
increase in protein produced. It is possible that there may be some defect in budding 482
for the rMuV-NP-S542A virus, which might have caused the decrease in RNA and 483
increased protein in the cells. Less protein may be exported in progeny virions. In 484
PIV5, a closely related paramyxovirus, it is known that negatively charged residues in 485
the tail of NP are important for NP-M interaction and virus budding (27). The impact of 486
the mutation at S542 may also be attributed to the transient nature of phosphorylation at 487
this site. This is consistent with the mass spectrometry data that shows S542 was only 488
significantly phosphorylated in the P-associated sample. rMuV-NP-S520A had a lower 489
virus titer when compared to MuV(wt), suggesting that there was some defect in virus 490
growth, although there were only modest decreases in the amount of genomic RNA and 491
mRNA produced. We were also unable to find any phosphorylation at this site by mass 492
spectrometry and saw no decrease in phosphorylation when the residue was 493
substituted for alanine. It is possible that phosphorylation at this residue per se does not 494
have an impact on virus life cycle, but the residue itself is important for the virus life 495
cycle. When amino acid residues at positions S94, T183, or S298 were substituted with 496
alanine, there was a large decrease in minigenome activity. Viruses containing these 497
mutations were not obtained after multiple attempts, likely due to the low level of 498
replicative activity seen in the minigenome system, suggesting that these residues play 499
important roles in the virus life cycle. It is surprising that viruses containing mutations at 500
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S67 and T387 were not obtained since these mutations did not affect minigenome 501
activity. It is likely that these residues, not necessarily their phosphorylation status, may 502
play a role beyond viral RNA synthesis. Interestingly, mutations at the unstructured C-503
terminus of NP allowed rescue of infectious virus and we were unable to rescue 504
infectious viruses containing mutations at the N-terminal of NP, suggesting that residues 505
in the more structure N-terminus need to be preserved. 506
507
Understanding the roles of phosphorylation of MuV NP will not only contribute to our 508
knowledge on viral RNA synthesis, but also aid design of novel anti-virals and the next 509
generation of vaccines. Since MuV is not known to encode its own kinase to 510
phosphorylate NP, the host kinases responsible for NP phosphorylation may be viable 511
drug targets. While host kinase responsible for phosphorylation of NP’s S439 residue is 512
not likely a good target for antiviral drug development, kinases responsible for 513
phosphorylation of N-terminal of NP may be good targets since mutating these residues 514
resulted in difficulties in obtaining infectious viruses. Identifying of the host kinases 515
responsible for phosphorylating these critical sites of NP may lead to development of 516
small molecule inhibitors of the kinases as anti-MuV drugs, which do not exist at 517
present. Preventing phosphorylation at NP-S439 was able to increase viral replication 518
in Vero cells. This mutation can be incorporated into vaccine viruses enabling faster 519
growth and higher titer viruses, which will reduce the cost of future vaccines. 520
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ACKNOWLEDGEMENTS 521
522
We appreciate the helpful discussion and technical assistance from all members of Dr. 523
Biao He’s laboratory. This work was supported by grants (R01AI097368 and 524
R01AI106307) from the National Institutes of Health. 525
526
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Figure Legends 527
Figure 1. Analysis of NP phosphorylation by mass spectrometry. (A) 528
Phosphorylation of NP in infected or transfected cells. Vero cells were infected with 529
MuV-IA and HEK293T cells were transfected with NP and P. After 24 hr, proteins were 530
labeled with 35S-met or 33P-Orthophosphoric acid. Cells were lysed and 531
immunoprecipitated with anti-NP mAb. The samples were resolved by SDS-PAGE. (B) 532
Immunoprecipitiation of NP by anti-NP and anti-P. Vero cells were infected by MuV-IA 533
and lysate was immunoprecipitated with an anti-P mAb (sample 1). Unbound protein 534
was immunoprecipiatated with and anti-NP mAb (sample 2). The samples were 535
resolved by SDS-PAGE followed by visualization using Coomassie blue and NP band 536
was excised for analysis by LC-MS/MS. The P1 and P2 bands of MuV P were excised 537
for analysis in another study. (C) Coverage of MS of NP after anti-P IP. (D) Coverage 538
of MS of NP after anti-NP IP. Phosphorylated positions are in bold and positions not 539
covered are struck through. Phosphorylation was considered significant with a random 540
probability score of less than 5%. 541
542
Figure 2. Phosphorylation of NP mutants in transfected cells. (A) Detection of NP 543
mutant phosphorylation. Residue S439 was found to be the major phosphorylation site 544
in NP. HEK293 cells were transfected with plasmids encoding either wt or NP with S/T 545
residues mutated to A followed by 35S-Met/Cys or 33P-Orthophosphoric acid. 546
Immunoprecipitation was performed with an anti-NP mAb and samples were resolved 547
by SDS-PAGE. A representative gel is shown along with data from three separate 548
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experiments. (B) Summary of quantified NP phosphorylation. The relative density of 549
the phosphorylated versus total NP was calculated for each experiment. All data was 550
normalized to wt NP. (One-way ANOVA with Holm-Sidak multiple comparison test, N=3, 551
*p<0.001) 552
553
Figure 3. Effects of NP mutants on the MuV minigenome system. (A) Peak 554
minigenome activity of the NP mutants. A MuV minigenome assay was performed 555
using plasmids encoding NP with possible phosphorylation sites mutated to alanine. 556
The amount of NP plasmid was varied (12.5, 25, 50, 100ng/well). The ratio of Renilla 557
luciferase to firefly luciferase activity was normalized to wt for each sample and the 558
peak titer is reported. Mutating position S439 was found to significantly increase 559
minigenome activity. (n=3, ANOVA with Dunnett’s multiple comparison test, *p<0.01, 560
**p<0.001) (B) Representative activity curves for the minigenome assay. The 561
minigenome activity for wt and S439A NP are shown at each concentration tested. (C) 562
The expression of NP was assessed by western blot. All mutant proteins were shown 563
to be expressed at similar levels, as seen by blotting for NP using an NP specific mAb. 564
565
Figure 4. Phosphorylation of NP mutants in infected cells. (A) Detection of 566
phosphorylation of NP mutants. Vero cells were infected with MuV (wt) or mutant 567
viruses. Radioactive labeling was performed and lysates were immunoprecipitation with 568
an anti-NP mAb. Samples were resolved by SDS-PAGE. A representative gel is 569
shown. (B) Summary of NP phosphorylation in infected cells. MuV-NP-S439A was 570
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found the have significantly reduced phosphorylation in infected cells. (N=3, student t-571
test, *p<0.01) 572
573
Figure 5. Growth kinetics of MuV mutants. In each experiment, cells were infected 574
with MuV(wt), S439A, S520A, or S542A. Media was collected at various time points. 575
The titer of virus in the media was determined by plaque assay using Vero cells (A) 576
Vero cells infected at an MOI of 5. (B) Vero cells infected at an MOI of 0.01. (C) HeLa 577
cells infected at an MOI of 5. (D) HeLa cells infected at an MOI of 0.01. (For all growth 578
curves: n=3, ANOVA with Dunnett’s multiple comparison test, *p<0.05) 579
580
Figure 6. Protein production in infected Vero cells. In each experiment, cells were 581
infected with MuV wt, S439A, S520A, or S542A. (A) Total protein production in Vero 582
cells infected at an MOI of 5 after 24 hours. Samples were resolved by SDS-PAGE and 583
NP was quantified by western blot. (B) Summary of total protein production in Vero 584
cells infected at an MOI of 5. Average density was calculated over multiple experiments 585
and 439A was found to have increased protein production. (C) Protein production in 586
Vero cells infected at an MOI of 0.1 after 6 hours. Cells were collected and stained 587
using anti-NP (A488) and anti-P (APC) for flow cytometry. The mean fluorescence 588
intensity (MFI) was calculated for the stained population. (D) Protein production in Vero 589
cells infected at an MOI of 5 after 24 hours. Cells were treated as in (C). (One-way 590
ANOVA with Holm-Sidak multiple comparison test, n=3, *p<0.05) 591
592
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Figure 7. Genomic RNA and mRNA levels in infected Vero cells. Vero cells were 593
infected at an MOI of 0.1 with MuV(wt), S439A, S520A, or S542A. Total RNA was 594
extracted from biological replicates (n=3). Real-time PCR was performed on each 595
samples using a using a MuV-F specific FAM-tagged probe. (A) Levels of genomic 596
RNA. Genome replication was calculated after normalization to genomic RNA levels at 597
0 hpi. (B) Levels of viral mRNA. mRNA production was calculated after normalization 598
to genomic RNA levels at 0 hpi. (C) Quantification of relative levels of mRNA to 599
genomic RNA. The ratio of mRNA to genomic RNA was calculated at each timepoint. 600
(Multiple T-tests with Holm-Sidak multiple comparison test, n=3, *p<0.05) 601
602
Figure 8. Interaction between MuV NP and P. (A) Interaction between NP and P in 603
transfected cells. HEK293T cells were transfected with wt and mutant NPs and P. 604
Proteins were labeled with 35S-Met/Cys and co-immunoprecipitation was performed 605
using antibodies specific to NP. No difference was detected in the amount of NP or P 606
pulled down. (B) Interaction between NP and P in transfected cells. Using the same 607
samples as (A), co-immunoprecipitation was performed using antibodies specific to P. 608
No difference was detected in the amount of NP or P pulled down. (C) Interaction 609
between NP and P in infected cells. Vero cells were infected with MuV (wt), S439A, 610
S520A, S542A, or mock infected. Total protein was labeled with 35S-Met/Cys and co-611
immunoprecipitation was performed using antibodies specific to NP or P. (D) The mean 612
of the NP to P ratio for the anti-P immunoprecipitation is graphed with the SEM shown. 613
There was less NP co-immunoprecipitated with P, during infection with rMuV-NP-614
S439A. (n=3, student t-test, *p<0.05) 615
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616
Table 1. Phosphorylation of MuV NP by in silico prediction and mass 617
spectrometry. Phosphorylation site prediction was performed using the NetPhos 2.0 618
Server (http://www.cbs.dtu.dk/services/NetPhos/). Values >0.5 were considered to likely 619
be phosphorylated and are highlighted. Phosphorylation sites found by mass 620
spectrometry (as described in figure 1) are shown in the two right columns. The random 621
probability score for each site is listed, with a score of <0.05 considered likely to be 622
phosphorylated and are highlighted. 623
624
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Table 1 Summary of phosphorylation site prediction in MuV NP
Amino Acid NetPhos
(in silico prediction) LC-MS/MS Phosphorylation Anti-P IP Anti-NP IP
T12 0.689 N/A N/A S25 0.071 0.0024 29 T30 0.950 N/A N/A T42 0.891 N/A N/A S67 0.990 N/A N/A S94 0.996 0.28 0.29
T183 0.091 N/A 8.4 S191 0.749 N/A N/A S226 0.510 N/A N/A S298 0.042 N/A 0.059 T368 0.816 N/A N/A T387 0.565 0.0004 0.0032 T395 0.500 N/A 150 S439 0.992 0.0074 0.00036 S520 0.979 N/A N/A
S542 0.028 0.00015 3.4
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