Newly Identified Minor Phosphorylation Site Threonine-279 ofMeasles Virus Nucleoprotein Is a Prerequisite for NucleocapsidFormation

Akihiro Sugai,a Hiroki Sato,b Kyoji Hagiwara,b* Hiroko Kozuka-Hata,c Masaaki Oyama,c Misako Yoneda,b Chieko Kaia,b

International Research Center for Infectious Diseases,a Laboratory Animal Research Center,b and Medical Proteomics Laboratory,c Institute of Medical Science, Universityof Tokyo, Minato-ku, Tokyo, Japan

Measles virus nucleoprotein is the most abundant viral protein and tightly encapsidates viral genomic RNA to support viral transcrip-tion and replication. Major phosphorylation sites of nucleoprotein include the serine residues at locations 479 and 510. Minor phos-phorylation residues have yet to be identified, and their functions are poorly understood. In our present study, we identified nine puta-tive phosphorylation sites by mass spectrometry and demonstrated that threonine residue 279 (T279) is functionally significant.Minigenome expression assays revealed that a mutation at the T279 site caused a loss of activity. Limited proteolysis and electron mi-croscopy suggested that a T279A mutant lacked the ability to encapsidate viral RNA but was not denatured. Furthermore, dephosphor-ylation of the T279 site by alkaline phosphatase treatment caused deficiencies in nucleocapsid formation. Taken together, these resultsindicate that phosphorylation at T279 is a prerequisite for successful nucleocapsid formation.

Measles virus (MV), a member of the Morbillivirus genus inthe Paramyxoviridae family, is an important human patho-

gen that causes disease characterized by fever, cough, coryza,conjunctivitis, and a maculopapular rash. Although the use ofeffective vaccines has decreased global mortality from measles,it remains a major cause of high mortality among children indeveloping countries (1–3). MV has a nonsegmented negative-stranded RNA genome containing six structural genes encodingnucleoprotein (N), phosphoprotein (P), matrix (M) protein, fu-sion (F) protein, hemagglutinin (H) protein, and large (L) protein(4), and the P gene produces two accessory proteins, known as Vand C (5, 6). N proteins encapsidate viral genomic RNA to sup-port viral transcription and replication by an RNA-dependentRNA polymerase (RdRp) L protein. The P protein is a multifunc-tional protein (7, 8) that assists with viral transcription and repli-cation as a cofactor of the L protein (9, 10). The C and V accessoryproteins suppress host immune responses (11, 12). The M proteinhelps virus assembly, and the F and H proteins are required formembrane fusion and binding to the host cellular receptor, re-spectively (13).

N protein is the most abundant viral protein in infected cells(14) and is mainly required for viral transcription and replication.N proteins tightly associate with the viral genome and antigenometo form an N-RNA complex with a herringbone-like structure (15,16). N proteins associate with every 6 bases of the 15,894-nucleo-tide viral genome and fully cover the genome RNA (17). This tightencapsidation allows the viral genome to be resistant to RNasesand small interfering RNAs (18, 19). Viral transcription and rep-lication occur on the N-RNA complex in association with viralRdRp (vRdRp), composed of L and P proteins. This complex ofN-RNA, P protein, and L protein is called the nucleocapsid (NC)and comprises 2,649 copies of the N protein, about 300 copies ofthe P protein, and about 20 to 30 copies of the L protein (20–22).Singly expressed N proteins associate with cellular RNA to formNC-like structures through a nonspecific association (23–25).vRdRp first transcribes the RNA genome, and viral structuralgenes are expressed. When a sufficient quantity of N proteins has

accumulated, the function of vRdRp shifts from transcription toreplication and the RNA genome is replicated exponentially (21).During the replication step, nascent growing viral RNA is imme-diately encapsidated by N protein and full-length viral antigeno-mic RNA (positive-sense strand) is produced and serves as a tem-plate for genome (negative-sense strand) replication (26).

We previously reported that the major phosphorylation sites ofthe MV N protein were S479 and S510 (27). Additionally, thefunctional significance of the major phosphorylation sites of Nprotein to the viral life cycle includes viral gene expression, viralgenome RNA stability, and regulation of P-protein phosphoryla-tion (28, 29). However, despite double mutation of the majorphosphorylation sites of MV N protein, it was still phosphory-lated, albeit to a lesser extent. The N-protein minor phosphoryla-tion sites have not been identified and are poorly understood. Inthe present study, we predicted nine minor phosphorylation siteswithin the N protein by mass spectrometry (MS) analysis andinvestigated these putative phosphorylation sites. Furthermore,we identified a functionally indispensable minor phosphorylationsite at threonine-279 (T279) and examined the role of threoninephosphorylation in viral reproduction.

MATERIALS AND METHODSCells, plasmids, and antibodies. Cos7, 293, 293T, and Vero cells werepropagated in Dulbecco’s modified Eagle’s medium (DMEM; Sigma)supplemented with 5% fetal bovine serum (JRH Bioscience), 2 mM L-glu-tamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37°C in 5%

Received 6 June 2013 Accepted 2 November 2013

Published ahead of print 6 November 2013

Address correspondence to Chieko Kai, ckai@ims.u-tokyo.ac.jp.

* Present address: Kyoji Hagiwara, Viral Infectious Diseases Research Unit, RIKEN,Wako, Saitama, Japan.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.01718-13

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CO2. COBL cells were maintained in RPMI 1640 medium (Sigma) withthe same supplements described above. pCAGGS mammalian expressionvectors (30) encoding wild-type (wt) N, N mutants, and P-protein genesfrom the HL strain of MV (31) were prepared as described previously (27).The generation of a polyclonal anti-MV N antibody has also been de-scribed previously (27).

MALDI-TOF/TOF MS analysis. MV-infected COBL cells were lysed,and NC-associated N proteins were purified by cesium chloride (CsCl)gradient centrifugation as described below. The N protein was separatedby SDS-PAGE and stained with Coomassie brilliant blue. The band cor-responding to the N protein was excised from the gel and destained fivetimes with 50 mM NH4HCO3 in 50% methanol. The N protein was thensubjected to in-gel digestion with trypsin or V8 protease at 37°C for 16 h aspreviously described (27, 32). The digested peptides of N protein wereseparated by nanoflow liquid chromatography (LC; KYA TechnologiesCorporation) and analyzed by matrix-assisted laser desorption ioniza-tion–time of flight (MALDI-TOF)/TOF MS (4700 proteomics analyzer)as described previously (27). Detected peptides were identified by a data-base search using MASCOT, version 2.0 (Matrix Science).

Immunoprecipitation assay. Cos7, 279T, and Vero cells were trans-fected with plasmids containing wt N and N mutants. At 24 h posttrans-fection, 0.38 mCi/ml of 32P (phosphorus-32 radionuclide; PerkinElmer)or 0.06 mCi/ml of 35S (EasyTag Express protein labeling mix; Perkin-Elmer) was added to the medium, and the cells were incubated at 37°Cfor 24 h. Cells were harvested and lysed in lysis buffer consisting of 0.5mM EDTA and 0.5% Triton X-100 in phosphate-buffered saline (PBS)supplemented with protease inhibitor cocktail (BD Biosciences) andPhosSTOP phosphatase inhibitor cocktail (Roche) for 1 h at 4°C, andcell debris was removed from the lysate by centrifugation. The lysatewas incubated with protein A–Sepharose CL-4B beads (AmershamBiosciences) and anti-MV N polyclonal antibody for 16 h at 4°C. Thebeads were collected, washed 5 times with PBS, and suspended in 2�Laemmli’s sample buffer. Component proteins were separated bySDS-PAGE, and radioactive proteins were detected using an imagingplate and phosphorimager (FLA-5100; Fujifilm).

Minigenome expression assay. Precise methods for the minigenomeexperiment were described previously (27, 28). Briefly, 293 cells in 24-wellplates were transfected with N, P, and L protein expression plasmids usingLipofectamine LTX reagent (Invitrogen) and Plus reagent (Invitrogen).After 24 h, viral minigenomic RNA encoding the firefly luciferase gene wastransfected with Lipofectamine 2000 reagent (Invitrogen). On the follow-ing day, cells were lysed in passive lysis buffer (Promega), and luciferaseactivity was measured using a PicaGene luminescence kit (Tokyo InkManufacturing) according to the manufacturer’s instructions. Fluores-cence intensity was detected by use of a MiniLumat LB 9506 luminometer(Berthold).

N-P protein binding assay. Cos7 cells were transfected with either wtN or mutant N protein (T279A) expression plasmids and a P proteinexpression plasmid using Lipofectamine LTX reagent and Plus reagent.On the following day, cells were labeled with 35S (0.06 mCi/ml) at 37°C for24 h. The cells were lysed, and the lysate was incubated with proteinA–Sepharose CL-4B and anti-N-protein antibody. The precipitated pro-teins were separated by SDS-PAGE and detected with a phosphorimager(FLA-5100).

Indirect immunofluorescence assay. Cos7 cells were transfected withexpression vectors for wt N, N-protein mutants (T279A, T279D, T279E,S479A/S510A, ST8A, and ST11A), and P protein using the FuGENE 6transfection reagent according to the manufacturer’s instructions. At 24 hposttransfection, cells were fixed and permeabilized with 3% paraformal-dehyde-PBS and 0.5% Triton X-100 –PBS, respectively. The cells wereincubated with polyclonal anti-N-protein antibody and monoclonal anti-P-protein antibody, followed by incubation with a 1:2,000 dilution of anAlexa Fluor 488 F(ab=)2 fragment of goat anti-rabbit IgG (H�L) (Invit-rogen) and an Alexa Fluor 568 F(ab=)2 fragment of goat anti-mouse IgG(H�L) (Invitrogen) supplemented with Hoechst 33342 (Cambrex Bio

Science). Fluorescence was visualized by use of a confocal laser scanningmicroscope (Fluoview FV1000-D system; Olympus).

Nucleocapsid purification. Cos7 cells were transfected with the ex-pression plasmid for N protein or its mutants using the FuGENE 6 trans-fection reagent (Roche Applied Science) according to the manufacturer’sinstructions. At 48 h posttransfection, cells were lysed in TNE buffer (10mM Tris [pH 7.8], 150 mM NaCl, 1 mM EDTA) supplemented with 1%NP-40 and protease inhibitor cocktail at 4°C for 30 min. The lysate waslayered onto 1.5 ml each of 25%, 30%, and 40% (wt/vol) CsCl gradients inTNE buffer and centrifuged in a Beckman Sw55Ti rotor for 2 h at 55,000rpm. The fraction containing the nucleocapsid was diluted with TNEbuffer and centrifuged at 55,000 rpm to precipitate the nucleocapsid,which was resolved in an appropriate volume of PBS.

Western blot analysis. Expression plasmids for wt N and T279A weretransfected in Cos7 cells, and each cell lysate was subjected to Western blotanalysis to detect the expression level of the N protein relative to that of aninternal control, GAPDH (glyceraldehyde-3-phosphate dehydrogenase).The cell lysates were then separated by CsCl gradient centrifugation. TheNC fraction was collected and further centrifuged to precipitate the NC-like particles. The yields of the NC-like particles were also visualized andquantified by Western blotting. Briefly, the lysate of N-protein-trans-fected cells and purified nucleocapsids were subjected to 10% SDS-PAGE,and the separated proteins were transferred onto an Immobilon-P trans-fer membrane (Millipore). The membrane was blocked with BlockACEreagent (DS Pharma Biomedical) and then incubated with anti-N poly-clonal antibody for 1 h at 37°C. The membrane was washed five times with0.05% Tween-PBS and incubated with a 1:2,000 dilution of polyclonalgoat anti-rabbit horseradish peroxidase-conjugated immunoglobulins(Dako Cytomation) for 1 h at 37°C. Immunoreactive bands were detectedusing enhanced chemiluminescence Western blotting detection reagents(GE Healthcare). Scanning of the chemiluminescence was performedwith a luminescent image analyzer (LAS-1000UV minisystem; Fujifilm).

Electron microscopy. Cos7 cells were transfected with wt N or NT279A expression plasmids using the Plus reagent and LipofectamineLTX reagent, and self-assembled NC-like particles were purified at 2 daysposttransfection. Preparation of the samples for electron microscopy wasperformed as previously described (27). Briefly, purified NC-like particleswere spotted on a copper grid coated with collodion and incubated for 5min at room temperature. The samples were then negative stained with2% uranyl acetate and observed under an electron microscope (H7000;Hitachi).

Limited proteolysis. The purified NC fraction of wt N, N T279A, andheat-denatured (for 10 min at 95°C) wt N were equally divided into eighttubes, and each aliquot was subjected to 0, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, or10.0 �g/ml of trypsin treatment for 1 h at 37°C. The reactions werestopped by adding SDS sample buffer and heating for 5 min at 95°C, andthen the samples were subjected to SDS-PAGE. The proteolytic bandswere detected by immunoblotting using anti-N polyclonal antibodies.

Bacterial alkaline phosphatase (BAP) treatment and NC fraction-ation. Cos7 cells in 6-cm dishes were transfected with expression plasmidsfor wt N, N T279A, and ST10A using the FuGENE 6 transfection reagent.At 24 h posttransfection, the nucleocapsids were purified by CsCl gradientcentrifugation and dissolved in 200 mM Tris-HCl buffer (pH 8.0) supple-mented with Complete EDTA-free protease inhibitor (Roche Diagnos-tics). Each sample was incubated with or without 1.5 units of BAP andPhosSTOP phosphatase inhibitor cocktail for 2 h at 65°C, and then 500mM NaCl was added and the mixture was incubated for 1 h at 37°C.NC-like particles were separated by CsCl gradient centrifugation (0.6 mlof 30% [vol/vol] glycerol, 1.2 ml of 20% [wt/vol] CsCl, 1.4 ml of 30%[wt/vol] CsCl, and 1.4 ml of 40% [wt/vol] CsCl in TNE buffer) in a Beck-man Sw55Ti rotor for 16 h at 36,000 rpm, and the gradients were dividedinto seven fractions. The N proteins contained in each fraction were im-munoprecipitated with protein A–Sepharose CL-4B and anti-N poly-clonal antibodies and were subjected to SDS-PAGE and Western blotanalysis to quantify the N-protein levels.

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RESULTSIdentification of a minor phosphorylation site of MV N protein.Previously, we performed MALDI-TOF/TOF MS and electros-pray ionization-quadrupole-TOF MS analysis of MV N proteinand identified two major phosphorylation sites from the trypticpeptides of N protein purified from cells transfected with an Nprotein-expressing plasmid (27). However, we did not identifyminor phosphorylation sites of the N protein from these trypticpeptides. Therefore, in this study, we reexamined minor phos-phorylation of the N protein by MALDI-TOF/TOF MS analysisusing trypsin-digested N protein derived from MV-infected cells.Additionally, we investigated minor phosphorylation using V8protease-digested N protein. In the latter case, phosphorylatedpeptides were selectively detected and analyzed by MALDI-TOF/TOF MS. As a result, the peptides digested with trypsin or V8protease covered 70.5% of the N-protein sequence (Fig. 1A andB), and nine putative phosphorylation sites were predicted: T279,S294, S295, S407, S418, T454, S456, S457, and S460. We furtherinvestigated these putative phosphorylation sites to identify sitesresponsible for the minor phosphorylation of N protein. We pre-pared alanine substitution mutants for each putative phosphory-lation site of N protein and examined the phosphorylation levelsof each putative phosphorylation site by 32P labeling and immu-noprecipitation. The mutants used in this study are shown in

Fig. 2A. As a result, among these nine putative phosphorylationsites, the T279 site was found to be remarkably phosphorylated,with a phosphorylation rate of 14.3% for wt N protein in Cos7cells (Fig. 2B). We also investigated the phosphorylation rate ofT279 in Vero and 293T cells but did not observe a significantchange in the rate in these cells compared with that in Cos7 cells(Fig. 2C). This indicated that the minor phosphorylation at T279was not a cell-type-specific phenomenon. Phosphorylation at theeight remaining putative phosphorylation sites occurred at tracelevels, and therefore, these sites may not be phosphate group ac-ceptors. Additionally, phosphorylation signals were barely de-tected in the ST11A mutant, where two major and nine minorputative phosphorylation sites had alanine substitutions.

The Paramyxovirus N protein possesses a highly conserved do-main at amino acids (aa) 258 to 357 which is referred to as thecentral conserved region (CCR) (Fig. 2D) and is known to beimportant for N-protein function (33, 34). In a previous analysis(27) and the present MS analysis (Fig. 1), aa 250 to 278 and 298 to324 were not covered within the N-protein CCR. This region in-cludes six serine and threonine residues: T259, S268, T272, S298,T309, and S319. We generated additional alanine mutants, theST14A and ST17A mutants (Fig. 2E), and examined whether thelow phosphorylation levels detected in the ST11A mutant wasthe result of phosphorylation of these sites. The level of phosphor-ylation of the ST14A and ST17A mutants was similar to that of theST11A mutant (Fig. 2F), suggesting that these six sites within CCRare not phosphate group acceptors. The trace levels of phosphor-ylation detected in the ST11A, ST14A, and ST17A mutants ap-peared to be because of nonspecific phosphorylation that oc-curred at very low rates.

T279 is an indispensable phosphorylation site for N-proteinfunction. To identify the phosphorylation sites required for N-protein function from the nine putative sites, we prepared ala-nine-substituted mutants of N protein and measured the N-pro-tein activity of each one by a minigenome reporter assay. Inagreement with our previous reports, a mutant with mutations ofboth major phosphorylation sites, S479A and S510A, showed46.3% activity compared with that for the wt N protein (Fig. 3A),whereas an alanine substitution mutant with a mutation of theT279 site showed a complete loss of activity and the N protein didnot aid transcription and replication, indicating that the T279 siteis functionally important. A mutant N protein with alanine sub-stitution mutations at the eight putative phosphorylation sitesother than T279 (the ST8A mutant) showed no reduction in ac-tivity compared with that of wt N protein. Additionally, the activ-ity of the ST10A mutant (where the amino acids at two majorphosphorylation sites and eight putative phosphorylation sitesother than T279 were replaced by alanine) was reduced to 36.6%of that of wt N protein. There was no statistically significant dif-ference between the activities of the S479A/S510A and ST10A mu-tants. Thus, the eight putative phosphorylation sites other thanT279 were not required for N-protein function, but the T279 sitewas found to be functionally indispensable. Therefore, we inves-tigated the T279 site further using N-protein mutants with aspar-tic acid (D) and glutamic acid (E) substitutions at the T279site. The T279D and T279E mutants showed no activity, similar tothe result for the T279A mutant, in the minigenome assay (Fig.3B). Since the replacement of T279 with acidic residues did notmimic the phosphorylation of T279, constitutively charged T279may be undesirable for N-protein function.

FIG 1 Identification of the minor phosphorylation sites in the N protein byMS analysis. (A) Trypsin-digested peptides of N protein were separated bynanoflow LC and analyzed by MALDI-TOF/TOF MS. (B) Phosphorylatedpeptides from V8 protease-digested N proteins were selectively analyzed andidentified by MALDI-TOF/TOF MS. The analyzed peptides were identifiedwith a MASCOT (version 2.0) database search and are shown in red.

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FIG 2 Analysis of minor phosphorylation sites of N protein. (A) Schematic diagram of the alanine substitution mutants of the N protein used in this study. Eachphosphorylation site is indicated as a green square, and sites where the amino acid was with an alanine residue are shown as red squares with a letter A. (B)Identification of the minor phosphorylation site in the N protein by immunoprecipitation assay. The N protein or its phosphorylation mutants were radiolabeledwith 32P or 35S in Cos7 cells. The relative phosphorylation (32P-N/35S-N) of each N protein mutant was quantified. (C) Phosphorylation of wt N and the T279Amutant in 293T and Vero cells. (D) Multiple-sequence alignment of the N protein CCR (aa 258 to 357) from Paramyxoviruses: canine distemper virus (CDV;GenBank accession no. AAG30916), rinderpest virus (RPV; CAA48388), peste des petits ruminants virus (PPRV; ACN62116), Hendra virus (AAC83187), Nipahvirus (NIV; AAK50548), Sendai virus (SEV; AAB06278), mumps virus (CBA10117), and human parainfluenza virus type 1 (HPIV1; NP_604433). Black and redarrowheads, S or T residues untested by MS analysis and putative phosphorylation sites detected by MS analysis, respectively. (E) Schematic diagram of additionalmutants with mutations at the CCR. (F) Phosphorylation properties of the mutants.

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The N protein possesses a nuclear localization signal and nu-clear export signal for nucleocytoplasmic transport, and the Nprotein expressed alone in cultured cells localizes to the nucleus(35, 36). To investigate the significance of the T279 site, we exam-ined the localization of the putative phosphorylation site mutants,including a series of T279 mutants, by indirect fluorescent-anti-body (IFA) assay. The S479A S510A, ST8A, and ST10A N-proteinmutants showed a nuclear localization identical to that of wt Nprotein, while T279 mutants showed a diffuse cytoplasmic distri-bution (Fig. 4A and B). Thus, T279 mutants showed abnormalproperties by IFA assay. The P protein has been known to retain Nprotein in the cytoplasm and colocalizes with the N protein (36).In the presence of the P protein, the T279A mutant exhibited adotted colocalization pattern with the P protein in the cytoplasm,similar to that seen for the wt N protein (Fig. 4C).

P protein is a major binding protein of the N protein and isrequired for viral transcription and replication (37, 38). The Pprotein functions as a carrier of the N protein to nascent replicat-ing viral N-RNA complexes for efficient encapsidation (10), sta-bilizes NC by regulating the phosphorylation status of N protein(29), and prevents nonspecific binding of the N protein to hostcellular RNA (10). To probe the mechanism of deactivation forthe T279A mutant in a minigenome assay, we investigated theabilities of wt N protein and the T279A mutant N protein to bindto P protein by coimmunoprecipitation assay. Transiently coex-pressed wt N or the T279A mutant N protein with P protein waslabeled with 35S and immunoprecipitated using anti-N polyclonalantibodies. There were no differences in coprecipitation of the P

FIG 3 Functional analysis of minor phosphorylation sites. (A) Minigenomeassay using the N protein mutants whose phosphorylation sites were substi-tuted with alanine residue. 293 cells were transfected with minigenomic RNAand plasmids expressing the N, P, and L proteins. On the following day, theluciferase activity of the cell lysates was measured. (B) Minigenome assay usingN-protein mutants with acidic residue substitutions at the T279 site.

FIG 4 Localization and N-P binding ability of the T279A mutant. (A) T279 mutants showed a diffuse cytoplasmic distribution. Cos7 cells were transfected witha plasmid expressing wt N or T279A mutant N, and the localization of each protein was detected with anti-N polyclonal antibody. The localization of otherN-protein mutants (B) and the distribution of wt N and T279A mutant N in the presence of the P protein (C) were evaluated. (D) wt N or T279A mutant N wastransfected into Cos7 cells, along with the P protein. Proteins were labeled with 35S and coprecipitated with anti-N polyclonal antibody (pAb). The bar graphshows the relative quantity of P protein coprecipitated with N protein (P protein/N protein). Error bars indicate standard deviations. IP, immunoprecipitation.

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protein (Fig. 4D); this finding and the dotted colocalization withthe P protein (Fig. 4C) suggest that the T279A mutant possesses anormal ability to bind to P protein, and therefore, the loss ofactivity of the T279A mutant in the minigenome assay was inde-pendent of N-P interactions.

T279 phosphorylation site of N protein is a prerequisite forNC formation. The region around T279 of the MV N protein iscalled CCR and has been reported to function as an N-N andN-RNA interaction domain to form NCs (34, 39, 40). Multiple-sequence alignment of CCRs among the Paramyxoviridae revealedthat T279 is highly conserved (Fig. 2D). Therefore, we examinedwhether phosphorylation of the T279 site is required for NC for-mation. NC formation ability was tested by measuring and com-paring the amount of NC-like particles in cells transfected with aplasmid expressing wt N protein or the T279A mutant N protein.The levels of expression of wt N protein and the T279A mutant Nprotein in Cos7 cells were similar, while the levels of purified NC-like particles from T279A mutant-transfected cell lysates were sig-nificantly reduced compared with those from wt N protein-trans-fected cell lysates (Fig. 5A). Thus, the alanine substitution at T279caused a remarkable impairment of NC formation, and the T279mutants showed a loss of activity in the minigenome assay.

Of note, the T279A mutants were detected in the NC fractionin the CsCl gradient centrifugation experiment (Fig. 5A), al-though the T279A mutant lost activity in the minigenome assay(Fig. 3A). To clarify that the N proteins acquired from the NCfraction formed correct NC structures, we subjected them to elec-tron microscopy. The wt N protein showed a typical herringbonestructure, while the T279A mutant from the NC fraction did notform a correct NC structure and aggregates of various sizes weredetected (Fig. 5B), suggesting that the T279A mutant did not re-tain the self-assembly and/or RNA encapsidation ability.

Furthermore, we generated a plasmid containing the full ge-nome with the T279A mutation in the N protein and attempted torescue the T279A recombinant virus using a reverse genetics sys-tem that we had previously established (29, 41). However, we wereunable to rescue the T279A mutant virus. This seems to be attrib-uted to a loss of N-protein functions; the T279A mutant nucleo-protein could not correctly encapsidate the RNA (Fig. 5B) and didnot support viral transcription and replication (Fig. 3A and B).

Phosphorylation of T279 is required for NC formation. Nprotein consists of a highly structured N core domain (aa 1 to 400)and an intrinsically disordered N tail domain (aa 401 to 525) (35,38). A correctly folded N core domain is resistant to proteases,including trypsin (41, 42). We evaluated the tertiary structure ofthe T279A mutant by limited proteolysis to determine whether theloss of self-assembly was due to the decay of the N core conforma-tion or the absence of phosphorylation. Plasmids for wt N andT279A mutant N were transfected into Cos7 cells, and each Nprotein was purified from the NC fraction obtained by CsCl gra-dient centrifugation. The purified N proteins were subjected tolimited proteolysis with various concentrations of trypsin. Indeed,the 43.5-kDa core domain of the wt N protein was resistant totrypsin hydrolysis (Fig. 6A). Heat-denatured wt N protein showeda different degradation pattern, and various proteolytic productsof less than 43.5 kDa were detected (Fig. 6B). The T279A mutantshowed trypsin resistance, and its degradation pattern was identi-cal to that of wt N protein (Fig. 6C). These results imply that thetertiary structure of the core domain of the T279A mutant re-tained its structural integrity. Thus, the alanine substitution of

T279 did not cause denaturation of the tertiary structure of the Ncore domain, but the absence of T279 phosphorylation seemed tobe critical for NC formation.

To investigate the significance of T279 phosphorylation on NCformation, we performed dephosphorylation of T279 with BAPand examined whether this caused abnormalities in NC forma-tion. Previous reports demonstrated that deficient NC-like parti-cles in Sendai virus could be discriminated by CsCl centrifugation(39, 41, 43). Using the same conditions, we examined whether thismethod was applicable to MV N protein. Lysates of wt N- and

FIG 5 The T279A mutant N protein fails to form NC-like particles. (A) Cos7cells were transfected with plasmids carrying wt N or T279A mutant N, and theN-protein expression levels in cell lysates and NC fractions obtained by CsCldensity gradient centrifugation were determined and are shown here. Bargraphs indicate the relative levels of N-protein expression. Error bars indicatestandard deviations. (B) Electron microscopy of wt N and T279A mutant N inthe NC fraction obtained by CsCl density gradient centrifugation.

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T279A mutant-transfected cells were subjected to CsCl gradientcentrifugation to discriminate between normal and deficient NC-like particles. The gradients were divided into seven fractions, andthe N protein in each fraction was detected by Western blotting.The NC-like particles of wt N were mainly detected in fractions 6and 7 (Fig. 7A), while aggregates of the T279A mutant N proteinwere broadly detected from fractions 1 to 7 (Fig. 7B), indicatingthat the deficient NC-like particles formed aggregates of varioussizes. Thus, normal NC-like particles could be discriminated fromdeficient aggregates. To demonstrate that T279 phosphorylation isimportant in NC formation, we then evaluated the influence of de-phosphorylation of the ST10A mutant by examining whether theST10A mutant could form normal NC-like particles in the presenceor absence of BAP. The amino acids at all phosphorylation sites otherthan T279 were replaced by alanine residues in the ST10A mutant(Fig. 2A). Since there are no significant phosphorylation sites otherthan the 11 sites (Fig. 2B, E, and F), the influence of BAP treatment onthe ST10A mutant was equivalent to that of T279 dephosphorylation.

NC-like particles from the ST10A mutant were mainly detected inNC fractions 6 and 7, similar to the findings for intact NC-like parti-cles, suggesting that the mutant ST10A formed normal NC-like par-ticles (Fig. 7C). The ST10A mutant treated with BAP showed a widedistribution from fractions 2 to 7 (Fig. 7D), indicating that dephos-phorylation of T279 caused a deficiency in NC formation. Further-more, addition of a phosphatase inhibitor abrogated the NC defi-ciency caused by BAP treatment (Fig. 7E), indicating that it was notcaused by binding of BAP to N protein but by dephosphorylation atT279. Taken together, these results suggest that phosphorylation atT279 is a prerequisite for NC formation.

DISCUSSION

We previously demonstrated that the major phosphorylation sitesof N protein (S479 and S510) are involved in various stages of theviral life cycle (28, 29). Additionally, we found that a mutant withmutation of both major phosphorylation sites remained phos-phorylated but to a lesser extent, suggesting that unidentified

FIG 6 The T279A mutant N protein showed the same proteolytic pattern as wt N protein. The wt N, T279A mutant N, and heat-denatured wt N proteins weretreated with various concentrations of trypsin. High-contrast images of the low-molecular-mass region (�37 kDa) are shown for low-density degradationproducts. Arrowheads, the various degradation products of the denatured N protein.

FIG 7 Dephosphorylation of T279 by BAP treatment impairs NC formation. NC-like particles of the wt N or N-protein mutants were separated by CsCl gradientcentrifugation. Gradients were divided into 7 fractions, and the N protein in each fraction was detected. (A) wt N; (B) T279A mutant N; (C) ST10A mutant N;(D) ST10A mutant N with BAP treatment; (E) ST10A mutant N with BAP and phosphatase inhibitor treatment. Bar graphs show the percentage of N-proteincontent in each fraction per the total amount of N protein in the 7 fractions.

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phosphorylation sites exist within the N protein. Here, we pre-dicted the existence of nine putative minor phosphorylation sitesof N protein by MS analysis. Moreover, we demonstrated thatamong the putative minor phosphorylation sites, a threonine res-idue at location 279 was notably phosphorylated and was a pre-requisite for NC formation.

Since the late 1970s, it has been known that N protein under-goes phosphorylation at both serine and threonine residues andthat the phosphorylation intensity of phosphoserine was strongerthan that of phosphothreonine (44, 45). Additionally, in the1990s, it was reported that the phosphorylation intensities ofphosphoserine and phosphothreonine were 88% and 12%, re-spectively, of the total N phosphorylation intensity (46). In agree-ment with these reports, we demonstrated that the phosphoryla-tion intensity of a mutant with mutations of both of the majorphosphorylation sites (S479A and S510A) was 15.7% compared tothat of wt N protein and the phosphorylation intensity of the T279site was 14.3% of the total N phosphorylation intensity. This in-dicated that the intensity of major phosphorylation at serine res-idues was 84.3% and that almost all of the remaining 15.7% phos-phorylation was due to phosphorylation of T279. Furthermore,specific phosphorylation was not detected in the ST11A mutant,suggesting the existence of no significant phosphorylation sitesother than the 11 sites already identified. In addition, alanine sub-stitution at putative minor phosphorylation sites other than T279did not have an influence in a minigenome expression assay.Taken together, these data indicate that the functionally signifi-cant phosphorylation sites of N protein are limited to the twomajor phosphorylation sites (S479 and S510) and the minor T279phosphorylation site.

It was reported that free N protein and NC-associated N pro-tein showed different antigenicities toward antibodies, and thisfinding was explained to be the result of a conformational differ-ence between the free N protein and NC-associated N protein(47–49). This suggests that conformational changes to N proteinare required for NC formation. Furthermore, free N protein isphosphorylated only on serine residues, whereas the NC-associ-ated N protein is phosphorylated on both serine and threonineresidues (46), implying that threonine phosphorylation and sub-sequent conformational changes of N protein are prerequisites toNC formation. In agreement with these studies, we demonstratedby electron microscopy that the T279A mutant did not form NC-like particles. Additionally, our data also suggested that an absenceof phosphorylation at the threonine residue resulted in a struc-tural abnormality in the NC-like particle. Taken together, thesedata indicate that NC formation requires a conformationalchange of N protein that is switched by phosphorylation at T279.However, wt N protein and T279A mutant N protein harvestedfrom NC fractions of CsCl density gradients did not show anydifference in proteolytic peptide patterns after trypsin digestion,indicating that the T279A mutant retained a normal tertiary struc-ture similar to that of the wt N protein. Thus, the conformationalchange required for NC formation was quite small and could notbe discriminated by the use of trypsin digestion patterns. Indeed,trypsin digestion of free N protein and NC-associated N proteinresulted in similar degradation patterns (46). Moreover, a mutantwith double mutations of the MV N CCR domain whose SL (res-idues 228 and 229) amino acids were replaced by QD (NQD) didnot show any differences from the wt in the results obtained bytrypsin digestion analysis, immunoprecipitation, and circular di-

chroism (CD) spectroscopy, while the mutant did not form nor-mal NC-like particles, as determined by electron microscopy (34).As the conformational change required for NC formation couldnot be detected by CD spectroscopy, the change was not accom-panied by a change in secondary structure. Thus, in the NC for-mation step, very small conformational changes are required forcorrect encapsidation.

Since the CsCl gradient centrifugation assay suggested thatthe ST10A mutant formed normal NC-like particles, the majorphosphorylation sites of N protein are dispensable for NC for-mation. This is consistent with a previous report demonstrat-ing that the N tail region is not required for NC formation (43).Here, we identified the T279 site as a new phosphate groupacceptor site that is indispensable for NC formation. However,the phosphorylation intensity of T279 was low, and this minorphosphorylation was only 14.3% of the total N phosphoryla-tion. Moreover, replacement of T279 with acidic residues, suchas aspartic acid and glutamic acid, also abrogated the pheno-type of wt N protein in a minigenome assay and an IFA assay.Thus, the phosphorylation at T279 is not constitutive, and Nprotein is functionally inactive under circumstances where allN proteins are constantly phosphorylated at T279. Moreover,although the threonine residue of free N protein was not phos-phorylated (46), 14.3% of the NC-associated N proteins werephosphorylated at T279. This indicates that one-seventh of theNC-associated N proteins are phosphorylated when N proteinsincorporate into the nascent N-RNA complex. Whether N pro-teins were expressed by a plasmid or during viral replication(46), the phosphorylation rate of the threonine residue in NCwas almost the same. Thus, T279 phosphorylation of the Nprotein is required for the maintenance of the herringbone-likestructure of NC, and T279 is different from the major phos-phorylation sites that take part in the functional regulation ofviral gene expression (27–29). Electron microscopy demon-strated that NC has a helical structure with approximately 13 to14 N proteins per turn (15, 50, 51). Therefore, N proteins maychange their own conformation through the phosphorylationof T279 at a ratio of 1 per 7 to cancel the structural distortion ofthe NC.

In the present study, we demonstrated that phosphorylation atT279 is a prerequisite for NC formation. However, the relation-ship between T279 phosphorylation and conformational changesto NC-associated N proteins remains to be clarified. Further anal-ysis of the conformational change in N protein might reveal theprecise mechanism of NC formation.

ACKNOWLEDGMENTS

This study was supported by grants-in-aid from the Ministry of Educa-tion, Science, Sports, and Culture, Japan.

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