Determination of a phosphorylation site in Nipah
virus nucleoprotein and its involvement in virus
Mingshu Huang,1Hiroki Sato,1Kyoji Hagiwara,1Akira Watanabe,2
Akihiro Sugai,1Fusako Ikeda,1Hiroko Kozuka-Hata,3Masaaki Oyama,3
Misako Yoneda1and Chieko Kai1,2
Received 18 March 2011
Accepted 23 May 2011
1Laboratory Animal Research Center, Institute of Medical Science, University of Tokyo, 4-6-1
Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
2International Research Center for Infectious Diseases, Institute of Medical Science, University of
Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
3Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, 4-6-1
Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Many viruses use their host’s cellular machinery to regulate the functions of viral proteins. The
phosphorylation of viral proteins is known to play a role in genome transcription and replication in
paramyxoviruses. The paramyxovirus nucleoprotein (N), the most abundant protein in infected
cells, is a component of the N–RNA complex and supports the transcription and replication of
virus mRNA and genomic RNA. Recently, we reported that the phosphorylation of measles virus N
is involved in the regulation of viral RNA synthesis. In this study, we report a rapid turnover of
phosphorylation in the Nipah virus N (NiV-N). The phosphorylated NiV-N was hardly detectable in
steady-state cells, but was detected after inhibition of cellular protein phosphatases. We
identified a phosphorylated serine residue at Ser451 of NiV-N by peptide mass fingerprinting by
electrospray ionization–quadrupole time-of-flight mass spectrometry. In the NiV minigenome
assay, using luciferase as a reporter gene, the substitution of Ser451 for alanine in NiV-N resulted
in a reduction in luciferase activity of approximately 45% compared with the wild-type protein.
Furthermore, the substitution of Ser451 for glutamic acid, which mimics a phosphoserine, led to a
more significant decrease in luciferase activity – approximately 81%. Northern blot analysis
showed that both virus transcription and replication were reduced by these mutations. These
results suggest that a rapid turnover of the phosphorylation of NiV-N plays an important role in
virus transcription and replication.
Nipah virus (NiV) is a recently emerged zoonotic virus that
causes encephalitic and respiratory illness in humans and
livestock, with a high mortality rate (40–70%) in humans
(Chua et al., 2000; WHO, 2004). Since its identification in
1998–1999 in Malaysia, NiV has been associated with several
outbreaks of human fetal viral encephalitis in South Asia
to-person transmissions (Hsu et al., 2004; WHO, 2004).
NiV is a negative-stranded, ssRNA virus that belongs to the
genus Henipavirus in the family Paramyxoviridae (Mayo,
2002a, b) and is composed of six structural proteins:
nucleoprotein (N), phosphoprotein (P), matrix protein
(M), fusion protein (F), glycoprotein (G) and large protein
(L). The N protein encapsidates the genomic RNA and
forms a nucleocapsid. This serves as a template for virus
replication and transcription, which are catalysed by an
RNA-dependent RNA polymerase (RdRp) that is com-
posed of P and L proteins (Wang et al., 2001).
Paramyxovirus N proteins possess a highly conserved
structure. The N-terminal 80% of the protein forms a
globular body, whereas the C-terminal 20% appears to be a
tail extending from the N-terminal body (Lamb & Parks,
2006). The genus Henipavirus is related closely to the genus
Morbillivirus within the family Paramyxoviridae. Measles
virus (MV), which belongs to the genus Morbillivirus, has a
well-characterized N protein. MV-N is also divided into two
regions: (i) an N-terminal domain, N-CORE, which has a
well-conserved sequence(aa 1–400), and (ii)a hypervariable
C-terminal domain, N-TAIL (aa 401–525) (Heggeness et al.,
1980, 1981; Longhi et al., 2003). MV-N-CORE contains all
of the necessary components for self-assembly and RNA
binding, as N proteins that are composed only of the core
Journal of General Virology (2011), 92, 2133–2141
032342G2011 SGM Printed in Great Britain2133
region can encapsidate newly synthesized RNA into
nucleocapsid-like particles. MV-N-TAIL contains the region
responsible for N–P binding and is consequently required
for virus transcription (Longhi et al., 2003).
NiV-N is composed of 532 aa, slightly larger than MV-N
(Wang et al., 2001). A recent study revealed that deletion of
¡128 aa from the C terminus of NiV-N does not impair
the formation of the NiV-N herringbone-like nucleocapsid
structure (Ong et al., 2009). However, deletion of 129 aa or
more from the C terminus completely abolishes the
formation of the herringbone-like particles and the protein
aggregates into spherical particles (Ong et al., 2009).
Furthermore, one of the binding sites of the P protein was
mapped to the 29 aa (aa 468–496) C-terminal region of
NiV-N (Chan et al., 2004). It is assumed that NiV-N has a
similar function to MV-N.
It has been reported that MV-N has five phosphorylated
serine and threonine residues (Gombart et al., 1995; Segev
et al., 1995). Recently, we identified the two major phos-
phorylation sites of MV-N by matrix-assisted laser desorp-
tion/ionization–tandem time-of-flight (MALDI-TOF/TOF)
and electrospray ionization–quadrupole time-of-flight (ESI-
Q-TOF) mass spectrometry analyses (Hagiwara et al., 2008).
Both phosphorylation sites were in the C-terminal region,
where phosphorylation was required for efficient virus
transcription in the MV minigenome assay system.
In the present study, we determined that NiV-N is also
phosphorylated. However, there is a rapid turnover of
phosphorylation, which is detectable only after inhibition of
cellular protein phosphatases. In addition, we identified the
phosphorylation site as Ser451 and examined the role of
phosphorylation of NiV-N in the transcription and
replication of virus RNA.
NiV-N is dephosphorylated immediately by
cellular protein phosphatases
To examine whether NiV-N is phosphorylated in host cells,
NiV-N was expressed transiently in COS-7 cells in the
presence of32P-labelled phosphate. After cell lysis, NiV-N
was immunoprecipitated with antiserum against NiV-N,
and was subjected to SDS-PAGE and autoradiography.
However,32P-labelled NiV-N was not detected (Fig. 1a).
Recently, it has been reported that phosphorylation of the P
protein at Ser54 of the human respiratory syncytial virus
(HRSV), another member of the family Paramyxoviridae, is
detected only by inhibition of cellular protein phosphatases,
due to its immediate dephosphorylation (Asenjo et al.,
2005). Therefore, to confirm whether NiV-N is also
were performed in the presence of okadaic acid (OKA), an
inhibitor of cellular protein phosphatases. In the presence
of OKA, strong
(Fig. 1a). To investigate the kinetics of phosphorylation
32P labelling of NiV-N was observed
and dephosphorylation of NiV-N, time-dependent experi-
ments were performed. The amount of32P-labelled NiV-N
increased graduallyup to 6 h afterOKA administration(Fig.
1b), and decreased within 2 h after OKA removal (Fig. 1c).
These results indicate that NiV-N has a rapid turnover of
phosphorylated residues and that host cellular activity is
involved in regulating the phosphorylation of NiV-N.
NiV-N is phosphorylated at Ser451
To identify the phosphorylation sites for NiV-N, NiV-N was
expressed transiently in COS-7 cells and the nucleocapsid
complex was purified by caesium chloride density-gradient
centrifugation (Hagiwara et al., 2008; Oglesbee et al., 1989).
The obtained fraction was analysed by Coomassie brilliant
blue (CBB) staining (Fig. 2, lane 1) and Western blotting
using an antiserum against NiV-N (Fig. 2, lane 2), which
showed that NiV-N was purified homogeneously. The
purified NiV-N was trypsinized by in-gel digestion, and
the resultant peptides were analysed by ESI-Q-TOF MS,
followed by a database search with Mascot ver. 2.0 (Matrix
Science). The total sequence coverage was 82% (Fig. 3a). In
the MS/MS spectrum of the precursor ion that corresponds
to the peptide 449-EMSISSLANSVPSSSVSTSGGTR-471,
OKA additionOKA removal
Mock (+) (_)
Mock (+) (_)
Mock 0126 h0126 hMock
Fig. 1. Phosphorylation of NiV-N after OKA treatment. (a) NiV-N
protein was expressed in COS-7 cells and labelled with32P or35S
in the presence (+) or absence (”) of OKA. (b) Time-dependent
experiments of phosphorylation of NiV-N. In the presence of32P or
35S, 100 nM OKA was added to the culture medium. Cells were
harvested at several time points. (c) After exposure to OKA for
24 h in the presence of32P or35S, OKA was removed from the
culture medium and cells were harvested at several time points.
M. Huang and others
2134Journal of General Virology 92
five fragment ions (b3, b4, b7, b8 and b11) were
accompanied by their dephosphorylated ions (b3298,
b4298, b7298, b8298 and b11298, respectively), whereas
seven fragment ions (y13, y14, y15, y16, y17, y18 and y19)
were not. This spectrum indicated that Ser451 is a
phosphorylation site on NiV-N (Fig. 3b).
To confirm the predicted phosphorylation site, Ser451 in
NiV-N was substituted with alanine (A), which is unable to
be phosphorylated, and the resultant NiV-N mutant, NiV-
N S451A, was expressed in COS-7 cells in the presence of
32P and OKA. The S451A mutation greatly reduced the
phosphorylation of NiV-N (Fig. 4), which indicated that
Ser451 is the major phosphorylation site on NiV-N. Ser451
is located in the C-terminal region, as in MV-N.
NiV-N Ser451 is involved in efficient transcription
and replication of the NiV minigenome
NiV-N is a major component of the virus ribonucleopro-
tein (RNP) complex and is required for virus mRNA
transcription and genome replication. We reported pre-
viously that the phosphorylation of MV-N upregulates
the transcriptional activity of MV minigenomic RNA
(Hagiwara et al., 2008). Therefore, to examine the role of
rapid-turnover phosphorylation of NiV-N Ser451 in virus
transcription and/or replication, we developed an NiV
minigenome assay using firefly luciferase as a reporter gene,
flanked by the leader and trailer sequences of the NiV
genome. In addition to the NiV-N S451A mutant, we
constructed another mutant NiV-N, S451E, in which S451
was substituted with glutamic acid (E) as a phosphoserine
mimic (Wu et al., 2002).
HEK 293 cells were transfected with the negative-strand
minigenomic RNA and supporting plasmids that expressed
NiV-N [wild-type (wt), S451A or S451E], -P and -L. A
luciferase assay was performed at 24 h post-transfection.
Western blotting of cell lysates from each minigenome
assay showed that there was no significant difference in the
expression of NiV-N between wt, S451A and S451E (Fig.
5b). However, the luciferase activity in NiV-N S451A was
45% lower than that of wt NiV-N (Fig. 5a), which suggests
that phosphorylation at Ser451 is required for efficient
transcription and/or replication of virus RNA. The
luciferase activity in NiV-N S451E, which was a mimic of
phosphoserine, was 81% lower than that in wt NiV-N (Fig.
5a). Therefore, it can be assumed that retaining a state of
phosphorylation is undesirable for NiV, because it
suppresses replication and/or transcription.
To investigate the effect of NiV-N phosphorylation on
replication and transcription using the NiV minigenome
assay, the expression of luciferase mRNA and positive-
strand anti-minigenomic RNA was quantified by Northern
blotting. Minigenome assays were performed under the
same conditions as described above and total RNA was
extracted. Luciferase mRNA and positive-strand anti-
minigenomic RNA were detected with an antisense ribop-
robe located within the luciferase gene. Luciferase mRNA
appeared as the upper band, apparently .1000 nt longer
than the antigenomic RNA (1866 nt) (Fig. 6a). We
determined the length of the poly(A) tail in the luciferase
mRNA by 39 RACE; approximately 1200 nt poly(A) tail
was detected (Fig. 6b). Thus we performed quantitative
analysis of the luciferase mRNA and antigenomic RNA by
Northern blotting (Fig. 6c). For the S451A mutation, the
levels of anti-minigenomic RNA and mRNA were reduced
slightly compared with those for wt NiV-N. For the S451E
mutation, the level of mRNA was 26% lower than that for
wt, whereas the level of anti-minigenomic RNA decreased
by 68%. We further performed real-time PCR analysis to
detect mRNA and anti-minigenomic RNA levels; it showed
a similar pattern to Northern blotting (Fig. 6d), as reported
previously (Sleeman et al., 2008). These results suggest that
replication was affected more than transcription.
Mutation of Ser451 of NiV-N does not affect
nucleocapsid or N–P complex formation
It has been reported that the MV-N-TAIL region binds to
the P protein, a component of the RdRp composed of P
and L proteins, and thus supports virus transcription and
replication (Bankamp et al., 1996; Johansson et al., 2003;
Liston et al., 1997; Longhi et al., 2003). In our own studies
of MV-N phosphorylation, the loss of phosphorylation of
MV-N did not affect its binding to the P protein (Hagiwara
et al., 2008). To determine whether the reduction in
luciferase activity observed with the mutant proteins was
caused by the abrogation of nucleocapsid formation
and/or the loss of binding to RdRp, we first investigated
the efficiency of nucleocapsid formation of the mutants.
Fig. 2. SDS-PAGE and Western blotting of purified NiV-N.
Recombinant NiV-N was expressed in COS-7 cells and purified by
caesium chloride density-gradient centrifugation. Samples were
separated by SDS-PAGE (10% gel) and detected by CBB
staining (lane 1). Purified NiV-N was detected by Western blotting
with NiV-N-specific antibody (lane 2). An empty vector plasmid
was also transfected into the cells and the lysate was utilized as a
negative control (lane 3).
Phosphorylation of Nipah virus N protein
NiV-N-wt, S451A and S451E were expressed in cells and
then subjected to caesium chloride centrifugation. As
shown in Fig. 7, S451A and S451E formed nucleocapsid at
a rate equal to wt (lanes 4–6). Next, NiV-N-wt, S451A and
S451E were mixed with NiV-P and -L, and purified by
caesium chloride centrifugation. If they bound to NiV-N,
they were purified together as a nucleocapsid complex.
There was no significant difference observed in the amount
of NiV-N and co-purified NiV-P and -L among wt, S451A
and S451E (Fig. 7, lanes 1–3). Solely expressed NiV-P (Fig.
7, lane 7) and -L (data not shown) were not detected in the
fraction. In addition, we confirmed that S451A and S451E
formed soluble N–P complex, similarly to N-wt, by using a
co-immunoprecipitation assay (data not shown). These
results indicate that mutation of Ser451 of NiV-N does not
affect either nucleocapsid or N–P–L complex formation.
Many viruses have evolved mechanisms to use cellular
machinery to modify their proteins and regulate their
functions. Recently, we have identified the phosphorylation
sites on the MV-N protein and revealed that phosphoryla-
tion is required for efficient transcription of minigenomic
RNA (Hagiwara et al., 2008). Phosphorylation of the N
protein has also been reported in Sendai virus (Hsu &
Kingsbury, 1982) and mumps virus (Naruse et al., 1981) of
Fig. 3. ESI-Q-TOF MS of NiV-N. (a) Digested
peptides were analysed by ESI-Q-TOF MS,
followed by a database search using Mascot
ver. 2.0 (Matrix Science). Identified peptides
are shown in red. (b) The spectrum was
interpreted using the software DataExplore.
The sequences from the N and C termini, and
the position of the modified residues, were
determined based on b and y ions, respec-
tively. A 98 Da neutral loss of ions is shown
above the sequences as b”98.
Mock wtwt S451AS451AMock
Fig. 4. Radioisotope labelling and immunoprecipitation of wt and
mutant N proteins. Wt N protein and mutants were expressed in
COS-7 cells and labelled with32P or35S in the presence of OKA.
After immunoprecipitation with NiV-N-specific antibody, samples
were resolved by SDS-PAGE (10% gel) and detected with an
FLA-5100 imaging system (Fujifilm).
M. Huang and others
2136 Journal of General Virology 92
the family Paramyxoviridae. Likewise, the N proteins of
rabies virus (RV) (Wu et al., 2002), Marburg virus
(MBGV) (Becker et al., 1994) and Ebola virus (Elliott
et al., 1985), which belong to the order Mononegavirales,
have also been reported to undergo phosphorylation. In
the case of RV, it has been reported that both virus
transcription and replication are reduced when the RV-N
protein is not phosphorylated (Wu et al., 2002). In the case
of MBGV, it has been reported that only the phosphory-
lated N protein can form nucleocapsid complexes and
interact with the genomic RNA (Becker et al., 1994). Thus,
phosphorylation of the virus N protein is a very important
step in the life cycle of the virus.
In the present study, we revealed that NiV-N also undergoes
phosphorylation at Ser451 (Fig. 3). However, the phosphor-
ylation state of the NiV-N protein is different from that of
MV-N, phosphorylation of which is stably detected in cells
(Hagiwara et al., 2008). However, we also found that
phosphorylation of NiV-N was not stable, but showed rapid
turnover (Fig. 1), suggesting that NiV-N phosphorylation/
dephosphorylation is regulated by a cellular kinase–
phosphatase system during the NiV life cycle.
Hendra virus (HeV) is also a member of the genus
Henipavirus. Identity at the amino acid level between the N
proteins of HeV and NiV is 92.1% (Wang et al., 2001).
Interestingly, HeV-N also possesses a Ser at aa 451,
suggesting that HeV-N also undergoes phosphorylation.
Further study on HeV-N protein phosphorylation should
be carried out.
We have ascertained previously that MV-N is phosphory-
2008). The tail region of MV-N possesses three conserved
regions that are enriched in hydrophobic residues: box-1,
box-2 and box-3, which correspond to MV-N aa 401–420,
489–506 and 517–525, respectively (Zhang et al., 2005).
Thus, the phosphorylation sites of MV-N are located in the
neighbourhood of box-2, which contains the binding site for
the MV-P protein X domain, where the P protein tethers
the viral polymerase to the nucleocapsid in support of
transcription and genome replication (Zhang et al., 2005).
Our previous study indicated that phosphorylation of MV-
N does not influence its binding to MV-P, but instead
upregulates the transcriptional activity of minigenomic
RNA (Hagiwara et al., 2008). In the case of NiV-N, it has
been shown that the NiV-N C-terminal region contains
a P-binding domain at aa 468–496 (Chan et al., 2004).
Therefore, the phosphorylation site of NiV-N that we
identified in this study, Ser451, is also located close to the
P-binding region, similar to that of MV-N. As shown in Fig.
7, phosphorylation-deficient NiV-N bound P to the same
extent as the wt NiV-N protein. Thus, phosphorylation
might not influence the direct interaction between NiV-N
and -P, as is the case for MV.
To clarify the role of phosphorylation of NiV-N, we utilized
the NiV minigenome assay system. We observed that virus
transcription and replication were reduced when NiV-N was
not phosphorylated in the minigenome system (Fig. 5). In
the case of RV, the N protein contains one phosphorylation
site, and virus transcription and replication are also reduced
in this assay when using the minigenome and a recombinant
virus with the phosphorylation site substituted with alanine
(Wu et al., 2002). Interestingly, the phosphoserine mimic of
the RV-N protein shows equivalent minigenome activity
and growth rate of the recombinant virus to that with wt
RV-N protein. Similarly, our previous study using an MV
minigenome assay demonstrated that when MV-N is not
phosphorylated,luciferase activityis reducedcompared with
phosphorylated N (Hagiwara et al., 2008), whilst the
phosphoserine mimic of MV-N restores the activity to
some extent (unpublished data). In contrast, in the case of
NiV-N, the mutant S451E, which has a similar negative
charge to NiV-N, shows lower luciferase activity than the
S451A mutant (Fig. 5). This suggests that the short duration
of phosphorylation of NiV-N is important for optimal
transcription/replication activity. These results are consist-
ent with the observation that phosphorylated NiV-N was
hardly detected in steady-state cells (Fig. 1a). In particular,
Northern blot analysis and real-time PCR showed that the
replication efficiency decreased in S451E, whilst replication
and transcription decreased to a similar level in S451A
(Fig. 6). These results imply that the rapid dephosphoryla-
tion of NiV-N enhances mainly this replication step. It
remains a possibility that the E mutation itself caused the
decrease in replication, but the S451E mutant retained both
Fig. 5. Minigenome assay. (a) Negative-strand minigenomic RNA
containing the ORF of the luciferase gene was transfected into
HEK 293 cells expressing wt or mutant N protein, together with P
and L proteins. Luciferase activity of cell extracts was measured
using a Mini Lumat LB 9506 (Berthold Technologies). The data
represent means±SD for triplicate samples. Statistical analysis
was performed using Student’s t-test; *P,0.05 compared with
NiV-N-wt. (b) Protein expression was detected by Western
blotting with NiV-N-specific antibody and glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) antibody.
Phosphorylation of Nipah virus N protein
nucleocapsid-formation and P- and L-binding activities
(Fig. 7). Thus, it suggests strongly that the replication-
specific downregulation by S451E was not caused by the
mere introduction of the mutation, but rather the
constitutive negative charge on the S451 residue. In
paramyxoviruses, it is believed that the RdRp travels from
upstream to downstream on the nucleocapsid, which
encapsulates the genomic RNA during transcription and
replication, and that the efficient transfer of P protein
between adjacent N proteins correlates with the efficiency of
virus RNA synthesis (Zhang et al., 2005). The phosphoryla-
tion of NiV-N S451 introduces a negative charge to the site.
Thus, rapid turnover of phosphorylation at S451 might
bring about change in the conformation and/or activities of
the N protein and the ribonucleocapsid–RNA polymerase
complex. The results of this study shed light on the
mechanism of the regulation of virus RNA synthesis of
NiV, which nonetheless remains somewhat unclear.
Previous studies have revealed that the HRSV-P protein
possesses a rapid-turnover phosphorylation site at Ser54,
and that the phosphorylation is not involved in the
formation of the P–N or P–L complexes (Asenjo et al.,
2005). Recently, using a recombinant HRSV, Asenjo et al.
(2008) demonstrated that the rapid-turnover phosphoryla-
tion of the HRSV-P protein is required for the virus
uncoating step in the early stage of infection, during which
the virus RNP particle is liberated from the M protein and
the virus proceeds to a rapid growth cycle. In the case of
NiV-N, we found that rapid turnover of N-protein
phosphorylation at S451 is not involved in N–P complex
formation in vitro (Fig. 7), but is instead implicated in
efficient virus genome replication and transcription in the
NiV minigenome assay (Fig. 6). However, additional
function(s) of the phosphorylation of the N protein,
important for the life cycle of NiV in vivo, may exist. In a
previous study, we established an NiV reverse-genetics
system (Yoneda et al., 2006). Using this system, rescued NiV
was shown to be similar to wt virus and to retain severe
pathogenicity in an animal model by experimental infection
(Yoneda et al., 2006). This system will be used for a future
study that will aim to elucidate the mechanism of rapid-
turnover phosphorylation of NiV-N and its role in the virus
S451A S451E L(_)
wt S451A S451E L(_)
Fig. 6. (a) Detection of minigenomic transcript and anti-minigenomic RNA with an antisense luciferase (Luc) probe. Total RNA
was prepared from HEK 293 cells by minigenome assay with NiV-N-wt, NiV-N-S451A and NiV-N-S451E, and was hybridized
with the antisense Luc probe. The respective anti-minigenomic RNA and mRNA are indicated. Total RNA was also hybridized
with a b-actin probe. (b) Confirmation of the length of the poly(A) tail of the luciferase mRNA in the minigenome assay. Total
RNA (described above) was subjected to 39 RACE as described in Methods, and amplified cDNA of the poly(A) tail was
separated in agarose gel. (c) Quantitative analysis of (a). The amounts of minigenomic transcripts and anti-minigenomic RNA
products in relation to those for wt were quantified as described in Methods. (c) Relative quantification of mRNA and anti-
minigenomic RNA by semi-quantitative real-time PCR. Total RNA prepared from the minigenome assay was reverse-transcribed
with oligo(dT) primer for mRNA or with a primer specific for NiV trailer sequence. The cDNA was subjected to real-time PCR, as
described in Methods. For normalization, GAPDH mRNA was used as an internal standard. These data represent means±SD
for triplicate samples. Statistical analysis was performed using Student’s t-test; *P,0.05 compared with NiV-N-wt.
M. Huang and others
2138Journal of General Virology 92
Cells. COS-7 (monkey kidney) and HEK 293 (human kidney) cells
were maintained in Dulbecco’s modified Eagle’s medium (DMEM;
Sigma) containing 5% FBS (Sigma-Aldrich), 100 U penicillin G ml21
and 100 mg streptomycin ml21(Invitrogen).
Construction of plasmids. TheN, P andL gene ORFs were amplified
by PCR using KOD Plus ver. 2 (Toyobo) primers, including SalI/
BamHI (N), MluI/BsmI (P) and MluI/NotI (L) sites, and an NiV-
supporting plasmid that encodes the NiV-N, -P and -L genes as a
template (Radecke et al., 1995; Yoneda et al., 2006). Amplified PCR
products were purified by using a MinElute PCR Purification kit
(Qiagen), inserted between SalI/BamHI (N), MluI/BsmI (P) and MluI/
NotI (L) sites in the pCAGGS mammalian expression vector (Tokui
et al., 1997) and designated pCAGGS-NiV-N, -P and -L, respectively.
Mutant NiV-N plasmids S451A and S451E were generated by
mutagenesis using Pfu DNA polymerase (Stratagene) according to
the manufacturer’s instructions.
Mammalian expression of NiV-N and purification of the
nucleocapsid. COS-7 cells (1.06106) were transfected with
6.0 mg pCAGGS-NiV-N by using FuGene 6 transfection reagent
(Roche), according to the manufacturer’s instructions. Forty-eight
hours after transfection, cells were lysed with 500 ml lysis buffer that
contained 10 mM Tris/HCl (pH 7.8), 150 mM NaCl, 1 mM EDTA,
1% NP-40, 1 mM Na3VO4, 50 mM NaF and protease inhibitor
cocktail (BD Biosciences) at 4 uC for 30 min. The lysate was
centrifuged at 20400 g for 10 min and the supernatant was layered
onto a discontinuous gradient that contained 25, 30 and 40% (w/w)
caesium chloride prepared in lysis buffer without NP-40. After
centrifugation at 55000 r.p.m. for 30 min with a SW55Ti rotor
(Beckman), the band material that contained NiV-N was collected.
Finally, NiV-N was pelleted by ultracentrifugation at 70000 r.p.m.
for 10 min with a TLA100.3 rotor (Beckman) and suspended in
20 ml PBS.
Production of NiV-N-specific antibody. Antiserum against NiV-N
was raised in a rabbit by immunization with approximately 120 mg
purified N protein mixed with complete Freund’s adjuvant (Difco).
Serum was collected 6 weeks after injection. The animal experiment
was approved by the Animal Ethics Committee in our institute and
conducted according to the institutional guidelines for animal
SDS-PAGE and Western blotting. Purified proteins were separated
by SDS-PAGE (10% gel) under reducing conditions and stained with
CBB. Western blotting was performed according to a standard method.
In brief, after separation by SDS-PAGE, proteins were transferred to an
Immobilon-P membrane (Millipore) and incubated with a 1000-fold
dilution of rabbit antiserum against NiV-N. The membrane was then
incubated with a 1000-fold dilution of HRP-conjugatedanti-rabbitIgG
antibody (DakoCytomation). Detection was carried out using ECL
Western blotting Detection Reagents (Amersham Biosciences) and the
LAS-1000UV mini system (Fujifilm).
Detection of phosphorylated NiV-N by autoradiography after
OKA treatment. COS-7 cells (1.56105) were transfected with 1.6 mg
pCAGGS-NiV-N plasmid for wt or phosphorylation-site mutants, as
describedabove. Twenty-four hours after transfection,32P (PerkinElmer)
or35S (EXPRESS35S Protein Labeling Mix; PerkinElmer) and 100 nM
OKA (Calbiochem) were added to the medium and cultured for 6–24 h.
The cells were lysed in lysis buffer that contained 5 mM EDTA, 0.5%
Triton X-100, 1 mM Na3VO4, 50 mM NaF and protease inhibitor
cocktail in PBS at 4 uC for 1 h, followedby clarification by centrifugation
at 20400 g for 30 min. Rabbit antiserum against NiV-N protein and
protein–G Sepharose 4 Fast Flow (GE Healthcare) were added to the
supernatant and incubated at 4 uC for 16 h with gentle rotation.
Afterwards, immunoprecipitation samples were resolved by SDS-PAGE.
The amount of labelled N protein was estimated by using the FLA-5100
imaging system (Fujifilm).
In-gel digestion of N protein by trypsin. Purified NiV-N was
subjected to SDS-PAGE and stained with a SeePico CBB Stain kit
(Benebiosis). A single band of approximately 58 kDa was excised
from the gel and then destained five times with 50 mM NH4HCO3in
50% methanol. After drying the gel in 100% CH3CN, 2.5 pmol
trypsin (Trypsin Gold Mass Spectrometry Grade; Promega) in 50 ml,
10 mM Tris/HCl (pH 8.5) was added and in-gel digestion was carried
out at 37 uC for 16 h. Digested peptides were extracted by treating the
gel twice with 0.1% trifluoroacetic acid (TFA) in 50% CH3CN, and
finally extracted once more by 0.1% TFA in 80% CH3CN for
ESI-Q-TOF MS. Digested peptides were applied to a high-resolution
nanoflow reversed-phase capillary LC (DiNa; KYA Technologies)
coupled with an electrospray source and Q-TOF tandem mass
spectrometer (Q-Tof-2; Micromass Ltd), as described previously
(Oyama et al., 2004, 2007). The acquired MS/MS mass spectral data
were processed using Mascot ver. 2.0 (Matrix Science) against each
database, with maximum tolerance of 100 p.p.m. in MS data and
0.15 Da in MS/MS data. The candidate peptide sequences were
screened using probability-based MOWSE scores that exceeded their
Minigenome assay. The NiV minigenome encoding luciferase was
constructed essentially as described by Halpin et al. (2004), with the
modification that the firefly luciferase gene was used instead of the
chloramphenicol acetyltransferase gene. The resulting minigenome
fragment was sequenced and cloned into EagI–BsmI sites of pMDB1
(Baron & Barrett, 1997). In vitro transcription was performed using
T7 RiboMAX Large-Scale RNA Production systems (Promega),
according to the manufacturer’s instructions, and negative-strand
Fig. 7. Nucleocapsid and N–P–L complex formation assay. NiV-N-
wt (lane 4), NiV-N-S451A (lane 5), NiV-N-S451E (lane 6) and
NiV-P (lane 7) were expressed individually in COS-7 cells and
subjected to caesium chloride density-gradient centrifugation.
Alternatively, cell extracts of NiV-P and NiV-L were mixed with NiV-
N-wt (lane 1), NiV-N-S451A (lane 2) or NiV-N-S451E (lane 3),
and the N–P–L complexes were purified by centrifugation in the
same manner. Each sample was resolved by SDS-PAGE and
detected by (a) CBB staining or (b) Western blotting using NiV-N-,
-P- and -L-specific antibodies, respectively.
Phosphorylation of Nipah virus N protein
minigenomic RNA was purified using MicroSpin G50 columns
HEK 293 cell monolayers in 24-well plates, grown to 90% confluence,
were infected with recombinant vaccinia virus vTF7-3 at an m.o.i. of
3. After 30 min infection, cells were washed with PBS and transfected
with NiV minigenome luciferase RNA (150 ng) that carried the
luciferase reporter gene, together with supporting plasmids that
carried N (312.5 ng per well), P (200 ng mg21) and L (100 ng per
well) (Halpin et al., 2004; Yoneda et al., 2006), using DMRIE-C
reagent (Invitrogen) according to the manufacturer’s protocol. After
4 h transfection, medium was removed. Cells were washed with
OPTI-MEM (Invitrogen GIBCO) and incubated with DMEM with
5% FBS for 20 h. Cells were lysed and subjected to the reporter assay
using Pickagene reagent (Toyo) and a Mini Lumat LB 9506
luminometer (Berthold Technologies), according to the manufac-
Preparation of a minigenome-specific RNA probe. A PCR
fragment of luciferase sequences from nucleotide positions 521 to 901
was amplifiedbyPhusion High-Fidelity
(Finnzymes). The T7 promoter sequence was also appended to the
reverse primer for RNA transcription in vitro. The PCR product was
extracted by the Wizard SV Gel and PCR Clean-Up system (Promega)
after agarose gel electrophoresis. DNA was extracted by phenol/
chloroform/isoamylalcohol extraction. The purified template DNA
was transcribed in vitro using T7 RNA polymerase by a DIG RNA
Labelling kit (Roche Applied Science), according to the manufac-
Northern blot analysis of minigenomic RNA synthesis. Total
RNA was extracted after transfection for the minigenome assay with
ISOGEN (Nippon Gene), following the manufacturer’s instructions.
RNA was extracted and separated on a 1.5% MOPS–formaldehyde
agarose gel at 100 V for 90 min at 4 uC, and transferred onto a
Hybridization of the membrane with a minigenome-specific RNA
probe was performed at 68 uC for 18 h. Signals from the hybridized
probes were detected by using a DIG DNA/RNA Labelling and
Detection kit (Roche Applied Science) and CDP-Star detection
reagent (GE Healthcare), according to the manufacturers’ instruc-
tions, and visualized by exposure to LAS-1000 mini (Fujifilm). Each
band was quantified by software from Science Lab 2001 Image Gauge
(Fujifilm) and the density of each band was compensated with its
background, which contains an equivalent area from this lane with no
signals. The value of density for the wt samples was set as 100%, and
the relative density of mutant samples was calculated by dividing the
density value of mutant samples by that of the controls.
Measurement of poly(A) tail length. Total RNA extracted from the
minigenome assay described above was subjected to 39 RACE for
determination of the length of the poly(A) tail in the luciferase mRNA
using an ALL-TAIL kit For Extreme 39 RACE (Bioo Scientific) in
accordance with the manufacturer’s instructions. In brief, an adeny-
a T4 RNA ligase, and then reverse-transcribed using a primer specific to
the adenylated adaptor. The cDNA was amplified by PCR with a primer
designed near the 39 end of the luciferase gene (59-GATCCTC-
ATAAAGGCCAAGAAGG-39) and the primerspecifictothe adenylated
adaptor. The PCR product was separated on an agarose gel.
Relative quantification of minigenomic RNA synthesis by real-
time PCR. Total RNA extracted from the minigenome assay
described above was transcribed with PrimeScript reverse transcrip-
tase (TaKaRa) using oligo(dT) primer for mRNA or trailer-specific
primer for anti-minigenomic RNA (59-ACCGAACAAGGGTAAA-
GAAG-39). Comparisons of the expression levels of luciferase mRNA
and anti-minigenomic RNA were performed by real-time PCR using
SYBR Premix Ex Taq (TaKaRa) and a primer pair specific for the
luciferase gene (59-AATCCATCTTGCTCCAACACC-39 and 59-CGT-
CTTTCCGTGCTCCAA-39). All data were normalized by the
corresponding level of GAPDH mRNA using a specific primer pair
(59-CCACCCATGGCAAATTCCATGGA-39 and 59-TCTAGACGGC-
mutants), pCAGGS-NiV-P and pCAGGS-NiV-L were expressed
individually in COS-7 cells using FuGene 6 transfection reagent, as
described above. After lysis in 250 ml lysis buffer, an equal volume of
cell lysate expressing N (or N mutants) was mixed with P and L, and
incubated for 30 min at 4 uC. The supernatant of each sample was
layered onto a discontinuous gradient containing 25, 30 and 40%
(w/w) caesium chloride and was then centrifuged as described above.
The band material was collected and pelleted by ultracentrifugation.
Each sample was subjected to SDS-PAGE and detected by Western
blotting using antiserum against NiV-N, -P and -L, respectively.
complexformation assay. pCAGGS-NiV-N (or N
This work was supported by Grants-in-Aid for Scientific Research
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