The
Phosphorylation of NS Protein of Wheat Rosette Stunt Virus
XIE Bao-Tong, YE
Yong-Jun, GONG Zu-Xun*
( Key
Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai
Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai
200031, China )
Abstract The
genome of wheat rosette stunt virus (WRSV), a plant rhabdovirus, is a single
negative strand RNA. It encodes five viral structural proteins: the
glycoprotein (G), the matrix protein (M), the nucleocapsid protein (N), the
large protein (L) and the non-structural protein (NS), which was later proved
to be a viral structural protein too and existed in a variety of
phosphorylation forms in case of vascular stomatitis virus (VSV). In this paper
we demonstrated that NS protein of WRSV, either bound with the viral
nucleocapsid or expressed in bacteria could be in vitro phosphorylated
in presence of viral nucleocapsid. We concluded that the NS protein of WRSV was
a phosphorylated protein and it might exist in both phosphorylated and
dephosphorylated forms in virions. Our results excluded the possibility that
the NS protein could be autophosphorylated. The L protein, the major component
of viral RNA dependent RNA polymerase is associated with the protein kinase for
phosphorylation of NS protein.
Key words wheat rosette stunt
virus; NS protein; phosphorylation; protein kinase
Wheat rosette
stunt virus (WRSV) is a plant rhabdovirus. The virus was first found in North
China at late 1970s. The virus genome is a single negative strand RNA and
encodes 5 viral structural proteins as in the case of an animal rhabdovirus ―
vascular stomatitis virus (VSV): the surface glycoprotein (G), the matrix
protein (M), the nucleocapsid protein (N), the large protein (L) and the
non-structural protein (NS), which was later proved to be a viral structural
protein too. Later studies indicated that NS protein, either present in mature
virions or in VSV-infected cells existed in a variety of phosphorylated forms.
Therefore, the NS protein was also named P protein instead of NS protein in
many recent references. We had reported previously that WRSV also contained
five viral structural proteins, as G, M, N, NS and L proteins. The latter three
proteins were bound with viral RNA and together formed the viral
nucleocapsid[1]. However, so far there was no report to describe the
phosphorylation form of NS protein of plant rhabdoviruses.
In previous
works we had reported the viral morphology and series biochemical
characteristics of WRSV[1－4]. More recently, the nearly-full length of cDNA library of WRSV
was successfully constructed and the nucleotide sequences of M and NS genes and
viral 5′-trails were also analyzed[5－8]. In the present paper we demonstrate that the NS protein
expressed in bacteria or the NS protein that exists in nucleocapsid (NP) could
be phosphorylated in vitro. Therefore, as for VSV, the phosphorylation
and dephosphorylation of NS protein may play important roles in the viral
genome regulation and multiplication.
1 Materials
and Methods
1.1 Expression
of NS of WRSV
A recombinant
vector pEGX-3X-NS containing the cDNA of WRSV NS gene was constructed,
expressed in E. coli DE3, and purified as reported previously[7].
1.2 Purification
of WRSV nucleocapsid
The concentrated
purified WRSV preparation was diluted with 20 mmol/L Tris-HCl buffer (pH 8.0)
to final volume of 3.5 mL. 0.45 mL 50% glycerol, 0.1 mL 200 mmol/L Tris-HCl
buffer (pH 8.0), 45 μL 100 mmol/L DTT, and 225 μL 20% Nonidet P40 were added to
this diluted virus preparation. The solution was votexed, and 180 μL 2.5 mol/L NaCl was slowly added.
All procedures were carried out at 4 ℃. The virus preparation was kept at 4 ℃ for 1 h and then added on top of 0.6 mL pre-prepared noncontinuous
40%－50% glycerol
gradients. The upper gradient was 0.3 mL of 40% glycerol and the lower was 0.3
mL of 50% glycerol. The glycerol was prepared by dilution with the buffer
containing 20 mmol/L Tris-HCl buffer (pH 8.0), 1 mmol/L DTT, 0.1% NP40 and 0.1
mol/L NaCl. The glycol gradient with the virus preparation was centrifuged at
50 000 g and at 4 ℃ for 2 h. The supernatant containing the viral membrane fraction was
discarded and the pellet was suspended in 1 mL 20 mmol/L Tris-HCl buffer (pH
8.0). The solution was clarified with low-speed centrifugation for 15 min. The
supernatant was kept at 4 ℃ for use. The protein concentration was determined by Bradford
method[9]. The purity of viral nucleocapsid preparation was examined by
electron microscopy.
Fig.1 The electron
micrograph of purified nucleocapsid of WRSV ( 20 000× )
Insert (lower right): partial
enlargement ( 30 000× )
1.3 In
vitro phosphorylation of viral nucleocapsid
One microliter
of [γ-32P]-ATP (10 μCi), 1 μL mixture of 10 mmol/L GTP, CTP and UTP, and the
viral nucleocapid preparations with different concentrations (0, 100 ng, 500
ng, 1 μg, 2 μg) were added to 2 μL of 10× phosphorylation reaction buffer [1
mol/L Tris-HCl buffer, (pH 8.0), 1 mol/L NaCl, 3 mmol/L DTT, 0.5 mol/L MgCl2].
Then ddH2O was added to reach the final volume of 20 μL. The mixture solution was
incubated at 30 ℃ for 2 h. The
mixture solution was electrophoresised with 12% SDS-PAGE. After electrophoresis
the dried gels were subjected to autoradiography at －70 ℃.
1.4 In
vitro phosphorylation of GST-NS fusion protein
The reaction
buffer consisted of 2 μL of 10× phosphorylation reaction buffer, 1 μL
[γ-32P]-ATP (10 μCi), 1 μL mixture of 10 mmol/L GTP, CTP and UTP, 1 μL of
purified viral nucleocapsid (100 mg/L), and 1 μL 100 ng purified GST-NS fusion
protein. Finally, ddH2O was added to the buffer to make the final
volume equal to 20 μL. Other steps of the phosphorylation assays were the same as
described above.
2 Results
2.1 Preparation
of WRSV viral nucleocapsid
It had been
observed by electron microscopy that the preparation contained viral
nucleocapsids of different lengths in high purity (Fig.1). It was shown
previously that the viral nucleocapsid of WRSV contained three structural
proteins: L, N and NS with molecular weights of 120 kD, 44 kD and 40 kD,
respectively[1].
2.2 The
expression of WRSV NS protein in E. coli DE3
The expression
vector pGEX-3X encodes the
GST fragment with size of 26 kD. After purification only one
band of GST-NS fusion protein appeared in SDS-PAGE with moleculer weight of 66
kD (Fig.2). The band was proved to be the GST-NS fusion protein by Western
blotting in our previous studies[7].
2.3 In
vitro phosphorylation of viral nucleocapsid
In vitro phosphorylation assays of purified viral nucleocapsid showed a
single band by SDS-PAGE with moleculer weight of 40 kD, when the amount of
nucleocapsid reached to 1 μg and 2 μg in the assays (Fig.3).
2.4 In
vitro phosphorylation of GST-NS fusion protein
In vitro phosphorylation assays of purified GST-NS fusion protein with
nucleocapsid (100 ng) showed that the fusion GST-NS protein could be in
vitro phosphorylated, whereas all the controls, the GST-NS fusion protein
or the nucleocapsid alone, and GST with nucleocapsid were not (Fig.4).
Fig.2 SDS-PAGE of
expressed and purified WRSV GST-NS fusion protein
M, protein marker; 1, WRSV
GST-NS fusion protein.
Fig.3 In vitro
phosphorylation of NS protein bound with WRSV nucleocapsid
M, protein marker; 1, 50 ng
nucleocapsid; 2, 100 ng nucleocapsid; 3, 1 μg nucleocapsid; 4, 2 μg
nucleocapsid.
Fig.4 In vitro
phosphorylation of GST-NS fusion protein
M, protein marker; 1, GST-NS
fusion protein+nucleocapsid (NP); 2, GST-NS fusion protein; 3, nucleocapsid
(NP); 4, GST+nucleocapsid (NP); 5, ddH2O.
3 Discussion
WRSV belongs to
the Rhabdoviridae family with VSV as the type member of the Vesiculovirus
genus. Both the genome organizations and major viral structural protein
compositions are similar for the two viruses. The VSV NS protein is a key
subunit of the viral RNA-dependent RNA polymerase complex[10]. The NS protein
in VSV virions and in VSV-infected cells exists in a variety of phosphorylated
forms. The protein is phosphorylated at multiple sites in two different domains.
Although the exact role of constitutive phosphorylation in NS protein function
is not well elucidated yet, it had been suggested that phosphorylation might be
important for the transcriptional activity of NS protein itself and of the
virus, as a whole[11－13]. More recently, it had been shown that specific serine and
threonine residues within the amino-terminal acidic domain I of NS protein must
be phosphorylated for transcription activity, whereas the optimal replication
activity of VSV RNA polymerase required phosphorylation of residues at
carboxy-terminal domain II of VSV-NS protein[14, 15]. Rabies virus is the
prototype of the Lassavirus genus within the Rhabdoviridae family. Rabies virus
N protein is phosphorylated, but VSV-N is not. Wu et al.[16] demonstrated that
both viral transcription and replication are reduced when the rabies virus N
protein is not phosphorylated. Recently, Mathur et al.[17] indicated
that the phosphorylation of VSV-NS protein might initiate a structural change
within the NS protein allowing GTP to bind, thus manifesting biology function
to the transcription factor. We had reported in our previous work that WRSV NS
protein was also required for in vitro viral RNA-dependent RNA
polymerase activity[18]. Therefore, the WRSV-NS protein might serve as one of
the subunits of viral RNA polymerase as in the case of VSV, but there was no
evidence in the elucidation of phosphory-lation of NS protein of plant
rhabdoviruses. Is the NS protein of plant rhabdovirus also a phosphorylated
protein? Does NS protein of plant rhabdovirus possess an autophosphorylation
activity that might be responsible for constitutive phosphorylation?
We had succeeded
in expressing WRSV NS protein in bacteria, since bacterial protein kinases are
generally specific for their natural substrates and are not as prevalent as in
eukaryotes. Therefore, the NS protein synthesized in bacteria might be
completely free of phosphates. Our results of in vitro phosphorylation
assays indicate that for WRSV the NS protein is also a constitutive
phosphorylation protein in virions. Since the NS protein bound to viral
nucleocapsid isolated from the virions could be in vitro phosphorylated,
it implicates that NS protein of virions might exist in both phosphorylation
and dephosphorylation forms.
The deduced
amino acid sequence of WRSV NS protein showed that it contains 17 Thr and 50
Ser residues, all of which are putative phosphorylation sites, although in VSV
two conservative Ser236 and Ser242 were already determined as the
phosphorylation sites for its NS protein[7,19]. The results of in vitro
phosphorylation assays of viral nucleocapsid alone and of expressed NS fusion
protein with nucleocapsid implicated that the L protein bound together with the
viral N protein-RNA complex might function as the protein kinase for the
phosphorylation of NS protein, although the L protein of WRSV was known as a
major component of the RNA-dependent RNA polymerase. There were some evidences
that suggested L protein of VSV was associated with protein kinase[19]. Our
results exclude the possibility that the NS protein could be
autophosphorylated. The in vitro phosphorylation of NS protein requires
L protein, and the reaction is also concentration-dependent on L protein. The
phosphorylation sites of WRSV NS protein and protein kinase activity of L
protein and study on the role of phosphorylation and dephosphorylation of NS
protein in viral transcription and replication regulation are currently
underway.
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Received: March 13, 2003 Accepted: March 31,
2003
This work was supported by a grant from the
National Natural Science Foundation of China (No. 30080005)
*Corresponding author: Tel, 86-21-54921220;
Fax, 86-21-54921125; e-mail, [email protected]