ISSN 0582-9879 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2003, 35(6): 518-521 CN 31-1300/Q
The Phosphorylation of NS Protein of Wheat Rosette Stunt Virus
( 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. 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.
1.1 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.
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. 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.
2.1 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.
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.
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).
M, protein marker; 1, WRSV GST-NS fusion protein.
M, protein marker; 1, 50 ng nucleocapsid; 2, 100 ng nucleocapsid; 3, 1 μg nucleocapsid; 4, 2 μg nucleocapsid.
M, protein marker; 1, GST-NS fusion protein+nucleocapsid (NP); 2, GST-NS fusion protein; 3, nucleocapsid (NP); 4, GST+nucleocapsid (NP); 5, ddH2O.
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. 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. demonstrated that both viral transcription and replication are reduced when the rabies virus N protein is not phosphorylated. Recently, Mathur et al. 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. 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. 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.
1 Zheng QX, Xu WJ, Zhu BM, Shen XR, Kung TH (Gong ZX). Purification and some properties of the nucleocapsid of wheat rosette stunt virus. Acta Biochim Biophys Sin, 1983, 15: 561－567
2 Gong ZX, Zheng QX. Studies on the N-protein of a plant rhabdoviruses. Acta Biochim Biophys Sin, 1985, 17: 650－652
3 Kung TH (Gong ZX), Zheng QX. The partial purification and the protein composition of the plant rhabdovirus-wheat rosette stunt virus. Acta Biochim Biophys Sin, 1985, 17: 248－252
4 Sun W, Peng H, Gong ZX. Studies on wheat rosette stunt virus RNA. Chinese Journal of Virology[KG-*3], 1986, 2: 60－67
5 Shen JL, Gong ZX. Construction of a near full-length cDNA library of wheat rosette stunt virus (WRSV). Virologica Sinica, 1997, 2: 155－161
6 Ye YJ, Gong ZX. Cloning, expression and identification of matrix protein gene of wheat rosette stunt virus (WRSV). Acta Biochim Biophys Sin, 1998, 30: 520－524
7 Shen JL, Ye YJ, Gong ZX. Cloning and sequencing of the gene encoding NS protein of wheat rosette stunt virus. Acta Biochim Biophys Sin[KG-*3], 1998, 30: 515－519
8 Ye YJ, Gong ZX. Sequence analysis of the trailer region of wheat rosette stunt virus genomic RNA. Acta Biochim Biophys Sin[KG-*3], 1999, 31: 93－96
9 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976, 72: 248－254
10 Banerjee AK, Barik S. Gene expression of vesicular stomatitis virus genome RNA. Virology, 1992, 188(2): 417－428
11 Kingsbury DW, Hsu CH, Morgan EM. A role for NS protein phosphorylation in vascular stomatitis vesiculovirus transcription. In: Bishop DHL, Compans RW eds. The Replication of Negative-strand Viruses, New York: Elsevier/North-Holland Publishing Co., 1981, 821－827
12 Kingsford L, Emerson SU. Transcriptional activities of different phosphorylated species of NS protein purified from vesicular stomatitis virions and cytoplasm of infected cells. J Virol, 1980, 33(3): 1097－1105
13 Barik S, Banerjee AK. Cloning and expression of the vesicular stomatitis virus phosphoprotein gene in Escherichia coli:Analysis of phosphorylation status versus transcriptional activity. J Virol, 1991, 65(4): 1719－1726
14 Pattnaik AK, Hwang L, Li T, Englund N, Mathur M, Das T, Banerjee AK. Phosphorylation within the amino-terminal acidic domain I of the phosphoprotein of vesicular stomatitis virus is required for transcription, but not replication. J Virol, 1997, 71(11): 8167－8175
15 Hwang LN, Englund N, Das T, Banerjee AK, Pattnaik AK. Optimal replication activity of vesicular stomatitis virus RNA polymerase requires phosphorylation of a residue(s) at carboxy-terminal domain II of its accessory subunit, phosphoprotein P. J Virol, 1999, 73(7): 5613－5620
16 Wu X, Gong X, Foley HD, Schnell MJ, Fu ZF. Both viral transcription and replication are reduced when the rabies virus nucleoprotein is not phosphorylated. J Virol, 2002, 76(9): 4153－4161
17 Mathur M, Banerjee AK. Novel binding of GTP to the phosphoprotein (P) of vesicular stomatitis virus. Gene Expr, 2002, 10(4): 193－200
18 Sun W, Gong ZX. In vitro studies on the nucleocapsid-associated RNA polymerase of wheat rosette stunt virus. Intervirology, 1988, 29(3): 154－161
19 Chattopadhyay D, Banerjee AK. Phosphorylation within a specific domain of the phosphoprotein of vesicular stomatitis virus regulates transcription in vitro. Cell, 1987, 49(3): 407－414
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@example.com