| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
1 Laboratory of Virology, National Agricultural Research Center, Tsukuba, Ibaraki 305-8666, Japan
2 Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan
Correspondence
Toshihiro Omura
toomura{at}affrc.go.jp
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). The outer shell of the RDV capsid is composed of P2 and P8, while the core consists of P1, P5 and P7 proteins enclosed by the P3 core capsid protein. The P3 core and P8 outer capsid proteins constitute the major framework of the architecture of RDV. P7, a nucleic acid-binding protein, which also binds to P3, is an important contributor to the conformation of RDV particles (Ueda & Uyeda, 1997). Results of a recent study of the structure of RDV suggested that some of the P8 trimers, anchored by interactions between P3 and P8 at positions on the threefold axes of symmetry, might form the seeding centre for assembly of the symmetry-mismatched inner and outer shells (Wu et al., 2000). A study of transgenic rice that expressed P3 and P8 also suggested that the P3–P8 interaction is necessary for the formation of a structure that closely resembles that of RDV particles (Zheng et al., 2000). However, the ability of the major core protein P3 to produce a single-shelled core and the mechanisms for sorting and organizing capsid proteins and segments of genome dsRNA remain to be clarified. In this study, we expressed the P3, P7 and P8 proteins in insect cells using a baculovirus system to determine the way in which the basic structural proteins of the RDV particle are generated by these major structural proteins.
Virus was propagated in our laboratory and viral dsRNA was purified from virions with a QIAquick PCR Purification Kit (Qiagen), according to the manufacturer's instructions. To generate the cDNAs from S3, S7 and S8, which included full-length open reading frames for the P3, P7 and P8 proteins of RDV, respectively, we used the following pairs of primers: 5'-CCATGGATGGACAGTACCGGCCGAGCATATGACG-3' and 5'-CCATGGTCATAGAGGCGGCTGATCTCCATTCGG-3' (S3F-NcoI/S3R-NcoI); 5'-AGATCTTTGGCGCCCGACATG-3' and 5'-CCATGGCGGTCGTAATGACCAAA-3' (S7F-BglII/S7R-NcoI); and 5'-AGATCTATGTCACGCCAGATGTGGTTAGACAC-3' and 5'-AAGCTTCTAATTTGGTCGATAGTATCTTCCAAATAC-3'(S8F-BglII/S8R-HindIII). The primers were based on the terminal RNA sequences of the S3, S7 and S8 segments, respectively (Kano et al., 1990; Nakashima et al., 1990; Omura et al., 1989) and restriction sites are underlined. cDNAs were generated by RT-PCR as described previously (Hagiwara et al., 2001). After digestion by appropriate restriction enzymes, the products of PCR were ligated into the transfer vector pBlueBacIII (Invitrogen). Restriction analysis and DNA sequencing were performed to confirm that the coding sequence of each segment was oriented appropriately with respect to the baculovirus polyhedrin promoter.
We found that the amino acid sequences predicted from the nucleotide sequences of S3 and S7 cDNAs contained some substitutions when compared with previously reported sequences (Kano et al., 1990; Nakashima et al., 1990). These alterations were located at residues 183 (His to Gln), 184 (Gly to Arg) and 1014 (Gly to Glu) in S3, and at residue 231 (Ala to Thr) in S7. The S8 cDNA encoded a protein with an amino acid insertion (Ala) at position 179 when its product was compared with the sequence of S8 reported previously (Omura et al., 1989).
Linearized AcNPV DNA and resultant transfer plasmid prepared to express P3 were used to co-transfect cultured Sf-9 cells in the presence of CellFECTIN in accordance with the manufacturer's instructions (Invitrogen). To solubilize the expressed P3 protein, recombinant baculovirus-infected Sf-9 cells were mixed with BugBuster Protein Extraction Reagent (Novagen) and incubated for 30 min at 25 °C; the supernatant was collected after centrifugation for 5 min at 30 000 g. After 10–40 % sucrose density gradient centrifugation (SDGC) of the supernatant for 70 min at 94 500 g, the material at a position close to the predicted position of RDV cores was collected and pelleted by centrifugation for 60 min at 155 000 g. Many core-like particles were observed in this fraction after expression of P3 alone (Fig. 1a). To confirm that the core-like particles were constructed from P3 protein, immunogold staining of the particles was performed using P3-specific antibody according to the method described by Lin (1984). As shown in Fig. 1(b), core-like particles were specifically labelled with gold particles in electron microscopy. These results indicate that P3 protein itself has the ability to form a single-shelled core without the assistance of any other structural proteins of RDV.
|
, double-shelled virus-like particles that resemble in both size and appearance RDV particles purified from RDV-infected rice plants (Fig. 1d) were observed by electron microscopy. Co-expression of P3 and P8 in Sf-9 cells also produced similar structures (data not shown); however, there were no such structures when P8 alone was expressed. These results suggest that recombinant P3 and P8 have the ability to generate double-shelled particles both in vivo and in vitro.
To determine whether core protein P7 could be enclosed inside the core particles, recombinant P7 was co-expressed with P3 in insect cells; P8 was then added and virus-like particles were purified as mentioned above. The proteins in the purified particles were analysed by SDS-PAGE (10 % polyacrylamide) and Western blotting with P3-, P7- and P8-specific antibodies. As shown in Fig. 2, P3 [lanes 4 in (a) and (b)], P7 [lanes 4 in (a) and (c)] and P8 [lanes 4 in (a) and (d)] were clearly detected in the purified virus-like particles. It has been reported that proteins with mobility higher than P8 are also encoded by RDV segment 8 (Suzuki & Sugawara, 1991; Mao et al., 1998), suggesting that the smaller bands detected in Fig. 2(d) may be such a protein. The band with molecular size bigger than P7 in Fig. 2(c) could be a band specific for baculovirus expression, but no details are known.
|
). To further rule out the possibility that the expressed free P7 was located at the same position as double-shelled virus-like particles in SDGC, particles purified by SDGC were further subjected to 30 % CsCl equilibrium centrifugation for 24 h at 190 400 g. After such centrifugation, a single band formed at the middle portion of the centrifugation tube. Single-shelled core-like particles were observed under the electron microscope in this band. This result suggests that a large proportion of the P8 outer capsid proteins were removed, leaving the P8 that had bound strongly to core particles (T-trimer; Wu et al., 2000) in CsCl equilibrium centrifugation. As shown in Fig. 3, P7 was detected in the re-purified particles, suggesting that the protein is not simply co-purifying in SDGC. Our results thus support the hypothesis that P7 is packaged inside viral particles with the genomic RNA, since P7 binds to RNA (Ueda & Uyeda, 1997).
|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Kano, H., Koizumi, M., Noda, H., Mizuno, H., Tsukihara, T., Ishikawa, K., Hibino, H. & Omura, T. (1990). Nucleotide sequence of rice dwarf virus (RDV) genome segment S3 coding for 114 K major core protein. Nucleic Acids Res 25, 6700.
Lin, N.-S. (1984). Gold–IgG complexes improve the detection and identification of viruses in leaf dip preparation. J Virol Methods 8, 181–190.[Medline]
Mao, Z., Li, J., Xu, Y., Zheng, H. H., Schiemann, J., Casper R. & Chen, Z. L. (1998). The 42K protein of rice dwarf virus is a post-translational cleavage product of the 46K outer capsid protein. Arch Virol 143, 1831–1838.[CrossRef][Medline]
Nakashima, K., Kakutani, T. & Minobe, Y. (1990). Sequence analysis and product assignment of segment 7 of the rice dwarf virus genome. J Gen Virol 71, 725–729.
Omura, T., Ishikawa, K., Hirano, H., Ugaki, M., Minobe, Y., Tsuchizaki, T. & Kato, H. (1989). The outer capsid protein of rice dwarf virus is encoded by genome segment S8. J Gen Virol 70, 2759–2764.
Omura, T. & Jan, J. (1999). Role of outer capsid proteins in transmission of Phytoreovirus by insect vectors. Adv Virus Res 54, 15–43.[Medline]
Ueda, S. & Uyeda, I. (1997). The rice dwarf phytoreovirus structural protein P7 possesses non-specific nucleotide acids binding activity in vitro. Molecular Plant Pathology [On-Line] http://www.bspp.org.uk/mppol/1997/0123Ueda/.
Suzuki, N. & Sugawara, M. (1991). Outer capsid protein heterogeneity of rice dwarf phytoreovirus. J Gen Virol 72, 2239–2342.
Zheng, H., Yu, L., Wei, C., Hu, D., Shen, Z., Chen, Z. & Li, Yi. (2000). Assembly of double-shelled, virus-like particles in transgenic rice plants expressing two major structural proteins of Rice dwarf virus. J Virol 74, 9808–9810.
Wu, S., Hammar, L., Xing, L., Markarian, S., Yan, J., Iwasaki, K., Fujiyoshi, Y., Omura, T. & Cheng, H. (2000). Phytoreovirus T=1 core plays critical roles in organizing the outer capsid of T=13 quasi-equivalence. Virology 271, 18–25.[CrossRef][Medline]
Received 15 October 2002;
accepted 22 November 2002.
This article has been cited by other articles:
|
|
 |
|
  S. Zhu, F. Gao, X. Cao, M. Chen, G. Ye, C. Wei, and Y. Li The Rice Dwarf Virus P2 Protein Interacts with ent-Kaurene Oxidases in Vivo, Leading to Reduced Biosynthesis of Gibberellins and Rice Dwarf Symptoms Plant Physiology, December 1, 2005; 139(4): 1935 - 1945. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Cao, P. Zhou, X. Zhang, S. Zhu, X. Zhong, Q. Xiao, B. Ding, and Y. Li Identification of an RNA Silencing Suppressor from a Plant Double-Stranded RNA Virus J. Virol., October 15, 2005; 79(20): 13018 - 13027. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Namba, K. Hagiwara, H. Tanaka, Y. Nakaishi, K. T. Chong, E. Yamashita, G. E. Armah, Y. Ono, Y. Ishino, T. Omura, et al. Expression and Molecular Characterization of Spherical Particles Derived from the Genome of the Hyperthermophilic Euryarchaeote Pyrococcus furiosus J. Biochem., August 1, 2005; 138(2): 193 - 199. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Li, Y. M. Bao, C. H. Wei, Z. S. Kang, Y. W. Zhong, P. Mao, G. Wu, Z. L. Chen, J. Schiemann, and R. S. Nelson Rice Dwarf Phytoreovirus Segment S6-Encoded Nonstructural Protein Has a Cell-to-Cell Movement Function J. Virol., May 15, 2004; 78(10): 5382 - 5389. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Hagiwara, T. Higashi, N. Miyazaki, H. Naitow, R. H. Cheng, A. Nakagawa, H. Mizuno, T. Tsukihara, and T. Omura The Amino-Terminal Region of Major Capsid Protein P3 Is Essential for Self-Assembly of Single-Shelled Core-Like Particles of Rice Dwarf Virus J. Virol., March 15, 2004; 78(6): 3145 - 3148. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
