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Short Communication |
1 Laboratory of Virology, National Agricultural Research Center, Tsukuba, Ibaraki 305-8666, Japan
2 Division of Bio-Crystallography Technology, RIKEN Harima Institute, Sayo-gun, Hyogo 679-5148, Japan
Correspondence
Kyoji Hagiwara
kyoji{at}affrc.go.jp
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ABSTRACT |
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), and one recently proposed genus, Seadornavirus (Attoui et al., 2000), only cypoviruses have single-shelled particles. The three-dimensional structure of cypovirus particles has been determined using cryo-electron microscopy (Hill et al., 1999; Zhang et al., 1999) and a recent publication suggested that the turret (spike) plays an important role in maintaining capsid integrity (Zhang et al., 2002). However, it is not known whether the major capsid protein of cypoviruses has the ability to assemble into single-shelled particles. Bombyx mori cypovirus 1 (BmCPV-1), a type member of the genus Cypovirus, has six structural proteins, VP1, VP2, VP3, VP4, VP6 and VP7, encoded by segments S1, S2, S3, S4, S6 and S7, respectively (Hagiwara & Matsumoto, 2000; Hagiwara et al., 2002). In this study, we expressed VP1 in insect cells using a baculovirus system to determine the ability of VP1 to assemble into virus-like particles.
The cDNA of genome segment S1 of BmCPV-1 cloned into pBluescript KS+ (Hagiwara et al., 2002) was subcloned into the pBlueBacIII transfer vector for baculovirus expression. Restriction enzyme analysis and DNA sequencing were performed to confirm that the coding sequence of the S1 segment was correctly oriented with the baculovirus polyhedrin promoter. The resulting transfer plasmid and linearized AcNPV DNA were co-transfected into Sf9 cultured cells using CellFECTIN, according to the manufacturer's instructions (Invitrogen), and recombinant VP1 was expressed in the insect cells.
To detect expression of VP1, antibody against intact virus was produced. BmCPV-1 particles were purified from polyhedra and two BALB/c mice were immunized with 100 µg of these particles, as described previously (Hagiwara et al., 1998). The serum was collected 4 days after the last immunization. In SDS-PAGE and Western blot analysis using BmCPV-1-specific antibody, major bands were detected in recombinant baculovirus-infected Sf9 cells (Fig. 1, lane 3) at the same position as VP1 of purified BmCPV-1 particles (Fig. 1, lane 1), indicating that the major capsid protein VP1 was correctly expressed by the baculovirus system.
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b-1). To confirm that the virus-like particles were constructed from VP1 protein, immunogold staining of the particles was performed using BmCPV-specific antibody against intact particles according to the method described by Lin (1984). As shown by electron microscopy [Fig. 2(a-2 and b-2)], intact BmCPV-1 and VP1 particles were specifically labelled with gold particles. SDS-PAGE and Western blot analyses of purified VP1 protein (Fig. 1
, lane 4) also confirmed that the particles were composed of expressed VP1. These results indicate that VP1 protein has the ability to form a single-shelled particle without the assistance of any of the other structural proteins of BmCPV-1.
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). VP1 particles were completely broken down by pH 4·2 at 37 °C for 5 min, while intact particles were completely broken down by pH 3·7 at 37 °C for 5 min (Table 1). Under alkaline conditions the stability of the two particle types was almost the same: both BmCPV and VP1 particles retained their structural integrity when incubated in 20 mM PBS, pH 12·3, at 37 °C for 5 min (Table 1). These results suggest that other structural proteins play an important role in stabilizing the particles under acidic conditions.
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). Our study has demonstrated that these exterior and interior proteins are not required for construction of the VP1 shell, but that they may influence the stability of virus-like particles, suggesting that attachment of these proteins on the fivefold axes may play an important role in maturation of the virus particles. It seems that both exterior (spikes) and interior proteins (such as RNA polymerase) recognize the icosahedral fivefold symmetry when they attach to the shell. Thus, the ability of VP1 to form a single-shelled particle independent of other structural proteins is an extremely important function for assembly of cypovirus particles.
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REFERENCES |
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Hagiwara, K. & Matsumoto, T. (2000). Nucleotide sequences of genome segments 6 and 7 of Bombyx mori cypovirus 1, encoding the viral structural proteins V4 and V5, respectively. J Gen Virol 81, 1143–1147.
Hagiwara, K., Tomita, M., Nakai, K., Kobayashi, J., Miyajima, S. & Yoshimura, T. (1998). Determination of the nucleotide sequence of Bombyx mori cytoplasmic polyhedrosis virus segment 9 and its expression in BmN4 cells. J Virol 72, 5762–5768.
Hagiwara, K., Rao, S., Scott, S. W. & Carner, G. R. (2002). Nucleotide sequences of segments 1, 3 and 4 of the genome of Bombyx mori cypovirus 1 encoding putative capsid proteins VP1, VP3 and VP4, respectively. J Gen Virol 83, 1477–1482.
Hill, C. L., Booth, T. F., Prasad, B. V. V., Grimes, J. M., Mertens, P. P. C., Sutton, G. C. & Stuart, D. I. (1999). The structure of a cypovirus and the functional organization of dsRNA viruses. Nat Struct Biol 6, 565–568.[CrossRef][Medline]
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]
Mertens, P. P. C., Arella, M., Attoui, H. & 41 other authors (2000). Family Reoviridae. In Virus Taxonomy. Classification and Nomenclature of Viruses. Seventh Report of the International Committee on Taxonomy of Viruses, pp. 395–480. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. San Diego: Academic Press.
Zhang, H., Zhang, J., Yu, X., Lu, X., Zhang, Q., Jakana, J., Chen, D. H., Zhang, X. & Zhou, Z. H. (1999). Visualization of protein–RNA interactions in cytoplasmic polyhedrosis virus. J Virol 73, 1624–1629.
Zhang, H., Yu, X.-K., Lu, X.-Y., Zhang, J.-Q. & Zhou, Z. H. (2002). Molecular interactions and viral stability revealed by structural analysis of chemically treated cypovirus. Virology 298, 45–52.[CrossRef][Medline]
Received 11 March 2003;
accepted 19 May 2003.
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