Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands1
Author for correspondence: Just Vlak. Fax +31 317 484820. e-mail just.vlak{at}medew.viro.wau.nl
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Abstract |
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The characterization of the structural proteins and their genomic sequence is of major importance to determine the taxonomic position of the virus. Furthermore, the structure and interaction of the WSSV virion proteins may explain the unique morphological features of this virus. Finally, diagnostic tests could be designed based on one or more of these structural proteins. VP28 and VP26, present in the envelope and nucleocapsid, respectively, were identified previously and showed no homology with sequences available in GenBank (Van Hulten et al., 2000b ). Here we report the identification of a third major structural protein, VP24, and the surprising relatedness of this protein to the previously identified WSSV structural proteins.
Purified WSSV was used to infect shrimp, Penaeus monodon, by intramuscular injections in the lateral area of the fourth abdominal segment. Virions were purified from haemolymph of infected P. monodon as described by Van Hulten et al. (2000b) . As a negative control, haemolymph was taken from uninfected P. monodon. The preparations were analysed by electron microscopy for the presence and purity of WSSV virions (not shown). The viral envelope was removed from the nucleocapsid by treatment with 1% NP-40 (Van Hulten et al., 2000b
). In the intact WSSV virions purified from P. monodon (Fig. 1a
, lane 3), four major polypeptide species were identified with apparent molecular masses of 28 kDa (VP28), 26 kDa (VP26), 24 kDa (VP24) and 19 kDa (VP19). From the SDSPAGE analysis (Fig. 1a
) it can be seen that VP26 and VP24 are the major proteins present in the purified nucleocapsids (Fig. 1a
, lane 4). VP28 and VP19 are removed by the NP-40 treatment and therefore associated with the viral envelope or tegument (Van Hulten et al., 2000b
). A schematic presentation of the WSSV virion is shown in Fig. 1(b)
.
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VP24 isolated from P. monodon was transferred from an SDSPAGE gel onto a PVDF membrane by semi-dry blotting. The 24 kDa band, derived from two separate WSSV preparations, was excised from the membrane and each was sequenced by Edman degradation as described previously (Van Hulten et al., 2000b ). The first N-terminal sequence obtained was MHMWGVYAAILAGLTLILVVIdI, of which the aspartic acid at position 22 was uncertain. From the second VP24 band more than 40 residues were sequenced (bold font in Fig. 2
) giving the sequence MHMWGVYAAILAGLTLILV VISIVVTNIELNKKLDKKDKdA, in which a serine residue was found at position 22 and an uncertain aspartic acid at position 40.
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The complete vp24 ORF, encompassing 627 nucleotides, and the promoter region of this gene, were found on the 18 kbp BamHI fragment (Fig. 2). The translational start codon was in a favourable context (AAAATGC) for efficient eukaryotic translation initiation (Kozak, 1989
). In the promoter region stretches of A/T-rich sequence, but no consensus TATA box, were found. A poly(A) signal overlapped the translation stop codon. The vp24 ORF encoded a putative protein of 208 amino acids with an amino acid sequence containing the experimentally determined N-terminal sequence of VP24. VP24 has a theoretical size of 23 kDa and an isoelectric point of 8·7. Four potential sites for N-linked glycosylation (N-{P}-[ST]-{P}), one site for O-glycosylation (Hansen et al., 1998
) (Fig. 2
) and nine possible phosphorylation sites ([ST]-X-X-[DE] or [ST-X-[RK]) were found within VP24, but it is not known whether any of these modifications do occur. No other motifs present in the PROSITE database were found in VP24. Computer analysis of the 208 amino acids showed that a strong hydrophobic region was present at the N terminus of VP24 (Fig. 3a
), including a putative transmembrane
-helix formed by amino acids 6 through 25. The algorithm of Garnier et al. (1978)
predicted several other
-helices and
-sheets along the protein. It is remarkable that VP28, VP26 and VP24 roughly have the same size (
206 amino acids) but have distinct electrophoretic mobilities. This may be due to differences in isoelectric points, conformational differences or post-translational modifications.
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As there is a high homology at the amino acid level among the three structural WSSV proteins, and conserved domains are present, there is reason to believe that their structures are similar. The presence of the hydrophobic domain indicates that these proteins most probably are capable of forming homo- and heteromultimers. Studies on the interaction of these proteins and their location in the virion are required to substantiate this hypothesis.
A way to explain the high degree of amino acid similarity of the three structural WSSV proteins is to assume that these genes have evolved by gene duplication and divergence. Nucleotide comparisons supported this hypothesis, as significant homology was found. Alignment of vp24, vp26 and vp28, revealed that vp24 has 40% nucleotide identity with vp26 and 43% with vp28, whereas vp26 has 48% nucleotide identity with vp28. The data presented here strongly suggest that these three WSSV structural protein genes share a common ancestor.
The most surprising observation might be that these proteins have evolved to give proteins with different functions in the WSSV virion, i.e. in the nucleocapsid and the envelope. Such a situation is unusual in animal DNA viruses, although a parallel may exist for the virion glycoproteins of alphaherpesviruses as their genes might have evolved by duplication and divergence (McGeoch, 1990 ). However, the homology of these genes is considerably lower than the homology among the WSSV virion genes. Also, the function of the alphaherpesvirus genes has not diverged. Gene duplication and functional divergence, however, can be observed in the plant-infecting closteroviruses, where a minor regulatory protein, VP24, appears to be a diverged copy of the coat protein (Boyko et al., 1992
). Also, in the animal rhabdoviruses, a structural and a non-structural glycoprotein may have evolved from a common ancestral gene (Wang & Walker, 1993
).
Structural proteins are well conserved within virus families. However, the three WSSV structural proteins identified so far have no homology to structural proteins of other viruses. The unique feature of the homologous structural virion proteins further supports the proposition that WSSV might be a representative of a new virus genus (Whispovirus) or perhaps a new family (Whispoviridae) (Van Hulten et al., 2000a , b
; Van Hulten & Vlak, 2000
).
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Footnotes |
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References |
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Boyko, V. P., Karasev, A. V., Agranovsky, A. A., Koonin, E. V. & Dolja, V. V. (1992). Coat protein gene duplication in a filamentous RNA virus of plants.Proceedings of the National Academy of Sciences, USA 89, 9156-9160.[Abstract]
Durand, S., Lightner, D. V., Redman, R. M. & Bonami, J. R. (1997). Ultrastructure and morphogenesis of white spot syndrome baculovirus (WSSV).Diseases of Aquatic Organisms 29, 205-211.
Flegel, T. W. (1997). Major viral diseases of the black tiger prawn (Penaeus monodon) in Thailand.World Journal of Microbiology and Biotechnology 13, 433-442.
Garnier, J., Osguthorpe, D. J. & Robson, B. (1978). Analysis of the accuracy and implications of simple method for predicting the secondary structure of globular proteins. Journal of Molecular Biology 120, 97-120.[Medline]
Hameed, A. S. S., Anilkumar, M., Raj, M. L. S. & Jayaraman, K. (1998). Studies on the pathogenicity of systemic ectodermal and mesodermal baculovirus and its detection in shrimp by immunological methods.Aquaculture 160, 31-45.
Hansen, J. E., Lund, O., Tolstrup, N., Gooley, A. A., Williams, K. L. & Brunak, S. (1998). NetOglyc: prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility.Glycoconjugate Journal 15, 115-130.[Medline]
Kozak, M. (1989). The scanning model for translation: an update.Journal of Cell Biology 108, 229-241.[Abstract]
McGeoch, D. J. (1990). Evolutionary relationships of virion glycoprotein genes in the S regions of alphaherpesvirus genomes.Journal of General Virology 71, 2361-2368.[Abstract]
Nadala, E. C. B., Tapay, L. M. & Loh, P. C. (1998). Characterization of a non-occluded baculovirus-like agent pathogenic to penaeid shrimp.Diseases of Aquatic Organisms 33, 221-229.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Research 22, 4673-4680.[Abstract]
Van Hulten, M. C. W. & Vlak, J. M. (2000). Genetic evidence for a unique taxonomic position of white spot syndrome virus of shrimp: genus Whispovirus. Proceedings of the Fourth Symposium on Diseases in Asian Aquaculture. Edited by C. Lavilla-Pitogo and others (in press).
Van Hulten, M. C. W., Tsai, M. F., Schipper, C. A., Lo, C. F., Kou, G. H. & Vlak, J. M. (2000a). Analysis of a genomic segment of white spot syndrome virus of shrimp containing ribonucleotide reductase genes and repeat regions.Journal of General Virology 81, 307-316.
Van Hulten, M. C. W., Westenberg, M., Goodall, S. D. & Vlak, J. M. (2000b). Identification of two major virion protein genes of white spot syndrome virus of shrimp.Virology 266, 227-236.[Medline]
Wang, Y. & Walker, P. J. (1993). Adelaide River rhabdovirus expresses consecutive glycoprotein genes as polycistronic mRNAs: new evidence of gene duplication as an evolutionary process.Virology 195, 719-731.[Medline]
Wang, Q., Poulos, B. T. & Lightner, D. V. (2000). Protein analysis of geographic isolates of shrimp white spot syndrome virus.Archives of Virology 145, 263-274.[Medline]
Wongteerasupaya, C., Vickers, J. E., Sriurairatana, S., Nash, G. L., Akarajamorn, A., Boonsaeng, V., Panyim, S., Tassanakajon, A., Withyachumnarnkul, B. & Flegel, T. W. (1995). A non-occluded, systemic baculovirus that occurs in cells of ectodermal and mesodermal origin and causes high mortality in the black tiger prawn Penaeus monodon.Diseases of Aquatic Organisms 21, 69-77.
Yang, F., Wang, W., Chen, R. Z. & Xu, X. (1997). A simple and efficient method for purification of prawn baculovirus DNA.Journal of Virological Methods 67, 1-4.[Medline]
Received 17 April 2000;
accepted 3 July 2000.