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1 School of Life Science, Xiamen University, 178 Daxue Road, Xiamen 361005, People's Republic of China
2 Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, Xiamen, People's Republic of China
Correspondence
Feng Yang
mbiotech{at}public.xm.fj.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). It is a major pathogen in shrimp aquaculture and can also infect most species of crustacean (Chou et al., 1995; Lo et al., 1996; Chang et al., 1998; Wang et al., 1998; Chen et al., 2000; Corbel et al., 2001; Sahul Hameed et al., 2001). First appearing in the 1990s in Taiwan, WSSV spread quickly to South-East Asia, the Indian subcontinent and Central and Latin America, causing catastrophic economic losses. Electron-microscopy studies reveal that WSSV is an enveloped, non-occluded and bacilliform virus (Wang et al., 1995; Wongteerasupaya et al., 1995). The virus contains a double-stranded, circular DNA of about 300 kb, which has been sequenced completely in three WSSV isolates (van Hulten et al., 2001a; Yang et al., 2001; Chen et al., 2002). Approximately 180 open reading frames (ORFs) are revealed by the analysis of the WSSV genomic DNA sequence (GenBank accession no. AF332093
[GenBank]
).
In previous studies, major structural proteins of WSSV virions were separated by gel electrophoresis and analysed by mass spectrometry, and more than 30 polypeptides matching WSSV ORFs were identified with a molecular mass range of 7–660 kDa (Huang et al., 2002a; Tsai et al., 2004; Zhang et al., 2004). At present, more attention has been paid to the viral envelope proteins, as these commonly play key roles in the primary-infection phase. So far, about 10 genes of WSSV have been identified to encode envelope proteins by using Western blotting or immunoelectron microscopy (van Hulten et al., 2000b, 2002; Huang et al., 2002b; Zhang et al., 2002a, b, 2004; Li et al., 2005, 2006; Zhu et al., 2005, 2006). Some of these envelope proteins, such as VP28, VP68, VP281 and VP31, were believed to be involved in WSSV infection by in vivo neutralization experiments (van Hulten et al., 2001b; Li et al., 2005; Wu et al., 2005) and VP26 was supposed to help the viral nucleocapsid to move toward the nucleus by interacting with actin or cellular actin-binding proteins (Xie & Yang, 2005). However, due to the lack of an established shrimp-cell line, little is known about the molecular events underlying the WSSV life cycle and mode of infection.
VP24, the product of the wsv002 gene of WSSV (Yang et al., 2001), is a major structural protein of WSSV. Our initial inspection of the VP24 gene sequence suggested that it might be a component of the viral envelope. However, VP24 was thought to be a nucleocapsid protein (van Hulten et al., 2000a). In this report, a more precise localization of VP24 within WSSV virions was determined. In addition, far-Western and neutralization experiments were conducted to further characterize the possible role of VP24 in WSSV infection.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of antibody.
The purified proteins were used as antigen to immunize mice by intradermal injection once every 10 days. Antigen (20 µg) was mixed with an equal volume of Freund's complete adjuvant (Sigma) for the first injection. Subsequent injections were conducted by using 20 µg antigen mixed with an equal volume of Freund's incomplete adjuvant (Sigma). Four days after the last injection, mice were exsanguinated and antisera were collected. The immunoglobulin (IgG) fractions were purified by protein A–Sepharose (Amersham Biosciences) and stored at –70 °C.
Virus purification and detergent extraction of intact virus.
WSSV inoculum was prepared from Penaeus japonicus shrimp with pathologically confirmed infection. The infection of healthy crayfish, Procambarus clarkii, and the purification of virus were performed as described previously (Xie et al., 2005). Briefly, the tissues of infected crayfish, excluding the hepatopancreas, were homogenized in TNE buffer (50 mM Tris/HCl, 400 mM NaCl, 5 mM EDTA, pH 8.5) and then centrifuged at 3500 g for 5 min at 4 °C. After filtering by nylon net (400 mesh), the supernatant was centrifuged at 30 000 g for 30 min at 4 °C. Then, the upper loose pellet was rinsed out carefully and the lower white pellet was suspended in 10 ml TN buffer (20 mM Tris/HCl, 400 mM NaCl, pH 7.4). After centrifugation at 3500 g for 5 min, the virus particles were sedimented by centrifugation at 30 000 g for 20 min at 4 °C and then resuspended and kept in 1 ml TN buffer.
Viral envelope was separated from the nucleocapsids as described by van Hulten et al. (2000b). Briefly, intact virions were mixed with an equal volume of 2 % Nonidet P-40 (NP-40) and incubated for 30 min at room temperature with gentle shaking. The extract was separated into soluble (envelope) and insoluble pellet (nucleocapsid) fractions by centrifugation at 30 000 g for 20 min at 4 °C.
For the Triton X-114 extraction (Bordier, 1981), intact virions were mixed with an equal volume of 2 % Triton X-114 and incubated at 4 °C for 30 min. The mixture was sedimented at 15 000 g for 5 min to separate the phases. Both phases were subjected to a second round of Triton X-114 extraction. All samples were concentrated by acetone precipitation at –20 °C and analysed by SDS-PAGE, transferred to a membrane and detected with anti-VP24 serum.
Immunoelectron microscopy.
Purified WSSV virion and nucleocapsid suspensions were mounted on Formvar- or carbon-coated nickel grids (300 mesh), respectively, and incubated for 15 min at room temperature. Then, the grids were blocked with 2 % BSA in PBS for 1 h, followed by incubation with anti-VP24 serum (1 : 1000 dilution in 2 % BSA) for 1 h. After washing three times with PBS, the grids were incubated with goat anti-mouse IgG conjugated to 10 nm colloid gold (Sigma) for 1 h. Subsequently, the grids were washed three times with PBS and briefly negatively stained with 2 % phosphotungstic acid (PTA; pH 7.0) for 1 min. Specimens were examined under a transmission electron microscope (JEM 100 cx
). For control experiments, pre-immune mouse serum was used to replace the primary antibody indicated above.
Gel electrophoresis and Western blot.
Proteins dissolved in loading buffer were separated by standard SDS-PAGE (Laemmli, 1970) with a 12 % resolution gel and a 4 % stacking gel and then transferred onto a PVDF transfer membrane (Amersham Biosciences). The blot was immersed in incubation buffer (20 mM Tris/HCl, 150 mM NaCl, 0.05 % Tween 20, 3 % non-fat milk, pH 7.5) at 4 °C overnight followed by incubating with anti-VP24 serum (1 : 5000 dilution) for 2 h. Subsequently, alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (Promega) (1 : 7500 dilution) was used as the secondary antibody and detection was performed with a substrate solution containing 4-chloro-1-naphthol and X-phosphate (Roche).
Biotinylation of rVP24 protein and far-Western experiment.
Purified rVP24 protein was dialysed against PBS and adjusted to a concentration of 1 mg ml–1. Two hundred micrograms of EZ-Link sulfo-NHS-LC-biotin (Pierce) was added directly to 1 ml rVP24 protein solution. The mixture was incubated at room temperature for 30 min and dialysed against PBS to remove the unreacted biotin reagent.
The viral structural proteins were separated by SDS-PAGE, transferred to a PVDF membrane and renatured gradually at 4 °C overnight in HEPES buffer (20 mM HEPES, 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1 % Tween 20, 10 % glycerol, pH 7.5) containing 5 % non-fat milk. The blot was washed and incubated with 0.5 µg biotinylated rVP24 in 5 ml incubation buffer for 1 h at room temperature. The blot was subsequently washed three times and incubated with AP-conjugated streptavidin (Promega) for 1 h at room temperature. After washing three times, the detection was performed with a substrate solution containing 4-chloro-1-naphthol and X-phosphate. Biotinylated glutathione S-transferase (GST) was incubated with viral structural proteins as a control.
Coimmunoprecipitation experiments.
The viral envelope proteins were dialysed against PBS and labelled with EZ-Link sulfo-NHS-LC-biotin as described above. The biotinylated envelope proteins were incubated with anti-VP24 serum (1 : 250 dilution) overnight at 4 °C. Subsequently, 10 µl protein A–Sepharose beads was added to the mixture and incubated for 1 h at 4 °C. The Sepharose beads were collected by centrifugation and washed five times with 500 µl PBS. The bound proteins were dissociated from the antibody by boiling in loading buffer for 5 min and separated on 12 % SDS-PAGE gels. The separated proteins were transferred onto a PVDF membrane and the biotinylated proteins were detected as described above.
In vivo neutralization assay.
Virus concentration was quantified by competitive PCR as described by Xu et al. (2001). The in vivo neutralization experiment was performed as described by van Hulten et al. (2001b). Prior to injection, 100 µl virions (equivalent to 106 copies) was incubated with anti-GST IgG (group 2, 20 µg) or with various amounts of anti-VP24 IgG (group 3, 5 µg; group 4, 10 µg; group 5, 20 µg). Then, the mixture of antibody and WSSV was injected intramuscularly into crayfish. At the same time, a negative control (0.9 % NaCl, group 6) and a positive control (WSSV only, group 1) were included in the injection. For each treatment, 20 crayfish were used. Crayfish mortality caused by WSSV was monitored daily.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), consistent with the hydrophilic analysis (van Hulten et al., 2000a). For the convenience of purification of the soluble VP24, a truncated form of VP24 without the N-terminal transmembrane domain from aa 1 to 25 was cloned into a bacterial expression vector upstream of a (His)6 tag. rVP24 was expressed as a soluble protein and purified through an Ni–NTA affinity column.
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, following NP-40 treatment, the typical envelope proteins (VP28 and VP19) were present exclusively in the envelope fraction and the nucleocapsid protein (VP15) was found completely in the nucleocapsid fraction, as expected. VP24 was only found in the envelope fraction, which was validated by Western blot. When the virions were subjected to treatment with other detergents, such as Triton X-100, VP24 also partitioned entirely in the envelope fraction, consistent with the literature data reported previously (Xie et al., 2005).
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). As envelope proteins contain hydrophobic sequences that anchor them in the envelope, this phase-separation technique can be used operationally to separate envelope proteins from soluble proteins. After this extraction technique, VP24 was found completely in the detergent phase, indicating that it behaves as a hydrophobic protein (Fig. 3).
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), whereas no gold particles could be observed on the viral nucleocapsids when using the same antibody (Fig. 4b). The control experiments showed that no gold particles were found on the envelopes of WSSV virions (Fig. 4c) when using pre-immune mouse serum as the primary antibody. All of these studies demonstrated that VP24 is an envelope protein of WSSV.
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). No signal was detected in the GST control.
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, VP28 was immunoprecipitated with the anti-VP24 antibody. The results of the Western analysis (Figs 2 and 3) showed that there was no cross-reactivity of anti-VP24 antibody with VP28, so VP28 was pulled down by interacting with VP24.
VP24 antibody neutralization in vivo
The anti-VP24 IgG was used in an in vivo neutralization assay. A constant amount of WSSV was incubated with various amounts of anti-VP24 IgG and injected into crayfish. The results (Fig. 6) showed that the positive-control group (WSSV only, group 1) displayed 100 % mortality by day 7 post-infection, whereas the negative-control group (0.9 % NaCl, group 6) showed no mortality. Addition of anti-GST IgG (group 2) resulted in a small initial delay of mortality, which reached 100 % by day 9. This could be due to compounds injected in crayfish stimulating the host-defence system. The groups injected with various amounts of anti-VP24 IgG mixed with virions exhibited 100 % mortality by days 12, 15 and 19 for groups 3–5, respectively. The above experiments were performed once again and the same result could be obtained. Therefore, it could be concluded that the infection of WSSV was truly delayed or neutralized by the antibody against VP24.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 2002; Zhang et al., 2002a, b; Xie & Yang, 2005) and VP15 with the nucleocapsid (van Hulten et al., 2002; Witteveldt et al., 2005). VP24 was initially thought to be a nucleocapsid protein (van Hulten et al., 2000b), but its localization was not confirmed by immunoelectron microscopy. Recent data in the literature showed that VP24 was present exclusively in the envelope fraction when high-purity virions were subjected to non-ionic-detergent treatment (Li et al., 2005; Xie & Yang, 2005; Xie et al., 2005), indicating that it might be an envelope protein. Furthermore, examination of the predicted protein sequence also suggested that VP24 is sufficiently hydrophobic to be envelope-associated (van Hulten et al., 2000a). Therefore, we decided to undertake a detailed biochemical characterization of VP24.
Using our highly purified virus, we used a method described by van Hulten et al. (2000b) to separate viral envelopes from the nucleocapsids and obtained cleaner extraction, such that VP24 and the major envelope proteins, e.g. VP28 and VP19, were found exclusively in the envelope fraction (Fig. 2). However, we could not exclude the possibility that different methods and protocols used for separating the envelope or nucleocapsid protein might produce different results. Therefore, we used another detergent (Triton X-100) to extract envelope proteins, obtaining the same results. In addition, Triton X-114 extraction and immunoelectron microscopy confirmed that VP24 behaves as an envelope protein (Figs 3 and 4). According to our current data, this would mean that VP24 is probably envelope-associated.
To date, the function of VP24 in the life cycle of the virus is still unknown. Amino acid analysis of VP24 and VP28 indicated that these two proteins have about 43 % amino acid identity (van Hulten et al., 2000a) and it was reported that VP28 could bind to shrimp cells as an attachment protein and help the virus to enter the cytoplasm (Yi et al., 2004). Therefore, we were originally interested in examining whether VP24 is involved in the process of virus entry, as is VP28. First, a cell-binding assay in vitro using biotinylated rVP24 was performed. The result indicated that VP24 could not bind to host-cell membranes (data not shown). Subsequently, in order to study further whether VP24 is involved in WSSV infection, an in vivo neutralization experiment with anti-VP24 IgG was conducted. The results revealed that infection by WSSV could be delayed significantly by anti-VP24 IgG, suggesting that VP24 plays a role in the WSSV infection. In addition, we have provided evidence for the direct interaction of VP24 and VP28 and we further postulate that VP24 and VP28 form a protein complex and participate in virus infection together.
Besides participation in virus infection, envelope proteins often play vital roles in virus assembly and budding (Chazal & Gerlier, 2003). Although during the last decade, intensive efforts have been undertaken for characterization of the structural genes, the initial steps in WSSV morphogenesis are poorly understood. Crucial to the understanding of the molecular events is the need to identify the many molecules involved. In this study, additional in vitro experiments provided evidence for the direct interaction of VP24 and VP28, the first time that the interaction between these structural proteins has been identified in WSSV. It was reported that protein–protein interactions are needed for viral morphogenesis of Vaccinia virus (VACV) (Szajner et al., 2003). VACV G7L protein interacts with the A30L protein and the stability of each was dependent on its association with the other. Both structural proteins are required for the association of dense viroplasm with viral membranes. Although, at present, we could not conclude whether VP24 and VP28 are required for WSSV morphogenesis, the identification of the VP24–VP28 interaction might provide insight to further investigations of the mode of morphogenesis.
Further research is required to reveal the exact role of VP24 in WSSV infection or morphogenesis. Efforts to identify additional proteins associated with VP24 are in progress. We anticipate that further exploration of the functions of envelope proteins, including VP24, will facilitate a better understanding of the molecular mechanism underlying WSSV infection and assembly and may be helpful for the diagnosis and control of virus infection.
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ACKNOWLEDGEMENTS |
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Received 30 September 2005;
accepted 22 February 2006.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
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); group 2, virus incubated with anti-GST IgG (20 µg) (
); groups 3–5, virus incubated with various amounts of anti-VP24 IgG [5 µg (
), 10 µg (x) or 20 µg (*), respectively]; group 6, negativecontrol (
). In all groups, virions injected into crayfish were equivalent to 106 copies.