Multiple envelope proteins are involved in white spot syndrome virus ...

Arch Virol (2006) 151: 1309–1317 DOI 10.1007/s00705-005-0719-2

Multiple envelope proteins are involved in white spot syndrome virus (WSSV) infection in crayfish L. J. Li, J. F. Yuan, C. A. Cai, W. G. Gu, and Z. L. Shi State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, P.R. China Received October 10, 2005; accepted December 21, 2005 c Springer-Verlag 2006 Published online February 20, 2006

Summary. White spot syndrome virus (WSSV) is a devastating viral pathogen of cultured shrimp worldwide. Previous studies have shown that the intact virion consists of at least 39 structural proteins and, among them, six were identified as envelope proteins involved in the virus infection. In this paper, the structural proteins VP36A, VP36B and VP31 (J Virol 2004; 78: 11360–11370), containing the RGD motif, were expressed in Escherichia coli and used to produce specific antibodies. Western blot confirmed that VP36A is a newly reported envelope protein. A neutralization assay with these three antibodies demonstrated that VP36A, VP36B and VP31 could significantly delay the initial infection of crayfish, but mortality still reached 100% at day 11 post-injection. However, a neutralization assay with the combination of antibodies against different envelope proteins showed that a combination of VP36B and VP31 antibodies could strongly inhibit WSSV infection in crayfish. These results revealed that multiple envelope proteins are involved in WSSV infection in crayfish and that VP36B and VP31 play a key role during this process. Introduction White spot syndrome virus (WSSV), one of the major pathogens in the cultured shrimp, has, since 1993, caused significant economic losses to the shrimp-farming industry worldwide. It causes high mortality and has a broad host-range among crustacean species [1, 11, 12]. The unique genomic structure and low homology of WSSV to known proteins, as well as other characteristics such as histopathology, cytopathology and morphology classified this virus in a new family, the Nimaviridae (http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/fr-fst-g.htm). Because there is no permissive cell line for WSSV, recent work has focused mainly on structural proteins. To date, 39 proteins have been identified as WSSV

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structural proteins by powerful proteomic methods [6, 16, 24], and at least nine have been confirmed as envelope proteins [5–7, 10, 17, 21, 23, 25]. Moreover, in vivo neutralization experiments with anti-envelope protein antibodies demonstrated that at least six envelope proteins are involved in WSSV infection [7, 10, 17, 21]. Nevertheless, the molecular mechanism of these proteins’ participation in the infection is unknown. In this paper, three structural proteins, VP36A, VP36B and VP31 [16], containing the RGD motif, were expressed to produce antibodies and investigate their function during infection. Materials and methods Crayfish The crayfish Procambarus clarkii was used in viral proliferation and neutralization assays. Crayfish were purchased from Wuhan market (Hubei Province, China); each was 25–30 g in weight and about 10 cm in length. They were reared for at least 3 days and, before experimental infection, confirmed to be WSSV-free by PCR with WSSV-specific primers. Virus proliferation and purification Proliferation and purification were conducted as described previously [7]. Briefly, an inoculum was prepared from WSSV-infected crayfish tissue (gills and pleopods). Tissue was homogenized in TN buffer (0.02 M Tris–HCl, 0.4 M NaCl, pH 7.4) at a ratio of 1:10 (w/w). After centrifugation at 1400 × g for 15 min, the viral suspension was passed through a 0.22-µm filter and injected into healthy crayfish (at a dilution of 1:4–1:10). Seven days post-injection (p.i.), fresh haemolymph was collected from the moribund crayfish and layered immediately onto 10–50% (w/w) continuous sucrose gradients prepared with CN buffer (0.0272 M citrate sodium, 0.072 M NaCl, pH 7.4), then centrifuged at 110 000 × g for 1 h at 4 ◦ C. The virus band was removed and precipitated at 110 000 × g for 45 min at 4 ◦ C. The virus pellet was resuspended in TNE buffer (0.5 M Tris–HCl, 0.1 M NaCl, 0.01 M EDTA, pH 7.4). Expression of VP36A, VP36B and VP31 in E. coli Expression plasmids were generated in fusion with a GST tag for the three proteins. For the vp36A gene, the forward primer was: 5 ATA GGA TCC ATG GCA TTA CAG GAA 3 , containing a BamHI site, and the reverse primer was: 5 GGC AAG CTT TCA AAC TAC TAC TAT 3 , containing a Hind III site. For the vp36B gene, the forward primer was: 5 TAA GGA TCC ATG GCG GTA AAC TTG 3 , containing a BamHI site, and the reverse primer was: 5 GGC AAG CTT TTA TGT CCA ACA ATT 3 , containing a Hind III site. For the VP31 gene, the forward primer was: 5 AGC GGA TCC ATG TCT AAT GGC GCA 3 , containing a BamHI site, and the reverse primer: 5 CGA GTC GAC TTA CTC CTC CTT 3 , containing a Sal I site. All these genes were amplified from genomic WSSV DNA by PCR and then ligated to the pGEM-T Easy Vector (Promega). The recombinant plasmids were confirmed by DNA sequencing before ligation to the expression vectors. All three genes were ligated into the pGEX-KG vector after digestion with restriction enzymes and expressed in E. coli DH5α. Expression and purification of protein were performed following a standard protocol [14]. Briefly, recombinant plasmids were transformed into competent E. coli DH5α and over-expressed by induction with of 1 mM IPTG (Isopropyl-β-D-thiogalactopyranoside) at 37 ◦ C. After incubation for 4 h, the induced bacteria were spun down at 4 ◦ C, suspended in ice-cold phosphate-buffered saline and sonicated for 10 min on ice. The insoluble debris

Multiple envelope proteins of WSSV

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containing the fusion protein was collected by centrifugation at 5,000 g and washed three times using PBS. Polyclonal antibodies against VP36A, VP36B, VP31 and WSSV virions The purified fusion protein was subjected to SDS–polyacrylamide gel electrophoresis (SDS– PAGE) analysis, then cut from the gel and used for antibody production, according to the protocol described by Huang et al. [7]. In addition to the recombinant VP36A, VP36B and VP31, an antibody against purified virions was also prepared according to standard protocol. Titers of antisera were determined by Western blot assay. Immunoglobulin (IgG) antibodies were purified by protein A-Sepharose (Promega) and stored at −20 ◦ C. Localization of VP36A Separation of envelope and nucleocapsids was performed as described previously [7]. Briefly, a volume of 250 µl resuspended virions was treated with 1% NP-40, 10 mM Tris–HCl, pH 8.5, for 2 h at room temperature with gentle agitation, then layered onto 6 ml 30% (v/v) glycerol (10 mM Tris–HCl, pH 8.5) and centrifuged at 150 000 × g for 60 min at 4 ◦ C. The envelope protein was recovered from the top of the gradient, then acetone-precipitated and dissolved in TNE after being centrifuged at 14000 × g for 30 min. The pelleted nucleocapsids were resuspended in TNE. The purified intact virions, nucleocapsids and envelope proteins were subjected to SDS– PAGE and then transferred to a nitrocellulose membrane. The membrane was blocked in 3% (w/v) BSA in TBS (0.2 M NaCl, 50 mM Tris–HCl, pH 7.5) for 2 h, followed by incubation with polyclonal rabbit anti-VP36A IgG diluted 1:1000 in Solution 1 (Can Get Signal Immunoreaction Enhancer Solution; TOYOBO) for 1 h at room temperature. After washing three times with TBS-T (0.1% Tween 20 in Tris-buffered saline), the membrane was incubated with Table 1. Composition of the injected solutions Group

Type

Constitution of injection

1 2 3 4 5 6

positive control preimmune serum VP36A antiserum VP36B antiserum VP31 antiserum VP36A + VP36B antisera

7

VP36A + VP31 antisera

8

VP36B + VP31 antisera

9

VP36A + VP36B + VP31 antisera

10

VP36A + VP36B + VP31 + VP76a antisera WSSV antiserum negative control

100 µl WSSV + 100 µl TNE 100 µl WSSV + 100 µl preimmune serum 100 µl WSSV + 100 µl VP36A antiserum 100 µl WSSV + 100 µl VP36B antiserum 100 µl WSSV + 100 µl VP36A antiserum 100 µl WSSV + 100 µl antisera of VP36A and VP36B (1:1) 100 µl WSSV + 100 µl antisera of VP36A and VP31 (1:1) 100 µl WSSV + 100 µl antisera of VP36B and VP31 (1:1) 100 µl WSSV + 100 µl antisera of VP36A, VP36B and VP31 (1:1:1) 100 µl WSSV + 100 µl antisera of VP36A, VP36B, VP36A and VP76 (1:1:1:1) 100 µl WSSV + 100 µl WSSV antiserum 200 µl TNE

11 12

aAntiserum

prepared by Huang et al. (2005) [7]

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alkaline phosphatase-conjugated goat anti-rabbit IgG diluted 1500-fold in Solution 2 (Can Get Signal Immunoreaction Enhancer Solution; TOYOBO). The membrane was washed as described above, and then detected using BCIP and NBT. Virus quantification and neutralization assay Virus concentration was determined by real-time PCR with SYBR Green chemistry. The appropriate concentration of virion, which resulted in 100% mortality, was used as the positive control. The composition of the injected solutions is shown in Table 1. Before injection into crayfish, WSSV virions were incubated with antibodies for 2 h. An injection of TNE was used as the negative control. Ten crayfish were used for each group and three replicates were performed. Crayfish mortality was monitored twice every day.

Results Virus quantification The concentration of purified virus was 8.7×108 copies per µl by Real-time PCR. Infection with 103 –105 virus copies resulted in a mortality of 100% after 10 days, while administration of 1.06×106 virus copies resulted in 100% mortality after 3–7 days (data not shown). Therefore, a WSSV inoculum of 1.06×106 virus copies was chosen as the dose for further experiment. Expression of VP36A, VP36B and VP31 and preparation of antibodies The WSSV ORFs encoding VP36A, VP36B and VP31 were expressed as GST fusion proteins in E. coli. Bands corresponding to the three fusion proteins were observed at the expected theoretical molecular weights (Fig. 1). Antigenic specificity and sensitivity of each antiserum against the fusion proteins and intact virions were detected by Western blotting (data not shown).

Fig. 1. Expression of VP36A, VP36B and VP31 in E. coli. 1: low molecular mass protein markers; 2: pGEX-KG, non-induced; 3: pGEX-KG, induced; 4: pGEX-KG-VP36A, noninduced; 5: pGEX-KG-VP36A, induced; 6: pGEX-KG-VP36B, non-induced; 7: pGEX-KGVP36B, induced; 8: pGEX-KG-VP31, non-induced; 9: pGEX-KG-VP31, induced


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Fig. 2. Localization of VP36A by Western blot. a SDS–PAGE of WSSV structural proteins; b Western blot of WSSV structural proteins withVP36A antibody. M, protein molecular marker; 1, intact virions; 2, envelope protein portion; 3, nucleocapsids

Western blot analysis and localization of VP36A in the virions To further characterize VP36A, purified intact WSSV virions, nucleocapsids and envelope proteins were subjected to SDS–PAGE (Fig. 2a), transferred onto nitrocellulose membranes and probed with rabbit anti-VP36A IgG. In the Western blot analysis, the anti-VP36A IgG (1:1000) showed a clear band in the intact virion and envelope protein fractions (Fig. 2b). WSSV neutralization The result of neutralization assays is shown in Fig. 3. No crayfish died in the negative control, i.e. injected with TNE only (group 12), while the crayfish injected with WSSV (group 1) showed 100% mortality at day 7 post-infection (p.i.). The group injected with mixed preimmune serum and WSSV (group 2) showed a similar result to that of the positive control. When the virus was pre-incubated with the antisera of VP36A, VP36B and VP31 (groups 3, 4 and 5, respectively), crayfish mortality was 100% at day 11, 10 and 10, respectively, but with an obvious initial delay in death. However, when the virus was pre-incubated with a combination of antisera against different envelope proteins, crayfish mortality significantly dropped (groups 6–10), especially with the combination of VP36B and VP31 antisera (group 8), which resulted in only 27% mortality at day 14 and had the same neutralization effect as whole-virion antiserum (group 11). The combination of VP36A and VP36B antisera, as well as VP36A and VP31 antisera (group 6 and 7), resulted in a slightly higher mortality of 57 and 67%, respectively, at day 14 p.i. Other combinations (groups 9 and 10) showed the same results as group 8.

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Fig. 3. In vivo neutralization of WSSV infection in crayfish. The antibodies used are listed in Table 1. Days post-infection are shown on the abscissa and cumulative mortality on the ordinate

Discussion In the WSSV genome there are six envelope proteins, identified, by neutralization assay, as being involved in WSSV infection. Any individual antibody against these envelope proteins could inhibit the infection efficiently. Shrimp mortality in these neutralization assays in all cases reached 100% [7, 10, 17, 21]. In this study, we chose three structural proteins, VP36A, VP36B and VP31, containing a RGD motif, of which two were characterized to be envelope proteins involved in WSSV infection [10, 21]. These three proteins were first expressed and used to produce specific antibodies, then VP36A was localized as a minor envelope protein by Western blot. A further in vivo neutralization assay with VP36A antibody revealed that VP36A is also involved in WSSV infection.


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However, the neutralization assay with a combination of antibodies against different envelope proteins gave an unexpected result. The combination of antibodies against VP31 and VP36B could significantly decrease crayfish mortality (only 27% at day 14 p.i.), and this result appeared only when an antibody against whole virions was used in the experiment. An assay with the combination of VP36A + VP36B and VP36A + VP31 antibodies also decreased the mortality of infected crayfish, but less efficiently than VP36B + VP31. Nevertheless, the addition of antibodies of more envelope proteins (VP76 and VP36A) to VP36B and VP31 did not result in a much stronger inhibition of WSSV infection. These results revealed that multiple envelope proteins are involved in WSSV infection in crayfish and that VP36B and VP31 play a key role during this process. Some viral envelope proteins mediate cell adhesion in an RGD-dependent manner. The RGD tripeptide mediates cell recognition and infectivity of a variety of pathogens, including adenovirus 2, coxsackievirus A9, human herpesvirus 8, hepatitis A virus and foot-and-mouth disease virus etc. [2, 4, 8, 13, 15, 19, 20]. In the WSSV genome, there are six structural proteins (VP36A, VP36A, VP36B, VP110, VP136 and VP664), which have a RGD motif [22]. All were proven to be structural proteins by proteomic methods [16]. VP31 and VP36B (VP281) were recently shown to be envelope proteins involved in virus infection [10, 21], and VP664 is a nucleocapsid protein [9]. However, whether the RGD motifs in these proteins play a key role needs to be elucidated through further research. Our results are useful in understanding the pathway of WSSV entry into the host cells and are a useful guide in controlling WSSV infection and transmission. The envelope proteins of WSSV, such as VP36A, VP36B and VP31, may serve as cell attachment proteins and play an important role in mediating virus–host interaction. Acknowledgements This work was supported by the Hi-Tech Research and Development Program of China (grant no: 2001AA620201 and 2003AA620201) and the Chinese Academy of Sciences (grant no: KSCX2-SW-302).

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