Identification and Characterization of a Prawn White Spot Syndrome Virus Gene That Encodes an Envelope Protein VP31

Virology 340 (2005) 125 – 132 www.elsevier.com/locate/yviro

Identification and characterization of a prawn white spot syndrome virus gene that encodes an envelope protein VP31

Li Li, Xixian Xie, Feng Yang*

Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, Xiamen, P.R. China

Received 13 April 2005; returned to author for revision 9 May 2005; accepted 3 June 2005 Available online 14 July 2005

Abstract

Based on a combination of SDS-PAGE and mass spectrometry, a protein with an apparent molecular mass of 31 kDa (termed as VP31) was identified from purified shrimp white spot syndrome virus (WSSV) envelope fraction. The resulting amino acid (aa) sequence matched an open reading frame (WSV340) of the WSSV genome. This ORF contained 783 nucleotides (nt), encoding 261 aa. A fragment of WSV340 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein with a 6His-tag, and then specific antibody was raised. Western blot analysis and the immunoelectron microscope method (IEM) confirmed that VP31 was present exclusively in the viral envelope fraction. The neutralization experiment suggested that VP31 might play an important role in WSSV infectivity. D 2005 Elsevier Inc. All rights reserved.

Keywords: White spot syndrome virus; VP31; Envelope protein; The neutralization experiment

Introduction VP292, and VP124 were identified as viral envelope proteins by immunoelectron microscopy (Huang et al., White spot syndrome virus (WSSV) is a major shrimp 2002a, 2002b; Zhang et al., 2002a, 2002b, 2004). As far pathogen that is highly virulent in penaeid shrimp and can as functional research is concerned, VP28, VP281, also infect most species of crustacean (Lo et al., 1996a, VP466, and VP68 were suggested to play an important 1996b; Chen et al., 1997; Flegel, 1997). The complete role in the systemic WSSV infection in shrimp by using sequence of WSSV genome has now been published for an in vivo neutralization assay (van Hulten et al., 2001b; three different isolates (Chen et al., 2002; van Hulten et al., Wu et al., 2005), and VP28 was used for detection of 2001a; Yang et al., 2001; GenBank Accession Nos. WSSV in crustaceans (Yoganandhan et al., 2004); VP281, AF332093, AF369029, AF440570 for viruses isolated from with a cell attachment RGD motif, was supposed to play China, Thailand, and Taiwan, respectively). Recently, the an important role in mediating WSSV infectivity (Huang International Committee on Taxonomy of Viruses approved et al., 2002a); VP26 was reported to be capable of a proposal to erect WSSV as the type species of the genus binding to actin or actin-associated proteins (Xie and Whispovirus, family Nimaviridae (www.ncbi.nlm.nih.gov/ Yang, 2005). ICTVdb/Ictv/index.htm). In this paper, the purified WSSV virions, viral envelope To date, some major structural proteins have been fractions were separated by SDS-PAGE. Among these identified (Huang et al., 2002b; Tsai et al., 2004). Among proteins, a 31 kDa protein (termed VP31) was analyzed those proteins, VP28, VP26/P22, VP281, VP466, VP68, using Nano-ESI MS/MS and further identified by Western blot and TEM (transmission electron microscopy) immuno- gold-labeling. In vivo neutralization assay was performed in * Corresponding author. this investigation to examine if VP31 can play a role in E-mail address: [email protected] (F. Yang). WSSV infection.

Results band in agarose gel at about 280 bp (Fig. 2A). Analysis of the products (Fig. 2B) revealed that the transcription Identification of WSSV-VP31 by MS initiation site was located at 94 nt upstream of the predicted ATG initiation codon, a putative TATA box The proteins of the WSSV virion, the envelope and was found at 37 nt upstream of the transcriptional nucleocapsid fraction were separated on 12% SDS-PAGE initiation sites. Amplification of the 3V region of the first- gel, respectively. As shown in Fig. 1A, the 31 kDa protein strand cDNA in 3V RACE yielded PCR products of about band (arrow indicated) was sequenced using Nano-ESI mass 320 bp (Fig. 2A). Although there was no putative spectrometry. Ten experimentally derived peptide masses polyadenylation signal (AATAAA), a possible polyadeny- were found to match the predicted peptide masses of the lation signal (AATAAT) (Caron et al., 2001; Halder et al., VP31 protein, covering 46% the amino acid sequence 1998) was found at 22 nt upstream of a poly (A) tail by (sequence coverage: 120 aa/261 aa = 46%, date not shown). sequencing 3V RACE fragments (Fig. 2B). Sequence alignment identified that the resulting amino acid sequence matched WSV340 of the WSSV genome ORF Temporal expression of vp31 transcription in WSSV-infected database. WSV340 is located from nucleotide position shrimp 196252–195510 on the genome and encodes a protein of RT-PCR was performed to detect vp31-specific tran- 261 amino acids, with a theoretical molecular mass of scripts at different infection stages (0 to 48 h p.i.). The result 29.6 kDa. showed that this gene transcript was first detected at 12 h p.i. and continued high transcription until 48 h p.i. (Fig. Characterization of WSSV vp31 gene 3A). This result suggested the vp31 gene was a late gene, just as most of WSSV late genes (Huang et al., 2002b; Rapid amplification of cDNA ends (RACE) Zhang et al., 2002a, 2002b). For the positive control, the The 5V and 3V regions of the vp31 transcript were results of RT-PCR with actin-specific primers were shown obtained by rapid amplification of the cDNA end (RACE). in Fig. 3B. When RNA was treated with RNase and then The RNA samples used in this study were prepared from subjected to RT-PCR with vp31-specific primers, no RT- the crayfish at 24 h p.i. In 5V RACE, after two rounds of PCR amplicon was seen, indicating that no virus genomic PCR amplifications, the PCR products formed a single DNA was left in the prepared RNA (negative control, date not shown).

Expression of rVP31c in E. coli BL21 (DE3) The C-terminal fragment of vp31 (vp31c) was cloned into the pET-GST vector and overexpressed as a GST fusion protein in BL21 (DE3) strain. A band corresponding to the GST-VP31c fusion protein was observed in the induced GST-VP31c as the expected size (date not shown). No protein band was found at the same position in uninduced GST-VP31c. The expressed fusion protein was purified by Ni-NTA resin under denaturing conditions. Purified rVP31c was used for antibody preparation.

Western blot analysis Purified WSSV virions, envelope proteins and nucle- Fig. 1. Coomassie brilliant blue stained 12% SDS-PAGE gel of purified ocapsid proteins were separated by SDS-PAGE, respec- WSSV virions, WSSV envelope and WSSV nucleocapsids and Western tively (Fig. 1A). Western blot results showed that the blot analysis. (A) SDS-PAGE of purified WSSV virions, WSSV envelope rVP31c polyclonal antiserum was reacted specifically with and WSSV nucleocapsids. Intact viral particles were obtained with VP31 in the purified viral envelope proteins, while no differential centrifugations. The virus envelope was removed from the binding could be found in the nucleocapsid proteins (Fig. virus particles by treatment with 0.2% Triton X-100. Then, the purified WSSV virions, WSSV envelope and WSSV nucleocapsids were assayed 1B). It confirmed that this protein should belong to viral by 12% SDS-PAGE with Coomassie staining. Lanes: M, low-molecular- envelope. weight protein marker. 1, purified WSSV virions. 2, purified WSSV envelope. 3, purified WSSV nucleocapsids. (B) Western blot analysis. The Localization of VP31 protein by IEM viral envelope and nucleocapsid proteins, after separation by SDS-PAGE, Purified WSSV virions were tested to be intact under were transferred onto a PVDF membrane followed by incubation with anti- rVP31c serum (diluted at 1:5000) and alkaline phosphatase-conjugated goat transmission electron microscopy (Fig. 4A). When both anti-mouse IgG, respectively. Lane 1, the viral envelope; Lane 2, the viral WSSV virions and nucleocapsids were incubated with nucleocapsids. rVP31c antiserum followed with gold-labeled secondary L. Li et al. / Virology 340 (2005) 125–132 127

Fig. 2. Nucleotide sequence of WSSV vp31 containing the 5V and 3V terminal region and the deduced VP31 protein sequence (one-letter code). (A) Agarose gel analysis of the 5V RACE and 3V RACE products. Lane M, 100 bp ladder DNA marker (Promega). After two rounds of PCR amplifications, the PCR products formed a single 280-bp band in 5V RACE and 320-bp band in 3V RACE. (B) The nucleotide sequence of the presumptive WSSV vp31 gene and the deduced VP31 amino acid sequence. The predicted TATA box, the possible polyadenylation signal (AATAAT), the start codons, two N-glycosylation sites (NLSE, NRTG) and FArg–Gly–Asp_ motif were boxed. Transcription start point and poly (A) were indicated with black dots. Primers used for 5V/3V RACE (VP31sp1, VP31sp2, VP31sp3) were underlined.

antibody, the gold particles were found to be located on the surface of the WSSV virions (Fig. 4D), while no particles could be found on the nucleocapsids (Fig. 4B). In the control experiments, rVP31c antiserum was replaced with a pre-immune mouse serum as the primary antibody, no gold particles could be seen for the WSSV virion too (Fig. 4C).

Neutralization assay

The results showed that the crayfish mortalities were very low for the negative control (0.9% NaCl). The crayfish from the positive control (WSSV only) displayed 100% Fig. 3. Temporal transcription analysis of WSSV-vp31 at different post- mortality at 8 days post-infection, and the crayfish from the infection times (h.p.i.). RT-PCR with the gene-specific primers was anti-GST IgG control displayed 100% mortality at 9 days performed using the total RNAs extracted from the infected crayfish. (A) post-infection. Whereas after the mixture of WSSV with RT-PCR with WSSV-vp31-specific primers. The transcript was first anti-rVP31c IgG was injected into crayfish, the crayfish detected at 12 h p.i. and continued to be highly transcribed until 48 h p.i.; (B) RT-PCR with h-actin-specific primers. No RT-PCR amplicon was mortality was significantly delayed (Fig. 5), indicating that observed. Lane M: 100 bp DNA ladder marker. Lane headings showed h the infection of WSSV could be delayed or neutralized by p.i. (0, 2, 4, 6, 8, 12, 18, 24 and 48 h p.i.). antibodies against rVP31c. Deceased shrimps were con- 128 L. Li et al. / Virology 340 (2005) 125–132

focused on the functional analysis of the gene products, especially on the functions of the viral envelope proteins. Recently, a simple and efficient method for purifying the virions and separating envelope proteins from virions has been developed in our laboratory (Xie et al., 2005), which facilitated the identification of the viral structural proteins with low abundance. By combining SDS-PAGE with mass spectrometry, vp31 was predicted to encode an envelope protein. This protein was also discovered by another study, in which the VP31 was shown to be a structural protein without further analysis on protein location and function (Tsai et al., 2004). In this study, Western blot showed that VP31 was in the envelope fraction. IEM further revealed that gold particles were present along the intact WSSV virions. This provided visual evidence that the vp31 gene encodes one of the envelope proteins of WSSV. Computer analysis showed that the predicted vp31 encoding protein lacks a predominant transmembrane domain, which suggested that VP31 might be a fusion protein that contained a domain involved in binding to other viral envelope protein for membrane anchoring. The same instance Fig. 4. Localization of VP31 in the WSSV virions by immunoelectron has been found at the vaccinia virus membrane protein, the microscopy (IEM) (Scale bar, 90 nm). (A) Electron micrograph of negatively stained intact WSSV virions (2% PTA); (B) Nucleocapsids of VV 14-kDa protein that is located on the surface of the WSSV incubated with anti-rVP31c antiserum as primary antibody followed intracellular mature virus form, and is essential for both the by gold-labeled secondary antibodies. (C) Intact WSSV virions incubated release of extracellular enveloped virus from the cells and with pre-immune mouse serum as primary antibody followed by gold- virus spread (Marı´a-Isabel et al., 1998). Such form had also labeled secondary antibodies. (D) Intact WSSV virions incubated with anti- been widely reported in some viral membrane fusion pro- rVP31c antiserum as primary antibody followed by gold-labeled secondary antibodies. Gold particles were indicated by arrows. teins, pathogen receptors and some cell adhesion molecules (Boyle and Compton, 1998; Jordens et al., 1999). These pro- firmed for the presence of WSSV by PCR detection (date teins could function to anchor the polypeptides to the not shown). membrane hydrophobic phase by means of locating aqueous activities or possibly interact with some membrane-spanning components (Stevens and Arkin, 2000). Therefore, we pre- Discussion sume that VP31 protein plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. White spot syndrome virus is a major pathogen of In addition, the cell attachment motif RGD (Ruoslahti cultured penaeid shrimp worldwide. After the completion of and Pierschbacher, 1986) occurred in vp31 gene sequence the WSSV genomic DNA sequencing, research has now (Fig. 3) suggested that the tripeptide recognition motif might

Fig. 5. Neutralization of WSSV infection in crayfish by specific antibodies. The solutions used for injection were shown at the bottom. For each treatment, 20 crayfish were used. After injections, the crayfish mortality was monitored daily at 25 -C. Days post-injection were shown on the abscissa and percentage mortality on the ordinate. L. Li et al. / Virology 340 (2005) 125–132 129 have an effect on VP31–host cells interactions. In previous and nucleocapsid fractions were evaluated by negative- studies, RGD structure has been reported widely to mediate staining transmission electron microscopy. the virus–host interaction (Senn and Klaus, 1993; Krezel et al., 1994; Verdaguer et al., 1995; Yingmanee et al., 2001; Mass spectrometric analysis of VP31 Mandl et al., 1989; Ruoslahti, 1996). Among WSSV proteins, six structure proteins, including VP31, have been The intact virions, envelope fractions and nucleocapsid reported to contain RGD motif (Tsai et al., 2004). Moreover, fractions were obtained as above. Purified WSSV virions VP31 possesses a threonine at the fourth position of the and the viral envelope fractions were separated using 12% RGD motif (RGDT), which was considered to be critical in SDS-PAGE (Laemmli, 1970) and stained with Coomassie interacting with integrin (Plow et al., 2000). brilliant blue R-250. A 31 kDa target protein band was Antibodies raised against individual viral envelope excised from the gel and dehydrated several times with protein have been used successfully in neutralization assay acetonitrile. After vacuum drying, the gel band was to identify envelope proteins involved in virus entry during incubated with 10 mM dithiothreitol in 100 mM ammonium infection (Martignoni et al., 1980; Schofield et al., 2000; bicarbonate (ABC buffer) at 57 -C for 60 min and van Hulten et al., 2001a, 2001b; Volkman and Goldsmith, subsequently with 55 mM iodoacetamide (Sigma) in 100 1985; Wu et al., 2005). In this study, a specific antiserum mM ABC buffer at room temperature for 60 min. Then, the against VP31 protein was prepared to test for neutralization gel was washed with 100 mM ABC buffer and dried. In-gel in vivo. The result suggested that VP31 should be involved protein digestion was performed using sequencing grade in the systemic infection of WSSV in crayfish by partici- modified porcine trypsin (Promega, Madison, WI) in 50 pating in virus attachment and membrane fusion. Future mM ABC buffer at 37 -C for 15 h. Digests were centrifuged experiments would be performed to demonstrate which part at 6000 Â g. The supernatants were separated, and the gel of VP31 was involved in the neutralization process and what pieces were extracted further first with 50% acetonitrile, 5% the role of VP31 in WSSV attachment and entry in the formic acid and then with acetonitrile. The extracts were systemic infection was. combined with the original digesting supernatants, vacuum- In summary, we identified a structure protein VP31 as an dried and redissolved in 0.5% trifluoroacetic acid and 50% envelope protein. VP31 might be a key protein in viral acetonitrile (Shevchenko et al., 1996). Subsequently, in-gel- invasion and could be a good candidate for screening digested sample was sequenced using Nano-ESI MS/MS receptors on shrimp cell surface. Study on VP31 will be mass spectrometry as described previously (Huang et al., helpful to realize the mechanism of the virus infection and 2002b). The resulting mass spectrometric data were to control the infection of WSSV in shrimp industry. searched against the theoretical ORF database of WSSV.

Computer analysis of the vp31 Materials and methods The potential sites for posttranslational modifications Virus purification and detergent extraction of intact virus were analyzed by the PROSITE database (Hofmann et al., 1999); The prediction of transmembrane domain and hydro- The WSSV isolate in this study originated from Penaeus phobic region of WSSV-VP31 was performed with TM-pred japonicus (Xiamen, China) and was proliferated in an program (Hofman and Stoffel, 1993) and DNAMAN alternate host, the crayfish (Procambarus clarkii). Prepara- software (Lynnon BioSoft, Vaudreuil, Canada), respectively. tion of WSSV virions and nucleocapsids was carried out as described previously (Xie et al., 2005). Brief, abundant viral Characterization of WSSV vp31 gene particles were obtained with only a few steps of conven- tional differential centrifugations, the upper loose pellet was Rapid amplification of vp31 cDNA ends (5V and 3V RACE) rinsed out, and purified virus in white pellet were Based on the nucleotide sequence of ORF340, the 5V and resuspended and kept in TM buffer (50 mM Tris–HCl, 10 3V ends of the cDNA encoding WSSV-VP31 were obtained mM MgCl2, pH 7.5). by 5V and 3V RACE using a commercial 5V/3V RACE kit The virus envelope was removed from the virus (Roche), according to the manufacturer’s recommendations. particles by treatment with Triton X-100. Generally, the The RNA samples used in this study were extracted from purified WSSV was mixed with an equal volume of 0.2% WSSV-infected crayfish 24 h p.i. and then treated with Triton X-100 and incubated for 1 h at room temperature. RNase-free DNase. For 5V RACE, the first-strand cDNA was The nucleocapsids were purified by centrifugation at synthesized using the specific primer sp1 (5V-GGCCGA- 20,000 Â g for 20 min at 4 -C. The envelope fraction TACCAATGTACAAG-3V), and then a poly (A) tail was was collected in the supernatant. The nucleocapsid fraction added to the cDNA products using terminal transferase in was subjected to a second round of Triton X-100 thepresenceofdATP.Theprimersp2(5V-CTTA- extraction to ensure the envelopes were removed com- CAGTGTTCTTCAAC-3V) and an oligo (dT) anchor primer pletely. The degree of purity of the intact virions isolated supplied with the kit were used for PCR. For 3V RACE, first- 130 L. Li et al. / Virology 340 (2005) 125–132 strand cDNA was synthesized using an oligo (dT) anchor column as described above. Final eluates were collected and primer. The primer sp3 (5V-GAGGATGTAGATGCAGTG- used as the negative control. 3V) and an anchor primer supplied with the kit were used for PCR. The PCR products from 5V and 3V RACE were each Preparation of antibody purified on a 2% agarose gel and subcloned into the Purified rVP31c protein was used as antigen to immunize pMD18-T vector (TaKaRa). Arbitrarily selected clones were mice by intradermal injection once every 10 days. Antigen sequenced and compared with the genomic DNA sequence (30 Ag) was mixed with an equal volume of Freund’s of WSSV. complete adjuvant (Sigma) for the first injection. Subse- quent three injections were conducted using 30 Ag antigen Transcriptional analyses of genes mixed with an equal volume of Freund’s incomplete adjuvant (Sigma). Four days after the last injection, mice The WSSV inoculum collected from virus-infected were exsanguinated, and antisera were collected. The titers shrimp P. japonicus was inoculated into the crayfish of the antisera were 1:15,000, as determined by ELISA. The according to the protocol described before (Li et al., IgG fraction was purified with rProtein A–Sepharose Fast 2004). Total RNAs at different times (i.e. 0, 2, 4, 6, 8, 12, Flow (Amersham). The optimal dilution of purified IgG, 18, 24 and 48 h post infection, h p.i.) were extracted from after serial dilutions, was 1:5000 as determined by ELISA. the hepatopancreas of WSSV-infected crayfish according to Horseradish-peroxidase (HRP)-conjugated goat anti-mouse the single-step RNA isolation method (Chomczynski and IgG was obtained from Promega. For a negative control, Sacchi, 1987). The obtained RNA was further purified using antigen was replaced with 1Â PBS. the SV Total RNA Isolation System (Promega) and quantified by spectrophotometry at 260 nm. The total Western blot RNA (2 Ag) was reverse transcribed with M-MLV RT [H-] Virus envelope fraction and nucleocapsid fraction dis- (Promega) using gene-specific reverse primer (GGCCGA- solved in loading buffer were separated by SDS-PAGE TACCAATGTACA). The cDNAs were subjected to PCR (Laemmli, 1970), respectively. These proteins were trans- using specific forward primer (5V-ATGAAGGATGCA- ferred onto a PVDF membrane (Amersham Pharmacia). The GAACTG-3V) and reverse primer (GGCCGATACCAATG- membrane was then immersed in blocking buffer (2% BSA, TACA). The PCR cycles were as follows: 94 -C for 2 min, 20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.5) at 30 cycles of 94 -C for 30 s, 55 -C for 30 s, 72 -C for 1 min room temperature for 30 min followed by incubation with followed by an elongation at 72 -C for 10 min. The anti-rVP31c serum (diluted 1:5000) for 1 h. Following this, crayfish h-actin gene was used as the internal control for alkaline-phosphatase-conjugated goat anti-mouse IgG RT-PCR with a gene specific primer set (5V-TCAT- (Promega) was used as the secondary antibody. Detection CAGGGTGTGATGGT-3V and 5V-TCTGAGTCATCTTCT- was performed using Western Blue Stabilized Substrate for CAC-3V). Total RNA from healthy crayfish was used as the Alkaline Phosphatase (Promega). negative control. Localization of VP31 protein by immunoelectron Partial expression of GST-VP31 fusion protein in microscopy Escherichia coli WSSV virion and nucleocapsid suspension were mounted The WSSV-vp31 gene C-terminal fragment (termed as on carbon-coated nickel grids (300 meshes) for 1 h at room vp31c) was amplified from WSSV genomic DNA using the temperature. Respectively, after washing with PBS, the grids forward primer (5V-CGGGATCC CAAGAAACTGGT- were blocked in 3% BSA for 1 h. Then, the grids were rinsed CAAGT-3V) and the reverse primer (5V-CGGAATTC with PBS followed by incubation with anti-rVP31c IgG as CTCCTCCTTAAAAGCAG-3V) that contained recognition the primary antibody (1:200, diluted in 3% BSA) for 2 h. sequences for BamHI and EcoRI restriction enzymes After washing four times with 1Â PBS, 10-nm-gold-labeled (underlined). The amplicon was cloned into the pET-GST anti-mouse goat IgG as the secondary antibody (Sigma) was vector (Gene Power Lab Ltd Shenzhen office). The added to the grids and incubated for 1 h at room temperature. recombinant plasmid was transformed into E. coli BL21 Grids were washed further four times with 1Â PBS and (DE3) cells. Liquid cultures were grown in a shaking stained with 2% phosphotungstic acid (PTA, pH 7.0) for 25 incubator (200 rpm) at 37 -C until the OD600 reached 0.7, min. Specimens were examined under a TEM. For control and these were then induced with 0.2 mM IPTG for 5 h at experiment, anti-rVP31c IgG was replaced with a pre- 37 -C. The cells were harvested by centrifugation at immune mouse serum as the primary antibodies indicated 4000 Â g for 5 min. The recombinant VP31c (termed above. rVP31c) was purified by Ni-NTA affinity chromatography under denatured conditions following methodology in the Neutralization assay QIAexpressionist handbook (Qiagen). The E. coli cells containing pET-GST vector were also induced with IPTG, Prior to injection into crayfish, WSSV virions were and total protein extracts were applied to the Ni-NTA incubated with anti-rVP31c IgG or the anti-GST IgG as a L. Li et al. / Virology 340 (2005) 125–132 131 control (final concentration 4 Ag IgG/ml, 107 virions/ml) for Krezel, A.M., Wagner, G., Seymour-Ulmer, J., Lazarus, R.A., 1994. 1 h at room temperature. 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