Identification and characterization of a prawn white spot syndrome virus gene that encodes an...

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Virology 340 (20

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 PR 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

White spot syndrome virus (WSSV) is a major shrimp

pathogen that is highly virulent in penaeid shrimp and can

also infect most species of crustacean (Lo et al 1996a

1996b Chen et al 1997 Flegel 1997) The complete

sequence of WSSV genome has now been published for

three different isolates (Chen et al 2002 van Hulten et al

2001a Yang et al 2001 GenBank Accession Nos

AF332093 AF369029 AF440570 for viruses isolated from

China Thailand and Taiwan respectively) Recently the

International Committee on Taxonomy of Viruses approved

a proposal to erect WSSV as the type species of the genus

Whispovirus family Nimaviridae (wwwncbinlmnihgov

ICTVdbIctvindexhtm)

To date some major structural proteins have been

identified (Huang et al 2002b Tsai et al 2004) Among

those proteins VP28 VP26P22 VP281 VP466 VP68

0042-6822$ - see front matter D 2005 Elsevier Inc All rights reserved

doi101016jvirol200506007

Corresponding author

E-mail address mbiotechpublicxmfjcn (F Yang)

VP292 and VP124 were identified as viral envelope

proteins by immunoelectron microscopy (Huang et al

2002a 2002b Zhang et al 2002a 2002b 2004) As far

as functional research is concerned VP28 VP281

VP466 and VP68 were suggested to play an important

role in the systemic WSSV infection in shrimp by using

an in vivo neutralization assay (van Hulten et al 2001b

Wu et al 2005) and VP28 was used for detection of

WSSV in crustaceans (Yoganandhan et al 2004) VP281

with a cell attachment RGD motif was supposed to play

an important role in mediating WSSV infectivity (Huang

et al 2002a) VP26 was reported to be capable of

binding to actin or actin-associated proteins (Xie and

Yang 2005)

In this paper the purified WSSV virions viral envelope

fractions were separated by SDS-PAGE Among these

proteins a 31 kDa protein (termed VP31) was analyzed

using Nano-ESI MSMS and further identified by Western

blot and TEM (transmission electron microscopy) immuno-

gold-labeling In vivo neutralization assay was performed in

this investigation to examine if VP31 can play a role in

WSSV infection

05) 125 ndash 132

L Li et al Virology 340 (2005) 125ndash132126

Results

Identification of WSSV-VP31 by MS

The proteins of the WSSV virion the envelope and

nucleocapsid fraction were separated on 12 SDS-PAGE

gel respectively As shown in Fig 1A the 31 kDa protein

band (arrow indicated) was sequenced using Nano-ESI mass

spectrometry Ten experimentally derived peptide masses

were found to match the predicted peptide masses of the

VP31 protein covering 46 the amino acid sequence

(sequence coverage 120 aa261 aa = 46 date not shown)

Sequence alignment identified that the resulting amino acid

sequence matched WSV340 of the WSSV genome ORF

database WSV340 is located from nucleotide position

196252ndash195510 on the genome and encodes a protein of

261 amino acids with a theoretical molecular mass of

296 kDa

Characterization of WSSV vp31 gene

Rapid amplification of cDNA ends (RACE)

The 5V and 3V regions of the vp31 transcript were

obtained by rapid amplification of the cDNA end (RACE)

The RNA samples used in this study were prepared from

the crayfish at 24 h pi In 5V RACE after two rounds of

PCR amplifications the PCR products formed a single

Fig 1 Coomassie brilliant blue stained 12 SDS-PAGE gel of purified

WSSV virions WSSV envelope and WSSV nucleocapsids and Western

blot analysis (A) SDS-PAGE of purified WSSV virions WSSV envelope

and WSSV nucleocapsids Intact viral particles were obtained with

differential centrifugations The virus envelope was removed from the

virus particles by treatment with 02 Triton X-100 Then the purified

WSSV virions WSSV envelope and WSSV nucleocapsids were assayed

by 12 SDS-PAGE with Coomassie staining Lanes M low-molecular-

weight protein marker 1 purified WSSV virions 2 purified WSSV

envelope 3 purified WSSV nucleocapsids (B) Western blot analysis The

viral envelope and nucleocapsid proteins after separation by SDS-PAGE

were transferred onto a PVDF membrane followed by incubation with anti-

rVP31c serum (diluted at 15000) and alkaline phosphatase-conjugated goat

anti-mouse IgG respectively Lane 1 the viral envelope Lane 2 the viral

nucleocapsids

band in agarose gel at about 280 bp (Fig 2A) Analysis of

the products (Fig 2B) revealed that the transcription

initiation site was located at 94 nt upstream of the

predicted ATG initiation codon a putative TATA box

was found at 37 nt upstream of the transcriptional

initiation sites Amplification of the 3V region of the first-

strand cDNA in 3V RACE yielded PCR products of about

320 bp (Fig 2A) Although there was no putative

polyadenylation signal (AATAAA) a possible polyadeny-

lation signal (AATAAT) (Caron et al 2001 Halder et al

1998) was found at 22 nt upstream of a poly (A) tail by

sequencing 3V RACE fragments (Fig 2B)

Temporal expression of vp31 transcription in WSSV-infected

shrimp

RT-PCR was performed to detect vp31-specific tran-

scripts at different infection stages (0 to 48 h pi) The result

showed that this gene transcript was first detected at 12 h

pi and continued high transcription until 48 h pi (Fig

3A) This result suggested the vp31 gene was a late gene

just as most of WSSV late genes (Huang et al 2002b

Zhang et al 2002a 2002b) For the positive control the

results of RT-PCR with actin-specific primers were shown

in Fig 3B When RNA was treated with RNase and then

subjected to RT-PCR with vp31-specific primers no RT-

PCR amplicon was seen indicating that no virus genomic

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 corre-

sponding 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-

ocapsid proteins were separated by SDS-PAGE respec-

tively (Fig 1A) Western blot results showed that the

rVP31c polyclonal antiserum was reacted specifically with

VP31 in the purified viral envelope proteins while no

binding could be found in the nucleocapsid proteins (Fig

1B) It confirmed that this protein should belong to viral

envelope

Localization of VP31 protein by IEM

Purified WSSV virions were tested to be intact under

transmission electron microscopy (Fig 4A) When both

WSSV virions and nucleocapsids were incubated with

rVP31c antiserum followed with gold-labeled secondary

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 FArgndashGlyndashAsp_ motif were boxed Transcription start point and poly (A) were indicated with black dots Primers used for 5V3VRACE (VP31sp1

VP31sp2 VP31sp3) were underlined

Fig 3 Temporal transcription analysis of WSSV-vp31 at different post-

infection times (hpi) RT-PCR with the gene-specific primers was

performed using the total RNAs extracted from the infected crayfish (A)

RT-PCR with WSSV-vp31-specific primers The transcript was first

detected at 12 h pi and continued to be highly transcribed until 48 h

pi (B) RT-PCR with h-actin-specific primers No RT-PCR amplicon was

observed Lane M 100 bp DNA ladder marker Lane headings showed h

pi (0 2 4 6 8 12 18 24 and 48 h pi)

L Li et al Virology 340 (2005) 125ndash132 127

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 (09 NaCl) The crayfish

from the positive control (WSSV only) displayed 100

mortality at 8 days post-infection and the crayfish from the

anti-GST IgG control displayed 100 mortality at 9 days

post-infection Whereas after the mixture of WSSV with

anti-rVP31c IgG was injected into crayfish the crayfish

mortality was significantly delayed (Fig 5) indicating that

the infection of WSSV could be delayed or neutralized by

antibodies against rVP31c Deceased shrimps were con-

Fig 4 Localization of VP31 in the WSSV virions by immunoelectron

microscopy (IEM) (Scale bar 90 nm) (A) Electron micrograph of

negatively stained intact WSSV virions (2 PTA) (B) Nucleocapsids of

WSSV incubated with anti-rVP31c antiserum as primary antibody followed

by gold-labeled secondary antibodies (C) Intact WSSV virions incubated

with pre-immune mouse serum as primary antibody followed by gold-

labeled secondary antibodies (D) Intact WSSV virions incubated with anti-

rVP31c antiserum as primary antibody followed by gold-labeled secondary

antibodies Gold particles were indicated by arrows


firmed for the presence of WSSV by PCR detection (date

not shown)

Discussion

White spot syndrome virus is a major pathogen of

cultured penaeid shrimp worldwide After the completion of

the WSSV genomic DNA sequencing research has now

Fig 5 Neutralization of WSSV infection in crayfish by specific antibodies The so

crayfish were used After injections the crayfish mortality was monitored daily

mortality on the ordinate

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 enco-

ding 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

has been found at the vaccinia virus membrane protein the

VV 14-kDa protein that is located on the surface of the

intracellular mature virus form and is essential for both the

release of extracellular enveloped virus from the cells and

virus spread (Marıa-Isabel et al 1998) Such form had also

been widely reported in some viral membrane fusion pro-

teins pathogen receptors and some cell adhesion molecules

(Boyle and Compton 1998 Jordens et al 1999) These pro-

teins could function to anchor the polypeptides to the

membrane hydrophobic phase by means of locating aqueous

activities or possibly interact with some membrane-spanning

components (Stevens and Arkin 2000) Therefore we pre-

sume that VP31 protein plays an important role in the biology

of the virus acting in virus-to-cell and cell-to-cell fusions

In addition the cell attachment motif RGD (Ruoslahti

and Pierschbacher 1986) occurred in vp31 gene sequence

(Fig 3) suggested that the tripeptide recognition motif might

lutions used for injection were shown at the bottom For each treatment 20

at 25 -C Days post-injection were shown on the abscissa and percentage


have an effect on VP31ndashhost cells interactions In previous

studies RGD structure has been reported widely to mediate

the virusndashhost interaction (Senn and Klaus 1993 Krezel et

al 1994 Verdaguer et al 1995 Yingmanee et al 2001

Mandl et al 1989 Ruoslahti 1996) Among WSSV

proteins six structure proteins including VP31 have been

reported to contain RGD motif (Tsai et al 2004) Moreover

VP31 possesses a threonine at the fourth position of the

RGD motif (RGDT) which was considered to be critical in

interacting with integrin (Plow et al 2000)

Antibodies raised against individual viral envelope

protein have been used successfully in neutralization assay

to identify envelope proteins involved in virus entry during

infection (Martignoni et al 1980 Schofield et al 2000

van Hulten et al 2001a 2001b Volkman and Goldsmith

1985 Wu et al 2005) In this study a specific antiserum

against VP31 protein was prepared to test for neutralization

in vivo The result suggested that VP31 should be involved

in the systemic infection of WSSV in crayfish by partici-

pating in virus attachment and membrane fusion Future

experiments would be performed to demonstrate which part

of VP31 was involved in the neutralization process and what

the role of VP31 in WSSV attachment and entry in the

systemic infection was

In summary we identified a structure protein VP31 as an

envelope protein VP31 might be a key protein in viral

invasion and could be a good candidate for screening

receptors on shrimp cell surface Study on VP31 will be

helpful to realize the mechanism of the virus infection and

to control the infection of WSSV in shrimp industry

Materials and methods

Virus purification and detergent extraction of intact virus

The WSSV isolate in this study originated from Penaeus

japonicus (Xiamen China) and was proliferated in an

alternate host the crayfish (Procambarus clarkii) Prepara-

tion of WSSV virions and nucleocapsids was carried out as

described previously (Xie et al 2005) Brief abundant viral

particles were obtained with only a few steps of conven-

tional differential centrifugations the upper loose pellet was

rinsed out and purified virus in white pellet were

resuspended and kept in TM buffer (50 mM TrisndashHCl 10

mM MgCl2 pH 75)

The virus envelope was removed from the virus

particles by treatment with Triton X-100 Generally the

purified WSSV was mixed with an equal volume of 02

Triton X-100 and incubated for 1 h at room temperature

The nucleocapsids were purified by centrifugation at

20000 g for 20 min at 4 -C The envelope fraction

was collected in the supernatant The nucleocapsid fraction

was subjected to a second round of Triton X-100

extraction to ensure the envelopes were removed com-

pletely The degree of purity of the intact virions isolated

and nucleocapsid fractions were evaluated by negative-

staining transmission electron microscopy

Mass spectrometric analysis of VP31

The intact virions envelope fractions and nucleocapsid

fractions were obtained as above Purified WSSV virions

and the viral envelope fractions were separated using 12

SDS-PAGE (Laemmli 1970) and stained with Coomassie

brilliant blue R-250 A 31 kDa target protein band was

excised from the gel and dehydrated several times with

acetonitrile After vacuum drying the gel band was

incubated with 10 mM dithiothreitol in 100 mM ammonium

bicarbonate (ABC buffer) at 57 -C for 60 min and

subsequently with 55 mM iodoacetamide (Sigma) in 100

mM ABC buffer at room temperature for 60 min Then the

gel was washed with 100 mM ABC buffer and dried In-gel

protein digestion was performed using sequencing grade

modified porcine trypsin (Promega Madison WI) in 50

mM ABC buffer at 37 -C for 15 h Digests were centrifuged

at 6000 g The supernatants were separated and the gel

pieces were extracted further first with 50 acetonitrile 5

formic acid and then with acetonitrile The extracts were

combined with the original digesting supernatants vacuum-

dried and redissolved in 05 trifluoroacetic acid and 50

acetonitrile (Shevchenko et al 1996) Subsequently in-gel-

digested sample was sequenced using Nano-ESI MSMS

mass spectrometry as described previously (Huang et al

2002b) The resulting mass spectrometric data were

searched against the theoretical ORF database of WSSV

Computer analysis of the vp31

The potential sites for posttranslational modifications

were analyzed by the PROSITE database (Hofmann et al

1999) The prediction of transmembrane domain and hydro-

phobic region of WSSV-VP31 was performed with TM-pred

program (Hofman and Stoffel 1993) and DNAMAN

software (Lynnon BioSoft Vaudreuil Canada) respectively


Rapid amplification of vp31 cDNA ends (5V and 3V RACE)Based on the nucleotide sequence of ORF340 the 5V and

3V ends of the cDNA encoding WSSV-VP31 were obtained

by 5V and 3V RACE using a commercial 5V3V RACE kit

(Roche) according to the manufacturerrsquos recommendations

The RNA samples used in this study were extracted from

WSSV-infected crayfish 24 h pi and then treated with

RNase-free DNase For 5VRACE the first-strand cDNAwas

synthesized using the specific primer sp1 (5V-GGCCGA-TACCAATGTACAAG-3V) and then a poly (A) tail was

added to the cDNA products using terminal transferase in

the presence of dATP The primer sp2 (5V-CTTA-

CAGTGTTCTTCAAC-3V) and an oligo (dT) anchor primer

supplied with the kit were used for PCR For 3VRACE first-


strand cDNA was synthesized using an oligo (dT) anchor

primer The primer sp3 (5V-GAGGATGTAGATGCAGTG-3V) and an anchor primer supplied with the kit were used for

PCR The PCR products from 5V and 3V RACE were each

purified on a 2 agarose gel and subcloned into the

pMD18-T vector (TaKaRa) Arbitrarily selected clones were

sequenced and compared with the genomic DNA sequence

of WSSV

Transcriptional analyses of genes

The WSSV inoculum collected from virus-infected

shrimp P japonicus was inoculated into the crayfish

according to the protocol described before (Li et al

2004) Total RNAs at different times (ie 0 2 4 6 8 12

18 24 and 48 h post infection h pi) were extracted from

the hepatopancreas of WSSV-infected crayfish according to

the single-step RNA isolation method (Chomczynski and

Sacchi 1987) The obtained RNAwas further purified using

the SV Total RNA Isolation System (Promega) and

quantified by spectrophotometry at 260 nm The total

RNA (2 Ag) was reverse transcribed with M-MLV RT [H-]

(Promega) using gene-specific reverse primer (GGCCGA-

TACCAATGTACA) The cDNAs were subjected to PCR

using specific forward primer (5V-ATGAAGGATGCA-GAACTG-3V) and reverse primer (GGCCGATACCAATG-

TACA) The PCR cycles were as follows 94 -C for 2 min

30 cycles of 94 -C for 30 s 55 -C for 30 s 72 -C for 1 min

followed by an elongation at 72 -C for 10 min The

crayfish h-actin gene was used as the internal control for

RT-PCR with a gene specific primer set (5V-TCAT-CAGGGTGTGATGGT-3V and 5V-TCTGAGTCATCTTCT-CAC-3V) Total RNA from healthy crayfish was used as the

negative control

Partial expression of GST-VP31 fusion protein in

Escherichia coli

The WSSV-vp31 gene C-terminal fragment (termed as

vp31c) was amplified from WSSV genomic DNA using the

forward primer (5V-CGGGATCC CAAGAAACTGGT-

CAAGT-3V) and the reverse primer (5V-CGGAATTC

CTCCTCCTTAAAAGCAG-3V) that contained recognition

sequences for BamHI and EcoRI restriction enzymes

(underlined) The amplicon was cloned into the pET-GST

vector (Gene Power Lab Ltd Shenzhen office) The

recombinant plasmid was transformed into E coli BL21

(DE3) cells Liquid cultures were grown in a shaking

incubator (200 rpm) at 37 -C until the OD600 reached 07

and these were then induced with 02 mM IPTG for 5 h at

37 -C The cells were harvested by centrifugation at

4000 g for 5 min The recombinant VP31c (termed

rVP31c) was purified by Ni-NTA affinity chromatography

under denatured conditions following methodology in the

QIAexpressionist handbook (Qiagen) The E coli cells

containing pET-GST vector were also induced with IPTG

and total protein extracts were applied to the Ni-NTA

column as described above Final eluates were collected and

used as the negative control

Preparation of antibody

Purified rVP31c protein was used as antigen to immunize

mice by intradermal injection once every 10 days Antigen

(30 Ag) was mixed with an equal volume of Freundrsquos

complete adjuvant (Sigma) for the first injection Subse-

quent three injections were conducted using 30 Ag antigen

mixed with an equal volume of Freundrsquos incomplete

adjuvant (Sigma) Four days after the last injection mice

were exsanguinated and antisera were collected The titers

of the antisera were 115000 as determined by ELISA The

IgG fraction was purified with rProtein AndashSepharose Fast

Flow (Amersham) The optimal dilution of purified IgG

after serial dilutions was 15000 as determined by ELISA

Horseradish-peroxidase (HRP)-conjugated goat anti-mouse

IgG was obtained from Promega For a negative control

antigen was replaced with 1 PBS

Western blot

Virus envelope fraction and nucleocapsid fraction dis-

solved in loading buffer were separated by SDS-PAGE

(Laemmli 1970) respectively These proteins were trans-

ferred onto a PVDF membrane (Amersham Pharmacia) The

membrane was then immersed in blocking buffer (2 BSA

20 mM Tris 150 mM NaCl 01 Tween 20 pH 75) at

room temperature for 30 min followed by incubation with

anti-rVP31c serum (diluted 15000) for 1 h Following this

alkaline-phosphatase-conjugated goat anti-mouse IgG

(Promega) was used as the secondary antibody Detection

was performed using Western Blue Stabilized Substrate for

Alkaline Phosphatase (Promega)

Localization of VP31 protein by immunoelectron

microscopy

WSSV virion and nucleocapsid suspension were mounted

on carbon-coated nickel grids (300 meshes) for 1 h at room

temperature Respectively after washing with PBS the grids

were blocked in 3 BSA for 1 h Then the grids were rinsed

with PBS followed by incubation with anti-rVP31c IgG as

the primary antibody (1200 diluted in 3 BSA) for 2 h

After washing four times with 1 PBS 10-nm-gold-labeled

anti-mouse goat IgG as the secondary antibody (Sigma) was

added to the grids and incubated for 1 h at room temperature

Grids were washed further four times with 1 PBS and

stained with 2 phosphotungstic acid (PTA pH 70) for 25

min Specimens were examined under a TEM For control

experiment anti-rVP31c IgG was replaced with a pre-

immune mouse serum as the primary antibodies indicated

above


Prior to injection into crayfish WSSV virions were

incubated with anti-rVP31c IgG or the anti-GST IgG as a


control (final concentration 4 Ag IgGml 107 virionsml) for

1 h at room temperature Then the 100 Al mixture of

antibody and WSSV was intramuscularly injected into

crayfish using a syringe with 29-gauge needle At the same

time a negative (09 NaCl) and a positive control (virus

only 107 virionsml) were included in the injections For

each treatment 20 crayfish were used After injections the

crayfish mortality was monitored daily at 25 -C Deceasedshrimps were examined for the presence of WSSV by PCR

detection

Acknowledgments

We thank Ping Chen for her technical assistance in the

transmission electron microscopy This work was sup-

ported by the lsquolsquo863rsquorsquo Program of China (2003AA626020)

National Natural Science Foundation of China (30330470

40276038) and Fujian Science Fund (2003F001)

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soluble form of human cytomegalovirus glycoprotein B J Virol 72

1826ndash1833

Caron H van Schaik B van der Mee M Baas F Riggins G van

Sluis P Hermus M-C van Asperen R Boon K Voute PA

Heisterkamp S van Kamper A Versteeg R 2001 The human

transcriptome map clustering of highly expressed genes in chromoso-

mal domainsmdashSupplementary material Science 291 1289ndash1292

Chen XF Chen C Wu DH Huai H Chi XC 1997 A new

baculovirus of cultured shrimp Sci China Ser C 40 630ndash635

Chen LL Leu JH 1 Huang CJ Chou CM Chen SM Wang CH

Lo CF Kou GH 2002 Identification of a nucleocapsid protein

(VP35) gene of shrimp white spot syndrome virus and characterization

of the motif important for targeting VP35 to the nuclei of transfected

insect cells Virology 293 44ndash53

Chomczynski P Sacchi N 1987 Single-step method of RNA isolation

by acid guanidinium thiocyanatendashphenolndashchloroform extraction Anal

Biochem 262 156ndash159

Flegel TW 1997 Special topic review major viral diseases of the black

tiger prawn (Penaeus monodon) in Thailand World J Microbiol

Biotechnol 13 433ndash442

Halder SK Takemori H Hatano O Nonaka Y Wada A Okamoto

M 1998 Cloning of a membrane-spanning protein with epidermal

growth factor-like repeat motifs from adrenal glomerulosa cells

Endocrinology 139 3316ndash3328

Hofman K Stoffel W 1993 TMbasemdashA database of membrane

spanning protein segments Biol Chem Hoppe-Seyler 374 166

Hofmann K Bucher P Falquet L Bairoch A 1999 The PROSITE

database its status in 1999 Nucleic Acids Res 27 215ndash219

Huang CH Zhang XB Lin QS Xu X Hew CL 2002a Character-

ization of a novel envelope protein (VP281) of shrimp white spot

syndrome virus by mass spectrometry J Gen Virol 83 2385ndash2392

Huang CH Zhang XB Lin QS Xu X Hu ZH Hew CL 2002b

Proteomic analysis of shrimp white spot syndrome viral proteins and

characterization of a novel envelope protein VP466 Mol Cell

Proteomics 1 223ndash231

Jordens R Thompson A Amons R Koning F 1999 Human dendritic

cells shed a functional soluble form of the mannose receptor Int

Immunol 11 1775ndash1780

Krezel AM Wagner G Seymour-Ulmer J Lazarus RA 1994

Structure of the RGD protein decorsin conserved motif and distinct

function in leech proteins that affect blood clotting Science 264

1944ndash1947

Laemmli UK 1970 Cleavage of structural proteins during the assembly

of the head of bacteriophage T4 Nature 227 680ndash685

Li Q Pan D Zhang J Yang F 2004 Identification of the thymidylate

synthase within the genome of white spot syndrome virus J Gen Virol

85 2035ndash2044

Lo CF Ho CH Peng SE Chen CH Hsu HC Chiu YL Chang

CF Liu KF Su MS Wang CH Kou GH 1996a White spot

syndrome baculovirus (WSBV) detected in cultured and captured

shrimp crabs and other arthropods Dis Aquat Org 27 215ndash226

Lo CF Leu JH Ho CH Chen CH Peng SE Chen YT Chou

CM Yeh PY Huang CJ Chou HY Wang CH Kou GH

1996b Detection of baculovirus associated with white spot syndrome

(WSBV) in penaeid shrimps using polymerase chain reaction Dis

Aquat Org 25 133ndash141

Mandl CW Guirakhoo F Holzmann H Heinz FX Kunz C 1989

Antigenic structure of the flavivirus envelope E protein at the molecular

level using tick-borne encephalitis virus as a model J Virol 63

564ndash571

Marıa-Isabel V German R David C Luis S Mariano E 1998

The vaccinia virus 14-kilodalton (A27L) fusion protein forms a triple

coiled-coil structure and interacts with the 21-kilodalton (A17L)

virus membrane protein through a C-terminal a-helix J Virol 72

10126ndash10137

Martignoni ME Iwai PJ Rohrmann GF 1980 Serum neutralization

of nucleopolyhedrosis viruses (baculovirus subgroup A) pathogenic for

Orgyia pseudotsugata J Invert Pathol 36 12ndash20

Plow EF Haas TA Zhang L Loftus J Smith JW 2000 Ligand

binding to integrins J Biol Chem 275 21785ndash21788

Ruoslahti E 1996 RGD and other recognition sequences for integrins

Annu Rev Cell Biol 12 697ndash715

Ruoslahti E Pierschbacher MD 1986 ArgndashGlyndashAsp a versatile cell

recognition signal Cell 44 517ndash518

Schofield DJ Glamann J Emerson U Purchell RH 2000 Identi-

fication by phage display and characterization of two neutralizing

chimpanzee monoclonal antibodies to the hepatitis E virus capsid

protein J Virol 74 5548ndash5555

Senn H Klaus W 1993 The nuclear magnetic resonance solution

structure of flavoridin an antagonist of the platelet GPIIbndash IIIa receptor

J Mol Biol 232 907ndash925

Shevchenko A Wilm M Vorm O Mann M 1996 Mass spectrometric

sequencing of protein silver-stained polyacrylamide gels Anal Chem

68 850ndash858

Stevens TJ Arkin IT 2000 Turning an opinion inside-out Rees and

Eisenbergrsquos commentary (Proteins 2000 38121ndash122) on FAre mem-

brane proteins lsquolsquoinside-outrsquorsquo proteins_ (Proteins 1999 36 135ndash143)

Proteins 40 463ndash464

Tsai JM Wang HC Leu JH Hsiao HH Wang AH Kou GH

Lo CF 2004 Genomic and proteomic analysis of thirty-nine

structural proteins of shrimp white spot syndrome virus J Virol 78

11360ndash11370

van Hulten MC Witteveldt J Peters S Kloosterboer N Tarchini

R Fiers M Sandbrink H Lankhorst RK Vlak JM 2001a

The white spot syndrome virus DNA genome sequence Virology

286 7ndash22

van Hulten MC Witteveldt J Snippe M Vlak JM 2001b White spot

syndrome virus envelope protein VP28 is involved in the systemic

infection of shrimp Virology 285 228ndash233

Verdaguer N Mateu MG Andreu D Giralt E Domingo E Fita

I 1995 Structure of the major antigenic loop of foot-and-mouth

disease virus complexed with a neutralizing antibody direct involve-

ment of the ArgndashGlyndashAsp motif in the interaction EMBO J 14

1690ndash1696

Volkman LE Goldsmith PA 1985 Mechanism of neutralization of


budded Autographa californica nuclear polyhedrosis virus by a

monoclonal antibody inhibition of entry by adsorptive endocytosis

Virology 143 185ndash195

Wu WL Wang L Zhang XB 2005 Identification of white spot

syndrome virus (WSSV) envelope proteins involved in shrimp

infection Virology 332 578ndash583

Xie XX Yang F 2005 Interaction of white spot syndrome virus VP26

protein with actin Virology 336 93ndash99

Xie XX Li HY Xu LM Yang F 2005 A simple and efficient

method for purification of intact White Spot Syndrome Virus (WSSV)

viral particles Virus Res 108 63ndash67

Yang F He J Lin X Li Q Pan D Zhang X Xu X 2001 Complete

genome sequence of the shrimp white spot bacilliform virus J Virol 75

11811ndash11820

Yingmanee B Pamela JH Farideh G Glyn S 2001 Argininendash

glycinendashaspartic acid motif is critical for human parechovirus 1 entry

J Virol 75 10000ndash10004

Yoganandhan K Syed MS Narayanan RB Sahul HAS 2004

Production of polyclonal antiserum against recombinant VP28 protein

and its application for the detection of white spot syndrome virus in

crustaceans J Fish Dis 27 517ndash522

Zhang X Huang CH Xu X Hew CL 2002a Transcription and

identification of an envelope protein gene (p22) from shrimp white spot

syndrome virus J Gen Virol 83 471ndash477

Zhang X Huang CH Xu X Hew CL 2002b Identification and

localization of a prawn white spot syndrome virus gene that encodes an

envelope protein J Gen Virol 83 1069ndash1074

Zhang X Huang C Tang X Zhuang Y Choy LH 2004 Identification

of structural proteins from shrimp white spot syndrome virus (WSSV)

by 2-DE-MS Proteins Struct Funct Bioinform 55 229ndash235


Results

Identification of WSSV-VP31 by MS

The proteins of the WSSV virion the envelope and

nucleocapsid fraction were separated on 12 SDS-PAGE

gel respectively As shown in Fig 1A the 31 kDa protein

band (arrow indicated) was sequenced using Nano-ESI mass

spectrometry Ten experimentally derived peptide masses

were found to match the predicted peptide masses of the

VP31 protein covering 46 the amino acid sequence

(sequence coverage 120 aa261 aa = 46 date not shown)

Sequence alignment identified that the resulting amino acid

sequence matched WSV340 of the WSSV genome ORF

database WSV340 is located from nucleotide position

196252ndash195510 on the genome and encodes a protein of

261 amino acids with a theoretical molecular mass of

296 kDa


Rapid amplification of cDNA ends (RACE)

The 5V and 3V regions of the vp31 transcript were

obtained by rapid amplification of the cDNA end (RACE)

The RNA samples used in this study were prepared from

the crayfish at 24 h pi In 5V RACE after two rounds of

PCR amplifications the PCR products formed a single

Fig 1 Coomassie brilliant blue stained 12 SDS-PAGE gel of purified

WSSV virions WSSV envelope and WSSV nucleocapsids and Western

blot analysis (A) SDS-PAGE of purified WSSV virions WSSV envelope

and WSSV nucleocapsids Intact viral particles were obtained with

differential centrifugations The virus envelope was removed from the

virus particles by treatment with 02 Triton X-100 Then the purified

WSSV virions WSSV envelope and WSSV nucleocapsids were assayed

by 12 SDS-PAGE with Coomassie staining Lanes M low-molecular-

weight protein marker 1 purified WSSV virions 2 purified WSSV

envelope 3 purified WSSV nucleocapsids (B) Western blot analysis The

viral envelope and nucleocapsid proteins after separation by SDS-PAGE

were transferred onto a PVDF membrane followed by incubation with anti-

rVP31c serum (diluted at 15000) and alkaline phosphatase-conjugated goat

anti-mouse IgG respectively Lane 1 the viral envelope Lane 2 the viral

nucleocapsids

band in agarose gel at about 280 bp (Fig 2A) Analysis of

the products (Fig 2B) revealed that the transcription

initiation site was located at 94 nt upstream of the

predicted ATG initiation codon a putative TATA box

was found at 37 nt upstream of the transcriptional

initiation sites Amplification of the 3V region of the first-

strand cDNA in 3V RACE yielded PCR products of about

320 bp (Fig 2A) Although there was no putative

polyadenylation signal (AATAAA) a possible polyadeny-

lation signal (AATAAT) (Caron et al 2001 Halder et al

1998) was found at 22 nt upstream of a poly (A) tail by

sequencing 3V RACE fragments (Fig 2B)

Temporal expression of vp31 transcription in WSSV-infected

shrimp

RT-PCR was performed to detect vp31-specific tran-

scripts at different infection stages (0 to 48 h pi) The result

showed that this gene transcript was first detected at 12 h

pi and continued high transcription until 48 h pi (Fig

3A) This result suggested the vp31 gene was a late gene

just as most of WSSV late genes (Huang et al 2002b

Zhang et al 2002a 2002b) For the positive control the

results of RT-PCR with actin-specific primers were shown

in Fig 3B When RNA was treated with RNase and then

subjected to RT-PCR with vp31-specific primers no RT-

PCR amplicon was seen indicating that no virus genomic

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 corre-

sponding 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-

ocapsid proteins were separated by SDS-PAGE respec-

tively (Fig 1A) Western blot results showed that the

rVP31c polyclonal antiserum was reacted specifically with

VP31 in the purified viral envelope proteins while no

binding could be found in the nucleocapsid proteins (Fig

1B) It confirmed that this protein should belong to viral

envelope

Localization of VP31 protein by IEM

Purified WSSV virions were tested to be intact under

transmission electron microscopy (Fig 4A) When both

WSSV virions and nucleocapsids were incubated with

rVP31c antiserum followed with gold-labeled secondary














pi (0 2 4 6 8 12 18 24 and 48 h pi)








(Fig 4C)























not shown)

Discussion

























































































mM MgCl2 pH 75)


































































of WSSV





















negative control


Escherichia coli






































Western blot














microscopy















above













detection

Acknowledgments






References



1826ndash1833








































1944ndash1947





85 2035ndash2044













564ndash571





10126ndash10137



















68 850ndash858








11360ndash11370




286 7ndash22








1690ndash1696
















11811ndash11820






























pi (0 2 4 6 8 12 18 24 and 48 h pi)








(Fig 4C)























not shown)

Discussion

























































































mM MgCl2 pH 75)


































































of WSSV





















negative control


Escherichia coli






































Western blot














microscopy















above













detection

Acknowledgments






References



1826ndash1833








































1944ndash1947





85 2035ndash2044













564ndash571





10126ndash10137



















68 850ndash858








11360ndash11370




286 7ndash22








1690ndash1696
















11811ndash11820




























not shown)

Discussion

























































































mM MgCl2 pH 75)


































































of WSSV





















negative control


Escherichia coli






































Western blot














microscopy















above













detection

Acknowledgments






References



1826ndash1833








































1944ndash1947





85 2035ndash2044













564ndash571





10126ndash10137



















68 850ndash858








11360ndash11370




286 7ndash22








1690ndash1696
















11811ndash11820



























































mM MgCl2 pH 75)


































































of WSSV





















negative control


Escherichia coli






































Western blot














microscopy















above













detection

Acknowledgments






References



1826ndash1833








































1944ndash1947





85 2035ndash2044













564ndash571





10126ndash10137



















68 850ndash858








11360ndash11370




286 7ndash22








1690ndash1696
















11811ndash11820
























of WSSV





















negative control


Escherichia coli






































Western blot














microscopy















above













detection

Acknowledgments






References



1826ndash1833








































1944ndash1947





85 2035ndash2044













564ndash571





10126ndash10137



















68 850ndash858








11360ndash11370




286 7ndash22








1690ndash1696
















11811ndash11820


























detection

Acknowledgments






References



1826ndash1833








































1944ndash1947





85 2035ndash2044













564ndash571





10126ndash10137



















68 850ndash858








11360ndash11370




286 7ndash22








1690ndash1696
















11811ndash11820































11811ndash11820

















Identification and characterization of a prawn white spot syndrome virus gene that encodes an...

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