JOURNAL OF VIROLOGY, Jan. 2005, p. 140–149
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 1
The Unique Stacked Rings in the Nucleocapsid of the White Spot
Syndrome Virus Virion Are Formed by the Major Structural
Protein VP664, the Largest Viral Structural Protein
Jiann-Horng Leu,1Jyh-Ming Tsai,1Han-Ching Wang,1Andrew H.-J. Wang,2,3
Chung-Hsiung Wang,4Guang-Hsiung Kou,1* and Chu-Fang Lo1*
Institute of Zoology1and Department of Entomology,4National Taiwan University, and Core Facilities for
Proteomics Research2and Institute of Biological Chemistry,3Academia Sinica, Taipei, Taiwan
Received 17 May 2004/Accepted 18 August 2004
One unique feature of the shrimp white spot syndrome virus (WSSV) genome is the presence of a giant open
reading frame (ORF) of 18,234 nucleotides that encodes a long polypeptide of 6,077 amino acids with a hitherto
unknown function. In the present study, by applying proteomic methodology to analyze the sodium dodecyl
sulfate-polyacrylamide gel electrophoresis profile of purified WSSV virions by liquid chromatography-mass
spectrometry (LC-MS/MS), we found that this giant polypeptide, designated VP664, is one of the viral
structural proteins. The existence of the corresponding 18-kb transcript was confirmed by sequencing analysis
of reverse transcription-PCR products, which also showed that vp664 was intron-less. A time course analysis
showed that this transcript was actively transcribed at the late stage, suggesting that this gene product should
contribute primarily to the assembly and morphogenesis of the virion. Several polyclonal antisera against this
giant protein were prepared, and one of them was successfully used for immunoelectron microscopy analysis
to localize the protein in the virion. Immunoelectron microscopy with a gold-labeled secondary antibody
showed that the gold particles were regularly distributed around the periphery of the nucleocapsid with a
periodicity that matched the characteristic stacked ring subunits that appear as striations. From this and other
evidence, we argue that this giant ORF in fact encodes the major WSSV nucleocapsid protein.
White spot syndrome virus (WSSV) is one of the most dev-
astating shrimp pathogens, and it has caused serious damage to
the worldwide shrimp culture industry. Although this virus can
infect several crustacean species, including shrimp, crab, and
crayfish (9, 12, 19, 24, 42), its virulence is particularly high in
infected penaeid shrimp, and mortality can reach 90 to 100%
within 3 to 7 days of infection (9, 46). The WSSV virion is a
nonoccluded, enveloped particle of approximately 275 by 120
nm with an olive-to-bacilliform shape, and it has a nucleocap-
sid (300 by 70 nm) with periodic striations perpendicular to the
long axis (42, 43). The most prominent feature of WSSV is the
presence of a tail-like extension at one end of the virion (10,
43), which gives this virus the family name Nimaviridae (27)
(“nima” is Latin for “thread”).
The complete genome sequences of three WSSV isolates
have been determined, and 532 open reading frames (ORFs)
that contain ?60 amino acids have been identified for the
Taiwan isolate (GenBank accession number AF440570). Ho-
mology searches against the NCBInr database suggest possible
roles or functions for only a few of these ORFs, and most of
the ORFs posted for the Taiwan isolate show no significant
similarity to other known proteins. Similar results have been
reported for the other two isolates (40, 44). Until recently,
?5% of the ORFs had been shown to encode either nonstruc-
tural proteins (ribonucleotide reductase, protein kinase, the
chimeric thymidine kinase/thymidylate kinase, and DNA poly-
merase [8, 22, 35, 36]) or structural proteins (VP28, VP26,
VP24, VP19, and VP15 [38, 39, 41, 47, 48, 49]). Recently,
however, thanks to the introduction of proteomic methods, the
total number of known viral structural proteins has increased
to 39 (17, 18, 21, 34). Tsai et al. (34) also reported a protein
that was much larger than the largest (220 kDa) marker pro-
tein, and by using liquid chromatography–nano-electrospray
ionization–mass spectrometry (LC–nano-ESI–MS/MS), they
found that this corresponded to a giant ORF that encodes a
long polypeptide of 6,077 amino acids with a theoretical mass
of 664 kDa. In this paper, we further investigate the gene
expression and function of this protein, designated VP664, and
present evidence that it is associated with one of the unique
features of the WSSV virion, i.e., the stacked ring subunits that
appear as striations on the WSSV nucleocapsid.
MATERIALS AND METHODS
Virus. The virus used for this study was isolated from a batch of WSSV-
infected Penaeus monodon shrimp collected in Taiwan in 1994 (42) and is now
known as the WSSV Taiwan isolate (25). The complete genome sequence
(307,287 bp) of this isolate is available in GenBank under accession number
Proliferation and purification of WSSV virions. Gill and epithelial tissues from
shrimp (Penaeus monodon; mean weight, 10 g) infected with the WSSV Taiwan
isolate were homogenized in TNE buffer (50 mM Tris-HCl, 0.1 M NaCl, and 1
mM EDTA, pH 7.5) at 0.25 g/ml. After centrifugation at 1,500 ? g for 10 min,
the supernatant was filtered (0.45-?m-pore-size filter) and injected (0.2 ml; 1:10
dilution in TNE) intramuscularly into healthy Procambarus clarkii crayfish. After
* Corresponding author. Mailing address: Institute of Zoology, Na-
tional Taiwan University, Taipei 106, Taiwan, Republic of China.
Phone: 886-2-23633562. Fax: 886-2-23638179. E-mail for Chu-Fan Lo:
email@example.com. E-mail for Guang-Hsiung Kou: ghkou@ntu
propagation of the virus in the crayfish for 4 to 6 days, the virions in the
hemolymph were purified as described previously (34). (Procambarus clarkii was
a convenient host for this study because it can sustain a heavy WSSV viral load
indefinitely, whereas in Penaeus monodon, once virus replication is triggered, the
disease progresses along a fixed course and ends in mortality in just a few days.)
Mass analysis. Identification of the WSSV structural protein by LC–nano-
ESI–MS/MS was performed as described previously (34).
Antibody preparation. PCR fragments representing six different coding re-
gions of vp664 were amplified by use of the primer sets listed in Table 1, digested
with restriction enzymes, and cloned into pET-28b(?). The resulting pET clones
were transformed into BL21 Codon Plus Escherichia coli cells (Stratagene). For
protein expression and purification, the cells were grown overnight at 37°C in
Luria-Bertani medium supplemented with 50 ?g of kanamycin/ml and 34 ?g of
chloramphenicol/ml. The cells were inoculated into new medium at a ratio of
1:300 and grown at 37°C for 1.5 to 2 h. Expression was induced by the addition
of 1 mM IPTG (isopropyl-?-D-thiogalactopyranoside), and incubation was con-
tinued for another 1.5 to 3 h. The induced bacteria were spun down at 4°C,
suspended in ice-cold phosphate-buffered saline containing 10% glycerol and a
protease inhibitor cocktail tablet (Roche Molecular Biochemicals), and soni-
cated for 30 s on ice. The insoluble debris was collected by centrifugation,
suspended in phosphate-buffered saline containing 1.5% sodium lauryl sarcosine,
and solubilized by shaking at 4°C for 2 h. The supernatant was clarified by
centrifugation and mixed with Ni-nitrilotriacetic acid-agarose beads on a rotating
wheel at 4°C for 16 h or overnight. The beads were then washed several times
with ice-cold wash buffer (1 M NaCl, 10 mM Tris-HCl, pH 7.5) to remove
unbound material. The fusion proteins were eluted directly from the beads with
sodium dodecyl sulfate (SDS) sample buffer and then were subjected to SDS-
polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The protein bands
containing the fusion proteins were sliced from the gel, minced, mixed with
Freund’s adjuvant, and used for antibody production.
Western blot analysis. Purified virions were subjected to SDS-PAGE, and the
separated proteins were then transferred to a polyvinylidene difluoride (PVDF)
membrane (MSI). The membranes were incubated in blocking buffer (1% bovine
serum albumin, 5% skim milk, 50 mM Tris, 200 mM NaCl, pH 7.5) at 4°C
overnight, followed by incubation with a polyclonal rabbit anti-VP664-1 antibody
(1:5,000 dilution in blocking buffer, or as indicated in Results) for 1 h at room
temperature. After the membrane was washed three times with TBS-T (0.1%
Tween 20 in Tris-buffered saline), it was incubated with a horseradish peroxi-
dase-conjugated anti-rabbit secondary antibody diluted 5,000-fold in blocking
buffer. The membrane was washed as described above, and proteins were visu-
alized by use of a chemiluminescence reagent (Perkin-Elmer, Inc.).
Localization of WSSV VP664 by immunoelectron microscopy (IEM). A puri-
fied WSSV virion suspension was adsorbed to Formvar-supported and carbon-
coated nickel grids (150 mesh) and incubated for 5 min at room temperature.
The primary antibody and preimmune rabbit serum were diluted 1:50 in incu-
bation buffer (0.1% Aurion Basic-c, 15 mM NaN3, 10 mM phosphate buffer, 150
mM NaCl, pH 7.4). The grids were blocked with blocking buffer (5% bovine
serum albumin, 5% normal serum, 0.1% cold water skin gelatin, 10 mM phos-
phate buffer, 150 mM NaCl, pH 7.4) for 15 min and then incubated with a diluted
primary antibody or preimmune rabbit serum for 1 h at room temperature. After
several washes with incubation buffer, the grids were incubated with a goat
anti-rabbit secondary antibody conjugated with 6- or 15-nm-diameter gold par-
ticles (1:40 dilution in incubation buffer) for 1 h at room temperature. The grids
were then washed extensively with incubation buffer, washed twice more with
distilled water to remove excess salt, and stained with 2% phosphotungstic acid
(pH 7.2) for 30 s. Specimens were examined with a transmission electron micro-
Time course analysis of vp664 by RT-PCR. Since temporal WSSV gene ex-
pression has already been widely studied with Penaeus monodon (e.g., see ref-
erences 7, 8, 22, 35, and 36), for a more direct comparison we continued to use
Penaeus monodon in this study for a time course analysis of vp664 by reverse
transcription-PCR (RT-PCR). Briefly, Penaeus monodon specimens were exper-
imentally infected by intramuscular injection with WSSV and subsequently col-
lected at the indicated times postinfection according to a procedure described by
Tsai et al. (35). Total RNAs were isolated from the pleopods (swimming legs) of
WSSV-infected shrimp or healthy shrimp by use of the TRIzol reagent according
to the manufacturer’s instructions (Invitrogen Corp.). The isolated RNAs (20
?g) were treated with DNase I (Roche Molecular Biochemicals) at 37°C for 1 h
and then recovered by phenol-chloroform-isoamyl alcohol extraction and etha-
nol precipitation. The total RNAs were reverse transcribed with SuperScript II
reverse transcriptase (Invitrogen Corp.) and an oligo(dT) anchor primer (Roche
Molecular Biochemicals). The first-strand cDNA products were subjected to
PCRs with the primer set 5?-CGGCGCAACAACAACAAGCA-3? and 5?-GTA
GTTGGGGGCTAAACACG-3? for WSSV vp664. The WSSV dna pol transcript
was amplified with the primer set 5?-CCCCCGGGATGCTTCACTTTAATGA
AAA-3? and 5?-CCCCCGGGCTTTTTGTAAGGGGTGAAAG-3?. The WSSV
vp28 transcript was amplified with the primer set 5?-ATCCTCGCCATCACTG
CTGT-3? and 5?-TTACTCGGTCTCAGTGCCAG-3?. The ?-actin transcript
was amplified with the primer set 5?-GAYGAYATGGAGAAGATCTGG-3?
and 5?-CCRGGGTACATGGTGGTGCC-3? and was used as an internal control
for RNA quality and amplification efficiency.
Full-length analysis of the 18-kb vp664 transcript by RT-PCR. Total RNAs
isolated from WSSV-infected shrimp at 36 h postinfection (hpi) were treated
with DNase I, extracted with phenol-chloroform-isoamyl alcohol, and precipi-
tated with ethanol. The total RNAs were primed with both an oligo(dT) anchor
primer (Roche Molecular Biochemicals) and a random primer (Promega) and
were reverse transcribed with SuperScript II reverse transcriptase (Invitrogen
Corp.). The first-strand cDNA products were subjected to PCRs with the 15
primer sets listed in Table 2.
5? and 3? RACE. The 5? and 3? untranslated regions of vp664 were determined
by use of a commercial 5?/3? random amplification of cDNA ends (RACE) kit
according to the instructions provided by the manufacturer (Roche Molecular
Biochemicals). Total RNAs were isolated and treated with DNase I as described
above. In the case of 3? RACE, the first-strand cDNA was synthesized by use of
an oligo(dT) anchor primer. The resulting cDNA was amplified with the specific
forward primer 5?-CGCCTCAACCCCTACATC-3? and the anchor primer. For
5? RACE, the first-strand cDNA was synthesized with the vp664 gene-specific
primer 1 (5?-TTCTGACGCAGCACGAAGAG-3?) and then purified by use of a
High Pure PCR product purification kit (Roche). The cDNA was given a 3? tail
of dTTPs by the use of terminal transferase and was subjected to a first round of
PCR with an oligo(dA) anchor primer and the vp664 gene-specific primer 2
(5?-CTGAATCGTATGTGGTCGTGGA-3?). The PCR product was diluted and
subjected to a second round of PCR with the anchor primer and the vp664
gene-specific primer 3 (5?-GATCCAGCACGAAGCTGCGATTG-3?). The final
TABLE 1. Primer sets for amplicons used to generate recombinant proteins for antibody production
Antibody Primer nameSequence (5?-3?)a
Size (bp) of
WSSV419-10.Hind III R
WSSV419-14 Hind III R
aUnderlining indicates the incorporated restriction enzyme sites at the 5? ends of the primers.
VOL. 79, 2005 VP664 IS THE MAJOR NUCLEOCAPSID PROTEIN OF WSSV141
PCR products of 5? and 3? RACE were cloned into the pGEM-T Easy vector
(Promega) and then sequenced.
Identification of WSSV VP664 by mass spectrometry. Figure
1A shows the protein profile of the WSSV virion, as analyzed
by gradient SDS-PAGE (8 to 18% acrylamide). At least 34
discrete bands were identified. These protein bands were ex-
cised from the gel and subjected to trypsin digestion. The
resulting peptides were then sequenced by LC–nano-ESI–MS/
MS. The peptide sequences obtained from the MS/MS data
were then analyzed against the NCBInr database by use of the
Mascot server, and 33 protein bands were identified as con-
taining WSSV-encoded peptides (31). The uppermost band in
Fig. 1A (indicated with an arrow) contained peptide sequences
encoded by vp664, with matching peptide sequences (Fig. 1B)
that covered 14% of this giant protein. As shown in Fig. 1B, the
matched sequences were evenly distributed along the entire
protein, from the N terminus to the C terminus, which con-
firms the expression of the full 6,077 amino acids of this giant
viral structural protein.
Preparation of VP664-specific antibodies and Western blot
analysis. To further characterize this giant structural protein,
we prepared antibodies against the coding region. A series of
pET expression plasmids harboring different parts of the cod-
ing regions of the WSSV vp664 gene were constructed. The
expressed proteins were purified, separated by SDS-PAGE,
extracted from the gel, and injected into a rabbit to produce
A total of six antibodies were produced (Table 1; Fig. 2).
The antigenic specificity of each antibody was determined by
Western blotting. Purified virions were subjected to SDS-
PAGE, transferred onto a PVDF membrane, and probed with
5,000-fold-diluted antibodies. As shown in Fig. 3, a protein
band with a relatively high molecular mass, far larger than the
180-kDa marker, was recognized by all six antibodies. The
VP664-10 antibody also identified a minor protein band with a
slightly lower molecular mass. A more complex pattern was
produced by the VP664-1 antibody: in addition to a smear
pattern in the upper part of the membrane blot, there were
also about 20 other immunoreactive bands. We interpreted
these as either degraded or C-terminally truncated VP664 pro-
teins, in all of which the N-terminal region was presumably
preserved intact. Decreasing the exposure time of the 664–1
blot revealed that the protein band with the strongest signal
intensity was the same size as those recognized by the other
antibodies (data not shown). Dilution of the 664–1 antibody
(10,000-, 50,000-, and 100,000-fold) produced a similar result
(Fig. 3B). The 664–1 antibody, which had a titer that was at
least 10-fold higher than those of the other antibodies, was
chosen for subsequent immunogold labeling analyses.
Localization of VP664 in the virion by IEM. The localization
of VP664 in the virions of WSSV was studied by IEM with the
664–1 antibody and a gold particle-conjugated secondary an-
TABLE 2. Primer sets used for RT-PCR analysis
Primer set Primer name Sequence (5?-3?)a
Size (bp) of
WSSV419-2 HindlII R
WSSV419-2 BamHI F
WSSV419-6 HindIII R
WSSV419-6 BamHI F
WSSV419-10 HindIII R
WSSV419-10 BamHI F
WSSV419-14 HindIII R
WSSV419-14 BamHI F
aUnderlining indicates the incorporated restriction enzyme sites at the 5? ends of the primers.
142 LEU ET AL.J. VIROL.
FIG. 1. (A) SDS-PAGE profile of purified WSSV virion. The uppermost band (VP664; arrow) was excised from the gel and subjected to
LC-MS/MS analysis. The peptide sequences suggested that this protein was the translated product of the vp664 ORF. Several other major
structural proteins, VP28, VP26, and VP24, are also indicated. (B) Amino acid sequence of VP664. The peptide sequences identified by LC-MS/MS
are indicated by underlining. Three bipartite nuclear targeting sequences are boxed. The RGD motif at positions 395 to 397 is indicated by double
VOL. 79, 2005VP664 IS THE MAJOR NUCLEOCAPSID PROTEIN OF WSSV143
tibody. During the procedures for virus purification and im-
munoelectron microscopy, some viral envelopes spontaneously
detached from their virions and released their nucleocapsids
into the preparation. Figure 4A shows a WSSV preparation
from Penaeus monodon (the WSSV isolate used to infect
Penaeus monodon for Fig. 4 was the same Taiwan isolate that
was used to infect the crayfish Procambarus clarkii in the other
parts of this study) that very clearly illustrates this phenome-
non, with examples of both entire and ruptured mature virions.
Figure 4B, on the other hand, shows an example of an imma-
ture naked nucleocapsid, i.e., a nucleocapsid that has not yet
become enveloped. Note that immature nucleocapsids can eas-
ily be distinguished from exposed mature nucleocapsids by
their shape: immature nucleocapsids are thin and rod-shaped
while mature nucleocapsids are fatter and rounder. These mor-
phological observations apply to both Penaeus monodon and
Procambarus clarkii. The IEM results for the crayfish prepara-
tions (Fig. 5) showed that the gold particles were almost all
associated with the viral nucleocapsids, and no gold particles
were found attached to the envelopes (Fig. 5A and B). The
gold particles were often distributed quite regularly along the
periphery of the nucleocapsids and were not confined to any
specific regions (Fig. 5C). In these immature nucleocapsids,
the periodicity of the gold particles also corresponded to the
periodicity of the stacked ring-like structures. Occasionally,
regularly spaced gold particles were also seen across the “top”
of a mature nucleocapsid in a pattern that appeared to follow
the periodicity of the stacked ring-like structures (Fig. 5D).
These results strongly suggest that VP664 is a nucleocap-
sid—as opposed to an envelope—protein. In the negative con-
trol (Fig. 5E), the preimmune rabbit antiserum showed limited
background cross-reactivity when tested against the WSSV
virions and nucleocapsids.
Time course analysis of vp664 transcript. The expression
profiles of the vp664 transcript in pleopods of adult Penaeus
monodon shrimp at various stages of infection with WSSV
were analyzed by RT-PCR. Two previously identified WSSV
genes, dna polymerase (dna pol) and vp28, were also included in
this assay for comparison. The results (Fig. 6) showed that a
small amount of vp664 transcript could be detected as early as
2 hpi and that the expression level remained unchanged until 8
hpi. After that, the amount of vp664 transcript dramatically
increased at 12 hpi and continued to increase until the end of
the analysis. The dna pol gene, an early expressed gene, was
transcribed as early as 2 hpi and steadily increased up to 36 hpi.
The transcript of vp28, a gene that encodes a WSSV viral
envelope protein, was also detected at 2 hpi, albeit at a low
level, and like vp664, this low level of expression remained
unchanged until 8 hpi. After that, the amount of vp28 tran-
script increased at 12 hpi and continued to increase until the
end of the analysis. The expression kinetics of the vp664 tran-
script were thus quite similar to those of vp28: they were both
highly expressed at the late stage and both translated into large
amounts of viral structural proteins.
Mapping the 5? and 3? ends of the transcript by using
RACE. The 5? and 3? ends of the vp664 transcript were deter-
mined by the RACE method. Due to the presence of a long
stretch of thymidines in the 5? upstream region, the usual 5?
RACE protocol (22, 35) was modified slightly, as described in
FIG. 2. Schematic diagram showing the regions of VP664 used for antibody preparation and RT-PCR analysis. The separate shaded boxes
represent the amino acid sequences corresponding to the recombinant proteins used for antibody production. The labeling of the RT-PCR
products shows the primer sets that were used for amplification (Table 2). The locations of two structural features, the RGD motif and bipartite
NLSs, are indicated on the protein, and the WSSV diagnostic PCR primer set developed by our laboratory, pms146F1 and pms146R1 (23), is also
FIG. 3. Specificities of six VP664 antibodies as tested by Western
blotting. (A) The purified virions were subjected to SDS-PAGE, and
the separated viral proteins were transferred onto a PVDF membrane
and probed with different antibodies diluted 5,000-fold. (B) Results of
probing with the 664–1 antibody at higher dilutions (10,000, 50,000,
144 LEU ET AL.J. VIROL.
Materials and Methods. The RACE products were cloned into
the pGEM-T Easy vector, six clones were randomly chosen for
sequencing, and the results are shown in Fig. 7. Two transcrip-
tion initiation sites of the vp664 transcript were located 196 and
199 nucleotides upstream of the translational start codon
(TAAC [initiation sites are shown in bold and underlined]). To
identify possible motifs that are important for transcriptional
regulation, we compared the 5? upstream region of vp664 with
the upstream regions of several structural protein genes, in-
cluding vp28, vp26, vp24, vp19, and vp15 (26). A consensus
sequence, AATAAC, was identified at the transcription initi-
ation sites of both vp664 and another major structural protein
gene, vp28, suggesting the importance of this consensus se-
quence in regulating the transcription of both of these highly
expressed (Fig. 1A) late genes. (The major transcription initi-
ation site of vp28 occurs at the C residue .) In vp664, the
consensus sequence also formed part of an inverted repeat
(boxed sequence in Fig. 7). Another sequence feature shared
by both genes is the presence of a T track in the 5? upstream
region, although the importance of this T track remains to be
determined. In the 3? untranslated region, two polyadenylation
signals (AATAAA) were found 73 and 90 bp downstream of
the vp664 stop codon. 3? RACE analysis showed that the
poly(A) tail addition site was located 12 bp downstream of the
first polyadenylation signal.
Splicing events in the vp664 transcript. To demonstrate the
existence of the long vp664 transcript as well as to study
whether any splicing events had occurred, we conducted a
series of RT-PCR analyses to verify its structure. A total of 15
primer pairs were used to generate 15 overlapping RT-PCR
products (Fig. 2) that provided complete coverage of the entire
coding region. The locations of the primers and the sizes of the
predicted amplified products are shown in Table 2, and the
results of the analyses are shown in Fig. 8. The fact that no size
differences could be discerned between the PCR products am-
plified from cDNAs and those amplified from the virus
genomic DNA suggests that no splicing events occurred. The
absence of any amplified PCR products from the RNA-only
(negative control) sample means that the PCR products am-
plified from the cDNAs were not due to contamination by
DNA from the viral genome. Sequencing of the overlapping
amplicons (data not shown) also completely matched the pep-
tide sequence shown in Fig. 1B, further confirming that there
were no deletions and thus that the gene is intron-free.
Amino acid sequence analysis. After BestFit (32) was used
to confirm that the amino acid sequence of VP664 has no
repeat sequences, the entire sequence was analyzed by several
homology searches against various databases to identify the
functional motifs and structural features of this giant polypep-
tide. A search using TMpred against the TMbase database (15)
did not find any predicted transmembrane domains; a search
using SignalP V1.1 (28) against the SwissProt database (2) was
negative for signal peptide prediction; and the results of a
search using BLASTP showed that there was no significant
homology to any known proteins in the nonredundant NCBI
database. However, a search of the PROSITE database by the
use of InterProScan identified a cell attachment site signature,
the RGD motif, in the N-terminal region, at positions 395 to
397. This motif has been shown to be involved in virus binding
to cellular integrins during infection by several viruses, such as
rotaviruses (14), papillomaviruses (11), foot-and-mouth dis-
ease virus (13), and human herpesvirus 8 (1). Further research
will be needed, however, to investigate whether the RGD motif
in VP664 mediates the binding of WSSV to shrimp cells or to
some extracellular matrix proteins. Three bipartite nuclear lo-
calization signal (NLS) sequences were also identified from the
PROSITE database, located at positions 2160 to 2176, 2468 to
2484, and 4313 to 4329. Again, the functionality of these do-
mains will need to be determined experimentally, but since an
NLS serves to direct nuclear proteins into the nucleus and
FIG. 4. Electron micrographs of purified virions. (A) The white outlines indicate (i) a complete mature virion with a characteristic tail, (ii) a
ruptured mature virion with more than half of the nucleocapsid exposed outside of the envelope, and (iii) a completely exposed mature
nucleocapsid. (B) Immature, naked nucleocapsid prior to being enveloped.
VOL. 79, 2005 VP664 IS THE MAJOR NUCLEOCAPSID PROTEIN OF WSSV145
since the assembly of WSSV virus particles occurs in the nu-
cleus, we would expect this major viral nucleocapsid protein to
contain at least one NLS that was functional.
For this paper, we used LC–nano-ESI–MS/MS analysis to
confirm the existence of the giant polypeptide encoded by
vp664 (Fig. 1). A time course analysis of this viral structural
protein gene (Fig. 6) showed that vp664 was actively tran-
scribed at the late stage, suggesting that its product is involved
primarily in the assembly and morphogenesis of the virion.
Immunoelectron microscopy analysis showed that VP664 was
localized to the viral nucleocapsid, with numerous gold parti-
cles distributed fairly evenly along the periphery of the entire
nucleocapsid and not confined to any specific or preferred
region (Fig. 5). Transmission electron micrographs published
in earlier studies (10, 16, 42) have already shown that the
rod-shaped WSSV nucleocapsid is divided into about 15 to 16
vertical segments that are perpendicular to the long axis and
about 18 to 20 nm thick. It has been argued that each segment
is composed of double rows of 14 globular subunits of 8 nm in
FIG. 5. Immunoelectron microscopy analysis of purified virions probed with VP664 antibody. (A and B) The antibody specifically binds to the
nucleocapsid and not to the viral envelope. (C) Most of the gold particles are localized to the perimeter of the nucleocapsid. (D) Occasionally,
the gold particles are localized across the top of a nucleocapsid. (E) A preimmune rabbit antibody or gold-conjugated secondary antibody does
not bind to virions.
FIG. 6. Time course analysis of vp664 transcripts by RT-PCR. To-
tal RNAs were extracted from the pleopods of WSSV-infected shrimp
and were subjected to RT-PCR analysis with the indicated primers,
i.e., vp664, dna pol, and vp28. Shrimp actin was also included as a
template control. Lane headings show times postinfection (hours). M,
146 LEU ET AL. J. VIROL.
diameter (10). In the present study, it was very difficult to
confirm unequivocally the existence of these discrete globular
subunits. Each segment was characterized, however, by a pat-
tern that repeated itself approximately five times across the
width of the virion (Fig. 5C). Curiously, in some of the mature
virions, the gold particles had a similar periodicity to this re-
peating pattern (Fig. 5D), from which we hypothesize that this
pattern may in fact arise from the conformation and packaging
of the giant VP664 proteins. This hypothesis is further sup-
ported by the fact that when WSSV nucleocapsids are purified
by CsCl gradient centrifugation and subjected to SDS-PAGE,
the only major band (there are several much fainter minor
bands) corresponds to VP664 (data not shown), which implies
that VP664 is the major component of the nucleocapsid. Thus,
the giant polypeptide encoded by vp664 must account for the
stacked, patterned rings that characterize the naked WSSV
nucleocapsid, if only because there are no other candidate
proteins in the nucleocapsid to account for this feature. The
distribution of the gold particles further suggests that the N
terminus of this protein is left exposed and that each of these
gold particles is attached (via the linked VP664 N-terminus-
specific antibody) to the exposed N terminus of a single VP664
molecule. We note here, also, that in the immunoelectron
microscopy results for the other five antibodies (664–8, 664–9,
664–10, 664–12, and 664–14) (Fig. 3), bound gold particles
were not detected (data not shown). We interpret this as fur-
ther evidence that while the N-terminal region remains ex-
posed, the bulk of this giant molecule is folded in such a way
that the binding sites for these other antibodies are inside the
protein and thus inaccessible. Another curious observation is
that while gold particles were sometimes seen across the top of
a mature exposed nucleocapsid (Fig. 5D), gold particles were
hardly ever seen across the top of an immature nucleocapsid
(e.g., see Fig. 5A, B, and C). From this observation, we infer
that the antibody was less tightly bound to the immature nu-
cleocapsid and was thus more easily washed away. The reason
for this is unclear, but we speculate that it may be related to
changes in the conformation of VP664 as the nucleocapsid
Now that the complete WSSV genome is available, the ap-
plication of proteomic methodology has led to the identifica-
tion of many novel viral structural proteins (17, 18, 21; this
paper). Most of these viral structural proteins are associated
with the viral envelope, and only three, VP26, VP24, and
VP15, are considered viral nucleocapsid proteins. For three of
these proteins, this determination was made by treating the
virions with NP-40 to remove the envelope and then analyzing
them by SDS-PAGE or Western blotting (38, 39, 41). How-
ever, a more recent study using IEM showed that VP26 is
actually localized to the envelope (47). In fact, until now,
immunoelectron microscopy has only been successful in locat-
ing WSSV envelope proteins, and VP664 is the first WSSV
viral nucleocapsid protein to be directly localized by this tech-
nique. The present demonstration that the anti-VP664 anti-
body can be used with IEM to locate the protein in WSSV
virions suggests that this antibody will be an invaluable tool for
FIG. 7. Partial nucleotide sequences of 5? and 3? untranslated re-
gions of vp664. (A) Sequencing results for six RT-PCR clones showed
that there are two transcription initiation sites for vp664. The putative
transcription initiation sites are shown in bold and underlined. The
inverted repeats are boxed. (B) Underlining indicates the locations of
two polyadenylation signals (AATAAA) downstream of the stop
codon. The poly(A) addition site, as determined by 3? RACE, occurs
12 bp downstream of the first polyadenylation signal and is indicated
by a box.
FIG. 8. Analysis of the entire 18-kb vp664 transcript by RT-PCRs
with 15 sets of primer pairs. Lane headings show the primer pairs, as
listed in Table 2. Three reactions were carried out for each primer pair
as follows: RNA lanes, total RNA as a negative control; vDNA lanes,
viral genomic DNA as a positive control; and cDNA lanes, reverse-
transcribed cDNA as a PCR template.
VOL. 79, 2005 VP664 IS THE MAJOR NUCLEOCAPSID PROTEIN OF WSSV 147
studying the assembly and morphogenesis of WSSV in infected
Proteins with molecular masses of ?600 kDa have been
reported for vertebrates (3, 20, 30, 31, 33, 45, 50), invertebrates
(29, 37), and protists (4, 5, 6). The largest known polypeptide
identified so far is the titin molecule (38,138 amino acids and
4.2 MDa in humans ), which is found in striated muscle.
Some invertebrates also have giant molecules (0.5 to 2.0 MDa)
that are functionally related to titin although they are not true
homologs (29, 37). However, large proteins are much less com-
mon in viruses, and so far there are no other known viral
proteins whose size comes close to the 6,077 amino acids and
(estimated) 664 kDa of VP664. It is tempting to speculate that
this uniquely large viral protein is also related to another
unique aspect of WSSV, that is, the ability of the WSSV nu-
cleocapsid to change its physical form from a compact naked
nucleocapsid (Fig. 4B) to the olive-shaped structure of the
mature virion (the volume of which is approximately twice that
of the naked nucleocapsid) (Fig. 4A, panel i, and Fig. 5E) to
the even larger, loosely packed, exposed mature nucleocapsid
(Fig. 4A, panels ii and iii). VP664 is especially implicated in
these physical changes because, as argued above, its molecules
evidently form the subunits of the stacked rings (Fig. 5C and
D). We anticipate that an investigation of the conformational
changes in the VP664 protein during viral morphogenesis will
help to elucidate these phenomena.
This investigation was supported financially by National Science
Council grants (NSC92-2311-B-002-014, NSC92-2317-B-002-001, and
NSC-92-2317-B-002-018). Proteomic mass spectrometry analyses were
performed by the Core Facilities for Proteomics Research located at
the Institute of Biological Chemistry, Academia Sinica, which are
supported by a National Science Council grant (91-3112-P-001-009-Y).
We thank Shin-Jen Lin and Chia-Wei Chang for help with the
expression and purification of the recombinant VP664 protein. We are
indebted to Paul Barlow for his helpful criticism.
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