Complete Genome Sequence of the Shrimp White Spot Bacilliform Virus

0022-538X/01/$04.00

⫹

0

DOI: 10.1128/JVI.75.23.11811–11820.2001

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Complete Genome Sequence of the Shrimp White Spot

Bacilliform Virus

FENG YANG, JUN HE, XIONGHUI LIN, QIN LI, DENG PAN, XIAOBO ZHANG,

XUN XU*

The Third Institute of Oceanography, Xiamen 361005, People’s Republic of China

Received 11 June 2001/Accepted 1 August 2001

We report the first complete genome sequence of a marine invertebrate virus. White spot bacilliform virus

(WSBV; or white spot syndrome virus) is a major shrimp pathogen with a high mortality rate and a wide host

range. Its double-stranded circular DNA genome of 305,107 bp contains 181 open reading frames (ORFs). Nine

homologous regions containing 47 repeated minifragments that include direct repeats, atypical inverted repeat

sequences, and imperfect palindromes were identified. This is the largest animal virus that has been completely

sequenced. Although WSBV is morphologically similar to insect baculovirus, the two viruses are not detectably

related at the amino acid level. Rather, some WSBV genes are more homologous to eukaryotic genes than viral

genes. In fact, sequence analysis indicates that WSBV differs from all known viruses, although a few genes

display a weak homology to herpesvirus genes. Most of the ORFs encode proteins that bear no homology to any

known proteins, either suggesting that WSBV represents a novel class of viruses or perhaps implying a

significant evolutionary distance between marine and terrestrial viruses. The most unique feature of WSBV is

the presence of an intact collagen gene, a gene encoding an extracellular matrix protein of animal cells that has

never been found in any viruses. Determination of the genome of WSBV will facilitate a better understanding of

the molecular mechanism underlying the pathogenesis of the WSBV virus and will also provide useful

infor-mation concerning the evolution and divergence of marine and terrestrial animal viruses at the molecular level.

White spot bacilliform virus (WSBV) or white spot

syn-drome virus (WSSV) is a major shrimp pathogen that is highly

virulent in penaeid shrimp, the most important species used in

aquaculture, and can also infect most species of crustacean (15,

32). Infection of penaeid shrimp by WSBV can result in

mor-tality of up to 90 to 100% within 3 to 7 days (57). A major

outbreak of WSBV infection in 1993 resulted in a 70%

reduc-tion in shrimp producreduc-tion in China (14, 57) and has raised

major concerns in aquaculture around the world. Prevention

and inhibition of infection by this virus can be difficult due

largely to the ability of WSBV to survive for a long time in the

environment (2 years in a shrimp pond) and also due to a poor

understanding of this virus at the molecular level.

WSBV was originally classified as an unassigned member of

the

Baculoviridae

because of its rod-shaped, enveloped

mor-phology (20). However, it was recently excluded from the

bac-ulovirus family and is temporarily unclassified due to the lack

of molecular information (53). The virus is known generally as

white spot syndrome virus (WSSV) (31), and a new genus

name,

Whispovirus

, was proposed by Vlak et al. (48). Sequence

analysis of individual genes and proteins later showed that

most WSBV proteins bear poor sequence homology to

bacu-lovirus proteins but have repeated regions similar to those of

some baculoviruses. To understand the molecular basis of viral

replication and infection, we decided to sequence the whole

genome of WSBV.

MATERIALS AND METHODS

Isolation and sequencing of WSBV genomic DNA.Intact WSBV genomic

DNA was isolated from dead and moribund WSBV-infectedPenaeus japonicus

shrimp which were collected from shrimp ponds in Tongan, Xiamen, east China, in October 1996 as previously described (56). A whole-genome random sequenc-ing method (19) was used to obtain the complete genome sequence for WSBV. Genomic DNA was cloned by the shotgun method intoSmalI-linearized pUC18 vector, amplified, and sequenced using ABI BigDye Terminator chemistry on ABI 377 and ABI 3700 capillary sequencers. Large DNA fragments of 5 to 10 kb were also obtained by partial digestion withSau3A1 and cloned into the pBlue-script vectors (41). This was used to form a genome scaffold and to verify the orientation and integrity of the contigs formed from the shotgun library. A total of 5,770 sequences for sevenfold coverage were assembled using the InnerPeace software by Charles Lawrence based on the Phred, Phrap, and Consed program originally developed at the University of Washington.

The WSBV genome sequence was confirmed by comparison of the observed restriction fragments from seven restriction enzymes (BamHI,EcoRI,HindIII,

KpnI,PstI,SalI, andXbaI) to those predicted from the sequence data and was also confirmed by the genome scaffold produced by sequence pairs from 1,495 large-insert clones, which covered 90% of the main genome.

Gaps were closed by a combination of sequence-walking of shotgun and PCR large-fragment libraries.

DNA sequence analysis.Genome DNA composition, structure, repeats,

re-striction enzyme patterns, and translation were analyzed with the DNAMAN software (Lynnon BioSoft, Vaudreuil, Canada). Open reading frames (ORFs) consisted of more than 60 codons that are initiated with a methionine codon. For detection of potential protein-coding regions, the codon usage bias and posi-tional base preference were evaluated by determining the codon frequency of known WSBV genes or cDNA cloned from the WSBV cDNA library. Homology searches were performed with the FASTA (38) and BLAST programs (3). Pro-tein motifs were analyzed by using the PROSITE database, release 16 (25). Trans-membrane domains and signal peptides were predicted with ANTHEPROT (23).

Preparation and screening of a WSBV cDNA library.Poly(A) mRNA was

isolated from WSBV-infected shrimp tissue using the PolyATtract System 1000 kit (Promega). Double-stranded cDNAs were synthesized using the SUPER-SCRIPT plasmid system for cDNA synthesis and plasmid cloning (GIBCO BRL). WSBV cDNA clones were selected by hybridization with the digoxigenin (DIG)-labeled WSBV genomic DNA probe (DIG labeling kit; Boehringer Mannheim) and sequenced. The transcription of some ORFs was also verified by PCR on a cDNA cocktail using ORF-specific primers.

Nucleotide sequence accession number.The complete WSBV sequence can be

obtained from the GenBank database (accession no. AF332093).

* Corresponding author. Mailing address: The Third Institute of

Oceanography, Xiamen 361005, People’s Republic of China. Phone:

86-592-2195296. Fax: 86-592-2085376. E-mail: xxu@public.xm.fj.cn.

11811

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RESULTS AND DISCUSSION

General features of the WSBV genome.

We have previously

developed a unique method that enables us to highly purify the

WSBV virus from infected shrimp tissue (56). A random

shot-gun method was employed to sequence the entire genome of

WSBV; the sequence was subsequently confirmed by the

ge-nome scaffold formed by sequencing a large-fragment DNA

library. The complete WSBV genome is a double-stranded

circular DNA of 305,107 bp, similar to a previous estimate of

290 kb (56). Since the origin of replication was unknown, the

start of the largest

Bam

HI fragment was chosen to be base 1

(Fig. 1). Three percent of the WSBV genome is made up of

nine homologous regions (

hr

s), while the remaining 97% of the

sequences are unique (see description below). The genome has

a total G

⫹

C content of 41%.

A total of 531 putative ORFs were identified by sequence

analysis, among which 181 ORFs are likely to encode

func-tional proteins (Table 1). This corresponds to an average gene

density of one gene per 1.7 kb. Thirty-six of the 181 ORFs

annotated here either have been identified by screening and

sequencing a WSBV cDNA library (Table 1) or have been

reported previously to encode functional proteins (45, 46, 48,

49, 50). Transcription of another 52 ORFs was confirmed by

reverse transcription-PCR (RT-PCR; see Material and

Meth-ods) (Table 1). The relative positions of the ORFs and

hr

s in

the genome are shown in Fig. 1. For 80% of the putative 181

ORFs there is a potential polyadenylation site (AATAAA)

downstream of the ORF (Table 1).

WSBV ORFs encode gene products homologous to known

proteins.

Table 1 contains a list of the 181 predicted WSBV

ORFs. Among 181 ORFs, the proteins encoded by 18 ORFs

show 40 to 68% identity to known proteins from other viruses

or organisms or contain an identifiable functional domain.

These proteins include enzymes involved in nucleic acid

me-tabolism and DNA replication, a collagen-like protein, and

three viral structure proteins (for details, see below). Thirty

FIG. 1. Circular representation of the WSBV genome. Arrows, positions (outer ring) of the 181 ORFs (red and blue indicate the different

directions of transcription); green rectangles, 9

hr

s. B, sites of

Bam

HI restriction enzymes (inner ring; their positions are in parentheses).

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TABLE 1. Listing of potentially expressed ORFs in WSBV

ORF Product position

(length [aaa_]) Best matchb[source]

BlastP score

Identity (%)

Length (aa)

Predicted structure and/or functionc_(position)d

Poly(A) signalc

WSV001 300501–445 (1,684) L23982, collagen type VII [Homo sapiens] 930 42 1,336 Collagen, TM ⫹ⴱ

WSV002 1118–495 (208) Nucleocapsid protein VP24, TM, SP ⫹ⴱ

WSV004 1511–1200 (104) ⫹ⴱⴱ

WSV006 2425–1541 (295) U07025, chitinase [Janthinobacterium lividum] 63 54 53 Glycosyl hydrolase, Pro-rich cluster (147–175), TM SP

⫺

WSV008 1749–2360 (204) Ser/Gly-rich region (4–80), TM ⫹ⴱ

WSV009 2672–2388 (95) ⫹

WSV011 3051–6953 (1,301) AF128951, flagellin [Escherichia coli] 41 18 496 TM, SP, ⫹ⴱ

WSV013 3955–3716 (80) ⫹

WSV020 6604–6254 (117) ⫺

WSV021 7645–7046 (200) TM ⫺

WSV022 7250–7432 (61) ⫹

WSV023 8502–7645 (286) ⫺

WSV025 9248–8556 (231) Ser/Glu-rich region (36–114) and basic

region (130–200), TM

⫺

WSV026 13936–9332 (1,535) AE003491,snogene product [Drosophila

melano-gaster]

71 25 185 Acidic region (1406–1445), TM ⫹

WSV035 16983–14068 (972) X77514, pupal cuticule protein [Galleria mellonella] 46 26 111 TM, SP ⫺ⴱ

WSV037 17000–20839 (1,280) Glu-rich cluster (578–636) ⫹

WSV045 20784–23726 (981) Acidic region (284–360), ATP/GTP binding

motif, TM

⫺

WSV047 21688–22047 (120) Basic region (24–59) ⫹

WSV049 22759–22145 (205) TM, SP ⫺

WSV051 23710–24297 (196) ⫹ⴱⴱ

WSV053 24906–24664 (81) ⫹

WSV055 25153–24965 (63) SP ⫹

WSV056 25878–25201 (226) Cys2/His2-type zinc finger ⫹ⴱ

WSV059 26631–27254 (208) ⫹ⴱ

WSV063 29077–28334 (248) AF078683, Ring-H2 finger protein RHA1a

[Arabidopsis thaliana]

44 40 52 Cys2/Cys2-type zinc finger ⫹

WSV064 30861–29080 (594) TM ⫹

WSV067 31092–31958 (289) NP-001062, thymidylate synthetase [Homo sapiens] 392 67 287 Thymidylate synthase ⫺

WSV069 32125–32796 (224) Cys2/His2-type zinc finger ⫹ⴱⴱ

WSV073 32948–34213 (422) TM, SP ⫹ⴱ

WSV076 34218–35045 (276) ⫺

WSV077 35074–35964 (297) TM ⫹ⴱ

WSV078 37245–36052 (398) AE003485, CG11122 gene product [Drosophila

melanogaster]

45 26 238 ⫹ⴱ

WSV079 38917–37385 (511) AL022223, putative protein [Arabidopsis thaliana] 43 33 69 EF-hand calcium-binding motif; Ring finger protein-like; Ser/Asp-rich region (204–330)

⫹ⴱ

WSV083 40718–38976 (581) AC018363, putative protein kinase [Arabidopsis

thaliana]

42 27 102 Protein kinase, Ser/Thr protein kinase active-site signature, TM

⫹

WSV091 42054–45488 (1,145) Glu/Ser-rich region (626–737), TM ⫹ⴱ

WSV097 45175–45471 (99) ⫹

WSV100 45951–47822 (624) AC024128, putative CBP [Arabidopsis thaliana] 47 41 60 CBP; Cys2/Cys2-type zinc finger, TM ⫹ⴱ

WSV107 48635–48943 (103) TM, SP ⫹

WSV108 50300–49083 (406) NP-012284, cell surface flocculin [Saccharomyces

cerevisiae]

55 24 237 Membrane-associated protein, TM ⫹ⴱ

WSV112 51809–50427 (461) Q89662, dUTP pyrophosphatase [fowl adenovirus type 1]

90 37 161 dUTPase ⫺

WSV115 52007–54910 (968) TM ⫺

WSV119 55055–58186 (1,044) Acidic region (122–174) ⫹ⴱⴱ

WSV128 58948–60057 (370) Repeat region (23–325) ⫹

WSV129 58956–60026 (357) X73481, mst101(2) [Drosophila hydei] 80 30 302 Repeat region (20–322) ⫹

WSV130 60581–60132 (150) ⫹ⴱⴱ

WSV131 62127–60676 (484) ⫹

WSV133 62204–63016 (271) TM ⫺

WSV134 62991–63656 (222) ⫹

WSV136 63666–64049 (128) TM, SP ⫹

WSV137 65042–64014 (337) TM ⫹ⴱⴱ

WSV139 68659–65036 (1,208) ⫺ⴱ

WSV142 69118–68708 (137) ⫺

WSV143 69265–76203 (2,313) AB037755, KIAA1334 protein [Homo sapiens] 48 18 554 Repeat region (325–427); Asn-rich cluster (990–1094), TM

⫹ⴱⴱ

WSV146 75119–74922 (66) ⫹

WSV147 77653–76277 (459) ⫹ⴱ

WSV150 78365–77451 (305) ⫺

WSV151 79065–83372 (1,436) TM ⫹ⴱ

WSV161 85707–83431 (759) Glu-rich cluster (97–134), Asn/pro-rich

region (467–637)

⫹ⴱ

WSV166 88980–85765 (1,072) AE003593, CG10523 gene product [Drosophila

melanogaster]

49 34 69 Cys2/Cys2-type zinc finger, TM ⫹ⴱ

WSV172 91607–89064 (848) P21524, ribonucleoside-diphosphate reductase large chain [Saccharomyces cerevisiae]

728 48 790 Ribonucleotide reductase large subunit, TM ⫹ⴱ

WSV177 92964–92647 (106) ⫹

WSV178 93229–94134 (302) T29757, protein UNC-89 [Caenorhabditis elegans] 46 22 216 Repeat region (82–302), TM, SP ⫹ⴱⴱ

WSV181 94624–95739 (372) ⫺

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TABLE 1—

Continued

ORF Product position

(length [aaa_]) Best matchb[source]

BlastP score

Identity (%)

Length (aa)

Predicted structure and/or functionc_(position)d

Poly(A) signalc

WSV184 95744–97366 (541) Cys2/Cys2-type zinc finger, TM ⫹

WSV188 97548–98786 (413) AF117061, ribonucleotide reductase R2 subunit

[Aedes albopictus]

364 59 313 Ribonucleotide reductase small subunit, TM ⫹ⴱ

WSV191 98854–99786 (311) AJ133437, deoxyribonuclease I [Penaeus japonicus] 54 32 149 Nuclease, TM, SP ⫹ⴱ

WSV192 102885–99829 (1,019) TM ⫹ⴱ

WSV195 103071–103841 (257) TM, SP ⫹

WSV198 103844–104677 (278) ⫺

WSV199 104760–107327 (856) AF156271, Ring finger protein terf [Homo sapiens] 43 30 72 Ring-H2 finger motif, TM ⫹

WSV206 108550–109161 (204) ⫹ⴱ

WSV207 109261–110085 (275) Proline rich, TM ⫹ⴱⴱ

WSV209 114953–110136 (1,606) TM ⫹

WSV214 115053–115292 (80) L41834, nuclear protein [Ensis minor] 59 46 73 DNA-binding protein ⫹ⴱⴱ

WSV216 118987–115406 (1,194) Protein-splicing signature, TM ⫹ⴱ

WSV220 119057–121078 (674) ⫹

WSV222 121100–123631 (844) AK016037, putative [Mus musculus] 44 32 58 Ring-H2 finger motif, ATP/GTP binding motif, TM

⫹ⴱⴱ

WSV226 123758–126547 (930) TM ⫹

WSV230 126755–127000 (82) ⫹ⴱⴱ

WSV231 129006–127162 (615) TM ⫹ⴱ

WSV234 130290–129409 (294) ⫹ⴱ

WSV235 129611–129811 (67) ⫹

WSV236 130076–130306 (77) TM ⫹

WSV237 130566–131441 (292) ⫺ⴱ

WSV238 131481–132938 (486) 38 30 139 Gly-rich cluster (50–138), TM, SP ⫹ⴱ

WSV242 132994–133893 (300) TM ⫺

WSV244 133969–136341 (791) TM ⫹ⴱⴱ

WSV249 137589–139937 (783) AC024760, contains similarity to TR [

Caenorhab-ditis elegans]

72 27 202 Ring-H2 finger motif, repeat region (454–633) ⫹ⴱ

WSV252 140111–141613 (501) ⫹ⴱⴱ

WSV254 141696–142538 (281) ⫹ⴱ

WSV256 142545–143696 (384) TM, SP ⫹ⴱ

WSV259 143760–144686 (309) ⫹ⴱⴱ

WSV260 147517–144752 (922) Asp/Glu/Ser-rich region (344–485), TM ⫹ⴱⴱ

WSV267 148612–147770 (281) ⫹ⴱ

WSV269 150145–148679 (489) TM ⫹

WSV270 150675–150166 (170) ⫹

WSV271 150688–154341 (1,218) S59310, probable membrane protein YMR317w

[Saccharomyces cerevisiae]

46 20 369 Lys/Ser-rich region (455–526), TM ⫺

WSV277 154557–156929 (791) D86346, crystal protein [Bacillus thuringiensis] 41 23 249 TM ⫹ⴱ

WSV282 159352–161253 (634) Ser-rich cluster (13–129), SP ⫹ⴱ

WSV284 161263–161562 (100) TM, SP ⫺

WSV285 161718–165017 (1,100) AE003824, CG13185 gene product [Drosophila

melanogaster]

48 25 183 ATP/GTP binding motif, TM ⫹ⴱⴱ

WSV289 169814–165120 (1,565) NP-011856, serine/threonine protein kinase

[Saccharomyces cerevisiae]

46 25 245 Protein kinase, TM, SP ⫹ⴱ

WSV291 167278–167532 (85) TM, SP ⫹

WSV294 170113–170730 (206) ⫺

WSV295 170832–171458 (209) TM ⫹ⴱⴱ

WSV299 172439–171513 (309) TM, SP ⫹ⴱⴱ

WSV302 173075–172509 (189) ⫺

WSV303 173178–175850 (891) NP-069209, transcription initiation factor IID

[Archaeoglobus fulgidus]

44 23 140 TBP Cys2/Cys2-type zinc finger, TM ⫹

WSV306 175840–177096 (419) TM ⫹ⴱ

WSV308 177124–178521 (466) ⫹ⴱ

WSV310 178530–179345 (272) TM ⫹

WSV311 180036–179425 (204) Nucleocapsid protein VP26, TM, SP ⫹ⴱⴱ

WSV313 183817–180279 (1,180) Glu-rich region (37–358) and Pro-rich

cluster (462–492), TM

⫹

WSV321 184132–184482(117) TM, SP ⫺

WSV322 184499–185179(227) TM ⫺

WSV323 185082–184819(88) ⫹ⴱⴱ

WSV324 185434–185189(82) ⫹

WSV325 185433–186827(465) TM, SP ⫹

WSV327 190743–188176(856) TM ⫹ⴱⴱ

WSV331 190094–190306(71) ⫹

WSV332 190876–193233(786) ⫹ⴱⴱ

WSV333 191135–190932(68) TM, SP ⫹

WSV338 194629–193331(433) TM, SP ⫹ⴱⴱ

WSV339 195503–194655(283) ⫺

WSV340 196292–195510(261) ⫺

WSV342 196697–196398(100) ⫹

WSV343 209342–196803(4,180) TM ⫹ⴱⴱ

WSV344 197221–197517(99) TM ⫹

WSV349 199510–199779(90) TM, SP ⫹

WSV360 209616–227846(6,077) U96166, srpA [Streptococcus cristatus] 46 23 222 Leucine-zipper motif, TM ⫹ⴱⴱ

WSV386 228196–227993(68) TM, SP ⫹

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ORFs predicted proteins that show a partial homology (20 to

39% identity) to known proteins or contain one or two

se-quence motifs (versus a real functional domain). The

remain-ing 133 ORFs encode proteins with no homology to any known

proteins or motifs.

Enzymes involved in nucleotide metabolism.

Among the 18

ORFs encoding proteins that show extensive homologies with

previously identified proteins, WSV067, WSV112, WSV172,

WSV188, and WSV395 may encode the WSBV homologues of

enzymes involved in nucleic acid metabolism (Table 1). The

TABLE 1—

Continued

ORF Product position

(length [aaa_]) Best matchb[source]

BlastP score

Identity (%)

Length (aa)

Predicted structure and/or functionc_(position)d

Poly(A) signalc

WSV387 228375–230561(729) ⫺

WSV390 230617–231579(321) ⫹

WSV394 231422–231724(101) ⫹

WSV395 231603–232796(398) T41553, thymidylate kinase [Schizosaccharomyces

pombe]

157 41 200 Thymidylate kinase; ATP/GTP binding motif ⫹

WSV397 232819–233331(171) ⫺

WSV398 233383–233763(127) TM ⫹

WSV399 234330–233782(183) ⫹

WSV403 236679–238601(641) Ring-H2 finger motif, repeat region (435–494),

SP

⫹ⴱ

WSV406 238659–239435(259) T27927, hypothetical protein ZK593.8 [

Caenorhab-ditis elegans]

50 26 175 TM, SP ⫹ⴱ

WSV407 240139–239459(227) ⫹

WSV412 240713–241189(159) ⫹

WSV414 241637–241275(121) Asp-rich cluster (63–86), TM, SP ⫹ⴱⴱ

WSV415 241775–243406(544) TM ⫺

WSV419 243217–243795(193) ⫺

WSV421 244242–244853(204) Envelope protein VP28, TM, SP ⫹ⴱⴱ

WSV423 247143–244954(730) T22255, hypothetical protein F45H7.4 [

Caenorhab-ditis elegans]

45 26 165 Protein kinase, TM ⫹ⴱ

WSV427 249230–247362(623) EF-hand calcium-binding motif, TM ⫺

WSV432 249151–249456(102) ⫹

WSV433 249426–253208(1,261) Pro-rich cluster (29–71), TM ⫹

WSV440 253297–255117(607) ⫺

WSV442 255075–257474(800) ATP/GTP binding motif, TM ⫹ⴱ

WSV446 257552–259129(526) ATP/GTP binding motif, TM, SP ⫹ⴱ

WSV447 264975–259168(1,936) Z70204, similarity to yeast hypothetical helicase

[Caenorhabditis elegans]

42 40 52 Helicase, ATP/GTP binding motif, Asp-pro-tease motif, TM

⫹ⴱ

WSV455 265079–265597(173) TM, SP ⫺

WSV457 265606–266400(265) TM, SP ⫹ⴱ

WSV459 266838–266446(131) TM ⫹

WSV461 267400–266930(157) ⫺

WSV462 267399–267647(83) ⫹

WSV464 268584–267721(288) ⫹

WSV465 272423–268695(1,243) Cys2/Cys2-type zinc finger, TM ⫹ⴱ

WSV477 274527–275150(208) Cys2/Cys2-type zinc finger, ATP/GTP binding

motif

⫹ⴱ

WSV479 276736–275210(509) Glu-rich cluster (467–485), TM ⫹ⴱⴱ

WSV482 277035–277571(179) TM ⫹ⴱⴱ

WSV483 277705–278076(124) ⫹

WSV484 278423–277776(216) TM ⫹ⴱ

WSV486 278637–280973(779) AF154037, surface protein PspC [Streptococcus

pneumoniae]

46 24 194 Lysine-rich, TM ⫺

WSV489 281865–281131(245) ⫹ⴱ

WSV492 282176–282583(136) ⫹

WSV493 283360–282677(228) Acidic region (59–103) ⫹ⴱⴱ

WSV495 283754–284011(86) ⫹

WSV497 285773–284079(565) TM ⫹ⴱⴱ

WSV500 286706–286080(209) Cys2/Cys2-type zinc finger, ATP/GTP binding

motif

⫹ⴱ

WSV502 286606–289632(1,009) AL352992, ariadne-like protein [Leishmania major] 51 51 33 Cys2/His2, Cys2/Cys2-type zinc finger, ATP/ GTP binding motif, TM, SP

⫹ⴱ

WSV508 291298–289685(538) TM ⫹ⴱⴱ

WSV513 291720–292202(161) ⫺

WSV514 292190–298774(2,195) X61920, DNA polymerase III catalytic subunit [

Sac-charomyces cerevisiae]

52 24 201 DNA polymerase, TM ⫹ⴱⴱ

WSV518 293724–293275(150) SP ⫹

WSV524 298729–298526(68) ⫹

WSV525 299033–298821(71) TM, SP ⫹

WSV526 300432–299089(448) TM ⫹ⴱ

a

aa, amino acid.

b

Accession numbers are from the GenBank or SwissProt database.

c

Function was deduced from the degree of amino acid similarity to either products of known genes or Prosite signatures. TM, transmembrane domains; SP, signal peptides.

d

Positions of amino acid residues.

e_{⫹, polyadenylation signal present;⫺, signal absent.ⴱ, the transcription of the ORF was also verified by RT-PCR;ⴱⴱ, the ORF was confirmed by cDNA sequencing.}

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highest degree of homology (67% identity over 287 amino

acids) was detected between the product of WSV067 and the

human thymidylate synthase. The 29-amino-acid thymidylate

synthase prosite motif (PS00091), which contains the catalytic

cysteine residue, is 100% conserved in the product of WSV067.

In addition, WSV112 may encode a WSBV homologue of

dUTPase (37% identity over 161 amino acids) since the five

conserved regions of dUTPase, especially the highly conserved

substrate-binding residues, were identified in the product of

WSV112 (13, 35). dUTPase has been shown to be essential for

the replication of DNA viruses (5). Consistent with the

previ-ous reports by van Hulten et al. (48) and Tsai et al. (45, 46),

WSBV contains ribonucleotide reductases (products of

WSV172 and WSV188) and also thymidylate/thymidine kinase

(product of WSV395). Among these enzymes, thymidylate

syn-thase catalyzes the methylation of dUTP to yield the

nucleo-tide precursor dTMP. This is an important step in the de novo

pathway of biosynthesis of pyrimidine (12). Despite its

ubiqui-tous distribution in nature, a viral thymidylate synthase was

found only in a few herpesviruses (2, 10, 26, 39),

Melanoplus

sanguinipes

entomopoxvirus (MsEPV) (1),

Chilo

iridescent

vi-rus (CIV) (36), and bacteriophages (9). Most vivi-ruses do not

contain thymidylate synthase, as they depend mostly on the

host enzymatic machinery for the replication of their genomes

so as to keep the viral genome small (36). WSBV and other

thymidylate synthase-containing viruses may therefore exhibit

a considerable independence from the host

deoxyribonucleo-tide synthesis. This may represent a significant advantage for

viral genome replication that may ultimately lead to

persis-tence of infection and a broad host range for viral infection

(36). It is possible that WSBV acquires these

replication-re-lated genes from its host and/or from a coinfecting virus that

might occur at an earlier period in evolution. However, since

the shrimp homologues of these genes have not been cloned,

we are not able to test this hypothesis.

Proteins involved in DNA replication and transcription.

WSBV contains genes encoding proteins involved in DNA

replication such as DNA polymerase (product of WSV514).

The WSBV DNA polymerase was putatively identified by the

presence of three highly conserved motifs, YGDTDSVFC

(DNA polymerase family B signature PS00116), KLG

MNSMYG, and DMTSLYP (conserved amino acid residues

are underlined), that are found in most eukaryotic DNA

poly-merases (4) as well as in some viral polypoly-merases (18, 29, 43).

However, since the degree of amino acid similarity between the

product of WSV514 and known DNA polymerases is low (24%

identity over 201 amino acids), its putative activity as a DNA

polymerase still awaits future experimental verification.

Inter-estingly, the size of this putative WSBV polymerase (2,195

amino acids) is much larger than those of the regular

poly-merases found in other organisms.

Products of ORFs that show weak similarity (BlastP score,

⬍

100; identity,

⬍

20 to 39%) to known proteins include

puta-tive TATA-box binding protein (TBP) (product of WSV303,

containing partial conservation with transcription initiation

factor IID repeat signature PS00351) (Fig. 2A), the putative

CREB-binding protein (CBP) (product of WSV100) (Fig. 2B),

nuclease (product of WSV191, containing most residues of

DNA/RNA nonspecific endonuclease active site PS01070), the

putative helicase (product of WSV447), and protein kinases

(products of WSV083, WSV289, and WSV423). Most of them

play important roles in the regulation of gene transcription.

TBP and CBP, which have never been reported in a virus

genome, deserve special attention since they are critical basal

transcription regulators in eukaryotic cells (21, 51). However,

their functions in virus are yet to be determined.

Structure proteins.

A unique feature of WSBV is that it

contains a collagen-like gene, WSV001, which encodes a

pre-dicted 1,684-amino-acid protein and whose transcription has

been confirmed by RT-PCR. The product of this ORF displays

FIG. 2. Multiple amino acid sequence alignment of products of WSV303 and WSV100. The homology regions are shaded (black, 100%; pink,

⬎

75%; blue,

⬎

50%). The positions of the amino acid sequence are indicated on both ends. (A) Alignment of product of WSV303 with a known

TBP. Human,

Homo sapiens

, accession no. XP_004534; yeast,

Saccharomyces cerevisiae

, accession no. M26403; fly,

Drosophila melanogaster

,

accession no. A35615; At,

Arabidopsis thaliana

, accession no. AC005223; Metha,

Methanothermobacter thermautotrophicus

, accession no.

AE000921; Archa,

Archaeoglobus fulgidus

, accession no. AE001078; Halob,

Halobacterium

sp. strain NRC-1, accession no. AE005110. (B)

Alignment of product of WSV100 with the CBP. Human,

Homo sapiens

, accession no. U47741; mouse,

Mus musculus

, accession no. S39161; fly,

D. melanogaster

, accession no. U88570; At,

A. thaliana

, accession no. AC024128.

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the highest degree of homology to human collagen type VII

(42% identity over 1,336 amino acids) (Fig. 3). This is the first

time that an intact collagen gene has been reported in a virus

genome. The collagen-like protein of WSBV contains a typical

repeat of Gly-X-Y (X is mostly proline, and Y can be any

amino acid) that can form the triple-helical structure

charac-teristics of animal collagen fiber. The presence of this

collagen-like protein may help to protect the WSBV from

environmen-tal factors and may contribute to its ability to survive for a long

time in a shrimp pond.

Previously only a short segment of collagen-homologous

se-quence was found in the structural proteins of ectocarpus

sil-iculosus virus 1 (EsV-1) (16), hepersvirus saimiri (HVS) (2,

22), and bacteriophage PRD1 (6, 7) (Fig. 3). In EsV-1, the

collagen-like sequence was found in the N-terminal half of

both vp55 and vp74 (16), which were encoded by the EsV-1

genome and which are likely to be the components of the viral

core structure. In HVS, the Gly-X-Y motif is repeated 18 times

and is located in the central region of saimiri

transformation-associated protein (STP). These collagen-like repeats may

serve as a hinge to extend the active domain of STP to its site

of action (2). Finally, in bacteriophage PRD1, a minor capsid

protein was found to contain a short collagen-like region

(Gly-X-Y)

(7). All of the collagen-like segments present in these

proteins are short. These segments may play only a

supple-mentary role in protein functions.

In addition, WSV002 and WSV311 encode a nucleocapsid

protein, and the product of WSV421 shows characteristics of

an envelope protein. These proteins have recently been

puri-fied from the nucleocapsid and envelope of WSBV (49, 50).

WSV214 encodes a polypeptide with 44.2% basic amino acid

residues (Arg/Lys) and 24.6% Ser residues. This amino acid

composition is similar to that of the DNA-binding protein of

insect baculoviruses (34, 40, 55). Homologs of these

DNA-binding proteins have also been found in granulosis virus (47).

The basic residues of these DNA-binding proteins have a high

affinity for the phosphate backbone of DNA, enabling the

generation of a highly compact form of viral genomic DNA.

Upon entry into a host cell, the DNA-binding protein may

become phosphorylated by a protein kinase, resulting in the

unpacking of the viral DNA (54).

Protein motifs.

ORFs containing zinc finger and leucine

zipper motifs have been found in WSBV (Table 1). These

motifs have been shown to be involved in DNA-protein

inter-action and in regulation of transcriptional activation. Ring-H2

finger motifs, a variation of the Ring finger motif (30, 44)

found in proteins critical for virus survival and replication (11,

42), are also detected. Products of WSV079 and WSV427

contain an EF-hand calcium-binding motif (PS00018). Proteins

with these motifs are found in some prokaryotic and all

eu-karyotic organisms and play important roles in the regulation

and control of normal cellular functions. The detection of

these motifs in proteins of a marine virus suggests that some of

these basic regulatory activities are well conserved throughout

evolution.

The remaining 133 ORFs encode novel proteins of unknown

function. These novel genes obviously will provide ample

op-portunities for future research and for exploration of

molecu-lar mechanisms by which a virus and its host interact to survive

in the marine environment.

Among the 181 ORFs examined, the products of 96 have

potential transmembrane domains and 32 proteins contain

both signal peptide sequences and substantial hydrophobic

do-mains, suggesting that they may be membrane-associated

pro-teins and that they may play an important role in the

WSBV-host cell interaction and WSBV-host range determination. Other than

the putative signal sequences and hydrophobic domains, these

proteins are not obviously related to other known proteins.

Repetitive regions.

Three percent of the WSBV genome is

composed of highly repetitive sequences, and the repeats are

distributed throughout the genome. We found nine

hr

s with a

total of 47 repeated minifragments encompassing direct

re-peats, atypical inverted repeat sequences, and imperfect

pal-indromic sequences. The nine

hr

s vary in size from 0.76 to 3.62

FIG. 3. Multiple amino acid sequence alignment of the product of WSV001 with human (

Homo sapiens

) type VII collagen, accession no.

L23982; fruit fly (

Drosophila melanogaster

) collagen, accession no. P08120; sea urchin (

Strongylocentrotus purpuratus

) collagen, accession no.

A43426; brown alga virus (BAV; ectocarpus siliculosus virus) collagen-like protein, accession no. NP_077542; HVS strain 484-77) collagen-like

protein, accession no. P25050; and bacteriophage PRD1 (PRD1) coat protein, which contains a short collagen-like region, accession no. P22536.

The homology regions are shaded (black, 100%; pink,

⬎

75%; blue,

⬎

50%). Repeat sequence density is shown as a ratio of

a

/

b

, in which

a

indicates

the length of the typical repeat sequence and

b

indicates the full length of the protein.

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kb, and

hr

1 to

hr

9 are separated in the WSBV genome by about

49, 13, 15, 28, 20, 28, 46, 36, and 55 kb of DNA, respectively.

Each

hr

contains several repeated minifragments, each with a

size around 300 bp. These minifragments are referred here as

a, b, c, d, e, f, etc. (Table 2). The percentage of homology

among the consensus sequences within the same homologous

region is over 73%, while the identity among the

hr

s is 61.6%

(Table 2). A few sequence motifs were found to be present at

very high copy numbers. For example, sequences CCAGAAA

or TTTCTGG, AGNGGTCCACC, and AACTTGACAT are

repeated 219, 88, and 47 times, respectively.

As an example of such repetitive region, the homology

among the b minifragments of the nine

hr

s is shown in Fig. 4.

Both GC-rich sequences and AT-rich sequences are found in

the repeats. In the imperfect palindromic sequences, there are

2- or 3-bp mismatches that always exist in the same location

within every palindrome (Fig. 4), suggesting a functional

sig-nificance for the mismatch. Atypical inverted repeat sequences

that can form one or two hairpin loops are also found within

the repeat segments. The AT-rich elements, inverted repeat

sequences, and loop structures are reminiscent of the origin of

TABLE 2. Positions and identities of hrs in WSBV genome

hr Position Minifragment Identity (%)

Identity between

hrs (%)

hr

1

24528–28184

a, b, c, d, e, f, g

73.87

61.62

hr

2

77591–78859

a, b, c, d, e

87.98

hr

3

91832–92592

a, b, c,

88.85

hr

4

107335–108339

a, b, c, d

87.26

hr

5

136540–137301

a, b, c,

91.41

hr

6

157231–159211

a, b, c, d, e, f, g

74.35

hr

7

186876–188141

a, b, c, d, e,

89.65

hr

8

234231–236419

a, b, c, d, e, f, g

79.77

hr

9

272510–274432

a, b, c, d, e, f

80.86

FIG. 4. Alignment of partial consensus sequences within each

hr

. The consensus minifragments b are shown in order:

hr

1 to

hr

9. The

hr

s are

shaded (black, 100%; pink,

⬎

75%; blue,

⬎

50%), and the numbers on both ends refer to the positions of consensus sequences in the WSBV

genome. The direct repeat region, the atypical inverted repeat sequence that may contribute to the hairpin loop, the imperfect palindrome, and

GC-rich and AT-rich regions are shown.

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replication in eukaryotic cells and also in some of the viruses

(17, 37). The presence of

hr

s is a feature of many baculovirus

genomes. The

hr

s may serve as transcription enhancers and

origins of DNA replication and play a fundamental role in the

viral life cycle (24, 27, 28, 33). The presence of nine

hr

s

sug-gests that WSBV may contain multiple replication origins. This

may account for the fast replication and the growth rate of

WSBV. Furthermore, although the organization of WSBV

hr

s

is similar to that of baculovirus, no homology among most of

their ORFs is detected. Thus, future investigations are needed

to determine whether WSBV is a seawater baculovirus and

whether the ancestors of WSBV and insect baculoviruses

evolved by separate routes, acquiring genes independently in

different environments.

In summary, we have obtained the complete genome

se-quence of WSBV. This is the first complete genome sese-quence

from a marine invertebrate virus. It is also the largest animal

virus genome sequenced (8, 52). As the genomic data

demon-strated, more than 80% of WSBV proteins bear no homology

to previously identified proteins. This leads us to consider a

separate evolutionary origin for this virus. Among the proteins

that show homology with known proteins, most seem to be

related to eukaryotic proteins and relatively few seem to be

related to viral proteins (Table 1). Although a few genes show

weak similarities to genes of herpesvirus (data not shown), the

morphology and the double-stranded circular WSBV genome

differ significantly from those of herpesvirus, which contains an

icosahedral capsid and a linear double-stranded DNA

mole-cule. On the other hand, WSBV shares some complex

mor-phological traits with the insect baculovirus, and a pattern of

interspersed repetitive regions in WSBV is similar to that

found in some of the insect baculoviruses, but sequence

com-parison indicates that they are not detectably related at the

amino acid level. Unfortunately, until now there were no

ge-nome sequence data available for the nonoccluded

baculovi-rus. Based on genetic analysis, WSBV clearly should not be

included in any of the currently recognizable baculovirus

sub-families and perhaps should be classified in a new virus family.

It is possible that other WSBV-like viruses that can infect other

organisms may exist. As the sequence of a representative of a

marine DNA virus, the complete WSBV genome sequence

should provide valuable information to serve as the genetic

basis for future studies. Future work may shed more light on

the evolution of these viruses.

ACKNOWLEDGMENTS

We thank Mei He and Yun Ye for their assistance, and we

acknowl-edge the support of Mingwei Wang, Lin Zao, and Yan Shen. We thank

Mark Yandell, Jennifer R. Wortman, Chinnappa Kodira, P. W. Li, and

Z. Deng of Celera Genomics for coordinating the project at Celera.

We thank Kunxin Luo of Lawrence Berkeley National Laboratory and

UC Berkeley for data analysis and critical reading of the manuscript.

This work is funded by the Chinese High Tech “863” Program

(Z19-02-05-01), Fujian Science Fund (C97053), and Science

Founda-tion of the State Oceanic AdministraFounda-tion.

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