Sequence Analysis of the Plutella xylostella Granulovirus Genome

Virology 275, 358–372 (2000) doi:10.1006/viro.2000.0530, available online at http://www.idealibrary.com on

Sequence Analysis of the Plutella xylostella Granulovirus Genome Yoshifumi Hashimoto,* ,1 Tohru Hayakawa,† Yasumasa Ueno,* Tomonori Fujita,* Yoshitaka Sano,* and Tsuguo Matsumoto* *Laboratory of Environmental Microbiology, Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan; and †Laboratory of Molecular Life Science, Graduate School of Science and Technology, Niigata University, Ikarashi, Niigata 950-2181, Japan Received May 29, 2000; returned to author for revision June 30, 2000; accepted July 13, 2000 The Plutella xylostella granulovirus (PxGV) genome DNA was sequenced and the predicted open reading frames (ORFs) were compared to genes of the first-sequenced GV, Xestia c-nigrum GV (XcGV), and those from other baculoviruses and organisms. PxGV DNA has a size of 100,999 bp with a G ⫹ C content of 40.7%. The analysis predicted 120 ORFs with a size of 150 nucleotides or larger that showed minimal overlap. Blast searches followed by a comparison of ORF arrangement with those of completely sequenced baculovirus genomes showed the presence of 102 homologs to other genes in the database. Among them, 74 and 100 were homologous to genes of Autographa californica NPV (AcMNPV) and XcGV, respectively. A striking feature of the relationship between the genomes of PxGV and XcGV was the conservation of the order and orientation of homologous genes. Even though the XcGV genome is much larger than that of PxGV (178 vs 101 kb) and had many more predicted ORFs (181 vs 120) with an average amino acid sequence relatedness of 42%, the order and orientation of almost all homologous genes was conserved. The PxGV genome contained four homologous regions (hrs), each with 10 to 23 repeated sequences of 101 to 105 nucleotides containing a 15-bp imperfect palindrome in the center of the repeats. © 2000 Academic Press

INTRODUCTION

(Lymantria dispar) (LdMNPV) (Kuzio et al., 1999), the silkworm (Bombyx mori) (BmNPV) (Gomi et al., 1999), and the beet armyworm (Spodoptera exigua) (SeMNPV) (IJkel et al., 1999). These investigations have revealed genome sizes of 128–161 kb, G ⫹ C contents of 40–58%, and have led to the prediction of from 139 to 163 open reading frames (ORFs) in the different genomes. Many NPVs have had adapted to cell culture, which has resulted in extensive investigations on gene expression and function. In contrast, GVs are difficult to grow in cell culture (Winstanley and Crook, 1993) and information on the function of their genes has been limited. Therefore, sequences of GV genomes are particularly important in determining the genome content and will allow prediction of virus functions by the identification of homologs of NPV genes within GV genomes. A major advance in our understanding of GVs and the extent of baculovirus diversity was the first description of a granulovirus genome, the common cutworm Xestia c-nigrum (XcGV) (Hayakawa et al., 1999). At almost 179 kb and 181 predicted ORFs, it is by far the largest baculovirus genome reported to date. Although the genome of XcGV is large, the infection of X. c-nigrum is slow and requires 2 to 3 weeks to kill infected insects and shows tissue tropism limited to the fat body (Goto et al., 1985). In contrast, one GV of particular interest is pathogenic for the diamondback moth (Plutella xylostella) (PxGV) (Asayama and Osaki, 1969; Kondo and Yamamoto, 1998). The diamondback moth is

The Baculoviridae are a large family of viruses with circular, supercoiled, double-stranded DNA genomes of between 90 and 180 kb. They are pathogenic for arthropods, particular insects of the orders Lepidoptera, Diptera, and Hymenoptera. Viruses similar to baculoviruses are also pathogenic for a number of crustaceans. They are classified into two genera: the nucleopolyhedroviruses (NPVs) have many virions occluded into large crystalline occlusion bodies (OBs) (Rohrmann, 1999) and the granuloviruses (GVs), which usually contain a single virion per small granular OB (Winstanley and O’Reilly, 1999). Although the NPV pathogenic for the cabbage looper Autographa californica (AcMNPV) is widely used as an expression vector (O’Reilly et al., 1994) and baculoviruses have been incorporated into pest management programs (Abot et al., 1994), DNA sequence analysis has only recently begun to reveal the distinctive features of baculovirus genomes and the extent of their diversity. In addition to the AcMNPV sequence originally reported by Ayres et al. (1994), four NPV genome sequences have recently been described. These included NPVs pathogenic for the Douglas fir tussock moth (Orgyia pseudotsugata) (OpMNPV) (Ahrens et al., 1997), the gypsy moth

1 To whom correspondence and reprint requests should be addressed. Fax: ⫹81-75-724-7764. E-mail: [email protected].

0042-6822/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

358

SEQUENCE ANALYSIS OF THE PxGV GENOME

a major pest of cruciferous crops and is widely distributed throughout the world. The history of the control of this insect has reflected its exposure to pesticides; it has developed resistance to a number of chemical pesticides (Ho et al., 1983) and has also become resistant to strains of the bacterial insecticide Bacillus thuringiensis that have been used for its control (Ankersmit, 1975; Liu et al., 1981; Tabashnik et al., 1994). Viruses which are infectious to the P. xylostella have been suggested as an alternative means for its control. Comprehensive pathological studies of an isolate of PxGV have been performed over the last two decades (Asayama and Osaki, 1969, 1970; Asayama and Inagaki, 1975a,b; Asayama, 1975, 1976). Hashimoto et al. (1996) have demonstrated that PxGV is highly virulent for P. xylostella with LC 50s in the range of 9.75 ⫻ 10 5 to 9.55 ⫻ 10 6 (OBs/g diet), has a very short lethal time (LT 50 ⫽ 3.5 days for third instar larvae) and infects a wide array of tissues in infected larvae. Recently, we established a comprehensive physical map of DNA of a PxGV K1 strain (Hashimoto et al., 2000). At about 100 kb, the genome of PxGV K1 is significantly smaller than that of XcGV and the NPV genomes that have been sequenced. Therefore, because of both its potential as an insect control agent and the contributions it could make to understanding baculovirus diversity, we have undertaken a program to determine the sequence of the PxGV genome. Because of its small size and high degree of virulence to its host insect, we thought that its sequence would provide the basis for identifying the genes involved in its pathology. In this report we describe the genome organization of the PxGV genome and compare it to that of XcGV. RESULTS AND DISCUSSION Genome structure and overview of gene organization An isolate of PxGV was cloned and subsequently amplified in P. xystella larvae. DNA was extracted and used for the production of overlapping subclones in plasmid vectors that were employed as templates for nucleotide sequence determination. The PxGV genome was found to have a size of 100,999 bp and a G ⫹ C content of 41%. Major predicted ORFs with a size of 50 amino acids and larger that showed minimal overlap were identified and are shown diagrammatically in a linear format in Fig. 1. This selection yielded 120 ORFs, which, as with other baculovirus genomes, showed no clear pattern of gene organization or orientation. Sixty-three (52.5%) of the ORFs were oriented to the right, whereas 57 (47.5%) were oriented to the left. The numbering of the ORFs was begun with the left side of the physical map of the PxGV genome, starting with the BamHI-J fragment (Hashimoto et al., 2000). As has been reported in all baculoviruses to date, the PxGV genome possesses homologous regions (hrs), each of which contains multiple copies of a repeat

359

unit and are located at four regions interspersed on the genome. Blast homology searches, using amino acid sequences deduced from each ORF, demonstrated that some ORFs are homologs of genes from baculoviruses or other organisms. In Fig. 1, the genes are numbered sequentially and if they show homology to a named gene, the name is shown in parentheses. In Table 1 each ORF is listed and its relationship to homologs from other baculoviruses is shown. ORFs, which did not show significant homology in Blast searches, were further examined in terms of their size, orientation, and relationship to other genes on the genome map. One hundred two homologs to genes in the database were identified, which represents 85.0% of the total ORF number of PxGV. Many of these genes likely have functions similar to their homologs from other baculoviruses. Overall the PxGV ORFs showed 42.4% average identity with the homologous ORFs (100) of XcGV and 33.1% with those (74) of AcMNPV. ORFs showing the highest amino acid identities to XcGV genes are homologs of ubiquitin (Px42), 88%, and granulin (Px4), 87%. Other highly conserved ORFs include Px52 (Xc79 [Ac38]) and Px40 (Xc50, [Ac106/ 107]) at over 73% and odv-e25 (Px74, Xc99 [Ac94]), Px86 (Xc120 [Ac81]), lef-9 (Px99, Xc139 [Ac62]), and lef-8 (Xc109, Xc148 [Ac50]) at over 60% identity to their XcGV homologs. Comparison of gene organization in PxGV, XcGV, and AcNPV genomes Although closely related NPV genomes such as AcMNPV, BmNPV, and OpMNPV show a high degree of colinearity of homologous genes in their genomes, this relationship is not preserved over many of the genomes of SeMNPV and LdMNPV (IJkel et al., 1999). To examine the conservation of the genome organization between PxGV and XcGV, the ORFs were compared in a graphical format (Fig. 2). In these analyses, the genes for each virus were plotted on the x or y axes and the relative position and orientation of its homolog are shown on the graph. The genes oriented in the same direction are shown in open circles and those in reverse orientation to one another are shown in shaded circles. The position of the granulin/polyhedrin gene shown in a closed square is in the lower-left corner of the graph. These comparisons enable an examination of the gene order and relative location of homologous and nonhomologous genes on each genome. Because the XcGV genome is much larger than that of PxGV, there are a number of gaps in the graph, which represent the lack of homologous genes between the viruses. The homologs consist of discontinuous clusters with two major gaps on XcGV genome, which correspond to Xc57 to Xc76 and Xc149 to Xc168. Only 8 of the 100 homologs are present in the opposite orientation on the genomes. Despite the fact

50

40 41 42 43 44 45 46 47 48 49

33328 33960 34108 34259 35721 36120 36847 37172 37630 37776 39609 42028

175 358 604 945 1685 2061 2882 4496 4971 5249 6442 7022 7291 7586 8955 9646 10723 10779 11816 12435 13172 14640 15910 16423 17589 18924 20587 20827 21475 22022 24055 24444 25366 27864 28157 28586 29841 30643 31746 32201

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

33 34 35 36 37 38 39

Left

ORF

Ⰶ

Ⰶ Ⰷ Ⰶ Ⰷ Ⰶ Ⰶ Ⰶ Ⰶ Ⰶ Ⰶ

33948 34121 34452 35503 36113 36878 37137 37633 37785 39512 41944 42435

375 609 912 1691 2080 2885 4992 5023 5252 6430 6984 7318 7566 8926 9605 10701 10899 11720 12118 13142 14134 15839 16305 17439 18884 20558 20826 21459 22029 24070 2444 25256 27748 28157 28558 29794 30631 31749 32207 33331

Ⰷ Ⰶ Ⰷ Ⰷ Ⰶ Ⰷ Ⰶ Ⰶ Ⰷ Ⰶ Ⰷ Ⰶ Ⰶ Ⰶ Ⰶ Ⰶ Ⰷ Ⰷ Ⰶ Ⰶ Ⰶ Ⰷ Ⰶ Ⰷ Ⰷ Ⰷ Ⰷ Ⰶ Ⰷ Ⰶ Ⰷ Ⰷ

Ⰶ Ⰶ Ⰶ Ⰷ Ⰷ Ⰷ Ⰷ

Right

Dir.

PxGV

23741 6444 12996 47245 15611 29420 11281 16567 5742 65522

11493 15658 47036 30073 41872 17944 44793

7592 8984 12311 28994 14846 32162 60291 20400 10813 46595 20304 11491 10160 51938 25267 38539 6979 36161 11617 27089 34449 47039 14750 39372 33139 63162 9539 24412 20685 77674 15277 32628

Mr

135 14973

206 53 114 414 130 252 96 153 51 578

97 133 402 263 368 153 376

66 83 105 248 131 274 536 175 93 393 180 98 91 446 216 351 58 313 100 235 320 399 131 338 431 544 79 210 181 682 129 270

aa

L — — (E) — — — L — L hr2 L

— L — L L L —

— — L L L L L E — — — L L L — L — L L L L (E) L E E — — L — L E —

Prom.

1 3 84 7 8 9 10 11 12 13 14 15 16

gran

pk

p74 83

77

39k lef-11 sod

p10

50 51 52 53 54 55 56 68

ubi

mmp glycogenin

lef-2 hr1 39 40 43 45 47

182

710

272 53 77 353 110 295 102 153

149 469 277 388 220

170 195 668 113 189

29 32 149 34 35

odv-e66

153 449 370 599

18 25 26 27

CpGV16L

187 386

17 19

302 540 187 86 484 196 99 83 453 229 353 71

248

84

aa

CpGV16L p10

ie-0 odv-e56

odv-e18

ie-1

5

ORF

p10

Name

54 (287) 45 (544) 25 (183) 35 (75) 31 (375) 30 (167) 54 (98) 54 (48) 39 (457) 25 (215) 50 (347) 33 (46)

48 (131) 47 (300) 48 (137) 31 (110) 27 (160) 34 (579) 33 (153) 47 (186) 46 (584) 47 (85) 44 (168)

Ⰷ Ⰷ Ⰶ Ⰷ Ⰶ Ⰷ Ⰶ Ⰶ Ⰶ Ⰶ Ⰶ Ⰷ

Ⰶ Ⰶ Ⰶ Ⰷ Ⰷ Ⰷ Ⰶ Ⰷ Ⰶ Ⰷ Ⰷ

22 (125)

43 (588)

Ⰷ Ⰷ

77 (184) 42 (53) 88 (76) 39 (353) 22 (55) 47 (214) 56 (89) 57 (145)

Ⰶ Ⰷ Ⰶ Ⰷ Ⰷ Ⰶ Ⰶ Ⰶ

24 (88) 35 (413) 51 (264) 52 (373) 37 (76)

87 (248)

Ⰷ

Ⰶ Ⰶ Ⰷ Ⰷ Ⰷ

25 (69)

Ⰷ

137

138

94

645

275 112 151

61 110 56 77 390

106 107 110 35 109 36 37 31

382

210

204 704

690

94

582 201 77 62 477 261 376 71

272 530

245

94

aa

22

6

115 46

23

137

147 146 145 143 142 141 148 29

10 119

8

137

Dir. %ID (range) ORF

XcGV

13 (100) 29 (83) 54 (149)

Ⰶ Ⰶ Ⰷ

Ⰷ

15 (78)

47 (558)

48 (56) 25 (56) 33 (52) 77 (77) 26 (375)

Ⰷ Ⰷ Ⰶ Ⰷ Ⰶ

Ⰶ

47 (375)

27 (144)

Ⰷ

Ⰷ

38 (188) 52 (626)

20 (377)

Ⰶ Ⰷ

Ⰷ

21 (76)

15 (140) 25 (154) 39 (66) 52 (33) 32 (479) 21 (107) 43 (359) 28 (53)

Ⰶ Ⰶ Ⰷ Ⰷ Ⰷ Ⰷ Ⰶ Ⰶ

Ⰷ

35 (248) 36 (555)

54 (240)

19 (73)

Ⰷ Ⰷ

Ⰷ

Ⰷ

133

134

24 23 29

107 111 25 109

20

6

115 50

21

133

145 144 142 140 139 138 146 39

1 119

3

133

Dir. %ID (range) ORF

AcMNPV

92

644

261 125 152

256 57 93 390

382

204

205 682

627

92

560 197 95 85 484 245 374 75

274 529

245

92

aa

Ⰷ

Ⰶ

Ⰷ Ⰷ Ⰶ

Ⰷ Ⰶ Ⰶ Ⰶ

Ⰷ

Ⰶ

Ⰶ Ⰷ

Ⰷ

Ⰷ

Ⰷ Ⰶ Ⰷ Ⰷ Ⰷ Ⰷ Ⰶ Ⰷ

Ⰶ Ⰷ

Ⰶ

Ⰷ

28 (72)

46 (559)

17 (204) 22 (91) 55 (149)

45 (174) 35 (52) 66 (91) 31 (366)

49 (372)

28 (153)

36 (182) 49 (680)

16 (367)

21 (78)

14 (145) 17 (190) 32 (93) 41 (46) 34 (476) 24 (83) 42 (358) 29 (55)

33 (239) 33 (552)

57 (240)

15 (61)

41

27

44 45 145

140 106 43 107

119

137

143 131

130

41

15 16 17 19 20 21 14 39

3 155

1

41

Dir. %ID (range) ORF

OpMNPV

Analysis and Homology Search of PxGV ORFs

TABLE 1

77

673

264 187 154

246 56 150 366

407

216

203 665

676

77

566 208 92 88 483 258 356 68

274 530

245

77

aa

Ⰷ

Ⰷ

Ⰶ Ⰶ Ⰷ

Ⰶ Ⰷ Ⰷ Ⰶ

Ⰷ

Ⰷ

Ⰶ Ⰷ

Ⰷ

Ⰷ

Ⰶ Ⰷ Ⰶ Ⰶ Ⰶ Ⰶ Ⰷ Ⰶ

Ⰷ Ⰷ

Ⰷ

Ⰷ

19 (72)

44 (559)

22 (69) 32 (93) 55 (153)

46 (193) 41 (51) 82 (76) 31 (362)

51 (371)

30 (99)

41 (188) 45 (565)

24 (462)

19 (72)

19 (216) 21 (194) 40 (90) 51 (41) 34 (466) 19 (124) 45 (356) 24 (41)

35 (244) 34 (552)

55 (240)

30 (71)

130

131

120 119 48

53 60 123 59

56 35

12

50 114

8

130

132 133 134 136 137 138 6 128

3 36

1

130

Dir. %ID (range) ORF

LdMNPV

88

653

317 103 151

222 59 80 356

283 413

209

214 685

656

88

714 200 92 80 460 244 371 136

295 526

246

88

aa

Ⰷ

Ⰶ

Ⰷ Ⰷ Ⰶ

Ⰷ Ⰶ Ⰶ Ⰶ

Ⰷ Ⰷ

Ⰷ

Ⰷ Ⰶ

Ⰷ

Ⰷ

Ⰶ Ⰷ Ⰶ Ⰶ Ⰶ Ⰶ Ⰷ Ⰶ

Ⰷ Ⰷ

Ⰷ

Ⰷ

21 (81)

42 (560)

19 (175) 46 (84) 53 (148)

40 (194) 45 (47) 78 (76) 32 (358)

52 (268) 50 (373)

31 (163)

39 (172) 27 (601)

23 (518)

11 (149)

17 (192) 20 (191) 33 (90) 39 (46) 33 (463) 22 (78) 41 (351) 22 (41)

41 (248) 33 (549)

55 (240)

28 (65)

Dir. %ID (range)

SeMNPV

Kuzio et al. (1984)

Kuzio et al. (1989)

Guarino and Smith (1990) Todd et al. (1995) Tomalski et al. (1991)

Guarino (1990)

Goto et al. (1998) Campbell and Cohen (1989)

Passarelli and Miller (1993a)

Hong et al. (1994)

Kang et al. (1997)

Kang et al. (1997) Kuzio et al. (1984)

Braunagel et al. (1996a) Chisholm and Henner (1988)

Braunagel et al. (1996b)

Guarino and Summers (1987)

Reilly and Guarino (1994)

Hooft van Iddekinge et al. (1983)

Kuzio et al. (1984)

References

360 HASHIMOTO ET AL.

95 96 97 98 99 100 101 102 103 104 105 106 107 108 109

82 83 84 85 86 87 88 89 90 91 92 93 94

51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81

42509 43679 44324 44819 45219 45978 46678 46929 47089 47259 47546 48153 48589 49725 50049 50527 51678 51866 52041 52602 53592 54064 57448 57911 58579 59051 59796 60208 61542 62554 63546 64066 65211 66237 66455 68028 68434 69050 69924 70196 71221 71757 72032 72461 75333 77299 78816 79678 80097 80675 81501 82996 83409 85068 85315 85528 86761 88012 89135 90797 91271

Ⰶ Ⰷ Ⰷ Ⰷ Ⰷ Ⰷ Ⰶ Ⰷ Ⰷ Ⰶ Ⰷ Ⰷ Ⰷ Ⰶ Ⰶ

Ⰶ Ⰶ Ⰶ Ⰷ Ⰷ Ⰷ Ⰷ Ⰷ Ⰶ Ⰷ Ⰶ Ⰶ Ⰷ

Ⰷ Ⰷ Ⰷ Ⰶ Ⰶ Ⰶ Ⰶ Ⰶ Ⰷ Ⰶ Ⰶ Ⰶ Ⰷ Ⰷ Ⰷ Ⰷ Ⰷ Ⰷ Ⰶ Ⰷ Ⰶ Ⰷ Ⰷ Ⰶ Ⰷ Ⰷ Ⰶ Ⰶ Ⰷ Ⰷ Ⰷ

43669 386 45450 44302 207 24844 44803 159 18159 45271 150 17220 45974 251 29930 46643 221 25881 46980 100 11718 47090 53 6375 47328 79 8990 47519 86 10247 48337 263 30396 48563 136 16065 49722 377 44281 50015 96 10898 50507 152 17936 51627 366 41501 51848 56 7190 52066 66 7383 52784 247 28549 53624 340 40184 54077 161 18343 57438 1124 132873 57924 158 16419 58555 214 23598 59049 156 18013 59803 250 30052 60203 135 15626 61506 432 50661 62504 320 36411 63417 287 34442 63869 107 12516 65120 66233 340 40136 66446 69 8296 68056 533 60608 68447 139 15638 69009 191 22241 69902 283 32523 70193 89 10181 71236 346 40214 71748 175 20874 72002 81 9165 72469 145 17206 75400 979 113403 77288 651 76748 78339 79709 297 34388 80064 128 14921 80681 194 23079 81520 281 32297 82985 494 57306 83412 138 16103 84980 523 61359 85253 61 7389 85515 66 7658 86718 396 46202 87969 402 48224 89148 378 43533 90445 436 49433 91213 138 15559 93787 838 98045

— — L — — L — — — — E L — — —

— EL L — L L L L — L L — —

E L L — — — — L L — E E L L — E L L L — L — L L EL L E — L L E 61 (220) 48 (118) 54 (246) 44 (443) 44 (329) 50 (284)

Ⰶ Ⰶ Ⰷ Ⰶ Ⰷ Ⰷ

110 111 112

lef-4 vp39 odv-ec27

45 (383) 52 (436) 64 (855)

Ⰷ Ⰷ Ⰶ

145 146 148

alk-exo hel-2

lef-8

859

457 455

26 (285) 40 (116) 23 (188) 37 (260) 63 (495) 51 (135) 43 (536) 28 (52) 30 (69) 31 (301)

Ⰶ Ⰷ Ⰷ Ⰷ Ⰷ Ⰷ Ⰶ Ⰷ Ⰷ Ⰶ

447 329 288

220 122 251

113 373 116 123 p91 118 741 tlp20 119 161 120 187 gp41 121 290 122 103 vlf-1 123 373 124 180 125 85 126 145 dnapol 132 1098 desmplakin 133 661 hr4 lef-3 134 351 135 120 136 171 iap 137 285 lef-9 139 493 fp 140 147 dna-lig 141 527 142 88 143 66 fgf 144 409

hr3

99 100 101

30 (353) 43 (63) 39 (247) 22 (134) 61 (183) 45 (289) 34 (103) 56 (342) 32 (177) 62 (81) 34 (144) 53 (1002) 23 (643)

54 (369) 41 (90) 20 (140) 46 (360) 57 (56) 32 (59) 52 (228) 50 (289) 36 (156) 48 (1157)

Ⰷ Ⰷ Ⰶ Ⰷ Ⰷ Ⰷ Ⰶ Ⰷ Ⰶ Ⰷ

91 372 92 120 169 144 93 372 94 60 87 1634 95 245 96 301 97 157 98 1159

Ⰶ Ⰷ Ⰶ Ⰷ Ⰷ Ⰷ Ⰶ Ⰷ Ⰶ Ⰷ Ⰷ Ⰶ Ⰷ

27 (75) 38 (61) 31 (258)

Ⰷ Ⰶ Ⰶ

164 99 277

87 88 89

57 (392) 73 (210) 54 (151) 39 (104) 51 (237) 32 (199) 33 (55)

Ⰷ Ⰷ Ⰷ Ⰶ Ⰶ Ⰶ Ⰶ

394 225 182 184 238 232 115

78 79 80 81 82 85 86

odv-e25

hel-1

lef-5

p6.9

lef-6 dbp

lef-1 fgf

p24

p47

181

32

50

876

419

286 516 214

27 62 61

133

386 192

84 133 984 808

76 75 65 66 67 68

847 180 233 409 109 379

464 347 290

83 82 81 80 78 77

90 89 144

19 (112)

Ⰶ

Ⰶ

49 (878)

36 (352)

25 (267) 53 (493) 38 (118)

Ⰷ Ⰷ Ⰶ

Ⰷ

20 (238) 38 (112)

35 (84) 19 (100) 33 (908) 16 (475)

Ⰶ Ⰶ Ⰶ Ⰷ Ⰶ Ⰷ

26 (539) 21 (85) 49 (182) 28 (292) 42 (26) 33 (342)

31 (464) 29 (325) 29 (276)

Ⰷ Ⰶ Ⰶ Ⰶ Ⰶ Ⰶ

Ⰷ Ⰶ Ⰷ

24 (345) 34 (44) 33 (43) 46 (250) 36 (304) 31 (163) 24 (873) 53 (139) 36 (222) 32 (115) 33 (255)

Ⰶ Ⰶ Ⰷ Ⰷ Ⰶ Ⰷ Ⰶ Ⰶ Ⰷ Ⰷ Ⰶ

101 361 100 55 145 77 99 265 98 320 96 173 95 1221 91 224 94 228 93 161 92 259

25 (56) 30 (69) 21 (256)

46 (348) 39 (204) 30 (125) 25 (145) 30 (247) 13 (342)

35 (378) 27 (63)

Ⰷ Ⰷ Ⰶ

Ⰶ Ⰶ Ⰷ Ⰶ Ⰶ Ⰶ

Ⰶ Ⰶ

99 173 316

401 216 198 327 266 181

387 122

103 102

150 28 25

40 38 129 13 14 32

411 112

95 138 300

399 209 192 320 243 205

54

131

27

35 65 64

72 73

79 78 70 71

86 85 84 83 81 80

91 90 141

884

424

205

268 489 208

373 131

84 130 985 875

819 155 218 367 105 374

457 351 297

102 354 101 51 142 95 100 263 99 313 97 172 96 1223 92 279 95 229 94 159 93 279

104 103

142 40 43

45 22 127 12 13 27

Ⰶ

Ⰷ

Ⰶ

Ⰶ Ⰷ Ⰶ

Ⰶ Ⰷ

Ⰶ Ⰶ Ⰶ Ⰷ

Ⰷ Ⰶ Ⰶ Ⰶ Ⰶ Ⰶ

Ⰷ Ⰶ Ⰷ

Ⰶ Ⰶ Ⰷ Ⰷ Ⰶ Ⰷ Ⰶ Ⰶ Ⰷ Ⰷ Ⰶ

Ⰶ Ⰶ

Ⰷ Ⰶ Ⰶ

Ⰶ Ⰷ Ⰷ Ⰶ Ⰶ Ⰷ

45 (889)

31 (359)

28 (76)

25 (264) 51 (491) 42 (118)

23 (77) 37 (107)

31 (82) 22 (90) 33 (913) 19 (126)

26 (505) 17 (78) 45 (183) 28 (244) 42 (26) 30 (340)

30 (457) 28 (323) 29 (295)

22 (324) 50 (48) 35 (43) 46 (239) 35 (299) 38 (113) 26 (720) 49 (138) 33 (228) 32 (115) 32 (189)

33 (400) 22 (91)

23 (79) 28 (68) 22 (223)

40 (394) 37 (203) 31 (125) 21 (44) 33 (232) 36 (33)

389 121

92 159 239

200 234 285

390 247

864 223 219 323 113 378

485 352 283

217 159 251

51

157 50

156

139 64 63 22

81 80

874

420 460

285

155 496 176 548

374 128

85 86 84 128 83 1014 82 778

91 90 89 88 87 86

93 92 18

96 95 94

102 381 101 99 17 92 100 278 99 322 98 183 97 1218

104 103

17 38 37

122 123 156

48 46

Ⰶ

Ⰶ Ⰷ

Ⰶ

Ⰷ Ⰷ Ⰶ Ⰷ

Ⰷ Ⰶ

Ⰶ Ⰶ Ⰷ Ⰶ

Ⰷ Ⰶ Ⰶ Ⰶ Ⰶ Ⰶ

Ⰷ Ⰶ Ⰶ

Ⰷ Ⰷ Ⰶ

Ⰶ Ⰶ Ⰶ Ⰷ Ⰶ Ⰷ Ⰶ

Ⰶ Ⰶ

Ⰷ Ⰷ Ⰷ

Ⰶ Ⰶ Ⰶ

Ⰶ Ⰶ

50 (877)

36 (390) 50 (419)

19 (110)

29 (73) 54 (492) 22 (45) 20 (489)

26 (72) 33 (97)

35 (80) 17 (99) 34 (990) 16 (416)

26 (544) 28 (97) 46 (187) 26 (234) 37 (35) 33 (345)

33 (462) 31 (324) 29 (275)

48 (217) 30 (153) 30 (248)

24 (334) 41 (56) 26 (47) 47 (233) 36 (306) 34 (167) 26 (815)

33 (391) 22 (91)

26 (82) 40 (38) 15 (59)

21 (146) 37 (227) 20 (51)

45 (392) 39 (206)

375 106

113 163 328

400 261 248 363 216 404

813 196 240 331 127 372

466 326 281

216 157 252

112

41

38

97 98

91 90

906

813

404

495 195

422 133

95 85 94 129 93 1063 92 704

77 78 79 80 81 82

74 75 135

71 72 73

64 388 65 75 134 92 66 279 67 300 69 170 70 1222

62 63

96 127 126

115 118 10 13 14 38

Ⰷ

Ⰶ

Ⰶ

Ⰶ Ⰷ

Ⰷ Ⰶ

Ⰶ Ⰶ Ⰷ Ⰶ

Ⰶ Ⰷ Ⰷ Ⰷ Ⰷ Ⰷ

Ⰷ Ⰷ Ⰶ

Ⰷ Ⰶ Ⰷ

Ⰷ Ⰷ Ⰶ Ⰶ Ⰷ Ⰶ Ⰷ

Ⰷ Ⰷ

Ⰷ Ⰶ Ⰷ

Ⰷ Ⰷ Ⰶ Ⰶ Ⰶ Ⰶ

48 (908)

34 (391)

16 (129)

53 (489) 39 (138)

16 (291) 34 (103)

39 (83) 17 (122) 35 (863) 15 (541)

25 (528) 24 (119) 50 (177) 31 (290) 48 (23) 18 (78)

32 (470) 30 (314) 27 (286)

47 (216) 35 (153) 32 (249)

23 (342) 47 (51) 29 (51) 49 (251) 35 (291) 29 (172) 24 (685)

38 (374) 14 (91)

24 (42) 21 (51) 28 (254)

46 (400) 40 (201) 31 (133) 28 (100) 38 (225) 19 (47)

Passarelli et al. (1994)

Oellig et al. (1987) Foury and Lahaye (1987)

Ayres et al. (1994)

Braunagel et al. (1992) Lu and Miller (1994) Beames and Summers (1989) Shuman and Schwer (1995)

Li et al. (1993)

Tomalski et al. (1988) Green et al. (1990)

McLachlin and Miller (1994)

Whitford and Faulkner (1992)

Russell and Rohrmann (1997) Raynes et al. (1994)

Passarelli and Miller (1993c) Thiem and Miller (1989) Braunagel et al. (1996b)

Lu and Carstens (1991)

Lu and Carstens (1991)

Lu and Carstens (1991)

Wilson et al. (1987)

Passarelli and Miller (1994) Mikhailov et al. (1998)

Passarelli and Miller (1993b) Ayres et al. (1994)

Wolgamot et al. (1993)

Carstens et al. (1993)

SEQUENCE ANALYSIS OF THE PxGV GENOME 361

Note. Selected PxGV ORFs are listed in the “PxGV” column by ORF number, start and end nucleotide (nt) number, direction on the linearized physical map, deduced amino acid residues, and M r. The presence of baculovirus early promoter (E: TATAWAW . . . [25–35 nt] . . . CAKT) within 80 nt downstream from mRNA start site is shown. Baculovirus or other homologous genes identified by PxGV ORFs during BlastP searches are shown in the “Name” column and their references are listed in the last column. In the columns “XcGV,” “AcMNPV,” “OpMNPV,” “LdMNPV,” and “SeMNPV” their homologous ORF properties, such as ORF number, size, direction on the linearized map, and homology identity percentage obtained from a range within the ORF, are shown. The homologs with no reference indicated are from Ayres et al. (1994), Ahrens et al. (1997), Kuzio et al. (1999), Hayakawa et al. (1999), and IJkel et al. (1999).

Knebel-Mo¨rsdorf et al. (1993) 23 (268) Ⰷ 390 7 28 (51) Ⰷ me53

180

325

Ⰷ

41 (313)

139

449

Ⰶ

21 (251)

137

455

23 23 (205) Ⰶ

342

36 (459) Ⰷ 523 27 39 (453) Ⰷ 125 34 (455) Ⰷ 489 14 38 (438) Ⰷ 15 egt

94235 94815 95399 95982 96311 97174 97430 98350 99643 100179 145 93891 94237 94980 95392 95982 96239 97251 97601 98354 99751 100218 110 111 112 113 114 115 116 117 118 119 120

Ⰷ Ⰷ Ⰷ Ⰶ Ⰶ Ⰷ Ⰷ Ⰷ Ⰶ Ⰷ Ⰷ

114 192 139 198 109 311 59 249 429 142 308

13193 21825 16266 23097 12758 36367 6700 28856 49330 16502 36652

— — L L — — E — EL E —

vp1054

506

31 (313) 54

365

Ⰷ

58

378

57 22 (312) Ⰷ

560

34 (285) Ⰶ 346 105 32 (325) Ⰷ

16 (99) Ⰶ 137 108 17 (114) Ⰷ 142 22 (113) Ⰷ 23 (113) 139

38 (130) 26 (68) 32 (38) 44 (311) 43 (60) 24 (234) Ⰷ Ⰶ Ⰶ Ⰷ Ⰶ Ⰷ 139 378 67 323 70 332 171 172 173 175 176 178

26 (111) 118 165

Ⰷ

53

aa Dir. %ID (range) ORF aa ORF Name Prom. Mr Right Left ORF

Dir.

aa

Ⰷ

56

Dir. %ID (range) ORF

aa

146

54

aa Dir. %ID (range) ORF

332

References Dir. %ID (range) Dir. %ID (range) ORF

aa

SeMNPV LdMNPV OpMNPV AcMNPV XcGV PxGV

TABLE 1—Continued

O’Reilly and Miller (1989)

HASHIMOTO ET AL.

Olszewski and Miller (1997)

362

that the two GV genomes show a high degree of sequence variability (average identity of 42%), with almost an 80-kb difference in genome size and gene content, the genes fall on a diagonal line, indicating that their relative location has been conserved. The maintenance of this conserved genome organization suggests that it may play a critical role in gene expression or genome replication. To further examine the relationship of gene organization among more diverse baculoviruses, the homologous genes from PxGV and AcMNPV were compared (Fig. 2b). Of the 74 homologous genes, 27, including 8 located within Ac75 to Ac83, 13 located within Ac89 to Ac103, and 6 located within Ac141 to Ac147, are clustered in a continuous diagonal line. A number of other small clusters of homologs are present, but are not likely to be continuous; rather, they represent clusters of homologs moved from one position to the other on the genome. A similar observation on the clustered homologs represented by three groups of AcMNPV genes has been confirmed in homologs between PxGV and other baculoviruses (Table 1) and may be a common feature among lepidopteran baculoviruses (Heldens et al., 1998; IJkel et al., 1999). Homologous regions A common feature of all the baculovirus genomes that have been sequenced is the presence of homologous regions, or hrs (Cochran and Faulkner, 1983; Majima et al., 1993). Hrs contain sequences of repeated DNA that are present in five to 13 different locations in the genome. In NPVs, they contain a core 30-bp imperfect palindrome that is located within a direct repeat (Kool et al., 1995). In several NPVs, they have been shown to act as enhancers of RNA polymerase II-mediated transcription of baculovirus early promoters (Theilmann and Stewart, 1992; Guarino and Summers, 1996) and also serve as origins of DNA replication in transient assays (Pearson et al., 1992; Arhrens et al., 1995; Kool et al., 1995). The PxGV genome contains four hrs, designated hr1, to hr4 (Fig. 3). The locations of the hrs are not conserved among baculoviruses in terms of their relationship to flanking genes, except one hr is often located near the homolog of Ac83 (Kuzio et al., 1999). This pattern appears to be loosely conserved in the PxGV genome because Px84, the Ac83 homolog, is located just two ORFs upstream of hr3 (Table 1). This likely reflects that hr3 is located in a region that is highly conserved among all sequenced lepidopteran baculovirus genomes (see above and Fig. 2b). The distances between PxGV hr1 and hr2, and hr3 and hr4 are similar to each other (Fig. 3). Both hrs 1 and 2 are about 2 kb distant, whereas hrs 3 and 4 are about 1 kb. Figure 3 shows alignments of the hr repeated sequences. The length of the repeat unit is 101 to 105 bp. Hr1 possesses 21 complete repeats and five partial re-

SEQUENCE ANALYSIS OF THE PxGV GENOME

363

FIG. 1. Organization of PxGV genome. A linear genome based on the circular physical map of PxGV previously reported is shown (Hashimoto et al., 2000). One hundred twenty ORFs are represented as numbered black arrows showing their direction on the genome. The names of ORFs homologous to characterized baculovirus genes are indicated in parentheses. Four PxGV homologous regions (hrs) are represented by open boxes and their names. Physical maps for HindIII, PstI, and XhoI and a scale bar are shown below the ORFs.

peats (four upstream and one downstream of the core region); hr2 possesses 23 repeats, including two incomplete upstream and two incomplete downstream of the core region; hr3 possesses 11 repeats, including three partial repeats (two upstream and one downstream); and hr4 possesses 10 repeats, within which two repeats are incomplete. The G ⫹ C content of the hrs is 28%, which is significantly lower than that of the genome (40%). The consensus sequences of repeat units indicate that hr1 and hr3, and hr2 and hr4 are in the same orientation to each other, but the two sets are oriented in opposite directions. The consensus sequences of the repeats are highly conserved and possess a 15-nucleotide palindrome near the center. This sequence is not clearly related to the palindromes of NPVs, although a distant

relationship cannot be ruled out. Compared to NPVs, the PxGV hrs are larger, contain more repeat units, but have a smaller palindromic region. However, the hrs of PxGV appear more similar to those from lepidopteran NPVs than to those of XcGV, which lacks a distinct palindromic core sequence. Although the presence of four hrs is the smallest number reported in a baculovirus genome to date, it may simply reflect the small size of the PxGV genome. Furthermore, with a size of about 6 kb, the PxGV hrs comprise a significantly larger percentage of the genome than those found in other baculoviruses. Genes specific to GVs and unique to PxGV Genes conserved in PxGV and XcGV but not in AcMNPV, BmNPV, OpMNPV, LdMNPV, and SeMNPV are

364

HASHIMOTO ET AL.

FIG. 2. Comparison of the PxGV gene organization between XcGV and AcMNPV. (a) Comparison of PxGV and XcGV ORFs. Homologs of PxGV and XcGV orfs (see Table 1) are plotted based on their relative location in the genome. PxGV and XcGV ORFs with no homologs are aligned beside the vertical and horizontal axes. Homologs with the same or opposite direction based on the physical map are shown as open and shaded circles, respectively. Granulin/polyhedrin genes are shown as closed squares. ORFs clustered in groups of three or more are indicated by the inclusive lines. Homologs that are in a distant location from clustered ones are represented by their names. (b) Comparison of PxGV and AcMNPV ORFs. The plots are organized as in (a), except for indication of homolog names.

listed in Table 2. These genes, 25 in all, include homologs of a gene reported from another granulovirus (Cydia pomonella GV [CpGV] Cp16L) (Kang et al., 1997). Two copies of Cp16L homolog are located contiguously

on the XcGV genome, but are separated by two genes in the PxGV genome. Px35 has homology to metalloproteinase (Brooks et al., 1996; Goto et al., 1998), which may contribute to the proteolysis of infected tissue, thereby

SEQUENCE ANALYSIS OF THE PxGV GENOME

facilitating release of virus OBs. Since a viral cathepsin (Slack et al., 1995) homolog is present in XcGV, but not in PxGV, the metalloproteinase homolog may play an important role in the breakdown of insect tissues during PxGV infection. The PxGV genome contains 18 ORFs that were not found in either XcGV or five NPVs that have been sequenced (Table 3A). These ORFs showed no significant homology to protein sequences in GenBank and, therefore, may be unique to PxGV. In contrast, the XcGV genome contains 53 ORFs with no homologs to sequences in the database. The regions of the genome coding for the ORFs that appear to be unique to PxGV accounted for about 11 kb, or 11% of the genome. This is much smaller than that for the unique genes of XcGV (44 kb, or 25% of the XcGV genome). The 101-kb PxGV genome is the smallest baculovirus genome so far sequenced and is about 78 kb shorter than XcGV and about 33 kb shorter than the AcMNPV genome. The limited number of unique ORFs appears to be a contributing factor to the compact size of the PxGV genome. Genes absence in GVs but present in five NPVs Fifteen genes are found in the genomes of AcMNPV, BmNPV, LdMNPV, and OpMNPV, and 16 in SeMNPV, but are not found in PxGV and XcGV (Table 3B). These genes may encode features unique to NPV replication, pathology, or structure, or they may have homologs in NPVs but have evolved to such an extent that their relatedness cannot be determined with confidence. There appears to be no homolog of p80/p87 capsid protein (Mu¨ller et al., 1990), which may reflect structural differences between occluded virions. The GVs also appear to lack a homolog of the polyhedron envelope/calyx protein (Russell and Rohrmann, 1997). During NPV infections a sequential rearrangement of the actin cytoskeleton occurs, which has been shown to involve the expression of a gene called arif-1 (Roncarati and Knebel-Morsdorf, 1997) and which appears to be absent in the GV genomes. It is not clear whether such an actin rearrangement occurs during GV infection. However, the merging of the nucleus with the cytoplasm during GV infection may result in a different mechanism of cytoskeletal involvement. Other ORFs lacking in the GV genomes include PKIP-1, which interacts with and stimulates the activity of a virus-encoded protein kinase (Fan et al., 1998) and which is present in GVs. Px39 may be an alternative since PKIP-1 and Px39 both have a kinase-anchoring domain. Other baculovirus genes not present in the PxGV genome PxGV lacks a homolog of gp64, the budded virus envelope fusion protein of a variety of NPVs, including AcMNPV, OpMNPV, and BmNPV (Blissard and Rohr-

365

mann, 1989; Whitford et al., 1989). Gp64 is also absent from the genomes of LdMNPV, SeMNPV, and XcGV (Kuzio et al., 1999; IJkel et al., 1999; Hayakawa et al., 1999). Recently an envelope fusion protein called Ld130 has been described for LdMNPV (Pearson et al., 2000). A homolog of Ld130, Px26, is present in the PxGV genome and has the properties predicted for a membrane protein, including a signal sequence at its N-terminus and a transmembrane domain near its carboxyl terminus. Similar to the XcGV domain, PxGV lacked a number of genes that have been shown to be required for late gene expression in AcMNPV (Li et al., 1993; Passarelli and Miller, 1993a–c; Lu and Miller, 1994; Morris et al., 1994; Passarelli et al., 1994; Todd et al., 1995, 1996). These include lef-7, -10, -12, p35, ie-2, and hcf-1. P35 is an inhibitor of apoptosis; this role may be accomplished by the inhibitor of apoptosis (iap) gene homologs (Px88 and Px118), which carry out this function in other baculoviruses lacking p35 (Crook et al., 1993; Birnbaum et al., 1994). Other genes that are absent in PxGV but often found in other baculoviruses include a gp37 homolog, which is related to a spindle protein-encoding gene of entomopoxviruses (Yuen et al., 1990), chitinase (Hawtin et al., 1995), and p26, whose function is unknown (Liu et al., 1986). PxGV also lacks homologs of baculovirus repeated genes such as bro (Kuzio et al., 1999), enhancin (Hashimoto et al., 1996), and xcrep1 and -2 (Hayakawa et al., 1999). Repeated genes present in PxGV Table 3C shows a list of repeated genes present in PxGV. Two copies of homologs of Cp16L are present (Px20 and Px23). These ORFs are also repeated in XcGV (Hayakawa et al., 1999). The function of these genes has yet to be determined. There are three apparent homologs of the p10 gene including Px2, Px21, and Px50. Previous studies on p10 have demonstrated that it is a component of fibrillar bodies that are associated with OB morphogenesis and is also involved in the lysis of cells in the late stage of infection (van Oers and Vlak, 1996). Although P10s are not highly conserved at the amino acid sequence level (Zuidema et al., 1993), a number of structural features in common to P10s have been described (Wilson et al., 1995). These include a heptad repeat of hydrophobic amino acids (hydrophobic residues, a and d, in a heptad) in the N-terminal region, a hydrophilic region rich in proline residues in the middle, an abundance of basic amino acid residues in the extreme C-terminus, and the absence of cysteine, tryptophan, and histidine residues. Px2 showed low but significant similarity to P10 of Spodoptera litura NPV (SlituNPV). A careful examination of the amino acid sequence of

366

HASHIMOTO ET AL.

SEQUENCE ANALYSIS OF THE PxGV GENOME

367

FIG. 3. Nucleotide sequences of PxGV hrs. Each hr sequence is represented by multiple repeats as oriented on the physical map showing the genome coordinates for the beginning and end. Based on a repeat of a maximum size, nucleotide gaps in partial repeats are shown by (-). Incomplete or partial repeats present in peripheral of hrs are considered as a part of hr. The consensus sequence for each hr (hr1–4 con) is represented at the bottom of each hr sequence. Uppercase and lowercase letters indicate completely and partially conserved nucleotides, respectively. A consensus total (con total) was obtained by using opposite strand of consensus sequences of hr2 and hr4 and is shown at the bottom of the figure. A palindrome with a stem of 7 bp is underlined.

Px2 showed two repeats, consisting of four noncontiguous heptads and four contiguous heptads in the Nterminal half and C-terminal half, respectively; the latter heptad repeat was predicted to form an ␣-helical coiledcoil structure. However, other structural features attributed to P10 amino acid sequences could not be detected in Px2 and the late gene transcription motif was absent within the sequence 160 bp upstream of the initiation codon of p10. Recently, the filament-associated late protein of entomopoxviruses (FALPE), which may have a function similar to the suggested role of P10 in OB morphogenesis, was described (Alaoui-Ismaili and Richardson, 1996). FALPE also shares some structural features with P10, including a predicted helix in the Nterminal region, a proline-rich central region, and a basic C-terminal tail, but no significant amino acid sequence similarity is evident. Px21 and Px50 also have a large domain predicted to adopt an ␣-helical coiled-coil structure and are considered potential p10 homologs. Similar and supporting observations have been reported in XcGV (Xc5, -39, and -83) (Hayakawa et al., 1999) and CpGV (CpORF17R) (Kang et al., 1997). Px12, Px59, and Px68 are homologs of Ac145/150. Blast searches demonstrated a number of homologs present in baculoviruses and entomopoxviruses, but not in vertebrate poxviruses. In entomopoxviruses, these ORFs contain a six-cysteine motif (Dall, 1998). Px59, Px68, Xc87, and Se96 are deficient of the first cysteine residue within the motif. Interestingly, a large number of insect proteins that have an apparent chitin-binding capacity also have the six-cysteine motif (Dall, 1998). Px56 and Px104 showed homology to human fibroblast growth factor 1 (hFGF1) (Sutherland et al., 1996) or its

baculovirus homologs (Ayres et al., 1994). Homology is more evident with Px104 than with Px56, which is closely related to Xc85 that was not identified as an hFGF1 homolog (Hayakawa et al., 1999). Genes indicating new sequence features In determining homologs of PxGV ORFs, some ORFs have been found to possess sequence features not previously described in baculovirus genes. Px52 has a mut-T domain signature (G-5aa-E-4aa-[STAGC]-[LIVMA]-1aaRE-[LIVMF]-1aa-EE), which is also retained in Xc79 (Maki and Sekiguchi, 1992). Proteins from microorganisms to humans have been identified that contain the mut-T domain and have been shown to possess the capacity to hydrolyze 7,8-dihydro-8-oxo-dGTP to monophosphate, thus avoiding the incorporation of 8-oxo-7,8-dihydroguanine into nascent DNA. This capacity reduces a frequent error in DNA replication caused by AT to GC transversions. The mut-T motif is present not only in Xc79, but also in NPV genes (Ac38, Bm29, Ld46, Op7, and 22) that are homologous to Px52. However, they possess an irregular mut-T motif; the second conserved residue, glutamic acid (E), is replaced by aspartic acid (D) and the following intervening sequence is five rather than four aa. The aa underlined in the preceding consensus sequence are conserved in the baculovirus genes. Revision of GV gene homologs The size, orientation, relationship to other genes on the PxGV and XcGV genome maps, and alignments of their amino acid sequences has led us to revise our conclusions regarding the relationships of three granu-

368

HASHIMOTO ET AL. TABLE 2

ORFs Conserved in PxGV and XcGV but Not in AcMNPV, BmNPV, LdMNPV, OpMNPV, and SeMNPV a PxGV

XcGV

8 (175) 9 (93) 20 (235) 23 (131) 24 (338) 25 (431) 28 (210) 31 (129) 34 (133) 35 (402) 38 (153) 44 (130) 57 (100) 65 (152) 82 (340) 83 (69) 90 (175) 97 (194) 102 (61) 103 (66) 110 (114) 113 (198) 114 (109) 116 (59) 117 (249)

7 (187) 8 (86) 17 (187) 18 (153) 25 (449) 26 (370) 29 (170) 34 (113) 39 (149) 40 (469) 47 (220) 54 (110) 86 (115) 169 (144) 113 (373) 116 (123) 124 (180) 136 (171) 142 (88) 143 (66) 165 (118) 172 (378) 173 (67) 176 (70) 178 (332)

Name

Cp16L Cp16L

metalloproteinase

a ORFs are represented by their numbers and amino acid residues in parentheses. Three ORFs homologous to previously described GV genes are shown in the last column.

lovirus genes determined from the XcGV genome analysis (Hayakawa et al., 1999). Px94 is located adjacent to a homolog of DNA polymerase (dnapol) (Px93) and shows homology (22% aa identity) to an internal region covering 382 aa of a human desmoplakin (desmoplakin I), which

is an essential constituent of intracellular junctions (Green et al., 1990). It is not clear what role Px94 may contribute to the viral infection, but homologous genes with relatively low identities are present next to dnapol in XcGV (Xc133), AcMNPV (Ac66), and other NPVs. Therefore, we believe that Px94, Xc133, and Ac66 are homologs. Px95 is also located in this highly conserved region in the position of the lef-3 gene. Px95 displays a low level of homology to a large region of lef-3 of AcMNPV (20% aa identity, over 238 aa in 385 aa), but did not show significant homology to lef-3 homologs in other NPVs. The Px95 homology in XcGV (Xc134) is also located in the same region of conserved genes. Therefore it is possible that Px95 and Xc134 are homologs of lef-3, but they have undergone extensive sequence divergence. LEF-3 has the properties of a single-stranded DNA-binding protein (Hang et al., 1995). In addition, Wu and Carstens (1998) demonstrated that lef-3 is required for the transport of helicase into the nucleus. The fact that the nuclear membrane appears to be eliminated during GV infections may have led LEF-3 to lose its transport function in these viruses. Finally, Px15 is a likely homolog of ie-0 (Ac141) (Kovacs et al., 1991). It is located in the same position relative to ie-1 of other baculoviruses and shows a low level of sequence identity to Ac ie-0. Xc14, the Px15 homolog, shows similar position and sequence relationships to Px15 and, therefore, we have concluded that both these genes are likely to be related to ie-0. Genome content comparison between PxGV and XcGV A comparison of the relative genome content of a number of categories of sequences in PxGV and XcGV demonstrated that PxGV has a relatively high content of

TABLE 3 Features of Baculovirus Gene Content A. 18 ORFs unique in PxGV a 1, 3, 5, 18, 19, 22, 27, 33, 39, 48, 58, 62, 77, 81, 105, 108, 111, 119 B. ORFs conserved in AcMNPV, BmNPV, LdMNPV, OpMNPV and SeMNPV, but not in PxGV and XcGV b Ac 17 Ac 18 Ac 19 Ac 20/21 (arif-1) Ac 24 (pkip-1) Ac 26 Ac 34 Ac 55 Ac 57 Ac 58/59 Ac 104 (p80/p87-Capsid) Ac 108 Ac 120 Ac 131 (pe/pp34/calyx) Ac 136 (p26) c C. Repeated ORFs of PxGV d p10 (2, 21, 50) Ac 145/150 (12, 59, 68) CpGV 16L (20, 23) fgf (56, 104) a

ORFs are represented by number. ORFs are represented as AcMNPV genes with their ORF numbers. Genes previously characterized are shown in parentheses. c SeMNPV has two copies of p26 homolog. d ORF numbers are shown in parentheses. b

SEQUENCE ANALYSIS OF THE PxGV GENOME

369

suggest that PxGV has retained genes that are the minimum essential for replication, which results in a highly virulent infection throughout many insect tissues. MATERIALS AND METHODS Virus, virus cloning, and insects The PxGV isolate, a gift from Dr. Kondo, was isolated from a diamondback moth larva from a cabbage field in Hikone City, Shiga Prefecture, in June 1994 (Kondo and Yamamoto, 1998). The virus was cloned by consecutive per os infection of a diluted OB suspension through diamondback moth neonates (Hashimoto et al., 1999). An isolate exhibiting a single genotype, which was confirmed by restriction endonuclease analyses, was designated as the PxGV K1 strain. Propagation of the virus was carried out by feeding an artificial diet (Miyasono et al., 1992) contaminated with PxGV OBs (0.1 ml equivalent to 10 8 OBs/g diet) to third instar larvae. Plasmid library construction and DNA sequence determination and analysis FIG. 4. A comparison of genome contents of PxGV and XcGV. Percentages represented by each sector show DNA consisting of ORFs conserved in PxGV, XcGV, AcMNPV, LdMNPV, OpMNPV, and SeMNPV (a); ORFs conserved only in PxGV and XcGV (b); ORFs unique to PxGV or XcGV, or shared between 2 GVs and 4NPVs (c); hrs (d); and non-hr intergenic sequence (e).

genes that are conserved in all baculoviruses (60%) (Fig. 4). This result is likely because of the limited size of the PxGV genome. The content of the non-hr intergenic sequence of the PxGV genome is much less (4%) compared with that of XcGV (9%). In contrast, the hr content of PxGV is much higher (7%) than that of XcGV (3%), despite a marked difference in genome sizes (80 kb). This suggests that these structures play a critical role in baculovirus biology and a reduction in their size may be deleterious to the virus. The genomes of PxGV and XcGV represent the extreme in sizes of baculoviruses that have been characterized to date. The size difference may be reflected in the infection cycles of the two viruses that are radically different in the insects with which they are associated. PxGV is highly virulent and infects many tissues. In contrast, XcGV is much less virulent for its apparent host, taking up to a month to kill infected larvae, and the infection appears to be limited to the fat body (Federici, 1997). It may be advantageous for a virus to evolve to become less lethal because it allows its host to grow, thereby providing a greater amount of tissue that can be devoted to virus replication. It is possible that many of the genes that XcGV contains in its genome have been accumulated because they modulate the infection and allow for the host insect to survive for a significantly longer time than those infected with PxGV. This would

Purification of OBs, extraction and restriction endonuclease analysis of viral DNA, and construction of the plasmid (pBluescript; pBS) library of the viral DNA were carried out as described by Hashimoto et al. (2000). The DNA templates were derived from EcoRI, HindIII, and PstI libraries and subclones generated using other enzymes. In these three libraries, the PstI-K fragment (3.2 kb, 44.6–47.4 map units [m.u.]) was missing. Plasmid clones used as templates for sequence determination were chosen randomly from the DNA libraries. DNA templates were purified by PEG precipitation. FITC-labeled primers including pBS forward, reverse, and internal primers were purchased (Cruachem, Kyoto, Japan). Sequencing reactions were carried out using a Thermo Sequenase fluorescent-labeled primer cycle sequencing kit (Amersham, Buckinghamshire, UK) and were analyzed using an automated sequencer (DSQ-1000L, Shimadzu, Kyoto, Japan). Each DNA strand was sequenced three or more times in each direction using different primers. The nucleotide sequence data were analyzed using Genetyx-Win ver. 3.02 (Software Development, Tokyo, Japan). ORFs described in this report were those predicted to have 50 or more amino acid residues and with minimal overlaps with other ORFs. Homology searches of amino acid sequences were carried out using the BLAST protocol (BlastP 2.0.7, released on December 21, 1998 (Altschul, 1990)) and recent protein databases (all nonredundant GenBank CDS translations ⫹ PDB ⫹ SwissProt ⫹ PIR ⫹ PRF on April 2000). Nucleotide sequence accession number The nucleotide sequence data reported in this study will appear in the GSDB, DDBJ, EMBL, and NCBI nucle-

370

HASHIMOTO ET AL.

otide sequence AF270937.

databases

under

accession

no.

ACKNOWLEDGMENTS We thank George F. Rohrmann for critical reading of the manuscript and assistance with the sequence analysis. This research was partially supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan (10306006, 10556009).

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