Journal of General Virology (2006), 87, 19171933

DOI 10.1099/vir.0.81792-0

Sequence analysis of the Choristoneura occidentalis granulovirus genome

Shannon R. Escasa,1 Hilary A. M. Lauzon,1 Amanda C. Mathur,1 Peter J. Krell2 and Basil M. Arif1
Correspondence Basil M. Arif barif@nrcan.gc.ca
1

Laboratory for Molecular Virology, Great Lakes Forestry Centre, Sault Ste Marie, ON P6A 2E5, Canada Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, Canada

Received 22 December 2005 Accepted 24 February 2006

The genome of the Choristoneura occidentalis granulovirus (ChocGV) isolated from the western spruce budworm, Choristoneura occidentalis, was sequenced completely. It was 104 710 bp long, with a 67?3 % A+T content and contained 116 potential open reading frames (ORFs) covering 88?4 % of the genome. Of these, 29 ORFs were conserved in all fully sequenced baculovirus genomes, 30 were GV-specic, 53 were present in some nucleopolyhedroviruses (NPVs) and/or GVs, three were common to ChocGV and Choristoneura fumiferana GV (ChfuGV) and one was so far unique. To date, ChocGV is the only GV identied that contains a homologue of the apoptosis inhibitor protein P35/P49, present in some group I NPVs. It is also the rst GV without a Xestia c-nigrum GV ORF 26 homologue. Five homologous regions (hrs)/repeat regions, lacking typical NPV hr palindromes were identied. ChocGV hrs were similar to each other but not to other GV hrs. A 1?8 kb repeat region with a high A+T content (81 %) and multiple repeats of 21210 bp was found between choc36 and 37. This area resembled the non-homologous region origin of DNA replication (non-hr ori) identied in Cryptophlebia leucotreta GV (CrleGV) and Cydia pomonella GV (CpGV). Based on the mean amino acid identities of homologous proteins, ChocGV was closest to fully sequenced genomes CpGV (52?3 %) and CrleGV (52?1 %). The closest amino acid identity was to individual ORFs from the partially sequenced ChfuGV genome (97?2 % in 38 ORFs). Phylogenetic analysis placed ChocGV in a clade with CrleGV and CpGV.

INTRODUCTION
Granuloviruses (GVs) have so far only been identied in Lepidoptera, while nucleopolyhedroviruses (NPVs) have been identied mainly in Lepidoptera, but also in Diptera and Hymenoptera (Blissard et al., 2000). GVs infect both agricultural and forest insect pests, making them potentially important as biological insecticides (Rashidan et al., 2004a). The sequencing of the Choristoneura occidentalis GV (ChocGV) genome brings the number of totally sequenced GVs to eight. The western spruce budworm, Choristoneura occidentalis, and the eastern spruce budworm, Choristoneura fumiferana, were previously considered to be the same species separated geographically by the Rocky Mountains. They are now considered to be different species. C. occidentalis is the most widely distributed and destructive defoliator of coniferous
The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is DQ333351. Comparison of the ChocGV genome with other granuloviruses is available as a supplementary table in JGV Online.

trees in Western North America. The rst recorded outbreak was in 1909 on Vancouver Island, Canada. The insects prefer new growth, damaging leaves, owers, cones and affecting tree survival and regeneration (Fellin & Dewey, 1999). Damage begins in the spring when larvae mine into needles and enter the swollen buds. Webs are spun on new foliage, where insects feed by chewing needles off at their base. Trees may recover unless repeated severe defoliation occurs over 35 years (http://www.pfc.forestry.ca). Previous work by Arif et al. (1986) describes comparisons of granuloviruses isolated from Choristoneura spp. Baculoviruses that have been isolated from C. occidentalis and C. fumiferana are cross-infective (Fellin & Dewey, 1999). This manuscript reports the complete sequencing and analysis of the ChocGV genome and compares it with other baculovirus genomes.

METHODS
Virus preparation and DNA extraction. ChocGV was propagated

in C. fumiferana larvae by placing third instar larvae on an articial diet (10 per cup), surface treated with 3?86108 occlusion bodies (OBs). Infected larvae were collected 3 weeks later, homogenized and stirred for 2 h in 0?5 % SDS. The homogenate was ltered and 1917

0008-1792 G 2006 SGM

Printed in Great Britain

S. R. Escasa and others centrifuged at 5000 r.p.m. (13.1 rotor; Beckman J2-21) for 15 min at 15 uC. The OB pellet was washed three times in deionized water, recentrifuged and suspended in 10 ml water. The sample was loaded on to a 45 % sucrose cushion and centrifuged (SW28 rotor; Beckman ultracentrifuge) at 8000 r.p.m. for 20 min at 15 uC. The OB pellet was resuspended in water and centrifuged at 5000 r.p.m. for 15 min at 15 uC. OBs were dissolved in an alkaline solution (1?0 M sodium carbonate and 0?4 M sodium thioglycollate) and placed on to a continuous 1045 % sucrose gradient and centrifuged at 20 000 r.p.m. (SW28 rotor; Beckman Ultracentrifuge) for 1?25 h at 4 uC. The banded virions were withdrawn (with a syringe), diluted with TE (10 mM Tris and 1?0 mM EDTA) and centrifuged at 22 000 r.p.m. (SW28 rotor; Beckman Ultracentrifuge) for 2 h at 4 uC. The pellet was suspended in 500 ml TE. Proteinase K (25 ml, 20 mg ml21) was added and incubated at 37 uC for 30 min followed by another 25 ml proteinase K with 1 % SDS (nal concentration) and incubated at 50 uC for 30 min. DNA was extracted with phenol/chloroform and dialysed against several changes of TE for 24 h at 4 uC.
DNA sequencing and analysis. ChocGV genomic DNA was nebulized, cloned into pUC18 and sequenced to yield 10 times coverage (Applied Biosystems; BGI Life Technology). Base calling was done using the software Phred (Ewing et al., 1998; Ewing & Green, 1998), pUC18 contaminations were removed with CrossMatch (Green, 2004), and the sequence was assembled using Phrap (Green, 1996). Gaps and regions with low quality data identied after preliminary assembly were lled in or rened by sequencing PCR amplied products.

baculovirus proteins from 27 genomes were used to determine phylogeny. A most parsimonious tree was constructed using CLUSTAL W protein alignments and PAUP 4.0b10 (Swofford, 2000) with maximumparsimony, heuristic search, step-wise addition option and bootstrap analysis (1000 replicates). Genomes used in phylogenetic analysis included those previously listed plus the following: HearNPV C1 (Zhang et al., 2005), Helicoverpa zea SNPV (HzSNPV; Chen et al., 2002), Mamestra congurata NPV A (MacoNPV A; Q. Li et al., 2002), MacoNPV B, (L. Li et al., 2002), Adoxophyes honmai NPV (AdhoNPV; Nakai et al., 2003), Choristoneura fumiferana MNPV (CfMNPV; de Jong et al., 2005), Neodiprion sertifer NPV (NeseNPV; GarciaMaruniak et al., 2004) and Culex nigripalpus NPV (CuniNPV; Afonso et al., 2001).

RESULTS AND DISCUSSION

ChocGV sequence analysis Approximately 11?5 times coverage of the genome was achieved by generating 1 209 500 nt of raw data from 1793 sequencing reads. An overall error rate of 0?009 % was estimated. The ChocGV genome contained 104 710 bp, slightly larger than the 99 kb estimated by restriction endonuclease analysis. The discrepancy in size was due to the presence of 10 HindIII fragments less than 1?0 kb identied by sequencing that were not seen on agarose gels. ChocGV is the third smallest GV sequenced to date, with PlxyGV (100 999 bp) and AdorGV (99 657 bp) being smaller (Supplementary Table S1 available in JGV Online). The A+T content of ChocGV was 67?3 %, second only to CrleGV with 67?7 % (Lange & Jehle, 2003). ChocGV contained 116 methionine-initiated ORFs, 62 (53?4 %) clockwise and 54 (46?6 %) counterclockwise, with respect to granulin that was designated ORF 1 (Table 1, Fig. 1). Choc41 (46 aa) was considered a potential ORF as it showed baculovirus homology (Table 1). There were minor overlaps between certain ORFs; the longest (139 bp) covered the 39 end of alkaline-exonuclease and the 59 end of helicase-2. Intergenic spaces and a non-homologous region origin of DNA replication Most intergenic spaces in the genome were less than 50 bp (48 spaces) or 50300 bp (36 spaces). Four were between 300 and 600 bp and three were greater than 1?0 kb. The rst large intergenic space was between choc21 and 22 (1018 bp) and the second between choc22 and 23 (1545 bp). These areas were expected to contain homologues to cp28 and cp30, respectively, but neither ORF was present (Fig. 1), and no BLAST matches with other baculovirus genomes were found. The third intergenic space (3441 bp) between choc36 and 37 contained a 1?8 kb region with multiple direct repeats that was extremely AT rich (81 %). Initial computer analysis suggested the presence of a 1144 aa ORF in this region with 27 leucine zippers. Leucine zippers contain leucine residues (L) at every seventh position over at least seven helical turns (Gattiker et al., 2002) that establish amphipathy to help stabilize a long a-helix (Landschulz et al., 1988). The leucine zipper motif is: [L(X6)L(X6)L(X6)L]
Journal of General Virology 87

Sequence analysis was carried out using NCBI open reading frame (ORF) nder (Wheeler et al., 2003), BLAST (Altschul et al., 1990), DNASTAR (Burland, 2000) and the Multi-purpose automated genomics project investigation environment (Magpie), available through the University of Calgary (Gaasterland & Sensen, 1996). Methionineinitiated ORFs potentially encoding 50 or more amino acids were considered for further analysis. Smaller ORFs with baculovirus homologues were also considered. DNA regions containing potential ORFs with unusually large repeats were deemed as non-coding and analysed separately. Repeated sequences were identied using REPuter (Kurtz & Schleiermacher, 1999) and Tandem Repeats Finder (Benson, 1999). ExPASy (Gasteiger et al., 2003), Prosite (Hulo et al., 2004) and SMART (Schultz et al., 1998) were used to identify protein motifs. Multiple sequence alignments and percentage amino acid identities were carried out using DNASTARs MEGALIGN CLUSTAL W (Thompson et al., 1994) with default conditions. GeneDoc was used to shade conserved sequences in aligned proteins (Nicholas & Nicholas, 1997). Seven GV genomes and 11 NPV genomes were used to identify gene conservation in ChocGV. The genomes included were from: Cydia pomonella GV (CpGV; Luque et al., 2001), Cryptophlebia leucotreta GV (CrleGV; Lange & Jehle, 2003), Adoxophyes orana GV (AdorGV; Wormleaton et al., 2003), Phthorimaea operculella GV (PhopGV; GenBank accession no. AF499596), Plutella xylostella GV (PlxyGV; Hashimoto et al., 2000), Xestia c-nigrum GV (XecnGV; Hayakawa et al., 1999), Agrotis segetum GV (AgseGV; GenBank accession no. NC_005839), Autographa californica multicapsid NPV (AcMNPV; Ayres et al., 1994), Helicoverpa armigera NPV (HearNPV G4; Chen et al., 2001), Spodoptera litura NPV (SpltNPV; Pang et al., 2001), Spodoptera exigua MNPV (SeMNPV; IJkel et al., 1999), Lymantria dispar MNPV (LdMNPV; Kuzio et al., 1999), Orgyia pseudotsugata MNPV (OpMNPV; Ahrens et al., 1997), Choristoneura fumiferana defective NPV, (CfDEFNPV; Lauzon et al., 2005), Epiphyas postvittana NPV (EppoNPV; Hyink et al., 2002), Rachiplusia ou MNPV (RoMNPV; Harrison & Bonning, 2003), Bombyx mori NPV (BmNPV; Gomi et al., 1999) and Neodiprion lecontei NPV (NeleNPV; Lauzon et al., 2004). Promoter regions were found using Promoter Scan (Prestridge, 1995) and InterPro (Mulder et al., 2003). Gene parity plots (Hu et al., 1998) compared the gene order of ChocGV with CpGV, CrleGV, AdorGV and AcMNPV. Concatamers of 29 conserved 1918

Sequence analysis of the ChocGV genome

(Bateman et al., 2004). This potential ORF did not have any baculovirus homologues. Sequencing errors are more common within repeat regions as frequent recombination can lead to differences in data between clones (Ahrens et al., 1997). Also, ChocGV was not plaque puried, increasing the possibility of clonal variation. Manual examination revealed variation among different clones covering the same area. Other fully sequenced GV genomes contain three smaller ORFs in this region (cp49, cp50 and cp51; Fig. 1). The presence of three ORFs between choc36 and 37 was suggested by BLASTN analysis. These potential ORFs had high identity to three ORFs from ChfuGV (GenBank accession nos AAN77189, AAN77190 and AAN77191). The beginning of the region also showed low nucleotide identity to cp50/ crle47 and its 39 region showed low identity to xecn47. Manual examination of the trace data showed several areas where sequencing errors were possible or where the addition or subtraction of a single base might cause a frameshift. Two of these potential changes involved areas with large strings of adenines. If bases were added to cause frameshifts, the rst potential ORF between choc36 and 37 would be larger than its ChfuGV homologue (GenBank accession no. AAN77189) as it would have three leucine zippers instead of one, suggesting that duplication of repeat regions has occurred in the ChocGV genome. The second potential ChocGV ORF had three repeats of 210 bp constituting 84 % of the ORF and the third lacked repeats but had a transmembrane domain at its 59 end. There were many factors to consider when interpreting the large space between choc36 and choc37: (i) the space had multiple repeats, (ii) it contained ambiguous bases in the trace data, (iii) ChocGV was not plaque puried and clonal variation existed in this region, (iv) no other baculovirus genomes contained homologues to the large potential protein between choc36 and 37, and (v) difculties arose in deciding where bases should be added or deleted to cause a frameshift producing three small ORFs. Because of the combination of these factors, decisions to have one large ORF or three smaller ORFs between choc36 and 37 were both rejected. Instead, DNA regions containing ORFs with large, unusual repeats were considered non-coding and were analysed separately as was done with the genome of CrleGV (Lange & Jehle, 2003). Intergenic spaces greater than 1?0 kb are found in other GVs. XecnGV has a 1473 bp intergenic region (Hayakawa et al., 1999) in approximately the same location as the rst large intergenic space in ChocGV. CrleGV has a 1?8 kb intergenic space between crle26 and crle27 that contains a 300 bp AT-rich region, direct repeats, short palindromes and is considered to be a potential non-homologous region origin of DNA replication (non-hr ori) (Jehle, 2002; Lange & Jehle, 2003). CpGV has a repeat region of 1?13 kb with six short imperfect repeats, three large imperfect repeats and an AT-rich section also considered to be a non-hr ori. The CpGV non-hr ori region, however, is located within three ORFs (cp25cp27) none of which have baculoviral
http://vir.sgmjournals.org

homologues (Huang & Levin, 1999). The non-hr ori repeats in CpGV and CrleGV are not found elsewhere in the genomes and are not similar to their homologous regions (hrs) (Luque et al., 2001; Lange & Jehle, 2003). Other baculoviruses with non-hr oris include: AcMNPV (Kool et al., 1994), OpMNPV (Pearson et al., 1993), SeMNPV (Heldens et al., 1997) and Spodoptera littoralis (SpliMNPV) (Huang & Levin, 1999). These regions are complex, contain direct and inverted repeats and are between 800 and 4000 bp long (Luque et al., 2001). The ChocGV 1?8 kb repeat region between choc36 and 37 showed similarities to the non-hr ori regions in CrleGV and CpGV as it had multiple repeats not found elsewhere in the genome, was not similar to hrs and was very AT rich. Hrs regions Typically, baculoviruses have many hrs interspersed throughout their genomes. These regions may act as enhancers of RNA transcription, as origins of DNA replication and as sites of recombination (Hayakawa et al., 2000). Most NPV hrs contain 30 bp palindromes within direct repeats, whereas GV repeat regions are more variable and often lack palindromes (Wormleaton et al., 2003). XecnGV contains nine hrs each with three to six direct repeats lacking a palindromic core (Hayakawa et al., 1999). PlxyGV has four large repeat regions centred on a palindrome that more closely resemble NPV hrs (Hashimoto et al., 2000), while AdorGV has nine repeat regions unlike NPV hrs (Wormleaton et al., 2003). ChocGV contained ve hrs/ repeat regions varying in size and number of direct repeats. The rst (hr-1), had three repeated sequences in a 157 bp area, hr-2, four repeated sequences within 129 bp, and hr-3, 10 repeated sequences within 288 bp. The fourth and fth hrs/repeat regions had four repeated sequences in a 106 bp area and 162 bp area, respectively (Fig. 2a). Direct repeats averaged 27 bp with the shortest being 19 bp and the longest 39 bp. These regions were not like NPV hrs and did not have embedded palindromes. Each hr was homologous to all other ChocGV hrs but not to hrs in other baculoviruses. Despite the differences between GV and NPV hrs, and between GV hrs themselves, the relative locations of hrs in ChocGV were similar to the locations of three of the hrs in CrleGV, and to eight of 13 repeats in CpGV (Fig. 2b). Gene content and organization ChocGV contained all 29 core genes found in baculovirus genomes sequenced to date. It was the rst fully sequenced GV lacking a xecn26 homologue. Of 116 total ORFs, 30 were found only in GVs, 53 were found in some GVs and/or NPVs, three only in ChocGV/ChfuGV (choc4, 10 and 11), and one (choc87) was so far unique. Since the ChfuGV genome has not yet been fully sequenced, a homologue to choc87 may still be identied. Of the ORFs found only in ChocGV/ChfuGV, choc4 and choc11 showed 100 % amino acid identity to their ChfuGV homologues (GenBank accession nos AAM60756 and AAM60754). Choc10 shared 80 % amino acid identity with its ChfuGV homologue (GenBank
1919

1920 Journal of General Virology 87

S. R. Escasa and others

Table 1. Characteristics of the ChocGV genome

Predicted ORFs are compared with homologues in eight GVs and AcMNPV. Direction of transcription is noted by the symbols: > (+ve strand) and < (2ve strand). Promoter motifs are identied as early (E) and late (L). Protein motifs and other homologues are shown in the Comments column. Late promoter motif (DTAAG), where D=A, G or T within 150 nt from start codon. ChocGV ORF Name Position Size (aa) CpGV ORF CrleGV AdorGV PhopGV PlxyGV XecnGV AgseGV ChfuGV GenBank no. aa id (%) AcMNPV ORF aa id (%) Promoter Comments

aa ORF aa ORF aa ORF aa ORF aa ORF aa ORF aa id id id id id id id (%) (%) (%) (%) (%) (%) (%) 1 93?2 2 36?2 3 61?5 1 94?4 2 30?8 3 57?6 1 91?2 2 25?2 3 59?7 4 85?5 5 19?7 6 45?1 1 88?4 2 23?6 3 45?7

1 2 3 4 5 6 hr-1 7 8 9 10 11 12 13 hr-2 14 15 16 17 18 19 20

granulin pk-1

1>747 744<1124 1105>1938 1993<2586 2642<3205 3195>3449 35243652 3687<5027 4863>5627 5670<5966 6162>7454 7580>8323 8355<8621 8628<9995 1012010283 10297<11358 11387<12496 12648>12818 12824<13315 13502>14494 14517>14921 14981<15574

248 126 277 197 187 84 446 254 98 430 247 88 455 353 369 56 163 330 134 197

1 94?8 2 29?1 3 56?1

1 88?8 AAC69544 99?6 2 23?6 AAG50436 100?0 3 50?4 AAL05839 100?0 AAM60756 100?0

8 10

53?7 L, E* L, ED 34?4 L E* L, E* ED

Protein kinase domain Transmembrane domain

4 52?7 5 42?0 7 37?1 8 45?1 9 66?7

4 46?8 5 50?6 6 38?9 7 47?4 8 72?7

4 31?9 5 41?7 6 37?4 7 45?8 8 56?6

4 53?7 5 31?8 6 34?2 7 41?3 8 61?6

8 17?6 9 30?6 10 26?1 11 30?4 12 61?6

7 33?0 8 27?1 9 24?4 10 26?4 11 56?6

6 43?8 AAM60757 100?0 7 30?6 AAM60759 100?0 8 33?1 AAL47676 92?5 9 38?3 AAM60755 97?8 10 54?5 AAM60758 100?0 AAM60753 80?1 147 146 145

ie-1

11?9 18?8 L, ED 33?3 L,ED L L 41?3 L 27?2 L 44?4 L, ED 19?7 L, Ed 26?3 ED L L L L

chtBD2 chitinbinding domain Translation initiation factor eIF-5

odv-e18 p49 odv-e56 p35/p49

14 77?6 15 53?5 18 73?7 19 33?3 20 75?6 22 64?4 23 71?9

13 86?0 14 55?1 17 71?2 19 40?4 20 74?4 23 68?2 24 63?0

10 79?5 11 47?1 12 67?2 15 22?8 16 55?5 17 61?0 18 54?3

12 83?3 13 40?0 16 71?6 17 40?4 19 66?5 20 60?7 21 60?0

13 40?4 14 40?0 16 65?9 17 38?6 20 54?9 21 53?0 23 50?8

12 61?9 13 37?9 15 52?0 16 26?3 17 55?0 19 53?5 18 51?1

AAM60754 99?6 11 62?9 AAL05838 100?0 12 47?9 AAL05837 100?0 15 57?5 AAM60760 100?0 16 28?1 18 59?1 19 59?8 AAM70480 20 56?3 83?8

143 142 148 136 29

Similar to p49, p35, AMV010 BaculoPEPN domain BaculoPEPN/ PEPC domain BaculoPEPN domain Ld7(27?6 %) CfDEF145 (29?8 %)

pep pep/p10 pep

Table 1. cont.
ChocGV ORF Name Position Size (aa) CpGV ORF CrleGV AdorGV PhopGV PlxyGV XecnGV AgseGV ChfuGV GenBank no. aa id (%) AcMNPV ORF aa id (%) Promoter Comments

http://vir.sgmjournals.org 1921

aa ORF aa ORF aa ORF aa ORF aa ORF aa ORF aa id id id id id id id (%) (%) (%) (%) (%) (%) (%) 26 17?3 28 17?4 30 58?8 32 40?4 9 21?4 34 35 36 38 39 40 42 43 44 51?6 64?5 63?5 50?3 45?8 38?4 20?4 40?0 69?5 21 19?5 23 43?8 25 33?3 40 15?9 26 28 29 32 33 36 37 38 39?9 55?8 51?0 41?5 31?0 36?4 33?1 59?5 25 13?6 27 55?4 29 40?5 31 33 34 37 38 39 40 41 42 42?7 58?2 59?6 47?0 36?9 42?0 22?0 34?5 59?9 24 13?3 26 43?7 28 33?2 29 30 31 32 34 35 36 25 15?8 27 31?5 29 24?6 23 14?6 25 52?0 27 27?1 29 33 34 35 36 40 41 42

21 22 23 hr-3 24 25 26 27 28 29 30 31 32 33 34 35 36 Non-hr ori like 37 38 39 40 41 42 43 44 45 46 47 48 hr-4 49 50

efp

15949<17022 18041>19291 20837>22597 2294123299 23907<24626 24667<25269 25359>25943 25998<28187 28226>28537 28518>29084 29068>29319 29348<29683 29680<30132 30185<31651 31630>32436 32464>33588 33515>33817 3453436324 37258<37869 37875>38024 38030<38317 38404>39435 39454>39594 39578<40333 40326<40604 40627<41112 41378<41692 41722<43716 43791<44153 44371<44979 4505445248 45306<45668 45729>46886

357 416 586 239 200 194 729 103 188 83 111 150 488 268 374 100

27 17?3 29 19?7 31 58?3 33 38?8 35 37 39 41 42 43 45 46 47 51?3 60?1 65?4 52?0 43?4 33?0 30?5 37?8 69?1

Zinc nger motif 23 L, Ed 11?6 ED L L 115 46 6 35?4 L, ED 53?2 L L, ED 19?0 L ED Coiled-coil motif ZnMc Glycosyl transferase family 8

ORF2 parvo-like virus(34?3 %)

odv-e66 lef-2

mp-nase p13 pif-2

47?8 32 38?5 47?7 149 39?5 31?7 34 36?5 33?3 35 32?3 36 32?1 25?4 39 15?3 27?3 40 26?6 53?0 43 49?8

43?8 35?9 AAM70479 36?5 35?7 33?3 30?1 32?0 62?4

70?2

Coiled-coil motif

L 22 50?7 L

48 72?4

45 71?9

39 59?2

44 62?4

37 56?4 45 52?5 152 23?8

43 53?3 AAN77187 100?0 AAN77188 100?0

ubq

39k lef-11 sod p10 p74

p47

203 49 95 343 46 251 92 161 104 664 120 202 120 385

52 53 54 55 56 57 58 59 22 60 67 62 68

60?3 57?1 83?2 51?7 46?8 33?1 57?0 64?7 20?2 62?7 59?1 42?1 64?5

50 51 52 53 54 55 56 57 23 58 64 60 61

69?6 50?0 85?7 58?4 48?9 30?1 55?9 64?5 14?4 61?8 48?7 48?6 64?2

43 44 45 46 47 49 50 51 13 53 56 57 58

70?4 40?8 76?8 57?3 42?6 33?3 44?1 51?6 8?7 51?2 53?5 23?9 57?5

47 48 49 50 51 52 53 54 20 55 59 60 61

71?1 50?0 78?9 47?4 48?9 34?1 53?4 59?3 24?0 62?4 59?6 43?8 62?4

40 41 42 43 44 45 46 47 21 49 50 51

59?3 38?0 70?8 44?5 6 ?4 17?0 43?0 57?1 19?2 51?1 34?7 52?6

50 51 52 53 54 55 56 68 5 77 78

61?3 26?0 84?6 41?3 27?7 20?4 49?5 63?0 20?0 42?3 53?6

46 47 48 49 51 52 54 55 56 57 59 60

60?8 71?9 56?1 46?8 34?1 47?3 62?4 25?0 49?2 53?0 36?4 56?2

AAN77192 AAN77193 AAN77194 AAN77195 AAN77196 AAN77197 AAN77198 AAN77199 AAN77200 AAN77201

99?0 100?0 100?0 100?0 100?0 100?0 100?0 100?0 100?0 100?0

106 110 35 109

32?3 10?0 74?4 L, EDd 27?9 L, ED L 29?0 56?6 33?0 ED 40?4 L, ED L

Sequence analysis of the ChocGV genome

37 31 137 138

PFAM: NPV_P10

L 40 37?0

Coiled-coil motif

Table 1. cont.
ChocGV ORF Name Position Size (aa) CpGV ORF CrleGV AdorGV PhopGV PlxyGV XecnGV AgseGV ChfuGV GenBank no. aa id (%) AcMNPV ORF aa id (%) Promoter Comments

1922 Journal of General Virology 87

S. R. Escasa and others

aa ORF aa ORF aa ORF aa ORF aa ORF aa ORF aa id id id id id id id (%) (%) (%) (%) (%) (%) (%) 62 73?4 63 65 66 67 68 69 60?7 30?9 62?4 67?2 47?3 31?0 59 70?2 60 61 62 63 64 65 51?0 15?0 56?0 60?6 37?5 30?7 62 69?9 63 65 66 67 69 70 47?0 22?8 54?3 53?2 42?4 25?7 52 58?7 53 54 55 7 56 57 55?0 33?8 51?3 53?4 27?0 10?0 79 59?2 80 81 82 84 85 86 42?6 22?2 47?9 45?1 23?7 8?9 61 60?1 62 63 64 65 66 50?6 29?9 56?8 36?0 25?9

51 52 53 54 55 56 57 58 p24 38?7kd lef-1 pif fgf-1

46918>47571 47574>48122 48119>48388 48424<48924 48905<49606 49621>51210 51213<51884 51931<52233

217 182 89 166 233 529 223 100

69 72?9 71 73 74 75 76 77 62?8 33?5 62?0 66?6 42?3 24?8

38 129 13 14 119

37?8 L, ED 23?0 L, ED E* 20?4 31?6 34?0 L L

PFAM: NUDIX motif

Zinc nger motif ChtBD2, transmembrane domain

59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83

lef-6 dbp

p48

p6?9 lef-5 19kd hel-1 odv-e25 p33 lef-4 vp39 odv-e27

vp91 tlp20 gp41

52398>52856 52849<53103 53152<53979 53951<54211 54147<54743 54767>55936 55967>56302 56351>57475 57499>57672 57736<58458 58405>59301 59294<59779 59763>63131 63163<63804 63831<64313 64366>65121 65122<66516 66582>67442 67503>68357 68560<69765 69721>70005 69986<71956 71925>72254 72121>72831 72791>73630

152 84 275 86 198 389 111 374 57 240 298 161 1122 213 160 251 464 286 284 401 94 656 109 236 279

79 80 81 82 82 83 84 85 86 87 88 89 90 91 92 93 95 96 97 99 100 101 102 103 104

35?3 38?8 50?0 35?6 34?5 74?6 44?5 59?7 66?0 70?5 59?5 57?4 53?9 81?3 55?3 63?9 53?3 61?2 61?1 41?5 33?7 42?0 43?6 79?2 72?5

70 71 72 73 73 74 75 76 77 78 79 80 81 82 83 84 86 87 88 90 91 92 93 94 95

39?9 35?3 50?0 35?6 25?1 73?6 41?4 60?5 55?2 68?5 58?5 51?2 53?9 81?3 47?2 66?1 53?8 59?5 60?7 42?9 31?6 45?6 38?2 79?3 69?6

66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88

36?2 45?9 49?3 66?7 40?2 54?7 73?2 59?8 61?2 44?4 52?9 72?0 36?6 55?2 52?5 62?0 57?2 32?3 15?1 41?0 35?5 64?4 48?6

71 72 73 74 74 75 76 77 78 79 80 81 82 83 84 85 87 88 89 92 93 94 95 96 97

34?6 42?4 30?1 8?0 18?2 65?3 26?6 51?5 54?5 64?3 58?9 48?1 43?0 73?4 42?2 61?5 53?8 48?4 51?6 30?1 29?5 40?9 36?4 66?1 59?6

59 60 61 63 64 66 67 69 70 71 72 74 75 76 78 79 80 82 83 84 85 86 87

26?3 31?8 19?4 50?5 25?8 43?1 50?9 56?0 44?1 34?0 39?4 66?8 41?4 53?4 48?0 32?1 39?6 29?9 26?1 36?7 26?4 64?6 43?2

87 88 89 90 90 91 92 93 94 95 96 97 98 99 100 101 110 111 112 113 116 118 119 120 121

30?1 36?5 17?0 6?9 14?6 55?0 28?6 45?6 50?0 49?0 51?5 47?5 38?6 64?0 35?8 50?4 42?9 38?3 38?2 27?5 26?3 33?2 20?9 58?0 49?3

67 68 69 70 71 72 73 74 75 76 77 78 79 81 82 83 85 86 87 88 90 91 93 94 95

17?4 34?9 25?0 20?7 23?1 60?5 40?2 52?1 50?0 53?1 50?0 40?1 41?6 66?8 36?6 58?7 46?9 37?6 43?5 29?1 22?1 34?9 31?8 56?1 55?4

150 28 25

15?3 L 11?8 L 18?5

AAO11741 100?0 AAN86838 87?9

AAL79608 100?0 AAY45882 95?5

103 102 101 100 99 98 96 95 94 93 92 90 89 144

31?2 16?5 19?6 33?9 42?7 41?5 30?9 22?0 37?4 22?4 36?5 28?0 27?9 24?2

L L L L, ED

L L L L, ED L, ED L L Coiled-coil motif

AAO16247

97?5

83 82 81 80

L 18?6 15?5 37?6 30?7 L

http://vir.sgmjournals.org 1923

Table 1. cont.
ChocGV ORF Name Position Size (aa) CpGV ORF CrleGV AdorGV PhopGV PlxyGV XecnGV AgseGV ChfuGV GenBank no. aa id (%) AcMNPV ORF aa id (%) 24?6 20?0 ED 30?7 L, ED Ed 24?7 L, E* 20?9 L 32?8 12?3 12?4 27?8 L 2 BIR motifs, RING nger 62 61 53?5 L 27?4 L L Promoter Comments

aa ORF aa ORF aa ORF aa ORF aa ORF aa ORF aa id id id id id id id (%) (%) (%) (%) (%) (%) (%) 16 47?7 96 37?6 97 62?0 89 42?0 90 29?4 91 59?9 86 26?8 98 41?2 99 62?7 98 20?2 137 25?0 88 27?1 122 25?9 89 46?4 123 51?1

84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 hr-5 100 101 102 103 104 105 106 107 108 109 110 111

iap-3

73611>74462 74488>74742

283

17 46?0

53 36?3 AAO43578 100?0 96 20?0 97 57?1

27 78 77

84 105 38?8 377 106 62?4

vlf-1

74696>75829 75826<76491 76680>76964 76985>77437 77450<80584 80574>82865 82917<83930 83902>84279 84329>84895 84948>85760 85735>87219 87229>87699 87734>88216 88235<89908 8996790031 90093>90248 90298>90516 90530<91684 91801>92082 92130>93341 93202>94587 94615<95745 95787<98369 98492<98662 98652>99053 99069<99956 99959<100138

2 BIR motifs, RING nger Transmembrane domain PFAM: phage integrase

dna pol desmop lef-3

221 94 107 67?1 98 67?9 150 108 58?4 99 60?0 1044 111 62?3 101 61?4 763 337 125 188 270 494 156 160 557 51 72 384 93 403 461 376 860 56 112 113 114 115 116 117 118 119 120 24?5 42?3 61?9 27?8 62?7 73?3 62?4 33?5 62?2 102 103 104 105 106 107 108 109 110 25?0 38?8 58?7 33?9 57?6 74?1 63?6 31?7 61?5

92 64?7 100 62?8 93 54?3 101 62?7 94 59?9 103 62?3 95 96 97 98 99 100 101 102 103 25?9 43?2 56?3 31?8 50?6 67?5 57?4 20?5 52?9 104 105 106 107 108 109 110 111 112 19?7 17?8 54?8 35?6 56?8

91 51?2 125 52?3 99 55?8 92 43?2 126 29?5 100 47?7 93 49?6 132 48?3 101 56?5 AAO26191 100?0 94 95 96 97 98 18?3 21?5 47?6 19?0 36?5 133 134 135 136 137 139 140 169 141 15?7 18?0 43?0 21?5 32?1 61?7 48?0 31?7 46?0 102 103 104 105 106 107 108 109 110 18?1 21?3 54?8 30?3 45?4 67?9 59?7 51?2 55?2

76 75 65 66 67 68

POLBc (DNA pol type B) Myosin tail

iap-5 lef-9 fp dna lig

70?9 99 64?8 61?8 100 58?3 29?8 65 21?1 55?4 101 47?9

121 28?8 111 34?6 118 21?2 102 19?2 142 23?1 111 38?5 122 58?2 112 52?2 104 39?1 117 44?8 103 30?3 143 26?9 112 31?0 123 124 125 126 130 131 133 33?0 62?8 52?6 55?7 31?7 72?9 36?8 113 114 115 116 118 119 39?0 58?5 50?8 52?5 27?8 70?7 105 106 107 108 109 110 22?4 36?2 39?1 37?7 22?7 67?5 116 115 114 113 117 121 36?1 56?4 49?3 53?9 25?4 71?3 104 106 107 108 109 21?8 44?9 48?1 36?0 65?7 144 145 146 148 170 18?2 39?9 45?2 62?0 33?3 113 115 116 117 118 121 26?0 45?0 45?3 29?1 68?2 31?6 32 133 14?3

E* L, E*

Transmembrane domain

Sequence analysis of the ChocGV genome

fgf-2 alk-exo hel-2 lef-8

31?9 ED L 49?0 L L 14?9 L ED L

BIR motif

50

Transmembrane domain

133 134 61?9 121 64?2 111 51?5 122 57?5 112 34?3 171 29?9 122 44?8 295 135 18?9 122 25?3 113 21?3 123 17?9 113 12?1 172 17?6 124 24?7 AAO16246 100?0 59 136 33?3 123 25?0 114 30?0 124 23?0 114 21?7 173 15?0 125 21?7

53

S. R. Escasa and others

*Early promoter (TATAWAW), with W+A,T and is 150 nt upstream from start codon with CAKT (K=G or T) 2040 nt downstream from TATA. DEarly promoter (TATAWTW), found 150 nt upstream from start codon. dEarly promoter with CACT initiator motif found 2040 nt from E* or ED. Homologues with low amino acid identity (<20 %), accepted based on BLASTP results and e-values.

448 141 51?2 128 53?5 118 42?6 129 46?0 118 42?6 129 50?1 AAG50437 100?0 310 143 55?6 129 52?3 119 48?0 130 49?3 120 37?3 180 31?5 131 40?3 AAC69543 88?9

72 137 50?7 124 54?8 125 32?9 174 24?3 126 38?4 330 138 55?3 125 57?7 115 50?8 126 48?3 115 40?1 175 42?3 127 46?2 299 140 38?3 127 45?1 117 34?9 128 36?3 117 21?6 178 22?7 128 33?8

aa ORF aa ORF aa ORF aa ORF aa ORF aa ORF aa id id id id id id id (%) (%) (%) (%) (%) (%) (%)

PlxyGV

accession no. AAM60753) and showed strong homology to conserved domains in translation initiation factors 5 and 2 (eIF5C and eIF2B_5) from Anopheles gambiae (GenBank accession no. EAA05225) (e=1?0 e-125, 55?5 % amino acid identity). Translation initiation factors are responsible for the binding of the initiator Met-tRNAi and mRNA to ribosomes (Das & Maitra, 2000). Choc36 had a 100 % amino acid identity to a ChfuGV ORF (GenBank accession no. AAN77188), which is listed in GenBank as a homologue to xecn152 (enhancin). Therefore, it would be expected that choc36 would also be a homologue to xecn152. However, BLASTP results showed that choc36 did not have a baculovirus match but had a high identity (9?0 e-10, 30?7 % amino acid identity) to a protein phosphatase regulatory subunit 15A in ORF 93 of Amsacta moorei entomopoxvirus (AmEPV; Bawden et al., 2000). CLUSTAL W alignments showed that ChfuGV (GenBank accession no. AAN77188) and choc36 shared 23?8 % amino acid identity with xecn152. Therefore, choc36 was accepted as a xecn152 homologue. Choc25 showed 21?4 % amino acid identity to a previously unique CrleGV ORF 9 and 34?3 % to a protein from a parvo-like virus [ORF 2 Yamanashi isolate from Bombyx mori densovirus (BmDNV-2; Bando et al., 1995)]. Other ChocGV ORFs had homologues in NPVs but not GVs. Choc20 shared 27?7 % amino acid identity with LdMNPV ORF 7 and 29?8 % with CfDEFNPV ORF 145. Choc53 shared a 24?4 % amino acid identity to LdMNPV ORF 111 and 27?8 % with MacoNPV A ORF 46. Choc62 and choc63 had BLASTP matches to cp82 at 35?6 and 25?6 % amino acid identity, respectively, but shared only 10?3 % amino acid identity with each other. The combined size of choc62 (261 bp) and choc63 (597 bp) was similar to that of cp82 (921 bp), suggesting that the ORFs corresponded to different regions of cp82. Choc62 aligned with the 39 end of cp82 (664921 bp), while choc63 aligned with the 59 end (89746 bp). The sequencing data did not reveal any sequencing errors that could link the two potential ORFs as one. The function of cp82 is not known. It is conceivable that low expression of a cp82 homologue could take place in ChocGV even if a computational frameshift had occurred resulting in choc62 and choc63. This suggestion is corroborated by the fact that low gene expression of lacZ was detected in AcMNPV p10 deletion mutants containing a translational frameshift in the lacZ region (Williams et al., 1989). Inhibitors of apoptosis Apoptosis plays an important role in virus replication and in cellular response to infection (OBrien, 1998). Viruses have acquired genes, the products of which evade or suppress programmed cell death. There are two types of baculovirus proteins that suppress apoptosis, P35/P49 homologues and inhibitors of apoptosis (IAPs) (Du et al., 1999). P35 acts as a direct inhibitor of proteases, while IAPs act upstream to prevent activation of the proteases (Vaux & Strasser, 1996). Functional homologues of P35 are found in some but not all NPVs, whereas IAPs are present in both NPVs and GVs.
Journal of General Virology 87

Promoter

AcMNPV

ORF

53a 24?7 E* 54 30?5 32 23?6 Ed 100131>100349 100219>101211 101377>102276 lef-10 vp1054 fgf-3 112 113 114

ChfuGV

CpGV

CrleGV

AdorGV

PhopGV

XecnGV

AgseGV

Size (aa)

ORF

GenBank no.

aa id (%)

Name

Table 1. cont.

ChocGV

ORF

1924

115 116

egt me53

102293<103639 103755>104687

Position

15 139

38?5 E* 17?0 L, E*

aa id (%)

Transmembrane domain UDPGT

Comments

Sequence analysis of the ChocGV genome

Fig. 1. Representation of ChocGV linear ORF map. The arrows indicate direction of transcription and relative size of ORFs. ChocGV ORF numbers and homologous ORFs in AcMNPV and CpGV are shown above the arrows and gene names below the arrows. Genome position is indicated by the scale (kb). ORFs are shaded according to the key.

ChocGV is the rst GV with a P35/P49 homologue. Choc15 shared the highest amino acid identity with SpliMNPV P49 (GenBank accession no. AJ006751) (27 %), SpltNPV ORF 55 P49 (25?7 %) and RoMNPV ORF 128 P35 (21 %). Choc15 showed a BLASTP match (1?0 e-07, 19?1 % amino acid identity) with a previously unreported P35 homologue from AmEPV (AMV010) (Bawden et al., 2000) (Fig. 3). While these values are not very high, functionality awaits further analysis. It is not yet known if choc15 functions as a P35 or as a P49 homologue as it shares conserved amino acids with both but its functionality and target of activity are yet to be determined. P35 proteins have a large loop domain known as the reactive site loop that contains the caspase recognition motif DQMDG at its apex, while P49 proteins contain the motif TVTDG (Pei et al., 2002). Choc15 and AMV010 appear to have different active site motifs than those of P35 and P49. Two motifs, 92DCSND96 and 86 VYNFD90 in choc15 and AMV010, respectively, were found in the same relative location as the active motifs of P35 and P49 (Fig. 3). It has been shown that AMV010 is an active apoptotic suppressor and that mutating the aspartic acid residue (D90) within the above motif results in an inactive protein (R. Clem, personal communication). Experiments are under way to verify the activity of choc15 and to ascertain the essentiality of D96 within its putative
http://vir.sgmjournals.org

active site motif. The molecular mass of choc15 was 42?6 kDa, which differs from P49 (49 kDa) and P35 (35 kDa) homologues. It is also interesting to note that the location of choc15 within ChocGV was similar to that of iap-3 in both CpGV and CrleGV (Fig. 2b). Five groups of iap genes have been identied in baculoviruses but not all are active suppressors of apoptosis (Ikeda et al., 2004). Homologues of iap-3, generally identied as active inhibitors of apoptosis, are found in both NPVs and GVs, but iap-5 homologues have so far been found only in GVs (Wormleaton et al., 2003). ChocGV contained two iap homologues; choc84 (iap-3) and choc95 (iap-5). Most IAP proteins contain baculovirus inhibitor repeats (BIRs) and a RING nger (Schultz et al., 1998). Both choc84 and choc95 contained two BIRs and one zinc nger ring. IAP and P35 are not homologues and are likely unrelated in ancestry and mechanism of action (Crook et al., 1993). Replication, translation and structural genes All genes involved in DNA replication or expression listed by Herniou et al. (2003) were found in ChocGV (Table 2). Of the replication- and transcription-specic genes present in some but not all baculoviruses (Hayakawa et al., 2000), ChocGV did not have late expression factor 7 (lef-7) or lef-12
1925

S. R. Escasa and others

Fig. 2. Alignment of ChocGV hrs/repeat regions. (a) ChocGV hrs/repeat regions are numbered from 1 to 5, with repeats designated by letter relative to their position in the genome. The direct repeat consensus sequence is shown below the alignment. Shading reects the degree of nucleotide conservation: black, 100 %; dark grey, >85 % and light grey, >60 %. (b) The location of ChocGV ve hrs/repeat regions is compared with that of CrleGV and CpGV. Homologous ORFs within each section are shaded the same colour.

typically found in group I NPVs. The genome also lacked immediate-early gene 2 (ie-2), proliferating cell nuclear antigen (pcna), pe38, ribonuclease reductase 1 (rr1), rr2 and dutpase. Homologues of all conserved baculoviral genes encoding structural proteins were found in ChocGV (Table 2). Granulin and ODV-E25 shared 90?9 and 72?2 % amino acid identity, respectively, with homologues from the seven sequenced GVs, making them two of the most highly conserved proteins in ChocGV. All three genes involved in per os infectivity were present in ChocGV, namely p74 (Faulkner et al., 1997) (choc46), per os infectivity factor-1 (pif-1) (Kikhno et al., 2002) (choc56) and pif-2 (Pijlman et al., 2003) (choc35). These three genes are conserved among all baculoviruses sequenced to date (Lauzon et al., 2004; Garcia-Maruniak et al., 2004). Three ChocGV ORFs contained sequences corresponding to the N terminus of the polyhedron envelope protein (PEP). These ORFs were found in similar locations and shared high
1926

similarity with other GV PEPs. ChocGV PEP proteins (choc17, 18 and 19) and CpGV PEP proteins (cp20, 22 and 23) averaged 70?6 and 68?5 % amino acid identity, respectively, with the PEP proteins from CrleGV (crle20, 23, 24). PEP is found on the surface of OBs and is important for the formation of polyhedra as it stabilizes and prevents them from fusing (Bateman et al., 2004). P10 proteins are characterized by having various common structural and functional domains, including a coiled-coil region, followed by a proline-rich area [(G/A/V/L/I) P (D/E/N) (V/L/I/P) P], a variable region and nally a basic region at the C-terminal. P10 proteins from different baculoviruses are characterized by the size differences among their shared domains and their sequences are generally poorly conserved (Van Oers & Vlak, 1997). Among the seven sequenced GV genomes, only three have P10. AdorGV and XecnGV each have one (ador13 and xecn5, respectively), while PlxyGV has three (plxy2, plxy21 and plxy50). The mean amino acid identity of choc45 with GV P10 proteins was only 14?5 %, but was 30 % with nine
Journal of General Virology 87

Sequence analysis of the ChocGV genome

Fig. 3. Choc15 alignment with P35 homologues. Proteins aligned include AmEPV (ORF 10), AcMNPV (ORF 136), BmNPV (ORF 112), RoMNPV (ORF 128), HycuNPV (GenBank accession no. AAO17287), LeseNPV (GenBank no. AAF78504), SpltMNPV (ORF 55) and SpliMNPV (GenBank no. AJ006751). Shading depicts conserved amino acids: black, 100 %; dark grey, >80 % and light grey, >60 %. The caspase recognition motifs in P35 homologues are boxed, underlined in P49 homologues and boxed in choc15 and AMV010.

NPV P10s (Fig. 4a), with the highest match being op133 (46?2 %), making choc45 closer to NPV P10s than to GV P10s. ChfuGV P10 (GenBank accession no. AAN77200), which is identical to choc45, clustered within group I NPV P10s in a phylogenetic tree (Lange & Jehle, 2003), and the gene arrangement of p10 in ChfuGV was identical to that of many NPVs (Rashidan et al., 2004b). ChocGV contained a second P10-like motif attached to a PEP protein (choc18). It contained a coiled-coil region with a heptad of hydrophobic and hydrophilic amino acids. The sizes of the domains for choc45 (P10) and the P10 region of choc18 are shown in Fig. 4(b). Along with their P10 proteins, AdorGV and XecnGV also have PEP/P10 homologues with a P10-like motif (ador17 and xecn19, respectively). Other GVs with PEP/P10 proteins include: CpGV (cp22), PhopGV (phop20), CrleGV (crle23) and AgseGV (agse19). The P10 domains within crle23 and choc18 shared 51?1 % amino acid identity and the PEP domains 87?8 %. The close association of P10 and the polyhedron envelope has been well documented in many NPVs and it is known that the presence of P10 is essential for the formation of the polyhedron envelope. In some GVs, the functional association of PEP and P10 might have been conserved in a single protein (Lange & Jehle, 2003). Further work must be done to fully understand the role of the different P10 homologues in ChocGV.
http://vir.sgmjournals.org

Entomopoxviruses produce cytoplasmic brils made of lament-associated late proteins (FALPE), while baculoviruses have laments in the nuclei and cytoplasm of infected cells made of P10 protein (Alaoui-Ismaili & Richardson, 1998). FALPE homologues share some of the structural features of P10 proteins (a proline-rich region and a basic C-terminal tail), but amino acid identity between FALPE and P10 proteins is low (Alaoui-Ismaili & Richardson, 1998). AMV032 (FALPE) and choc45 (P10), however, share a high amino acid identity (30?5 %) due to their identical proline-rich region, alternating proline (P) and glutamic acid (E) (Fig. 4c) not seen in other NPV or GV P10s to date (except ChfuGV). However, these EP repeats have been seen in Trypanosoma sp. procyclins (glycosyl phosphatidylinositol-anchored proteins that contain EP/ GPEET amino acid repeats at the C-terminal) (Rashidan et al., 2004b; Ruepp et al., 1999). In phylogenetic analysis (data not shown), FALPE and choc45 grouped with each other, suggesting a strong evolutionary relationship. Auxiliary genes Auxiliary genes are not essential for replication but may give the virus a selective advantage in nature (OReilly, 1997). ChocGV did not appear to contain baculovirus repeat orfs (bros), iap-1, chitinase, cathepsin and some other auxiliary genes present in some GVs (Table 2). Bro genes are highly
1927

S. R. Escasa and others

Table 2. ChocGV genes grouped according to function

Genes present in ChocGV are represented by an ORF# and those missing are represented by CpGV and/or XecnGV ORFs. Genes present in ChocGV Transcription Replication 39K(42), lef-11(43), p47(50), lef-6(60), lef-5(68), lef-4(75), vlf-1(86), lef-9(96), lef-8(107) ie-1(7), lef-2(29), lef-1(55), dbp(61), hel-1(71), dna pol(90), lef-3(92), dna ligase(99), hel-2(105), me53(116) granulin(1), pk-1(3), odv-e18(12), odv-e56(14), pep/p10(18), efp(23), odv-e66(27), pif-2(35), p10(45), p74(46), p24capsid(52), pif-1(56), p6?9(67), odv-e25(72), vp39(76), odv-e27(77), vp91(80), gp41(83), fp(97), 108, 109, 110, 111, vp1054(113) p35(15), mp-nase(33), ubq(39), sod(44), fgf-1(57), iap3(84), iap-5(95), fgf-2(102), alk-exo(104), lef-10(112), fgf-3(114), egt(115) 2, 5, 6, 8, 9, p49(13), 16, 17, 9, 20, 21, 22, 24, 25, 26, 28, 30, 31, 32, 34, 36, 37, 38, 40, 41, 47, 48, 49, 51, 53, 38?7kd(54), 58, 59, 62, 63, 64, 65, 66, 69, 19kd(70), 73, p33(74), 78, 79, tlp20(81), 82, 85, 88, 89, desmoplakin (91), 93, 94, 98, 100, 101, 103, 106 4, 10, 11 87 Genes missing in ChocGV pe-38(cp24) rr1(cp127), rr2(cp128)

Structural

ptp-2(cp66, cp98)

Auxiliary

chitinase(cp10) cathepsin(cp11), ctl(xecn127), gp37(cp13, xecn107)

Unknown

ChocGV/ChfuGV ChocGV unique

repetitive (16 present in LdMNPV) and conserved, but their function is not yet clear (Kuzio et al., 1999). Chitinases are directly involved in the degradation of insect cuticle during moulting, and cathepsin is involved with insect liquefaction (Slack et al., 1995; Hawtin et al., 1997). The absence of these two genes from ChocGV is manifested by the larvae not liquefying upon death. It is not yet clear as to why ChocGV lacks chitinase and cathepsin. ChocGV, however, had an ORF (choc9) with a coiled-coil domain, a chitin-binding domain (type2 ChtBD2), and a high amino acid identity with crle8 (72?7 %) and cp9 (66?7 %), which also have ChtBD2. Chitinbinding domains are extracellular domains that contain six cysteines needed to form three disulde bridges (Schultz et al., 1998). Auxiliary genes present in ChocGV are listed in Table 2. Ubiquitin (ubq) was the second most conserved gene in ChocGV, having a mean of 78?8 % amino acid identity to homologues in seven sequenced GVs. All GV genomes sequenced to date have three broblast growth factors (fgfs). ChocGV is no exception, with choc57, 102 and 114 in the same relative locations as fgfs in other GVs. FGFs are ubiquitous in nature and are highly conserved in structure and amino acid sequence. They are found throughout the genome and play important roles in cell proliferation, differentiation and tissue repair (Ornitz & Nobuyuki, 2001). Comparison of ChocGV with other baculoviruses Comparative analysis of baculoviruses can provide insight into their evolutionary history. Similarities and differences in amino acid sequences and in gene order help place
1928

baculoviruses in groups sharing gene characteristics and overall genome relatedness. ChocGV and ChfuGV are almost identical in size and restriction enzyme digestion proles. Comparison of DNA proles from both viruses cut with EcoRI, HindIII, KpnI and BamHI reveals only minor differences in band size ranging from 0?05 to 5 kb, with the largest difference being in HindIII-P. Comparison of structural proteins also reveals only minor size differences between the two viruses (Arif et al., 1986). CLUSTAL W alignment of individual ChocGV sequences with ChfuGV sequences revealed 100 % amino acid identity for 27 of 38 ChfuGV ORFs sequenced to date (Table 1), with the mean amino acid identity for all being 97?2 %. With similar restriction patterns, biological relationships and high genomic homology, ChocGV and ChfuGV may be variants of the same viral species. The fully sequenced baculoviruses with the highest mean amino acid identity with ChocGV were CpGV (106 homologous ORFs, 52?3 % amino acid identity), CrleGV (106 ORFs, 52?1 % amino acid identity) and PhopGV (103 ORFs, 48?1 % amino acid identity) (Supplementary Table S1 available in JGV Online). The NPV with the highest mean amino acid identity to ChocGV was HearNPV G4 (65 homologous ORFs averaging 31?2 %). Gene arrangement in baculoviruses is a reection of their evolutionary history, with more closely related viruses sharing a higher degree of gene collinearity (Hu et al., 1998). Gene parity plots compare the positions of homologous genes in different genomes and are used to illustrate conservation between baculovirus genomes (Herniou et al., 2003). Examination of the number of ORFs in the same
Journal of General Virology 87

Sequence analysis of the ChocGV genome

Fig. 4. P10 homologues. (a) Alignment of baculovirus P10 proteins: ChocGV_a (choc45), ChocGV_b (choc18), CrleGV (crle23 PEP/P10), AcMNPV (ORF 137), CfDEFNPV (ORF 131), OpMNPV (ORF 133), EppoNPV (ORF 120), CfMNPV (ORF 129), SeMNPV (ORF 130), SpltNPV (ORF 19), LdMNPV (ORF 41) and HearNPV_C1 (ORF 21). The coiled-coil region consists of heptad repeats shaded light grey (*), proline-rich regions in dark grey and the basic region in black. (b) This model, modied from Van Oers & Vlak (1997), shows how P10 domains vary between choc45 P10 and choc18 (P10/PEP). Shading in (b) corresponds to that in (a). (c) The alignment of choc45 and AmEPV ORF 32 (FALPE) show both have an identical proline-rich stretch at their carboxyl ends not found in any other baculovirus P10 (except ChfuGV). They share a 30?5 % amino acid identity, higher than choc45 with GV P10s.

order as ChocGV (diagonal line in Fig. 5), showed that ChocGV shared the highest gene order with CrleGV (88?8 % collinearity), CpGV (87?9 %), AdorGV (85?3 %) and AcMNPV (24 %) (Fig. 5). The positions of iap-3 and p10 were the most variable. In ChocGV, p10 was found immediately downstream of p74, as in most NPVs. However in most GVs, p74 is distal from p10. One drawback of using parity plot analysis as compared with phylogenetic trees is that they do not give quantitative information on genome relatedness (Herniou et al., 2001). Phylogenetic analysis Traditionally, phylogenies were determined by analysing the sequences of individual genes such as polyhedrin, granulin (Zanotto et al., 1993), dna polymerase (Bulach et al., 1999) or
http://vir.sgmjournals.org

ecdysteroid UDP-glucosyltransferase (Barrett et al., 1995) from several genomes. Recently, however, a concatenation of shared proteins has been shown to produce more reliable trees (Herniou et al., 2001). A most parsimonious tree produced using concatamers of the 29 conserved proteins from 27 fully sequenced baculovirus genomes (Fig. 6) reected the three major taxonomic divisions within the lepidopteran baculoviruses: group I NPV, group II NPV and GVs. ChocGV grouped in a clade with CpGV and CrleGV. This was expected, as they shared the highest mean amino acid identity to ChocGV. The Hymenopteran and Dipteran baculoviruses grouped in separate clades. A new baculovirus classication system has been proposed for these nonlepidopteran baculoviruses, as they do not group with lepidopteran NPVs or GVs (Herniou et al., 2003; Lauzon et al., 2004).
1929

S. R. Escasa and others

Fig. 5. Gene parity plot comparison of ChocGV with AcMNPV (a), CpGV (b), CrleGV (c) and AdorGV (d). Homologous genes are plotted based on their relative location in the genomes. Those without homologues are aligned on the x or y axes, respectively. The positions of ChocGV p10 and iap-3 relative to AcMNPV, CpGV, CrleGV and AdorGV are noted. Each diamond represents an ORF.

Fig. 6. The most parsimonious tree based on concatenation of 29 conserved proteins from 27 baculovirus genomes. Bootstrap values (%) for 1000 replicates are shown. Lepidopteran baculoviruses branched into three major groups: group I and II NPVs and GVs. The Hymenopteran baculoviruses NeleNPV and NeseNPV, and the Dipteran virus CuniNPV, formed their own branches. 1930 Journal of General Virology 87

Sequence analysis of the ChocGV genome

Based on sequence and phylogenetic analyses of complete genomes, ChocGV was most closely related to CpGV, but individual ORFs showed the highest identity to those in the partially sequenced ChfuGV genome. ChocGV, however, had some features that were closer to NPVs than to GVs. A P35/P49 homologue, previously found only in NPVs, was present in ChocGV. ChocGV P10 shared a higher amino acid identity with NPV P10s than with GV P10s and ChocGV contained two ORFs (choc20 and 53) previously found only in NPVs. The relationship of ChocGV P10 to AmEPV FALPE was also noteworthy, with choc45 being the only baculovirus ORF identied to date that contains a proline-rich region similar to that in FALPE. The common set of granulovirus genes has dropped, as ChocGV is the rst GV sequenced without a xecn26 homologue. As more baculovirus genomes are sequenced, more information will become available about genes that are essential for NPVs and GVs and on the evolution of the family Baculoviridae.

Bawden, A. L., Glassberg, K. J., Diggans, J., Shaw, R., Farmerie, W. & Moyer, R. M. (2000). Complete genomic sequencing of the Amsacta

moorei entomopoxvirus: analysis and comparison with other poxviruses. Virology 274, 120139.
Benson, G. (1999). Tandem repeats nder: a program to analyze

DNA sequences. Nucleic Acids Res 27, 573580.

Blissard, G. W., Black, B., Crook, N., Keddie, B. A., Posse, R., Rohrmann, G., Theilmann, D. & Volkman, L. (2000). Baculoviridae.

In Virus Taxonomy; Seventh Report of the International Committee on Taxonomy of Viruses, pp. 195202. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. San Diego: Academic Press.
Bulach, D. M., Kumar, C. A., Zaia, A., Liang, B. & Tribe, D. E. (1999).

Group II nucleoployhedrovirus subgroups revealed by phylogenetic analysis of polyhedrin and DNA polymerase gene sequences. J Invertebr Pathol 73, 5973.
Burland, T. G. (2000). DNASTARS Lasergene sequence analysis

software. Methods Mol Biol 132, 7191.

Chen, X., IJkel, W. F. J., Tarchini, R. & 8 other authors (2001). The

sequence of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus genome. J Gen Virol 82, 241257.

ACKNOWLEDGEMENTS
This research was supported by grants from Genome Canada through the Ontario Genomics Institute and the Canadian Biotechnology Strategy Fund. We would like to thank the University of Calgary (Department of Biochemistry and Molecular Biology) for the use of Magpie in aiding in genome sequence analysis. We thank Dr Rollie Clem for making some of his data on AMV010 P33 available to us.

Chen, X., Zhang, W. J., Wong, J. & 9 other authors (2002). Compara-

tive analysis of the complete genome sequences of Helicoverpa zea and Helicoverpa armigera single-nucleocapsid nucleopolyhedroviruses. J Gen Virol 83, 673684.
Crook, N. E., Clem, R. J. & Miller, L. K. (1993). An apoptosisinhibiting baculovirus gene with a zinc nger-like motif. J Virol 67, 21682174. Das, S. & Maitra, U. (2000). Mutational analysis of mammalian

translation initiation factor 5 (eIF5): role of interaction between the

b subunit of eIF2 and eIF5 in eIF5 function in vitro and in vivo. Mol

REFERENCES
Afonso, C. L., Tulman, E. R., Lu, Z., Balinsky, C. A., Moser, B. A., Becnel, J. J., Rock, D. L. & Kutish, G. F. (2001). Genome sequence

Cell Biol 20, 39423950.

de Jong, J. G., Lauzon, H. A. M., Dominy, C., Poloumienko, A., Carstens, E., Arif, B. M. & Krell, P. J. (2005). Analysis of the

of a baculovirus pathogenic for Culex nigripalpus. J Virol 75, 1115711165.

Ahrens, C. H., Russell, R. L., Funk, C. J., Evans, J. T., Harwood, S. H. & Rohrmann, G. F. (1997). The sequence of the Orgyia pseudotsugata

Choristoneura fumiferana nucleopolyhedrovirus genome. J Gen Virol 86, 929943.

Du, Q., Lehavi, D., Faktor, O., Qi, Y. & Chejanovsky, N. (1999).

Isolation of an apoptosis suppressor gene of the Spodoptera littoralis nucleopolyhedrovirus. J Virol 73, 12781285.
Ewing, B. & Green, P. (1998). Base-calling of automated sequencer traces using Phred. II Error probabilities. Genome Res 8, 186194. Ewing, B., Hillier, L., Wendl, M. C. & Green, P. (1998). Base-calling of

multinucleocapsid nuclear polyhedrosis virus genome. Virology 229, 381399.

Alaoui-Ismaili, M. H. & Richardson, C. D. (1998). Insect virus

proteins (FALPE and p10) self associate to form laments in infected cells. J Virol 72, 22132223.
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403410. Arif, B. M., Zhang, G.-Y. & Jamieson, P. (1986). A comparison of

automated sequencer traces using Phred. I. Accuracy assessment. Genome Res 8, 175185.
Faulkner, P., Kuzio, J., Williams, G. V. & Wildon, J. A. (1997). Analysis

three granulosis viruses isolated from Choristoneura spp. J Invertebr Pathol 48, 180186.
Ayres, M. D., Howard, S. C., Kuzio, J., Lopez-Ferber, M. & Possee, R. D. (1994). The complete DNA sequence of Autographa californica

of p74, a PDV envelope protein of Autographa californica nucleopolyhedrovirus required for occlusion body infectivity in vivo. J Gen Virol 78, 30913100.
Fellin, D. G. & Dewey, J. E. (1999). Western Spruce Budworm. Forest

insect and disease leaet 53. USDA forest service.

Gaasterland, T. & Sensen, C. W. (1996). Fully automated genome

nuclear polyhedrosis virus. Virology 202, 586605.

Bando, H., Hayakawa, T., Asano, S., Sahara, K., Nakagaki, M. & Iizuka, T. (1995). Analysis of the genetic information of a DNA

analysis that reects user needs and preferences. A detailed introduction to the MAGPIE system architecture. Biochimie 78, 302310.
Garcia-Maruniak, A., Maruniak, J. E., Zanotto, P. M. A., Doumbouya, A. E., Liu, J. C., Merritt, T. M. & Lanoie, J. S. (2004). Sequence analysis

segment of a new virus from silkworm. Arch Virol 140, 11471155.

Barrett, J. W., Krell, P. J. & Arif, B. M. (1995). Characterization,

sequencing and phylogeny of the ecdysteroid UDP-glucosyltransferase gene from two distinct nuclear polyhedrosis viruses isolated from Choristoneura fumiferana. J Gen Virol 76, 24472456.
Bateman, A., Coin, L. L., Eddy, S. R. & 10 other authors (2004). The

of the genome of the Neodiprion sertifer nucleopolyhedrovirus. J Virol 78, 70367051.

Gasteiger, E., Gattiker, A., Hoogland, C., Ivanyi, I., Appel, R. D. & Bairoch, A. (2003). ExPASy: the proteomics server for in-

Pfam protein families database. Nucleic Acids Res 32, D138D141. http://vir.sgmjournals.org

depth protein knowledge and analysis. Nucleic Acids Res 31, 37843788. 1931

S. R. Escasa and others

Gattiker, A., Gasteiger, E. & Bairoch, A. (2002). ScanProsite: a reference

implementation of a Prosite scanning tool. Appl Bioinformatics 1, 107108.

Gomi, S., Majima, K. & Maeda, S. (1999). Sequence analysis of the ge-

provides in vivo evidence for the utilization of baculovirus non-hr oris during replication. J Gen Virol 83, 20252034.
Kikhno, I., Gutierrez, S., Croizier, L., Croizier, G. & Ferber, M. L. (2002). Characterization of pif, a gene required for the per os

nome of Bombyx mori nucleopolyhedrovirus. J Gen Virol 80, 13231337.

Green, P. (1996). Documentation for Phrap (http://bozeman.mbt.

infectivity of Spodoptera littoralis nucleopolyhedrovirus. J Gen Virol 83, 30133022.

Kool, M., Goldbach, R. W. & Vlak, J. M. (1994). A putative non-hr origin of DNA replication in the HindIII-K fragment of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus. J Gen Virol 75, 33453352. Kurtz, S. & Schleiermacher, C. (1999). REPuter fast computation of maximal repeats in complete genomes. Bioinformatics Applications Note 15, 426427. Kuzio, J., Pearson, M. N., Harwood, S. H., Funk, C. J., Evans, J. T., Slavicek, J. M. & Rohrmann, G. F. (1999). Sequence and analysis of

washington.edu).
Green, P. (2004). CrossMatch (http://phrap.org/phrap.docs/general.

html).
Harrison, R. L. & Bonning, B. C. (2003). Comparative analysis of the

genomes of Rachiplusia ou and Autographa californica multiple nucleopolyhedrovirus. J Gen Virol 84, 18271842.
Hashimoto, Y., Hayakawa, T., Ueno, Y., Fujita, T., Sano, Y. & Matsumoto, T. (2000). Sequence analysis of the Plutella xylostella

granulovirus genome. Virology 275, 358372.

Hawtin, R. E., Zarkowska, T., Arnold, K., Thomas, C. J., Gooday, G. W., King, L. A., Kuzio, J. A. & Possee, R. D. (1997). Liquefaction

the genome of a baculovirus pathogenic for Lymantria dispar. Virology 253, 1734.
Landschulz, W. H., Johnson, P. F. & McKnight, S. L. (1988). The

of Autographa californica nucleopolyhedrovirus-infected insects is dependent on the integrity of virus-encoded chitinase and cathepsin genes. Virology 238, 243253.
Hayakawa, T., Ko, R., Okano, K., Seong, S. I., Goto, C. & Maeda, S. (1999). Sequence analysis of the Xestia c-nigrum granulovirus

leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 240, 17591764.
Lange, M. & Jehle, J. A. (2003). The genome of the Cryptophlebia leucotreta granulovirus. Virology 317, 220236. Lauzon, H. A. M., Lucarotti, C. J., Krell, P. J., Feng, Q., Retnakaran, A. & Arif, B. M. (2004). Sequence and organization of the Neodiprion

genome. Virology 262, 277297.

Hayakawa, T., Rohrmann, G. F. & Hashimoto, Y. (2000). Patterns of

genome organization and content in lepidopteran baculoviruses. Virology 278, 112.

Heldens, J. G. M., Broer, R., Zuidema, D., Goldbach, R. W. & Vlak, J. M. (1997). Identication and functional analysis of a non-hr origin

lecontei nucleopolyhedrovirus genome. J Virol 78, 70237035.

Lauzon, H. A. M., Jamieson, P. B., Krell, P. J. & Arif, B. M. (2005).

Gene organization and sequencing of the Choristoneura fumiferana defective nucleopolyhedrovirus genome. J Gen Virol 86, 945961.
Li, L., Donly, C., Li, Q., Willis, L. G., Keddie, B. A., Erlandson, M. A. & Theilmann, D. A. (2002a). Identication and genomic analysis of a

of DNA replication in the genome of Spodoptera exigua multicapsid nucleopolyhedrovirus. J Gen Virol 78, 14971506.
Herniou, E. A., Luque, T., Chen, X., Vlak, J. M., Winstanley, D., Cory, J. S. & OReilly, D. R. (2001). Use of whole genome sequence data to

second species of nucleopolyhedrovirus isolated from Mamestra congurata. Virology 297, 226244.
Li, Q., Donly, C., Li, L., Willis, L. G., Theilmann, D. A. & Erlandson, M. (2002b). Sequence and organization of the Mamestra congurata

infer baculovirus phylogeny. J Virol 75, 81178126.

Herniou, E. A., Olszewski, J. A., Cory, J. S. & OReilly, D. R. (2003).

The genome sequence and evolution of baculoviruses. Annu Rev Entomol 48, 211234.
Hu, Z. H., Arif, B. M., Jin, F., Martens, J. W. M., Chen, X. W., Sun, J. S., Zuidema, D. G., Goldbach, R. W. & Vlak, J. M. (1998). Distinct gene

nucleopolyhedrovirus genome. Virology 294, 106121.

Luque, L., Finch, R., Crook, N., OReilly, D. R. & Winstanley, D. (2001). The complete sequence of the Cydia pomonella granulovirus

genome. J Gen Virol 82, 25312547.

Mulder, N. J., Apweiler, R., Attwood, T. K. & 34 other authors (2003). The InterPro Database, 2003 brings increased coverage and

arrangement in the Buzura suppressaria single-nucleocapsid nucleopolyhedrovirus genome. J Gen Virol 79, 28412851.
Huang, J. & Levin, D. B. (1999). Identication and functional analysis

new features. Nucleic Acids Res 31, 315318.

Nakai, M., Goto, C., Kang, W., Shikata, M., Luque, T. & Kunimi, Y. (2003). Genome sequence and organization of a nucleopolyhedro-

of a putative non-hr origin of DNA replication from Spodoptera littoralis type B multinucleocapsid nucleopolyhedrovirus. J Gen Virol 80, 22632274.
Hulo, N., Sigrist, C. J., Le Saux, V., Langendijk-Genevaux, P. S., Bordoli, L., Gattiker, A., De Castro, E., Bucher, P. & Bairoch, A. (2004). Recent improvements to the PROSITE database. Nucleic Acids

virus isolated from the smaller tea tortrix, Adoxophyes honmai. Virology 316, 171183.
Nicholas, K. B. & Nicholas, H. B. (1997). GeneDoc: a tool for editing

Res 32, D134D137.

Hyink, O., Dellow, R. A., Olsen, M. J., Caradoc-Davies, K. M. B., Drake, K., Herniou, E. A., Cory, J. S. OReilly D. R. & Ward, V. K. (2002). Whole genome analysis of the Epiphyas postvittana

and annotating multiple sequence alignments. (Distributed by authors.) Version 2.6.002.

OBrien, V. (1998). Viruses and apoptosis. J Gen Virol 79, 18331845. OReilly, D. R. (1997). Auxiliary genes of baculoviruses. In The Baculoviruses, pp. 267300. Edited by L. K. Miller. New York: Plenum. Ornitz, D. M. & Nobuyuki, I. (2001). Protein family review: broblast

nucleopolyhedrovirus. J Gen Virol 83, 957971.

IJkel, W. F. J., van Strien, E. A., Heldens, J. G. M., Broer, R., Zuidema, D., Goldbach, R. W. & Vlak, J. M. (1999). Sequence and

growth factors. Genome Biol 2, 3005.13005.12.

Pang, Y., Yu, J., Wang, L. & 7 other authors (2001). Sequence

organization of the Spodoptera exigua multicapsid nucleopolyhedrovirus genome. J Gen Virol 80, 32893304.
Ikeda, M., Yanagimoto, K. & Kobayashi, M. (2004). Identication

analysis of the Spodoptera litura multicapsid nucleopolyhedrovirus genome. Virology 287, 391404.
Pearson, M. N., Bjornson, R. M., Ahrens, C. & Rohrmann, G. F. (1993). Identication and characterization of a putative origin of

and functional analysis of Hyphantria cunea nucleopolyhedrovirus iap genes. Virology 321, 359371.
Jehle, J. A. (2002). The expansion of a hypervariable, non-hr ori-like region in the genome of Cryptophlebia leucotreta granulovirus

DNA replication in the genome of a baculovirus pathogenic for Orgyia pseudotsugata. Virology 197, 715725. Journal of General Virology 87

1932

Sequence analysis of the ChocGV genome

Swofford, D. L. (2000). PAUP*. Phylogenetic analysis using parsi-

Pei, Z., Reske, G., Huang, Q., Hammock, B. D., Qi, Y. & Chejanovsky, N. (2002). Characterization of the apoptosis suppres-

sor protein P49 from the Spodoptera littoralis nucleopolyhedrovirus. J Biol Chem 277, 4867748684.
Pijlman, G. P., Pruijssers, A. J. P. & Vlak, J. M. (2003). Identication

mony (*and other methods), 4.0 edn. Sunderland, MA: Sinauer Associates.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W:

of pif-2, a third conserved baculovirus gene required for per-os infection of insects. J Gen Virol 84, 20412049.
Prestridge, D. S. (1995). Predicting Pol II promoter sequences using

improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specic gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.
Van Oers, M. M. & Vlak, J. M. (1997). The baculovirus 10-kDa

transcription factor binding sites. J Mol Biol 249, 923932.

Rashidan, K. K., Nassoury, N., Giannopoulos, P. N., Mauffette, Y. & Guertin, C. (2004a). Identication, characterization, and phylogenic

protein. J Invertebr Pathol 70, 117.

Vaux, D. L. & Strasser, A. (1996). The molecular biology of

apoptosis. Proc Natl Acad Sci U S A 93, 22392244.

Wheeler, D. L., Church, D. M., Federhen, S. & 8 other authors (2003). Database resources of the National Center for Biotechnology.

analysis of conserved genes within odvp-6e/odv-e56 gene region of Choristoneura fumiferana granulovirus. J Biochem Mol Biol 37, 206212.
Rashidan, K. K., Nassoury, N., Giannopoulos, P. N., Mauffette, Y. & Guertin, C. (2004b). Identication, characterization and phylogenic

Nucleic Acids Res 31, 2833.

Williams, G. V., Rohel, D. Z., Kuzio, J. & Faulkner, P. (1989). A

analysis of conserved genes within the p74 gene region of Choristoneura fumiferana granulovirus genome. J Biochem Mol Biol 37, 700708.
Ruepp, S., Kurath, U., Renggli, C. K., Drun, R. & Roditi, I. (1999).

cytopathological investigation of Autographa californica nuclear polyhedrosis virus p10 gene function using insertion/deletion mutants. J Gen Virol 70, 187202.
Wormleaton, S., Kuzio, J. & Winstanley, D. (2003). The complete sequence of the Adoxophyes orana granulovirus genome. Virology 311, 350365. Zanotto, P. M. A., Kessing, B. D. & Maruniak, J. E. (1993). Phylo-

Glutamic acid/alanine-rich protein from Trypanosoma congolense is the functional equivalent of EP procyclin from Trypanosoma brucei. Mol Biochem Parasitol 98, 151156.
Schultz, J., Milpetz, F., Bork, P. & Ponting, C. P. (1998). SMART, a

simple modular architecture research tool: identication of signaling domains. Proc Natl Acad Sci U S A 95, 58575864.
Slack, J. M., Kuzio, J. & Faulkner, P. (1995). Characterization of

genetic interrelationships among baculoviruses: evolutionary rates and host associations. J Invertebr Pathol 62, 147164.
Zhang, C. X., Ma, X. C. & Guo, Z. J. (2005). Comparison of the

v-cath, a cathepsin L-like proteinase expressed by the baculovirus Autographa californica multiple nuclear polyhedrosis virus. J Gen Virol 76, 10911098.

complete genome sequence between C1 and G4 isolates of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus. Virology 333, 190199.

http://vir.sgmjournals.org

1933