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1 Canadian Forest Service, Great Lakes Forestry Centre, Sault Ste Marie, ON P6A 2E5, Canada
2 Entomology and Nematology Department, University of Florida, Bldg 970, Natural Area Drive, Gainesville, FL 32611, USA
3 LEMB, Instituto de Ciências Biomédicas, USP, Av. Lineu Prestes 1374, CEP 05508-900, São Paulo, SP, Brazil
4 Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, PO Box 100245, Gainesville, FL 32610-0245, USA
5 Division of Biology, Imperial College London, Silwood Park, Ascot, Berkshire SL5 7PY, UK
6 Canadian Forest Service, Atlantic Forestry Centre, PO Box 4000, Regent Street, Fredericton, NB E3B 5P7, Canada
Correspondence
James E. Maruniak
jem{at}ifas.ufl.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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These authors contributed equally to this work. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Until recently, most fully sequenced baculovirus genomes were from lepidopteran hosts and were divided into either group I or II nucleopolyhedroviruses (NPVs) or granuloviruses (GVs) (Herniou et al., 2001). Sequencing of the Culex nigripalpus NPV (CuniNPV) genome showed that the dipteran baculoviruses are at a large evolutionary distance from the lepidopteran NPVs and GVs (Afonso et al., 2001; Herniou et al., 2003). Partial sequencing of the lef-8 and ac22 homologues from hymenopteran baculoviruses Neodiprion sertifer NPV (NeseNPV), Neodiprion lecontei NPV (NeleNPV) and Gilpinia hercyniae NPV similarly showed a division of the baculoviruses according to the order of their hosts (Herniou et al., 2004). The recent sequencing of the NeseNPV and NeleNPV genomes has shown clearly that the hymenopteran viruses are evolutionarily distant from the lepidopteran and dipteran baculoviruses and may belong to a novel genus of the family Baculoviridae (Garcia-Maruniak et al., 2004; Lauzon et al., 2004).
NeseNPV infects the European pine sawfly, N. sertifer, a serious defoliator of pine in many parts of Europe and Asia. This Old World insect was unknown in North America until its introduction to the US in 1925 and to Canada in 1939 (Brown, 1982), where it became an important pest of coniferous forests. NeseNPV was introduced to North America from Sweden in 1950 as a biological-control agent against N. sertifer (Bird, 1953; Brown, 1982), later becoming available as a registered product called Neochek-S (Huber, 1986). NeleNPV was first identified in Ontario in 1950 in the red-headed pine sawfly, N. lecontei (Bird, 1961). N. lecontei attacks young, natural pine stands, plantations and greenhouse cultures, causing defoliation, death of young trees, reduced growth and tree deformity (Cunningham et al., 1984). NeleNPV is also available as a registered product called Lecontvirus and has been used successfully for many years as a biological-control agent against N. lecontei (De Groot & Cunningham, 1983; http://www.glfc.cfs.nrcan.gc.ca/Lecontvirus.pdf).
Unlike the majority of lepidopteran baculoviruses, hymenopteran baculoviruses replicate only in the epithelial cells of the larval midgut. The gregarious nature of many sawflies, combined with the excretion of infective virus from the midgut prior to insect death, leads to the rapid spread of the viruses with insects, dying 4–7 days after infection (Federici, 1997). Dipteran baculoviruses are also restricted to midgut replication, but their feeding ecologies differ from those of sawflies. Hymenopteran and lepidopteran larvae feed on terrestrial plants, whereas mosquito larvae are aquatic (Afonso et al., 2001; Herniou et al., 2004).
NeseNPV and NeleNPV have smaller genomes than other sequenced baculoviruses, contain fewer ORFs and share limited similarity with lepidopteran NPVs, GVs and CuniNPV. NeseNPV and NeleNPV were related more closely to each other than to other baculoviruses, but even though they infect hosts from the same genus, they did not share as high a degree of sequence identity with each other as with some lepidopteran baculoviruses infecting hosts from different families (Lauzon et al., 2005). This paper compares the genomes of NeseNPV and NeleNPV and discusses their similarities and differences.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) and NeleNPV (Lauzon et al., 2004) were done by using CLUSTAL W from lasergene's dnastar MEGALIGN program (version 5.06) with default conditions. All remaining open reading frames (ORFs) were compared with those in GenBank by using the National Center for Biotechnology Information (NCBI) BLAST searches (Altschul et al., 1990, 1997), including standard protein–protein BLAST (BLASTP) searches. The Viral Orthologous Clusters (VOCs) program from the Viral Bioinformatics Resource Center (www.virology.ca) was also used with default settings to identify further homologues. New potential NeseNPV and NeleNPV matches were considered homologous if amino acid identity of complete ORFs was >20 %. If amino acid identity of proteins was <20 %, matches were noted, but were not included when calculating overall mean amino acid identities or in homologue totals. A syntenic map was prepared, in order to compare the collinearity of the genomes. The genomes were aligned with the BLAST2 sequence tool by using the TBLASTX program. They were translated in all six reading frames and the encoded peptides of one genome were compared with those of the other. The coordinates of all high-similarity pairs (HSPs) in the output were then used with the Artemis Comparative Tool (ACT) (http://www.sanger.ac.uk/Software/Artemis/) to generate a syntenic map showing the collinearity of conserved regions between genomes (not specifically ORFs). The HSP scores accepted for this analysis were from 27 to 405, with identities ranging from 27 to 67 %. ORFs were analysed with the Simple Modular Architectural Research Tool (SMART) (Schultz et al., 1998, 2000). Conserved protein domains were aligned by using CLUSTAL W and shading was done with GeneDoc. Phylogenetic reconstructions were done by using maximum likelihood with PHYML (global tree search) (http://www.lirmm.fr/mab/sommaire_english.php3) (Guindon & Gascuel, 2003), TREE-PUZZLE 5.2 (quartet method) (http://www.tree-puzzle.de/) and global parsimony by heuristic search using PAUP 4.0b10 (Swofford, 2003). Support for trees was obtained after 1000 non-parametric bootstrap replications with PHYML, by the number of quartets supporting a given node with TREE-PUZZLE and after 1000 bootstrap replications with PAUP. Only nodes with support above 50 % were annotated.
NeseNPV and NeleNPV trypsin-like serine protease three-dimensional (3D) modelling.
The 3D structures of the trypsin-like serine proteases from NeseNPV ORF 7 (nese7) and NeleNPV ORF 6 (nele6) were modelled by structural-homology methods utilizing the COMPOSER module in SYBYL 7.0 (Tripos Inc.). The pro-segment regions were removed and the sequences from aa 30 for nese7 and aa 31 for nele6 were used for modelling. Disulfide bonds were built manually based on sequence homology and known trypsin structures (nese7: Cys55–Cys71, Cys182–Cys198 and Cys209–Cys233; nele6: Cys56–Cys72, Cys183–Cys199 and Cys210–Cys234). The proteases were minimized by using the Powell method and Tripos force field to an energy gradient of 0.05 kcal mol–1 Å–1. Gasteiger–Hückel charges were used in the minimization (Purcell & Singer, 1967). Structural overlaps and figures were generated by using PyMOL (v0.98; Molecular Graphics System) (DeLano, 2002). Default settings were used with all programs.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Based on analysis of the individual genomes, each virus was reported to have 43 baculovirus homologues (Garcia-Maruniak et al., 2004; Lauzon et al., 2004). The NeseNPV genome contained a methyltransferase (mtase1) homologue (nese5/ac69) not reported in NeleNPV and NeleNPV had an ac76 homologue (nele41) not reported in NeseNPV. Comparative analysis showed 58.4 % amino acid identity between nele41 and nese44, inferring that nese44 is an ac76 homologue; however, no direct match was obtained between nese44 and ac76 homologues. No homologue of nese5 was found in NeleNPV, thus the latter still lacks an identified mtase1. The VOCs program identified nele79 as a Xestia-c-nigrum granulovirus ORF 164 homologue (20.4 % amino acid identity), but no homologue was found in NeseNPV. By adding the inferred NeseNPV ac76 homologue and the xecn164 homologue to NeleNPV, each genome now has 44 baculovirus homologues, with 43 of them shared with each other.
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, but were not included in homologue totals or amino acid means. A previously unreported similarity between nele25 and nele26 (25.6 % amino acid identity) was found, but neither ORF had homologues in NeseNPV. Counting only the ORFs with >20 % amino acid identity, NeseNPV had 21 ORFs that were not found in NeleNPV and NeleNPV had 20 ORFs not found in NeseNPV (Tables 1 and 2
).
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), but was lower than the mean amino acid identity shared between many lepidopteran baculovirus genomes (Lauzon et al., 2005). The mean amino acid identity for the 43 ORFS shared by NeseNPV and NeleNPV and present in other baculoviruses was 57.9 %, but for the 26 ORFs found only in NeleNPV and NeseNPV, it was 41.5 %, suggesting that there might be more selection pressure on the ORFs shared with other baculoviruses. Overall, the ORFs with the highest amino acid identity in NeseNPV and NeleNPV were ODV-E18 (84.5 %), POLYHEDRIN (82.1 %) and PIF-2 (74.3 %) (Table 2).
Non-syntenic regions (NSRs)
The most obvious differences between the two genomes were found in the regions between the polyhedrin and DNA-binding protein (dbp) genes (nese1–nese22 and nele1–nele14) (Table 2). Due to the lack of conserved synteny in these areas, they have been called NSRs. The NSRs are clearly seen in the syntenic map that compares the conserved regions between the two linearized genomes (Fig. 1). In the NSRs, NeseNPV had 15 ORFs that were not found in NeleNPV and NeleNPV had eight ORFs not found in NeseNPV. The discrepancy in the number of ORFs may be partially due to the duplication of ORFs. nese18 and nese19 were previously considered duplicate genes because they shared 71.2 % amino acid identity, were in the opposite orientation, lacked identifiable upstream promoters and each ORF was flanked by direct repeats (drs) or homologous regions (hrs) (Garcia-Maruniak et al., 2004). nele4 and nele8 had tentative matches to nese9, nese11, nese12, nese18 and nese19. The only previously known baculovirus homologues in the NSRs were the mtase1 homologue in NeseNPV (nese5/ac69) and the iap homologues (nele11/nese17). It is noteworthy, however, that the closest BLASTP matches for these ORFs were to insect proteins. The highest match for nese5 was to a honeybee (Apis mellifera) protein (GenBank accession no. XP_394722
[GenBank]
). The highest matches for nele11 were an A. mellifera SON DNA-binding protein (GenBank accession no. XP_396370
[GenBank]
) and an inhibitor of apoptosis (IAP) from Spodoptera frugiperda (GenBank accession no. AAF35285
[GenBank]
), and for nese17, a Bombyx mori IAP (GenBank accession no. AAK57560
[GenBank]
). Other ORFs in the NSRs, such as nele6/nese7 (trypsin-like serine proteases), also showed closest matches to insect proteins. It is conceivable that the NSRs arose by horizontal transfer of a gene cluster from an insect host(s) and only genes useful to the virus had enough selection pressure to be maintained or to prevent extensive mutations. This might account for the different ORF content and low similarity between ORFs in the NSRs. The G+C content of the NeleNPV and NeseNPV NSRs was also different, at 33.7 and 37.4 mol% G+C, respectively. Differences in nucleotide composition have been attributed to different levels of gene expression, differences in time of gene acquisition or codon usage of the host, a strategy to reduce the competition for nucleotides in viruses infecting the same host (Lange & Jehle, 2003), and insertion of DNA from a different origin (Desiere et al., 2001). NeseNPV had an inversion relative to NeleNPV between nele22 and nele34 and a high degree of collinearity with NeleNPV for the remainder of the genome (Table 2; Fig. 1).
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). The size difference between the two genomes may be partially attributed to the presence of six hrs in NeseNPV, accounting for 3669 bp. NeseNPV hr regions had a higher G+C content (49.8 mol%) than the overall genome (33.8 mol%), contributing to the slightly higher G+C content in NeseNPV compared with NeleNPV (33.3 %) and the higher G+C content of the NeseNPV NSR compared with the NeleNPV NSR. Without its hrs, the G+C content of NeseNPV was 33.0 mol%. The presence of repeats has been associated with major rearrangements, insertions and deletions in baculovirus genomes (Ahrens et al., 1997). Even in closely related baculoviruses, such as Rachiplusia ou MNPV and AcMNPV or Helicoverpa armigera NPV isolates C1 and G4, differences are mainly located near hr regions (Harrison & Bonning, 2003; Zhang et al., 2005). The presence of multiple repeats in the NSRs, therefore, may account for some of the variation between the two genomes in this region.
iap genes
The close relationship between baculoviral iap genes and insect iap genes suggests that baculoviral iap genes may have been acquired through gene transfer from host insects (Huang et al., 2000; Hughes & Friedman, 2003). NeseNPV and NeleNPV IAPs strengthen this hypothesis, as nese17 and nele11 showed top BLASTP matches to insect IAPs. They showed even closer matches to insect IAPs than to each other. nele11 contained two baculovirus IAP repeats (BIRs) and lacked a RING finger, whereas nese17 had one BIR and a zinc finger.
Membrane-fusion proteins
A striking feature of NeseNPV and NeleNPV is the lack of an identified membrane-fusion protein homologous to GP64 or to an F protein (Garcia-Maruniak et al., 2004; Lauzon et al., 2004). ld130 homologues, found in all group II NPVs and some group I NPVs, as well as in CuniNPV, may be the primordial baculovirus envelope-fusion proteins and GP64/67 homologues may be the functional replacements for ld130 homologues in group I NPVs (Rohrmann & Karplus, 2001). These proteins mediate the fusion of budded virus to cell membranes and the release of nucleocapsids (Pearson et al., 2000, 2001). GP64 may also be required for the spread of infection from the insect gut to the haemocoel (Monsma et al., 1996), a function perhaps not required in the hymenopteran viruses due to their restriction to the midgut. Membrane-fusion proteins generally have limited sequence similarity, making their identification difficult (Rohrmann & Karplus, 2001; Pearson & Rohrmann, 2002), but they usually contain a signal peptide, transmembrane domain, conserved cysteines and a furin-cleavage site (Kuzio et al., 1999; Pearson et al., 2000; Rohrmann & Karplus, 2001). There were no unidentified ORFs common to NeseNPV and NeleNPV that contained both signal peptides and transmembrane domains. nele18/nese26 and nele68/nese71 had transmembrane domains that overlapped potential signal peptides, but the ORFs were much shorter than ld130 or GP64 homologues and lacked conserved cysteines and a furin-cleavage site. Baculoviruses appear to be very adaptable and those lacking GP64 can utilize the envelope-fusion protein of Vesicular stomatitis virus as their fusion protein, suggesting that the ability of baculoviruses to enter cells could be accommodated by a variety of envelope proteins (Mangor et al., 2001; Pearson & Rohrmann, 2002). It is therefore possible that another hymenopteran baculovirus ORF(s) may act as a functional replacement for a membrane-fusion protein if one is required by hymenopteran baculoviruses.
Shared ORFs between NeseNPV and NeleNPV
Several ORFs not previously identified in lepidopteran or dipteran baculoviruses were reported independently in NeseNPV and NeleNPV and included a trypsin-like serine protease (nele6/nese7, 72.6 % amino acid identity), a zinc finger-like protein (nele49/nese52, 35.6 % amino acid identity), three proteins homologous to regulators of chromosome condensation proteins (RCC1) in NeleNPV and two in NeseNPV (nele69/nese72, 60.5 % amino acid identity; nele70/nese73, 43.1 % amino acid identity; nele71), a densovirus-like capsid protein (nele81/nese83, 67.8 % amino acid identity) and a phosphotransferase homologue (nele89/nese90, 39.1 % amino acid identity). Some of these ORFs may be specific to hymenopteran baculoviruses and provide them with a selective advantage.
Trypsin-like serine protease.
NeseNPV and NeleNPV are the first reported baculoviruses with trypsin-like serine proteases. Similar proteins have been identified in actinomycetes and bacteria and in many eukaryotes, including insects (Ross et al., 2003). nele6 contained the trypsin catalytic triad, histidine, aspartic acid and serine, as well as the six conserved cysteines reported in nese7 (Garcia-Maruniak et al., 2004). Both ORFs shared top BLASTP matches to similar proteins from insects. Hymenopteran trypsin-like serine proteases with BLASTP matches to nele6/nese7 included A. mellifera proteins (GenBank accession nos XP_394076 and XP_397087
[GenBank]
). The honeybee protein GenBank XP_394076 shared 46.2 % amino acid identity with nele6 and 47.1 % with nese7, values higher than the amino acid identities of most hymenopteran baculovirus ORFs to lepidopteran NPV homologues. All three phylogenetic analyses (maximum likelihood with PHYML, TREE-PUZZLE and maximum parsimony) grouped the hymenopteran baculovirus trypsins with the A. mellifera protein GenBank XP_394076 and showed relatedness to other insect trypsin-like proteins. The best-resolved consensus was obtained with the non-parametric bootstrap tree with PHYML using the Jones–Taylor–Thornton (JTT) substitution model for amino acids (Jones et al., 1992) and is shown in Fig. 2. Bootstrap values >50 % obtained with the other two methods are also shown in Fig. 2. All three methods supported the idea that the trypsin genes of the hymenopteran baculoviruses were acquired by an ancestral Neodiprion baculovirus via horizontal transfer, possibly from a host. The G+C contents of nele6 (43.2 mol%) and nese7 (43.5 mol%) were much higher than the overall G+C content of either genome. Differences in the G+C content of an ORF relative to a genome could be an indication of insertion of DNA from a different origin (Desiere et al., 2001). There is evidence that several baculovirus genes have been transferred horizontally from eukaryotes, possibly from their hosts or bacterial sources (Hughes & Friedman, 2003). Some genes originally derived from a host may have different functions in the virus due to viral adaptation, as was found recently for the baculovirus F protein (Lung & Blissard, 2005).
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-trypsin (PDB file 2PTC
[PDB]
) (Marquart et al., 1983) to compare the overall conformation of the molecules and the structure of their active sites. A C
overlap of nese7 and -trypsin gave a square root of mean square deviation (RMSD) of 2.3 Å (Fig. 3a). A comparison of a C overlap of nese7 and nele6 gave an RMSD of 1.3 Å (Fig. 3b). The structural-homology modelling of NeseNPV and NeleNPV trypsins showed that their conformations were similar to that of the bovine -trypsin. Fig. 3(c) shows that the predicted catalytic residues of nese7 (His70, Asp117 and Ser213) and nele6 (His71, Asp118 and Ser214) overlapped structurally with the catalytic triad of -trypsin, suggesting that the viral trypsins may be functional proteins. Experimental assays are under way to ascertain functionality.
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). C2H2 zinc fingers are composed of 25–30 amino acid residues, including two conserved cysteines and two conserved histidine residues in a C–2–C–12–H–3–H motif that binds a zinc ion (Knight & Shimeld, 2001). nele49 and nese52 contained four C2H2 zinc-finger domains between aa 2 and 22, 26 and 49, 54 and 75, and 81 and 101 in nele49 and aa 2 and 22, 28 and 51, 56 and 77, and 83 and 106 in nese52. They shared an overall amino acid identity of 35.6 %, but identity over the zinc-finger regions was much higher at 50 %. It has been found that a tandem array comprising a minimum of two zinc fingers is required for sequence-specific, high-affinity DNA binding (Böhm et al., 1997). Inference of evolutionary history is difficult for zinc-finger proteins, due to the conservation of key residues critical for the structure of the domain and the repetition of the C2H2 motif in individual genes. By using percentage amino acid identity over zinc-finger regions, it has been suggested that a similarity score >45 % indicates a relationship not due to baseline identity and that all sequences showing an identity score >55 % are in orthology groups (Knight & Shimeld, 2001). It is therefore likely that nese52 and nele49 are related. No significant virus matches were found for nele49 or nese52; instead, top matches included a zinc-finger protein from Drosophila melanogaster (GenBank accession no. NP_609448
[GenBank]
; 36 % amino acid identity, 40/109 aa) for nele49 and an Anopheles gambiae zinc-finger protein (GenBank accession no. EAA00343
[GenBank]
; 36 % amino acid identity, 38/104 aa) for nese52. They also showed significant BLASTP matches to several hymenopteran zinc-finger proteins, including A. mellifera proteins (GenBank accession nos XP_395651
[GenBank]
and XP_39083), with seven and 15 zinc fingers, respectively. An alignment of individual zinc fingers from the above sequences is shown (Fig. 4). The combined evidence of sequence identity between nele49 and nese52, the conserved number and position of zinc fingers and the similar location of both ORFs within the genomes suggest that these proteins may play a hitherto unknown role in the hymenopteran baculoviruses.
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). They bind to chromatin and interact with ran, a nuclear GTP-binding protein, to promote the loss of bound GDP and the uptake of fresh GTP, thus acting as a guanine nucleotide-dissociation stimulator. The interaction of RCC1 with ran probably plays an important role in the regulation of gene expression. In addition to being essential for DNA replication, the initiation of mitosis and the transition from mitosis to G1, the RCC1/ran proteins are also involved in protein import into the nucleus (Huang et al., 1997).
NeseNPV and NeleNPV have three RCC1 homologues located as sequential ORFs in their genomes and are the only viruses with RCC1/BLIP domains noted in the InterPro taxonomic coverage for RCC1 proteins, although several insect groups have these proteins (InterPro IPR000408). nele69/nese72 shared top BLASTP matches with several insect RCC1 proteins, including D. melanogaster BJ1 (GenBank accession no. P25171
[GenBank]
; nele69, 35.8 %; nese72, 40.3 % amino acid identity). The D. melanogaster RCC1 protein, BJ1, has been found to be functionally equivalent to the vertebrate RCC1 proteins (Ohtsubo et al., 1991). The top BLASTP match for nele70/nese73 was the RCC1 protein from A. gambiae (GenBank accession no. XP_310273; nele70, 28.4 %; nese73, 33.0 % amino acid identity). Insect RCC1 matches were also found with nele71 and nese74, but these ORFs shared low amino acid identity with each other (10.7 %) and were not considered homologues to each other, but both were considered RCC1 homologues. nele71 was previously identified as an RCC1 homologue (Lauzon et al., 2004), but nese74 was not (Garcia-Maruniak et al., 2004). By using TBLASTX, the nucleotide sequence from nele69 to nele71 and from nese72 to nese74 showed top match to an A. mellifera RCC1 protein (GenBank accession no. XM_394158
[GenBank]
).
RCC1 proteins contain seven tandem repeats of a domain composed of 50–60 aa. The repeats make up the major part of the length of the protein (Ohtsubo et al., 1989). Only one copy of the RCC1 repeat domains was found in each of the NeseNPV and NeleNPV RCC1 ORFs, making the ORFs smaller (55–136 aa) than other RCC1 proteins (D. melanogaster BJ1, 547 aa). The total size of the three potential RCC1 proteins in NeseNPV (295 aa) and NeleNPV (368 aa) was still smaller than other RCC1 proteins. Two signature patterns are present in RCC1 proteins. The first and most conserved is found in the N terminus of the second repeat and has the pattern G-x-N-D-x(2)-(AV)-L-G-R-x-T (PROSITE PS00625; Hulo et al., 2004). nele69 contained a perfect match to this consensus pattern and nese52 had a close, but imperfect match (Fig. 5). The second consensus pattern is derived from conserved positions in the C-terminal part of each repeat. Potential matches to the second consensus pattern were not as close in the NeseNPV and NeleNPV RCC1 homologues. The sequence of the repeated domain of RCC1 proteins appears to be well-conserved through evolution (Ohtsubo et al., 1991). The presence of RCC1-like genes in various insects and in the hymenopteran baculoviruses suggests the possibility of a horizontal transfer and a possible role in the biology of hymenopterans. The presence of three small ORFs each containing only one RCC1 repeat, however, might also mean that a mutation has occurred, resulting in a frameshift making the RCC1 homologues non-functional.
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). Both nele81 and nese83 showed strong BLASTP matches to densovirus structural proteins. Top BLASTP matches were to viral protein 1-4 from Casphalia extranea densovirus (GenBank accession no. NP_694840
[GenBank]
) (nele81, 30.2 %; nese83, 31.5 % amino acid identity) and a capsid protein from Bombyx mori densovirus (GenBank accession no. NP_694837
[GenBank]
) (nele81, 24.8 %; nese83, 29.5 % amino acid identity). nele81 and nese83, however, were significantly shorter than these densovirus proteins. Most densoviruses infect a variety of larval tissues, but not the midgut. Interestingly, densoviruses from B. mori and C. extranea are an exception and are found predominantly in the columnar cells of midgut epithelium (Fédière et al., 2002). The functionality of the densovirus homologues in the hymenopteran baculoviruses is not known, but work is presently under way to investigate this.
Phosphotransferase.
BLAST analysis showed that nele89 and nese90 had homology with the RNA 2'-phosphotransferase KptA/TPT family, a group of proteins involved in tRNA splicing. Phosphotransferases have not been reported previously in viruses, but are found in eukaryotes and a limited number of eubacteria and archaeal organisms. Eubacteria are not known to splice tRNA, suggesting an unknown function for this protein family (Spinelli et al., 1998, 1999). Phylogenetic analysis shows no evidence for a recent horizontal transfer of the phosphotransferase into eubacteria, but suggests that it has been present in this group since close to the time when the eukaryotes, eubacteria and archaea diverged (Spinelli et al., 1998). A most-parsimonious tree including nele89 and nese90 confirmed this hypothesis and showed that nele89 and nese90 group in the eukaryote clade (data not shown). After nese90, the top BLASTP match for nele89 was a phosphotransferase protein from D. melanogaster (GenBank accession no. NP_788477
[GenBank]
). This protein showed 36.7 % amino acid identity to nele89 and 32.7 % to nese90. Only one insect phosphotransferase was found, so it is not known whether a horizontal transfer could have occurred from an insect host or perhaps another eukaryote or an intermediary. Sequence blocks conserved in phosphotransferase proteins (Spinelli et al., 1998) were found in both nele89 and nese90, suggesting that they may share a common structure with known phosphotransferase proteins and might therefore be functional (Fig. 6).
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; Lauzon et al., 2004). Analysis with the VOCs program did not support nele18/nese26 (41.9 % amino acid identity) as being odv-e25 homologues, although BLASTP searches showed low-overlapping matches to envelope glycoproteins. nele53/nese56 (62.9 % amino acid identity) also did not appear as p10 homologues by using VOCs, although they shared features common to P10 proteins, including a late promoter, correct size range, a conserved A at position –3, location near p74 and helix-forming residues. The VOCs program indicated that nele21/nese29 may be desmoplakin homologues and nele40/nese43 may be ac75 homologues, but amino acid identities were <20 %. These ORFs were in the expected genomic location, so the possibility remains of them being baculovirus homologues. VOCs analysis suggested that nele9/nese16 were desmoplakin homologues, that nele35 was a repeated ORF similar to a BRO protein and that nele61/nese64 were Phthorimaea operculella GV ORF 117 homologues. These potential matches are listed under comments in Table 2, but were not considered as being clearly identified. Some of the remaining shared ORFs had high levels of amino acid identity (nele53/nese56, 62.9 %) or shared similar features (nele48/nese51, double-stranded RNA-binding motifs), implying that they may be important in the biology of hymenopteran baculoviruses. In conclusion, NeseNPV and NeleNPV appeared to be related much more closely to each other than to the lepidopteran baculoviruses or to CuniNPV. Both were small in size, were AT-rich, had fewer ORFs than other baculoviruses and their arrangement of ORFs was basically collinear. Neither had an identified membrane-fusion protein, previously considered a core baculovirus protein, and both shared many ORFs not found previously in lepidopteran or dipteran baculoviruses. A few of the unknown ORFs found only in NeleNPV and NeseNPV, such as the trypsin-like serine protease homologues, might have been transferred horizontally from insect hosts. NeseNPV and NeleNPV had NSRs between their polyhedrin and dbp genes that contained a large number of repeats and unique ORFs. The viruses differed from each other in that ORFs present in their NSRs were variable and their NSRs had different G+C contents. Across their genomes, NeleNPV contained 20 ORFs not found in NeseNPV and NeseNPV had 21 ORFs not present in NeleNPV. NeleNPV also had an inversion relative to NeseNPV between nele23 and nele34 and NeseNPV had drs and hrs, whereas NeleNPV had only drs. Although NeseNPV and NeleNPV were related more closely to each other than to other baculoviruses, their mean amino acid identity was not high, suggesting that NeseNPV and NeleNPV may be related more closely to other hymenopteran baculoviruses than to each other. Based on the comparison of the polyhedrin sequences from Neodiprion abietis NPV (NeabNPV), another New World sawfly baculovirus, NeabNPV appeared to be related more closely to NeleNPV than to NeseNPV. Perhaps some of the differences between NeleNPV and NeseNPV might be due to N. lecontei being a New World species and N. sertifer an Old World pest. The two viruses would have had little geographical overlap until the introduction of NeseNPV to North America. The genomic sequence of another hymenopteran baculovirus will help to confirm which ORFs may be unique to hymenopteran baculoviruses, provide further information on the evolution of hymenopteran baculoviruses and provide further support for a separate grouping of hymenopteran baculoviruses in the family Baculoviridae.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 30 November 2005;
accepted 31 January 2006.
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