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1 Laboratório de Evolução Molecular e Bionformática, Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, SP, Brazil
2 Laboratório de Virologia Molecular, Núcleo Integrado de Biotecnologia, Universidade de Mogi das Cruzes, Mogi das Cruzes, SP, Brazil
3 Entomology and Nematology Department, PO Box 110620, University of Florida, Gainesville, FL 32611-0620, USA
4 Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, Brazil
5 Embrapa Recursos Genéticos e Biotecnologia-Núcleo Temático de Controle Biológico (NTCB), Brasília, DF, Brazil
6 EMBRAPA-CNPSo, Londrina, PR, Brazil
Correspondence
Paolo Marinho de Andrade Zanotto
pzanotto{at}usp.br
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the sequence determined in this work is DQ813662.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) that have been used intensively as expression vectors (King & Possee, 1992; Kost et al., 2005), as models of genetic regulatory systems (Lu & Miller, 1997; Miller, 1999), as biological-control agents against insect pests (Podgewaite, 1985; Moscardi, 1999) and as potential non-human viral DNA vectors for gene delivery (Tani et al., 2003). Since the first baculovirus genome sequence from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was published (Ayres et al., 1994), the number of complete genomes available has risen to 30. The use of the Anticarsia gemmatalis MNPV (AgMNPV) in the control of velvetbean caterpillar, A. gemmatalis, has been the most successful example of a virus used as a biological pesticide (Moscardi, 1999). In Brazil, it is currently applied over approximately 2 million hectares per year, providing effective control of larvae of the key crop defoliator of soybean fields. Since the initial isolation of AgMNPV from infected larvae in Brazil (Allen & Knell, 1977; Carner & Turnipseed, 1977), its production, commercialization and field application have increased constantly (Moscardi, 1999). Comparisons among AgMNPV temporal isolates have shown that the viral heterogeneity of the commercial preparation has increased and that changes are concentrated at ‘hot spots' (Maruniak et al., 1999). AgMNPV isolate 2D (AgMNPV-2D) was cloned by plaque purification and chosen as the prototype (Maruniak, 1989; Maruniak et al., 1999) as it was the major genotype present among a wild-type population in 1972 (Allen & Knell, 1977). The UFL-AG-286 cell line (Sieburth & Maruniak, 1988a, b), established from A. gemmatalis, produces high titres of AgMNPV and is suitable for in vitro experiments with AgMNPV (Pombo et al., 1998; Castro et al., 2006). Moreover, genetic modification of AgMNPV has allowed studies of its pathology in A. gemmatalis larvae (Soares & Ribeiro, 2005), leading to the characterization of many A. gemmatalis larvae haemocytes, all of which have been shown to be susceptible to AgMNPV infection (da Silveira et al., 2003, 2004).
Several individual genes of AgMNPV-2D have been sequenced and analysed: polh (polyhedrin) (Zanotto et al., 1992), gp41 (Liu & Maruniak, 1999), egt (ecdysteroid UDP-glucosyltransferase) (Rodrigues et al., 2001), p10 (Razuck et al., 2002), gp64 (Pilloff et al., 2003; Slack et al., 2004), v-trex (viral 3' repair exonuclease) (Slack & Shapiro, 2004), p143 (helicase) (Lima et al., 2004), dnapol (DNA polymerase) (Dalmolin et al., 2005), iap-3 (Carpes et al., 2005) and p74 (Belaich et al., 2006). In addition, a homologous region (hr4) of AgMNPV-2D has been characterized (Garcia-Maruniak et al., 1996). Phylogenetic analysis of polh indicates that AgMNPV belongs in the group I NPVs (Zanotto et al., 1993) and comparisons of some genes have shown that AgMNPV is closely related to Choristoneura fumiferana defective MNPV (CfDefNPV) (Lima et al., 2004; Lauzon et al., 2005). AgMNPV-2D has been modified genetically and is a viable expression system for heterologous genes (Arana et al., 2001; Ribeiro et al., 2001). The need to sequence the complete genome of AgMNPV was justified, given the increase in basic research on this virus and its increasing importance as a biological control agent. In addition, AgMNPV is probably one of the most important baculovirus systems under study today, as it is one of the few viruses that will allow integrating studies from large-scale field application to the genomic and post-genomic levels. In this work, the complete genome sequence of AgMNPV-2D and its genetic organization are presented.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) was grown in UFL-AG-286 cells. Viral DNA was purified from infected A. gemmatalis larvae injected with the viral isolate, as described previously (Garcia-Maruniak et al., 2004).
AgMNPV-2D DNA library construction.
Our sequencing strategy took advantage of the availability of the complete AgMNPV-2D HindIII genomic library (Johnson & Maruniak, 1989; Maruniak et al., 1999). Subclones were obtained from HindIII clones with the four-cutter restriction enzymes HaeIII, RsaI and Sau3AI, cloned into pGEM-3Z (Promega). Clones were sequenced using primers binding to the T7 and SP6 promoter sequences. To create several sequencing start points, EZ : : TN transposons (Epicentre) were inserted in the large HindIII A, C, D and E clones, as described by Garcia-Maruniak et al. (2004).
DNA sequencing.
Internal sequences were obtained by: (i) primer walking using AgMNPV-2D-specific primers, (ii) further subcloning of smaller subfragments or (iii) by closing gaps or resolving ambiguities by PCR amplification of AgMNPV-2D DNA using specific primers. Sanger reactions were done by cycle sequencing using PTC-200 thermocycler machines (MJ Research) with an ABI PRISM Big Dye Terminator Sequencing Ready Reaction kit versions 2 and 3 (Applied Biosystems). Electrophoresis was carried out on ABI 3100 DNA sequencers.
DNA sequence assembly.
Each nucleotide position was sequenced at least three times on each strand. Base calling (Q=26) was done using the PHRED program (Ewing & Green, 1998; Ewing et al., 1998) and end clipping, cloning site, vector trimming and contig assembly were done using the ALIGNER program, version 1.3.4 (CodonCode Corp.).
Genome annotation.
The complete genome was compared with other baculovirus genomes using the Artemis Comparative Tool release 1 (The Sanger Centre) (http://www.sanger.ac.uk/Software/ACT/) and then annotated with the Artemis release 5 program (http://www.sanger.ac.uk/Software/Artemis/). The Tandem Repeats Finder (http://tandem.bu.edu/trf/trf.html; Benson, 1999) and Dotter (Sonnhammer & Durbin, 1995) programs were used to locate homologous regions (hrs) and direct repeats (drs). ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to find open reading frames (ORFs) of over 50 aa and their homology to sequences in GenBank was analysed using the link to BLASTP (Altschul et al., 1997). Alignment of homologous amino acid sequences to highlight conserved regions was done using the conserved domain database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml; Marchler-Bauer et al., 2005). DNA and protein sequences were aligned using CLUSTAL X (Thompson et al., 1997) with default settings.
Phylogenomics.
In order to establish the relationship of AgMNPV-2D to 27 sequenced baculoviruses and Heliothis zea virus 1 (HzV-1), pairwise distance matrices were calculated with the BLASTPHEN pipeline. For each pair of genomes, bit scores, S' (Altschul et al., 1990; Pertsemlidis & Fondon, 2001), were calculated for all local high-scoring pairs, which were collected to obtain a global genomic similarity either by central tendency statistics (median, mean and mode) or by comparing S' distributions. As S', being a measure of similarity, is obviously inversely proportional to evolutionary distance, we assumed 1/S' as a measure of evolutionary distance (k), and outputs were parsed with BLASTPHEN and the distributions of S' were obtained for each pair of genomes. These distributions were subjected to a range of data analyses prior to clustering and their efficacy in generating informative genome clusters was evaluated (P. M. A. Zanotto, M. A. C. Baccaro, R. N. Pereira & D. Krakauer, unpublished results). Dendrograms for complete baculovirus genomes were produced with ultrametric clustering algorithms (UPGMA, neighbour joining and their derivatives) (Li & Graur, 1991).
Gene content analysis.
In order to study gene acquisition and loss during baculovirus evolution, a gene-content dataset expanded from Herniou et al. (2001, 2003) was used, including 665 genes from all sequenced baculoviruses (except Chrysodeixis chalcites NPV, Agrotis segetum NPV and Trichoplusia ni SNPV). It also included a synthetic outgroup (i.e. a ‘basal’ root in which all genes are absent), in order to define gain and loss of genes in more detail among baculoviruses and other cellular organisms (both prokaryotes and eukaryotes) and DNA and RNA viruses.
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RESULTS AND DISCUSSION |
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; Lange & Jehle, 2003). Fig. 1 shows regions of the genome with higher than average G+C content, such as the largest non-coding region around 126 kbp, and regions that are distinctly AT-rich such as the hrs. AgMNPV-2D contained a total of 152 non-overlapping ORFs of 
50 aa, of which 81 (53 %) were oriented clockwise and 71 (47 %) anticlockwise (Fig. 1). The putative coding regions were numbered sequentially starting with the polh gene in the antisense direction as ORF1 (ag1). The 29 genes shared by all baculovirus genomes, including the dipteran and hymenopteran baculoviruses (Herniou et al., 2003; Garcia-Maruniak et al., 2004), were also found in the AgMNPV-2D genome. The phenogram shown in Fig. 2 summarizes the relationship of AgMNPV to 27 other complete genomes of baculoviruses and HzV-1. The topology of the genome phenogram was congruent with trees for individual genes (Zanotto et al., 1993) or trees for concatemers of the 29 baculovirus conserved genes (Garcia-Maruniak et al., 2004). Separation of the lepidopteran baculoviruses as NPVs of groups I and II and granuloviruses (GVs) was confirmed, as was the distant relationship that exists between the lepidopteran, hymenopteran and dipteran baculoviruses (Herniou et al., 2001, 2003; Garcia-Maruniak et al., 2004).
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to infer the most parsimonious reconstructions of the presence or absence of each gene (represented as discrete unordered characters). This was done to investigate patterns of independent gain and loss of orthologous genes (defined as sharing significant BLAST search scores) along lineages of baculovirus and several viral and cellular outgroups. The pattern of gene acquisition in group I NPVs is shown in Fig. 3. Group I NPVs had 26 synapomorphies (i.e. defining shared ORFs). Arrows pointing up in Fig. 3 represent synapomorphies that underwent secondary loss in later descendants [e.g. ep98 lost in Orgyia pseudotsugata MNPV (OpMNPV)]. Genes shared elsewhere are represented as arrows pointing down (e.g. gp64 is only present in group I NPVs and in Thogoto virus (Orthomyxoviridae) (Morse et al., 1992). Seven defining genes represented as boxes were unique to group I NPVs (e.g. odv-e26). Moreover, group I NPVs had two lineages: (i) the AcMNPV lineage (defined by nine synapomorphies), including AcMNPV, Rachiplusia ou NPV (RoMNPV) and Bombyx mori NPV (BmNPV), and (ii) the AgMNPV lineage (defined by eight synapomorphies) in which the CfDefNPV belongs as a sister group of AgMNPV and which included OpMNPV, Choristoneura fumiferana MNPV (CfMNPV) and Epiphyas postvittana NPV (EppoNPV). The division of group I NPVs into two different lineages has been delineated in recent studies (Jehle et al., 2006; Landais et al., 2006). ORFs for genes having orthologues involved in known functions were found and are discussed below.
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). Nevertheless, inversions of large portions of the genomes were observed in a region between the polh (ag1) and p47 (ag44) genes, shared by all four genomes. Two inversions differentiated AgMNPV-2D, CfDefNPV and OpMNPV from AcMNPV. The first inversion of 7.8 kbp was located between the first repeat region (comprising five copies of a 30 bp imperfect palindromic sequence that included residue 1 of the linearized genome) and the next two copies of a 30 bp imperfect palindromic sequence between nt 7747 and 7864 of the AcMNPV genome. The second, of 11.5 kbp, started near the pkip gene and ended at the beginning of the p47 gene. This inversion did not appear to be associated with repetitive elements, but contained the highly diverse sod locus flanked by hrs. Insertion of the AgMNPV-2D unique ag31 (PARP; see below) took place in this region of low synteny. Another significant inversion, located between the previous two mentioned above, differentiated AgMNPV-2D and CfDefNPV from all of the other group I NPVs. It comprised a region of 9.6 kbp flanked by hrs similar to hr1a in AcMNPV. Most of the other differences in organization among the four genomes could be explained by insertions and deletions (Fig. 4). The majority of the regions lacking synteny (70 %) in comparison with the other group I NPVs were near hrs or drs, supporting previous findings that DNA insertions and deletions occur in these regions, contributing to the plasticity of the viral population (Garcia-Maruniak et al., 1996; Li et al., 2002b).
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). Nine hrs were found in the AgMNPV-2D genome, but four (hr1–hr4) adjacent to baculovirus repeat ORFs (bro) have already been mapped (Garcia-Maruniak et al., 1996). The consensus sequence had 92 nt for hr1 (inverted in relation to the three other hrs) and hr2, 84 nt for hr3 and 127 nt for hr4 (Fig. 5a). The majority of the repeats were tandem repeats, except for hr3. A comparison of the 84 nt shared by the four consensus sequences (Fig. 5a) resulted in 82 % sequence identity, increasing to 90 % for the imperfect palindrome region comparison. Five other hrs (hr1a, -2a, -2b, -2c and -3a) were found that had imperfect palindrome sequences, but were not located in the core of tandemly repeated sequences, and this was similar to the structure of the hrs of CfDefNPV (Lauzon et al., 2005). Three imperfect palindromes were present in hr1a, -2a, -2c and -3a, whilst only one copy was found in hr2b (Fig. 5b). The relative position of the AgMNPV-2D hrs matched the location of nine of the 13 hrs found in CfDefNPV. AgMNPV hr2, hr2b, hr3a and hr4 were located downstream from the sod, p95, pif and p74 genes, respectively, in conserved locations relative to other group I NPVs (Lauzon et al., 2005).
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) to 17 in Spodoptera litura NPV (SpltNPV) (Pang et al., 2001). All group I NPVs have hrs, but six baculovirus genomes do not have typical hrs: Cydia pomonella GV (CpGV) (Luque et al., 2001), Adoxophyes orana granulovirus (AdorGV) (Wormleaton et al., 2003), Agrotis segetum NPV (AgseGV), Chrysodeixis chalcites NPV (ChchNPV) (van Oers et al., 2005), T. ni single NPV (Willis et al., 2005) and Neodiprion lecontei NPV (NeleNPV) (Lauzon et al., 2004). Moreover, five drs were also found in the AgMNPV genome. The locations of the hrs and drs are specified in Table 1.
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). They appear specifically to bind cis-regulatory host factors. AgMNPV-2D hrs had similarity to the 12-O-tetradecanoylphorbol-3-acetate (TPA) response elements (TREs), which belong to the family of bZIP motifs. The TRE motif TGA(C/G)TCA was present 18 times in AgMNPV-2D, nine (50 %) of which were 10–30 bases away from the palindromes in hr1, hr2, hr2a, hr2c and hr3 (Fig. 5). A second type of bZIP motif, the cyclic AMP response element (Landais et al., 2006), was not present in AgMNPV-2D hrs. According to the sole presence of TRE or the combination of both motifs, Landais et al. (2006) found two phylogenetic clusters within the group I NPVs, resembling the AcMNPV and AgMNPV lineages presented here (Figs 2 and 3).
AgMNPV genes encoding structural proteins
Twenty-four of the 152 ORFs identified encoded known viral structural proteins. These included the nine conserved structural genes found in all baculoviruses and the six additional structural genes conserved in lepidopteran baculoviruses (Herniou et al., 2003). These 24 genes are also present in CfDefNPV (Lauzon et al., 2005). Sequence similarity among the structural proteins of AgMNPV-2D and CfDefNPV was generally above 92 % (Table 1), suggesting that they may be structurally similar. Interestingly, the putative major DNA binding protein, p6.9 (ag96), of both viruses was identical, being more conserved than the polyhedrin protein. The p6.9 and polyhedrin proteins were highly conserved among all of the group I NPVs (about 90 % similarity). In contrast, the AgMNPV-2D 1629 capsid (ag152) protein showed the lowest levels of sequence conservation among the group I NPV structural proteins (Table 1). In AcMNPV, this structural protein, which is present in budded viruses and occlusion-derived viruses, is essential for virus replication (Possee et al., 1991). The putative p15 capsid protein (ag84), present only in group I NPVs, was 40–50 % smaller in size in both AgMNPV-2D and CfDefNPV.
Genes involved in replication and transcription
The AgMNPV-2D genome encoded the essential genes lef-1 (ag19), lef-2 (ag3), lef-3 (ag67), dnapol (ag65), helicase (ag92) and ie-1 (ag143) (Lu et al., 1997). It also had the DNA binding protein (dbp) (ag42) (Mikhailov et al., 1998), which unwinds DNA during replication, and lef-11 (ag24), which is associated with the expression of late genes but is also essential for DNA replication (Todd et al., 1995; Lin & Blissard, 2002). Moreover, both pe-38 (ag148 and ag149) and ie-2 (ag145) genes, which increase viral DNA replication (Kool et al., 1994; Ahrens & Rohrmann, 1995), were present (Table 1). Interestingly, pe-38 was split into two ORFs (Table 1). The pe-38 gene was found in group I NPVs and in an apical cluster of GVs that included CpGV, Phthorimaea operculella GV (PhopGV) and CrleGV (Fig. 2 and data not shown). The ie-2 gene was found only in the group I NPVs and therefore seems to have a more restricted distribution among the baculoviruses compared with pe-38 and ie-1. The late expression factor genes [lef-1 to lef-12 (ag45)], 39K (pp31) (ag25) and p47 (ag44) were also found (Table 1). The lef-4 (ag87), lef-8 (ag51), lef-9 (ag62) and p47 genes encode the DNA-dependent RNA polymerase and are found in all sequenced baculoviruses (Lu & Miller, 1997; Garcia-Maruniak et al., 2004). However, only lef-8, which also is present in HzV-1, was not unique to the baculoviruses, having homologues in both eukaryotes and prokaryotes, as indicated by our gene-content analysis (data not shown).
The lef-1, -2, -3 and -11, helicase and ie-1 genes were present in all baculoviruses but absent in HzV-1. Moreover, the typical baculovirus type B DNA polymerase was quite distinct from that found in HzV-1 (Garcia-Maruniak et al., 2004). HzV-1 shares sequence similarity with 10 baculovirus genes (Cheng et al., 2002), but has a distinct set of core functions involved in replication. Possibly, the shared functions allow it to infect insects but do not necessarily imply common ancestry with the Baculoviridae.
bro genes
Eight bro genes [bro-a (ag6), bro-b (ag7), bro-c (ag11), bro-d (ag33), bro-e (ag63), bro-f (ag105), bro-g (ag115) and bro-h (ag135)] were found in AgMNPV-2D (Table 1). It should be noted that bro-h is very small and seems to be the remnant of a bro gene. This gene was located near hr4 at a highly variable region (Garcia-Maruniak et al., 1996; Maruniak et al., 1999). Baculovirus bro genes are assumed to constitute a multigene family, composed of redundant ORFs dispersed along the genome. The number of bro genes is quite variable, ranging from none in GVs (Hashimoto et al., 2000) to 16 in LdMNPV (Kuzio et al., 1999). They may influence baculovirus genome diversity and be involved in recombination between baculovirus genomes (Li et al., 2002a, b, 2005) and may cause strain heterogeneity (Zhang et al., 2005). However, contradictory results have been reported on the role of bro genes in viral infection and replication, both in vivo and in vitro (Kang et al., 1999; Zemskov et al., 2000; Bideshi et al., 2003). Thus, their role is still not clear and not all bro genes appeared to be homologues upon close scrutiny during our analyses.
Anti-apoptotic genes
Three inhibitor of apoptosis protein genes [iap-1 (ag40), iap-2 (ag70) and iap-3 (ag34)] were found in AgMNPV-2D (Table 1). The iap-3 gene of AgMNPV-2D encodes a functional anti-apoptotic protein (IAP) (Carpes et al., 2005). IAPs were first discovered in baculoviruses (Crook et al., 1993) and later found in various animal species, including insects and humans (Clem, 1997; Uren et al., 1998). IAPs have ring fingers (zinc-binding motif) at the C terminus, which appear to be involved in their own ubiquitination (Yang & Li, 2000; Bergmann et al., 2003). Homologues of iap genes have been found in most sequenced baculovirus genomes to date with the exception of Culex nigripalpus NPV (CuniNPV) (Afonso et al., 2001). Analysis of the putative AgMNPV-2D-encoded IAPs showed that both IAP-1 and IAP-3 had two baculoviral iap repeat (BIR) motifs in the N-terminal region and a RING finger motif at its C-terminal end. However, IAP-2 had only one BIR motif at the N terminus and a RING finger motif at the C terminus.
In contrast, no homologues for the anti-apoptotic p35 protein (Clem & Miller, 1994) were present in AgMNPV-2D. So far, the p35 gene has been found in AcMNPV, BmNPV and RoMNPV, three closely related group I NPVs, and has homologues in SpltNPV (group II NPV) and in Xestia c-nigrum GV (XecnGV), as well as in the entomopoxviruses (Clem et al., 1991; Kamita et al., 1993; Du et al., 1999; Pang et al., 2001). p35 inhibits caspase activity, blocking apoptosis (Bump et al., 1995).
Auxiliary genes
Auxiliary genes are not essential for virus replication, but may provide a selective advantage (O'Reilly, 1997). Two auxiliary genes, cathepsin (Hill et al., 1995) and chitinase (Hawtin et al., 1995), found in most lepidopteran NPVs [with the exception of chitinase in Adoxophyes honmai NPV (AdhoNPV)] were not present in the AgMNPV-2D genome. The genomic region that harbours these two genes is highly conserved in group I NPVs. Previous studies by Slack et al. (2004) could not locate the genes around the AgMNPV gp64 locus and demonstrated a lack of enzymic activity in AgMNPV-infected cell cultures. The absence of both the chitinase and cathepsin genes may be responsible for the lack of liquefaction of A. gemmatalis larvae killed by AgMNPV. Nevertheless, other genes, such as the 25K gene, or host apoptotic responses may also be involved and may explain the liquefaction of T. ni by AgMNPV (Katsuma et al., 1999; Slack et al., 2004). When used as a biological pesticide, AgMNPV usually kills A. gemmatalis larvae within 7–10 days of virus application (Moscardi & Sosa-Gómez, 1992). The bodies of infected larvae usually remain intact for at least 2 days after death. After that period and under humid conditions, the larval body darkens and lyses due to putrefaction. However, under dry conditions, the bodies of the larvae dry up and shrink, keeping polyhedral inclusion bodies packed within them. Interestingly, the lack of liquefaction is of the utmost importance for the biological control programme using AgMNPV in Brazil (Moscardi, 1999). This is because it is necessary to collect dead and dying larvae for the preparation of future viral stocks, after applying viruses in the field (Moscardi & Sosa-Gómez, 1992; Moscardi, 1999). This has the benefit of reducing production costs to the farmers and may help in keeping fit viruses selected under natural conditions. The infectivity of AgMNPV and the lack of infected larvae liquefaction were probably key factors in allowing the expansion and current success of the biocontrol programme.
The AgMNPV-2D genome had several auxiliary genes that are also found in other baculoviruses (Table 1). Two ORFs encoding protein tyrosine phosphatases, ptp-1 (ag9) and ptp-2 (ag8), were found. PTP specifically removes phosphates from tyrosine residues and regulates tyrosine phosphorylation in concert with protein tyrosine kinases. They are associated with fibrillar structures in infected cells and can also be detected in budded viruses and occlusion-derived viruses, suggesting that they are a component of the viral capsid (Li & Miller, 1995). It has been shown that larvae of B. mori infected with BmNPV display enhanced locomotory activity due to a baculovirus-encoded PTP (Kamita et al., 2005). Whilst the ptp-1 gene was found in all other members of the group I NPVs, the ptp-2 gene was found only in CfDefNPV, CfMNPV and OpMNPV (Table 1).
The pnk/pnl gene (ag103) had 66.7 % identity with pnk/pnl of both AcMNPV (ac86) and RoMNPV (ro83), which may be an RNA ligase possibly involved with RNA repair (Martins & Shuman, 2004). In phages, pnk/pnl activity is encoded by two different genes. All three group I NPV pnk/pnl genes had a high level of sequence conservation (79.6 %) suggestive of structural and functional equivalence. Interestingly, this gene was found to be present only in these three genomes. Therefore, it was either acquired independently by the AgMNPV lineage and the ancestral lineage of both AcMNPV and RoMNPV, or several successive independent losses have taken place along the NPV radiation.
The superoxide dismutase (sod) gene was found in AgMNPV-2D (ag32). The AcMNPV sod gene represents part of the enzymic defence against oxygen toxicity and is present in almost all aerotolerant organisms (Tomalski et al., 1991). Homologues of the sod gene are found in several baculoviruses and in entomopoxviruses and insects. Although the function of the sod gene in baculoviruses remains unknown, it may protect occluded viruses in the environment from superoxide radicals generated by exposure to sunlight (Ignoffo & Garcia, 1994).
A putative ctl gene for a conotoxin-like peptide found in predatory marine snails was found (ag30), which had similarity mainly to the ctl-2 gene of OpMNPV (op30). For that reason, we named this gene ctl-2 (Table 1). OpMNPV also harbours the ctl-1 gene, which has high levels of similarity to the ctl gene of AcMNPV (Eldridge et al., 1992). The ctl gene is also present in the genome of other NPVs, GVs and entomopoxviruses. However, no ctl homologues are present in the CfDefNPV and EppoNPV genomes, indicative of independent secondary losses. The biological function of this protein is unknown, but it has been suggested that it may participate in the regulation of calcium levels during infection and induce some form of paralysis in infected insects (O'Reilly, 1997).
Ag122 encoded a putative 3' repair exonuclease (v-trex) (Slack & Shapiro, 2004), which showed homology to eukaryotic 3' exonuclease and was found in only two other closely related baculoviruses, CfDefNPV and CfMNPV.
A putative gene for ubiquitin (ubi) (ag26) was also found, as in all other lepidopteran baculoviruses sequenced to date. Ubiquitins are abundant in eukaryotic cells and mediate protein breakdown (Hershko & Ciechanover, 1992) and other cellular processes. However, little is known about their function in baculoviruses and they appear not to be essential for replication.
Genes with no homology to other baculovirus genes
Three AgMNPV-2D coding regions, ag31, ag64 and ag83, were unique and had no significant hits in the GenBank database. Ag64 and ag83 can encode 86 and 54 aa, respectively. Ag31 encoded a 369 aa polypeptide with significant similarity to the poly(ADP-ribose) polymerase (PARP) from several organisms, with a probability of being similar by chance to other known PARPs of <10–23. The C terminus is the most highly conserved region of PARP proteins. An alignment of the C terminus of this protein of AgMNPV-2D and five other organisms comprising representatives of insects, plants, birds and nematodes is shown in Fig. 6. The AgMNPV-2D PARP-like protein was smaller than the homologous insect PARPs, but was similar in size to that of vertebrates [e.g. chicken (Gallus gallus) with 358 aa]. The best match obtained by BLASTP was to the plant Oryza sativa with 37 % identity with a region of 212 aa from the total 655 aa protein. PARP-1 is a nuclear enzyme activated by nicked DNA molecules, possibly involved in DNA repair (D'Amours et al., 1999; Smith, 2001). PARP binds to damaged DNA and catalyses the formation of ADP-ribose polymers that attach to its own glutamic acid residues, as well as to other proteins (Althaus & Richter, 1987; Smulson et al., 1994). It is also thought to be a part of the base excision repair pathway (Oei & Ziegler, 2000), to promote transcription (Vispé et al., 2000) and to be involved in apoptosis (Smith, 2001). PARP-1 is required for retroviral integration in the host genome (Ha et al., 2001). In adenoviruses, inhibition of PARP-1 reduces viral infectivity (Dery et al., 1986). It has been shown that PARP binds to simian virus 40 capsid proteins VP1 and VP3 and that the latter protein stimulates PARP activity, possibly leading the infected cell towards a necrotic pathway (Gordon-Shaag et al., 2003). The function of the PARP-like protein in AgMNPV has not been studied and, as AgMNPV is the only baculovirus to date with this putative gene, further analysis is under way (T. S. Rizzi, B. M. Ribeiro, C. M. Romano, F. L. Melo, J. V. C. Oliveira & P. M. A. Zanotto, unpublished results).
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; Lauzon et al., 2005). Besides the unique genes found in AgMNPV-2D, there were other noteworthy differences between their genomes. AgMNPV-2D had additional bro genes (bro-h), a ctl-2 gene and the pnk/pnl gene. On the other hand, CfDefNPV harboured the chitinase and cathepsin genes. The position of these genes in the CfDENPV genome suggests that they were lost from the ancestor baculovirus lineage that resulted in the AgMNPV-2D genome. CfDefNPV also harboured additional putative genes, located at positions equivalent to ag21 and between ag113 and 114 (Table 1). Several of the differences observed between the two genomes coincided with highly variable regions previously detected among AgMNPV variants (Maruniak et al., 1999). For instance, the AgMNPV-2D ctl-2 and parp-1 genes were located in a variable region at 1.78 and 1.80 map units (m.u.), respectively. Likewise, the location of the expected chitinase and cathepsin genes (conserved in other baculoviruses) would correspond to another variable region of the AgMNPV-2D genome around 79.1 m.u. Moreover, ag21, near another variable region mapped at 13.32 m.u., seemed to be the combination of cfde19 and -20. Taken together, these data suggest that AgMNPV might indeed have hot spots that may favour evolutionary novelty. In conclusion, as AgMNPV is the most widely used biopesticide in the world, it was essential that its DNA be sequenced so that we can scrutinize its gene content and function, unravel its genetic regulatory network and monitor genetic changes and evolution of the genome. Our previous research indicated areas of genome change over the years of virus application in soybean in Brazil that will now be studied. One of the reasons that the programme may have been so successful was identified during sequencing of the genome. Neither chitinase nor cathepsin, present in all other baculoviruses sequenced to date, nor other ORFs that would potentially fulfil this role were found in AgMNPV-2D. This allows field production and harvesting of relatively intact insects full of virus needed for subsequent applications.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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