1 Insect Biocontrol Laboratory, USDA, Beltsville, MD 20852, USA
2 Departamento de Biologia Cellular, Universidade de Brasília, CEP 70910-900 Brasília DF, Brazil
3 Embrapa Recursos Genéticos e Biotecnologia Parque Estação Biológica, CEP 70770-900 Brasília-DF, Brazil
Correspondence
Jeffrey Slack
slackj{at}ba.ars.usda.gov
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ABSTRACT |
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INTRODUCTION |
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Baculoviruses are widely recognized as effective biological insecticides. The insecticidal properties of AcMNPV have been improved by adding foreign genes and/or by inactivating accessory viral genes (for review see Inceoglu et al., 2001). The knowledge from these studies is currently being applied to baculovirus species with specificity for economically important insect pests. Of these, AgMNPV has been most extensively and successfully applied for biological control of Anticarsia gemmatalis, especially in Brazil (Moscardi, 1999
). AgMNPV exhibits a high degree of genetic heterogeneity (Croizier & Ribeiro, 1992
). AgMNPV isolate 2D (AgMNPV-2D) is a prototype as its genomic profile, based on restriction endonuclease (REN) analysis, represents 40 % of field isolates (Maruniak et al., 1999
).
The AgMNPV-2D genome was previously mapped by restriction endonucleases (Johnson & Maruniak, 1989; Maruniak et al., 1999
). In the current study, we sequenced the 12·5 kbp BamHI-D REN fragment of AgMNPV-2D. A number of AgMNPV sequences have been published including homologous repeat (hr) regions (Croizier & Ribeiro, 1992
; Garcia-Maruniak et al., 1996
), the polyhedrin gene (Zanotto et al., 1992
) and the egt gene (Rodrigues et al., 2001
). A region of AgMNPV-2D common to this study was also published (Razuck et al., 2002
). That region contains homologues of the AcMNPV genes p94, p26, p10 and the 3' end of the p74 gene. The 5' end of an ORF homologous to the Choristoneura fumiferana (Cf)MNPV gene p22.2 (ORF 126) is also present. Most recently, the AgMNPV gp64 gene sequence was published (Pilloff et al., 2003
). Although the sequence was derived from the SF isolate of AgMNPV, it is identical to the corresponding sequence in our study. This study focuses on the genes downstream of gp64 that, to our knowledge, have not been previously described.
Comparative studies suggest that there are two evolutionarily divergent groups of NPVs; group I and group II (Bulach et al., 1999; Herniou et al., 2001
; Zanotto et al., 1993
). Single gene comparisons of AgMNPV suggest that AgMNPV belongs to the group I NPVs that include AcMNPV, CfMNPV, Bombyx mori NPV (BmNPV), Orygia psuedotsugata MNPV (OpMNPV), Epiphyas postvittana NPV (EppoNPV), Choristoneura fumiferna defective MNPV (CfDEF), Antheraea pernyi NPV (AnpeNPV), Hyphantria cunea NPV (HycuNPV), Anagrapha falcifera MNPV (AnfaMNPV), Rachoplusia ou NPV (RaouNPV) and Perina nuda NPV (PenuNPV).
To further define the evolutionary relationship of AgMNPV among other baculoviruses, we compared the AgMNPV BamHI-D REN fragment with the corresponding regions of six other group I NPVs: AcMNPV, BmNPV, OpMNPV, EppoNPV, HycuNPV and CfMNPV.
In this study, we focus on two features of this AgMNPV-2D BamHI-D REN fragment: (a) the presence of a baculovirus gene with a 3' repair exonuclease motif; and (b) the absence of ChiA and v-cath homologues downstream of gp64. We also report on a replication assay analysis of two hr-like repeat elements that were discovered during this study.
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METHODS |
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Southern blot hybridizations.
To locate the AgMNPV gp64 gene, a plasmid (pAcEcoRI-H) containing the EcoRI-H fragment from the AcMNPV-2D genome was digested with BamHI, which generates three fragments each containing portions of the gp64 ORF. The fragments were labelled with [32P]dCTP using random 9-mers (Rediprime kit, Amersham), and used to probe a plasmid library of AgMNPV-2D HindIII fragments by Southern blot hybridization. For Southern blot hybridizations, membranes containing REN digests of cloned AgMNPV-2D DNA fragments were incubated in prehybridization buffer (6x SSC, 0·5 % SDS, 5x Denhardt's solution, 100 µg denatured salmon sperm DNA) ml-1 for 2 h at 60 °C, and then hybridized with the 32P-labelled AcMNPV probe DNA overnight in prehybridization buffer+0·01 M EDTA. The membrane was then washed for 15 min at 60 °C in 50 ml 2x SSC, 0·1 % SDS, and for another 15 min in 0·1x SSC, 0·1 % SDS, and exposed to X-ray film for 72 h.
PCR protocol.
Vent polymerase (New England Biolabs) was used for PCR screening for a v-cath gene homologue in AgMNPV-2D. 100 ng of viral template DNA was combined with 250 µM dNTPs, 2 U Vent polymerase, 1 µM of each primer, 1x Thermpol buffer (New England Biolabs) in a final volume of 100 µl. PCR mixtures were topped with 30 µl mineral oil. v-cath primers used were: 5'-GGCTTGTGCGGCGCGTGCTGGC-3' and 5'-ACCCTRAAATAACCCTCCTCTCCCCA-3'. 30 PCR cycles were run: 95 °C 1 min, 40 °C 1 min, and 72 °C 1 min. Parallel PCR reactions were run with OpMNPV and AcMNPV template DNA to ensure the specificity of v-cath primers. PCR products were also sequenced to ensure that the v-cath ORF region was being amplified. PCR reactions with primers to the AgMNPV gp64 gene (5'-GGCGTGTGGGTGCCTGGTA-3' and 5'-GACCAGCCGCTGGCGTCTTT-3') were also included in parallel amplifications to ensure AgMNPV DNA template quality.
Cloning, sequencing and nucleotide sequence analysis.
The 12·5 kbp BamHI-D REN fragment of AgMNPV-2D was cloned into the BamHI site of plasmid, pBS(-) (Stratagene), resulting in the construct pBS-Ag/BamHI-D. Sequencing was performed using rhodamine terminator cycle sequencing kits (Applied Biosystems) and an ABI Prism 377 genetic analyser sequencing machine. Template pBS-Ag/BamHI-D plasmid DNA was purified by CsCl gradient. Both strands were sequenced with a total mean redundancy of approximately 24. The entire 12·5 kbp AgMNPV-2D region was conceptually translated and searched for homologous genes using BLASTX (http://www.ncbi.nlm.nih.gov). DNAStar MapDraw was used to locate remaining ORFs (greater than 50 amino acids). ORFs that overlapped identified homologues were excluded. AgMNPV genes were named using the same nomenclature as used for AcMNPV and where no gene names were previously assigned in AcMNPV, the AgMNPV gene (ORF) was named according to the AcMNPV ORF number with an Ac appended to signify the homologue (for example ORF119Ac).
Phylogenetic analysis.
Protein homologue sequences were aligned with AgMNPV sequences using DNAStar MegaAlign and the CLUSTAL W method (Thompson et al., 1994) (gap penalty=10, gap length penalty=0·2, PAM 350 series protein weight matrix). For phylogenetic tree generation, alignments were saved as NEXUS files. NEXUS files were linked together and analysed by maximum parsimony using the program PAUP* 4.0 (Swofford, 1998
). PAUP* 4.0 analysis employed a heuristic search for possible trees. Bootstrap values were calculated by performing 100 000 replicate heuristic tree searches with a parsimony optimality criterion. A bootstrap 50 % majority consensus tree was generated.
Assembly of the genomic segments of HycuNPV.
The genomic regions flanking the HycuNPV gp64 gene were derived by assembling contigs with DNAStar SeqMan and overlapping GenBank sequence submissions. GenBank accessions and corresponding HycuNPV (Taxon 28288) ORFs are as follows: AF121457 (ChiA), AF120926 (v-cath) and AF190124 (gp64).
Origin of replication assay.
AgMNPV repeat regions (R1 and R2), as well as AcMNPV hr5 were PCR-amplified and cloned by ligation into a pGEM-T plasmid (Promega). Amplified regions from AgMNPV corresponded to nt 8081024 (217 bp, includes repeat 1), nt 10021163 (162 bp, includes repeat 2) and nt 8081163 (366 bp, includes repeats 1 and 2). Infection-dependent plasmid replication assays were performed as described earlier (Kool et al., 1993, 1994
). Sf-9 cells (2x106) were transfected by calcium phosphate precipitation with 5 µg of a plasmid containing either R1, R2, R1 and R2 or hr5 (positive control). At 24 h post-transfection, transfected cells were infected with AgMNPV or AcMNPV at an m.o.i. of 10 p.f.u. per cell and incubated at 27 °C. At 4872 h post-infection, total DNA was extracted from cells (O'Reilly et al., 1992
), and 13 µg of DNA was digested with DpnI (1·5 U µg-1) at 37 °C for 12 h, followed by digestion with PstI (1·5 U µg-1) for 12 h. DNA fragments were separated on agarose gels, transferred to Genescreen Plus membrane, and hybridized with a 32P-labelled pGEM-T plasmid DNA probe (9x108 c.p.m. µg-1) generated by random hexamer priming (Prime-It Rmt kit, Stratagene). Blots were prehybridized for 2 h at 65 °C in prehybridization solution [5x SSC, 0·1 % laurylsarcosine, 0·02 % SDS, 10 % blocking reagent (Roche)]. Hybridization was carried out for 14 h at 65 °C, in prehybridization solution plus probe (1x106 c.p.m. ml-1). Membranes were washed for 10 min in two changes of 2x SSC/0·1 % SDS at room temperature, followed by two 15 min washes in 0·5x SSC/0·1 % SDS at 65 °C. Membranes were exposed to phosphorscreens (Amersham) and screens were scanned on a Storm phosphorimager machine (Amersham).
V-CATH protease assays.
Approximately 2x106 Tn5B1-4 (High-5) cells were infected for 1 h at an m.o.i. of 2·5 p.f.u. per cell with AgMNPV, AcMNPV, or an AcMNPV recombinant virus containing an inactivated v-cath gene (AcMNPVv-cath(-)) (Slack et al., 1995). We also similarly infected Ld652Y cells with OpMNPV. At 75 h post-infection, cells were harvested for proteinase assays. Cells were pelleted by centrifugation at 1000 g for 5 min, then resuspended, washed and pelleted twice in 5 ml PBS/EDTA (10 mM sodium phosphate, 120 mM NaCl, 2·7 mM KCl, 5 mM EDTA, pH 6·2). Cells were suspended in a final volume of 600 µl PBS/EDTA (pH 6·2), 5 µg Pepstatin A ml-1. Cells were lysed by freezing and thawing and stored at -70 °C. Protein concentrations of cell lysates were determined using the Bradford assay (Bradford, 1976
) and then lysates were diluted to a concentration of 70 ng µl-1 in PBS/EDTA. Cell lysate aliquots of 20 µl were combined with 50 µl of assay buffer (100 mM citrate, 100 mM sodium phosphate, 0·5 %, w/v, Tween, 5 mM DTT, 2 M urea). A pH series (pH 4·58·0) of assay buffers was made using NaOH or HCl. Each 70 µl lysate/assay buffer mixture was then added to 30 µl of substrate (0·5 %, w/v, azocasein; 10 µg Pepstatin A ml-1). The mixture was incubated at 37 °C for 4·5 h in a sealed 96-well plate. The assay was terminated by addition of 100 µl of 10 % (v/v) trichloroacetic acid. Undigested azocasein was precipitated overnight at 4 °C and pelleted by centrifugation at 1000 g for 15 min. Supernatants were transferred to a new 96-well plate and the A405 was determined on an ELISA plate reader.
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RESULTS AND DISCUSSION |
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Gene organization in the AgMNPV-2D BamHI-D REN fragment
The AgMNPV BamHI-D fragment was found to contain 17 baculovirus gene homologues including gp64. The arrangement of baculovirus gene homologues in the AgMNPV-2D BamHI-D fragment was compared to the corresponding known regions of the group I NPVs CfMNPV, EppoNPV, OpMNPV, BmNPV, RaouMNPV, AcMNPV, HycuNPV, AnfaMNPV, PenuNPV, AnpeNPV and CfDEF (Fig. 1). AgMNPV-2D gene composition in the BamHI-D region most closely resembled CfMNPV as there were two common gene homologues that were not found in other NPVs. Those homologues were v-trex (CfMNPV ORF 113) and p22.2Cf (CfMNPV ORF 126). The predicted amino acid sequence identity between v-trex homologues was 68·4 % and between p22.2Cf homologues was 61·6 %. EppoNPV and OpMNPV were also more similar to AgMNPV than BmNPV, RaouMNPV and BmNPV. AgMNPV-2D lacked protein kinase-2 (pk-2), p35 and hr5. Curiously, AgMNPV-2D contained a remnant of the 3' end to the p94 gene similar to BmNPV. AgMNPV-2D differed from all other group I NPVs that have been analysed by lacking ChiA and v-cath genes downstream of gp64.
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Repeat regions
Immediately downstream of ORF119Ac are found two short repeat sequences [Repeat 1 (R1) (102 bp: 891992) and Repeat 2 (R2) (80 bp: 10051084)] with resemblance to baculovirus homologous repeats (hrs) (Fig. 3a). R1 and R2 each contain approximately 40 bp of semi-palindromic sequence. It is notable that region 8971036 of our AgMNPV-2D sequence is 77 % identical to region 58197 of CfDEF (accession U72030) (Fig. 3b
). This homologous CfDEF region is found downstream of the CfDEF ChiA gene homologue (Fig. 1
). Both R1 and R2 are also similar to previously published AgMNPV-2D and AgMNPV-D7 repeat regions (Garcia-Maruniak et al., 1996
).
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v-trex gene
During our analysis of ORF homologues we noticed that the AgMNPV homologue to CfMNPV ORF 113 also had homology to a type of group III exonucleases called Three prime Repair EXonucleases (TREXs) (Mazur & Perrino, 1999). For this reason we suggest the nomenclature, v-trex gene and V-TREX protein. TREX proteins are the most abundant 3'5' DNA exonucleases found in mammalian cells (Mazur & Perrino, 1999
, 2001
) and they are different from other type III exonucleases in that they function independently from the DNA polymerase complex (Mazur & Perrino, 1999
, 2001
). This characteristic would facilitate the acquisition of a foreign trex gene by baculoviruses. The v-trex gene could have been acquired from an invertebrate host. Insect proteins from Anopheles gambiae PEST and Drosophila melanogaster are 39·0 % and 33·3 % identical to AgMNPV V-TREX (Fig. 4
a). Neither of the insect proteins has been characterized but they both contain common exonuclease motifs; Exo I, Exo II and Exo III. These motifs form the active sites of type III exonucleases (Bernad et al., 1989
; Koonin & Deutscher, 1993
). The Exo III motif of V-TREX contains the consensus sequence HXAXXD (Fig. 4b
), which is a hallmark of TREX type exonucleases (Mazur & Perrino, 1999
).
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ChiA and v-cath genes
The largest single variation of this AgMNPV-2D BamHI-D region from other group I NPVs is the absence of a conserved 3 kb region downstream of gp64. Other group I NPVs, including EppoNPV, OpMNPV, AcMNPV, RaouMNPV, BmNPV, CfMNPV, HycuNPV, CfDEF and AnpeNPV, have the ChiA and v-cath genes downstream of gp64 (Fig. 1). The PenuNPV gp64 gene submission (accession AF289055) also contains the 3' end of a downstream v-cath gene. Thus, AgMNPV-2D lacking the v-cath and ChiA genes downstream of the gp64 gene is unusual. Among the group II NPVs LdMNPV, SeMNPV, SpltNPV, MacoNPV-A, MacoNPV-B, HzNPV and HearSNPV that lack gp64, the v-cath and ChiA genes are not linked to each other and do not have conserved genomic locations. The v-cath and ChiA genes are absent from the group II NPV Culex nigripalpus (Cuni)NPV (Afonso et al., 2001
) and from the granuloviruses Plutella xylostella (Plxy)GV (Hashimoto et al., 2000
) and Phthorimaea operculella (Phop)GV (accession NC_004062). These facts begged the question of whether the ChiA and v-cath genes were absent from the AgMNPV-2D genome or were they simply relocated in the genome. In this study we made an effort to find evidence for an AgMNPV v-cath gene homologue.
Southern blotting was done to probe for an AgMNPV v-cath gene homologue. These experiments proved negative (data not shown). We also tried the approach of engineering PCR primers that could anneal to the conserved regions among v-cath gene homologues of the group I NPVs (Fig. 5a, b). We successfully used these primers to amplify v-cath gene fragments from OpMNPV and AcMNPV DNA but were unable to amplify any products from AgMNPV-2D DNA (Fig. 5c
).
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We were unable to detect any V-CATH proteinase activity in the lysates from AgMNPV-infected T. ni cells at 75 h post-infection (Fig. 5). By contrast, the lysates from AcMNPV-infected T. ni cells and OpMNPV-infected Ld652Y cells showed significant amounts of proteolytic activity.
Our results suggest that V-CATH-like proteinase activity is absent from AgMNPV-2D. To our knowledge, AgMNPV-2D may be the first example of a type I NPV that lacks a v-cath gene. Since the ChiA gene is functionally linked with v-cath (Hawtin et al., 1997; Hom & Volkman, 2000
), it could also be presumed that AgMNPV-2D is also lacking a ChiA gene homologue. If the ChiA and v-cath genes are indeed absent from the AgMNPV-2D genome, functional homologues may still exist. A metalloproteinase has been proposed to be the functional homologue of V-CATH in the v-cath gene-lacking baculovirus PxGV (Hashimoto et al., 2000
). Alternatively V-CATH and ChiA enzymes may simply not be necessary for the propagation of AgMNPV-2D in an insect population.
T. ni larvae injected with AgMNPV-2D BV died, liquefied and turned black as readily as AcMNPV BV-injected larvae (data not shown). For AcMNPV and other baculoviruses, this pathology has been attributed to the enzymic activities of ChiA and V-CATH (Dai et al., 2000; Hawtin et al., 1997
) and is presumed to aid in the dispersal of baculoviruses in the environment. The v-cath and chiA genes are not, however, exclusively linked to host liquefaction. For example, the 25K FP gene of BmNPV is also linked to host liquefaction (Katsuma et al., 1999
). Host-specific and environment-specific factors may also be at play in the liquefaction or melting of baculovirus-infected insects. Lack of V-CATH activity may be contributing to the success of AgMNPV as biological insecticide. AgMNPV-infected A. gemmatalis larvae frequently do not liquefy after death. This characteristic has facilitated the field collection of AgMNPV-infected cadavers in the mass production of biological insecticide. Absence of v-cath/ChiA in the gp64 locus of AgMNPV-2D could be an anomaly of isolate 2D. However, REN digest patterns of 24 genomic variants of AgMNPV (Croizier & Ribeiro, 1992
; Maruniak et al., 1999
) suggest that the region downstream of gp64 has been stable for at least 25 years. When AgMNPV or other baculoviruses are dispersed by human activity, the activities of V-CATH and ChiA become less required. Indeed, the field collection itself may have maintained the selective pressure necessary to prevent recombination of the ChiA and v-cath genes from other group I NPVs.
Conclusions
In this study, we have examined a genomic region that flanks the gp64 gene of AgMNPV-2D. While there is much similarity between AgMNPV-2D and other group I NPVs it is somewhat striking that AgMNPV-2D appears to be missing ChiA and v-cath genes. Absence of the proteinase activity is supportive of at least the absence of the v-cath gene. Most certainly the future completion of the AgMNPV-2D genome will clarify this. Future investigations by our group will pursue the functional role of the v-trex gene that is shared between AgMNPV and CfMNPV.
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ACKNOWLEDGEMENTS |
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Received 30 August 2003;
accepted 3 October 2003.