1 Saskatoon Research Centre, AAFC-Saskatoon, 107 Science Place, Saskatoon, Saskatchewan, Canada S7N 0X2
2 Southern Crop Protection and Food Research Centre, AAFC, London, Ontario, Canada
3 Pacific Agri-Food Research Centre, AAFC, Summerland, BC, Canada
Correspondence
Martin Erlandson
erlandsonm{at}agr.gc.ca
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ABSTRACT |
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Present address: Department of Molecular Genetics and Microbiology, PO Box 100266, School of Medicine, University of Florida, Gainesville, FL 32610-0266, USA.
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Introduction |
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Enhancin was originally described from GV granules that showed an ability to enhance the infectivity of NPVs. It was first described from the GV of the armyworm, Pseudaletia unipuncta (PsunGV), as an 126 kDa protein, synergistic factor, that was responsible for an increase in larval susceptibility to PsunNPV in mixed infections (Yamamoto & Tanada, 1978, 1980
; Zhu et al., 1989
). Since then, synergistic activity has been associated with several GVs and enhancin genes have been identified in PsunGV and Helicoverpa armigera GV (HearGV) (Roelvink et al., 1995
), Trichoplusia ni GV (TnGV) (Hashimoto et al., 1991
), Choristoneura fumiferana GV (CfGV) (NCBI database, accession #AAG33872) and Xestia c-nigrum GV (XecnGV) (Hayakawa et al., 1999
). However, to date, enhancin has been described in only one NPV, Lymantria dispar MNPV (LdMNPV), which carries two copies of the enhancin gene, en1 and en2 (Bischoff & Slavicek, 1997
; Kuzio et al., 1999
; Popham et al., 2001
). In LdMNPV, deletion of either the en1 or en2 gene caused a decrease in virus potency, suggesting that both en1 and en2 of LdMNPV are involved in infectivity (Bischoff & Slavicek, 1997
; Popham et al., 2001
).
Purified TnGV enhancin protein degrades high molecular mass peritrophic matrix (PM) proteins, mainly mucin, both in vivo and in vitro and has been demonstrated to be a metalloprotease (Hashimoto et al., 1991; Lepore et al., 1996
; Wang & Granados, 1997
). Enhancin facilitates baculovirus infection by disrupting the PM of the midgut thereby allowing virions greater access to the midgut epithelial cells, resulting in the insect's increased susceptibility to viral infection (Derksen & Granados, 1988
; Lepore et al., 1996
). There is also evidence that the enhancement effect of the enhancin protein may be achieved through the interaction of enhancin protein with both the viral envelope and the host-cell plasma membrane, thereby increasing the fusion of virions with midgut cells (Tanada, 1985
; Tanada et al., 1975
, 1980
; Wang et al., 1994
).
The bertha armyworm, Mamestra configurata (Lepidoptera: Noctuidae), is an important pest of cruciferous oilseed crops in western Canada. A baculovirus, M. configurata (Maco) NPV, has been isolated from field populations of M. configurata larvae and determined to have potential as a biological control agent for this pest insect (Erlandson, 1990). Recently the complete genome sequence of the MacoNPV-A isolate was determined [155 060 bp encoding 169 putative open reading frames (ORFs) (Li et al., 2002
)]. A putative enhancin gene was identified in the MacoNPV genome, which makes it only the second NPV to have an enhancin gene. The contribution of LdMNPV enhancins to the virulence of LdMNPV has been documented (Bischoff & Slavicek, 1997
; Popham et al., 2001
) and the synergistic effect of GV enhancins have all been demonstrated with NPVs; however, whether an enhancin from an NPV can enhance the virulence of a heterologous NPV is not clear. Here we report on the identification and characterization of the enhancin gene from MacoNPV and an analysis of its synergistic function by its expression in an Autographa californica MNPV (AcMNPV) recombinant.
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Methods |
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The Spodoptera frugiperda cell line Sf9 was maintained at 27 °C in Grace's complete medium (GibcoBRL) supplemented with 10 % foetal bovine serum. Stocks of AcMNPV E2 and AcMNPV-Ac360GAL (Dickson & Friesen, 1991
) viruses were propagated in Sf9 cells at the specified m.o.i. Infected cells were harvested at various times post-infection (p.i.) and pelleted by centrifugation (3000 g). The cell pellets were re-suspended in TE buffer (0·01 M Tris/HCl, 0·001 M EDTA, pH 7·5) and PIBs were isolated from cells by adding 1/10 vol. of conditioned Pronase (5 mg ml-1 in 0·01 M Tris, pH 7·5, pre-incubated at 37 °C for 2 h) and 10 % Triton X-100, incubating at room temperature for 1 h, and concentrating by centrifugation (3000 g).
Virion purification and viral DNA extraction essentially followed previously described methods (Erlandson, 1990; Li et al., 1997
). Briefly, the PIBs were further purified by isopycnic centrifugation on sucrose gradients and the occlusion derived virus (ODV) was released from PIBs by incubation in alkaline PIB dissolution buffer (0·1 M Na2CO3, 0·17 M NaCl, 0·001 M NaEDTA, pH 10·8) and purified by sucrose density-gradient centrifugation. Virus DNA was extracted from ODV using the method of Smith & Summers (1978)
.
Cloning the enhancin gene and AcMNPV-enhancin recombinant virus construction.
The entire MacoNPV enhancin gene is contained within the MacoNPV-A HindIII-J REN fragment, which was previously cloned in a pBS+ vector (Li et al., 1997). Two PCR primers, 5'-CAGCCTGCAGTGGGACAAATAT-3' and 5'-CGTCTAGATTGATTTGCCTGCGGACGCTGAT-3', were designed to amplify the enhancin gene with its intact native promoter. The pBS-MacoHind J plasmid DNA was used as template with these primers to amplify the enhancin gene using Pfu DNA polymerase (Stratagene). The PCR product was digested with XbaI and PstI and cloned into an AcMNPV transfer vector (pEVocc+) which contains a multiple cloning site upstream of the polyhedrin gene (Fig. 1
) (Dickson & Friesen, 1991
). The resulting plasmid, pEVocc+/enMP, was sequenced using two primers designed to sequence the junctions at both ends of the insert.
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DNA sequencing and sequence analysis.
Nucleotide sequences were determined using an ABI Prism BigDye Terminator Cycle Sequencing Kit. For each sequencing reaction, approximately 200 ng of plasmid DNA was subjected to dideoxynucleotide chain termination sequencing and resolved with an ABI 377 DNA Sequencer (PE Biosystems). Sequence data were analysed using AutoAssembler DNA Sequence Assembly software (PE Biosystems). The generated sequences were analysed with Wisconsin Genetics Computer Group programs (Devereux et al., 1984) and DNASTAR software. Homology searches were carried out with the updated GenBank/EMBL, SWISSPROT and PIR databases, via the NCBI nucleotide and protein database using the BLAST algorithm (Altschul et al., 1990
). All percentage identity estimates were based on the percentage of identical amino acid residues for the predicted protein sequences from two complete genes. Multiple sequence alignments were accomplished with DNASTAR, Clustal X and GeneDoc software with default gap penalty (10) and gap length penalty (10) in PAM 250 matrix. For phylogenetic analysis, sequences were aligned using Clustal X and the phylogeny inference package, PHYLIP (version 3.5), was used to estimate phylogenetic relationships. Bootstrap analysis with 100 replicates was used to estimate the reliability of phylogenetic trees.
RNA purification and Northern blot analysis.
Total RNA was isolated from NPV-infected Sf9 cells, harvested at specific times p.i., using TRIzol reagent (GibcoBRL) following the manufacturer's protocol. Total RNA (10 µg) was separated on a 1·2 % agarose gel and transferred to Hybond-N+ membranes (Amersham). The 2·6 kb XbaIPstI fragment from the pEVocc+/enMP plasmid, which contained the enhancin gene, was labelled with [-32P]dCTP using a random primer DNA labelling system (GibcoBRL) and used as a probe for Northern hybridization analysis (Church & Gilbert, 1984
). Briefly, the membrane was pre-hybridized in hybridization buffer (0·5 M sodium phosphate containing 7 % SDS) at 60 °C for 2 h and then hybridized with the labelled probe in the same buffer overnight at 60 °C. After washing twice in 2xSSC buffer at 60 °C for 10 min, twice in 1xSSC containing 0·5 % SDS at 60 °C for 20 min and once in 0·1xSSC at room temperature for 10 min, the membrane was exposed to XAR film (Kodak).
Insect bioassays.
Polyhedra were isolated from Sf9 cells infected with either AcMNPV-E2 or AcMNPV-enMP2 recombinant virus, purified and quantified as previously described (Li et al., 1997). A dose-response assay was conducted with 2nd-instar T. ni larvae using five dose levels, based on a twofold serial dilution series, for each virus. The larvae were infected by allowing them to feed for 12 h on a canola leaf disc (4 mm diameter) treated with known quantities of polyhedra. Larvae that consumed the entire leaf disc were included in the assay and transferred to fresh artificial diet for the remaining 7 days of the bioassay. Larval mortality was assessed daily for the dose-response assays and every 8 h in time-response bioassays used to determine survival time 50 % (ST50). Each deceased larva was checked microscopically to confirm infection. Day 7 mortality data were analysed using SAS-Probit (SAS version 8) to estimate lethal dose 50 % (LD50) values for each virus. The ST50 estimates were derived using the time-mortality model of Vistat (R. P. Hughes, BTI, Ithaca, NY, USA).
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Results |
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Characterization of the putative enhancin protein sequence
The MacoNPV ORF89, enhancin, encodes a protein of 847 aa (Fig. 2). This is smaller than the 901 aa enhancins of PsunGV, CfGV and TnGV, the 902 aa enhancin of HearGV, and the 867, 898 and 856 aa proteins encoded by XecnGV ORF 152, 154 and 166, respectively (Hashimoto et al., 1991
; Hayakawa et al., 1999
; Roelvink et al., 1995
). However, it is larger than the 824 aa enhancin encoded by XecnGV ORF150 and the 783 and 788 aa E1 and E2 of LdMNPV (Bischoff & Slavicek, 1997
; Hayakawa et al., 1999
; Kuzio et al., 1999
; Popham et al., 2001
). BLAST comparisons demonstrated that, overall, MacoNPV enhancin had 19 % and 20 % aa identity to E1 and E2 of LdMNPV, respectively, and 21 % to 23 % aa identities to enhancins of GVs, including HearGV(23 %), TnGV (22 %), PsunGV (22 %), CfGV (22 %), and XecnGV enhancins encoded by ORF 150 (22 %), 152 (21 %), 154 (23 %) and 166 (22 %).
To identify any similarities to potential biologically significant domains or motifs from existing protein families, the MacoNPV enhancin protein sequence was compared with proteins in the PROSITE database (Release 16.45, 30 August 2001). A zinc-binding signature domain, HEXXH, typical of the neutral zinc metalloprotease superfamily (Jongeneel et al., 1989; Murphy et al., 1991
; Jiang & Bond, 1992
) and similar to that found in LdMNPV and the GV enhancins was found in MacoNPV enhancin at residues 228232 (HEIAH) (Fig. 2
). This zinc-binding signature site also includes a conserved aspartic acid residue 25 aa downstream of the HEXXH sequence (Fig. 3
). With the exception of XecnGV ORF166, a zinc-binding signature typical of metalloproteases was found in all the baculovirus enhancins identified thus far, including enhancins of MacoNPV, LdMNPV, HearGV, TnGV, PsunGV, CfGV, XecnGV ORF 150, 152 and 154 (Fig. 3
).
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Northern blot analysis was performed to confirm the expression of enhancin mRNA in AcMNPV-enMP2-infected Sf9 cells. A time-course experiment was done in Sf9 cells infected with an m.o.i. of 20 TCID50 units per cell. At each time-point p.i., total RNA was extracted, electrophoresed and processed for Northern blot analysis. The MacoNPV-A enhancin gene was expressed from the recombinant virus AcMNPV-enMP2 from 24 to 72 h p.i. as an approximately 2·6 kbp transcript (Fig. 5). As AcMNPV replication initiates at 68 h p.i. in Sf9 cells, the initiation of enhancin at 24 h p.i. confirms that it is a late gene as predicted by the late gene promoter consensus sequence at the 5' end of the gene (Fig. 2
). No MacoNPV enhancin-related transcripts were identified in AcMNPV-E2-infected cells.
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Discussion |
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A phylogenetic analysis of all the enhancins described thus far from baculoviruses led to the separation of two well-defined clusters (Cluster I and II) (Fig. 4). The phylogenetic tree presented here differs somewhat from that proposed by Popham et al. (2001)
, in as far as LdMNPV E1 and E2 were previously grouped with XecnGV ORF152 (E2) in one cluster while the other enhancins from GVs were grouped in a second cluster. In our phylogenetic analysis, which includes MacoNPV enhancin, LdMNPV E1 and E2, as well as XecnGV ORF 150 (E1), 152 (E2) and 166 (E4) are grouped together in one cluster (Cluster II, Fig. 4
). Interestingly, enhancins in Cluster II fall in a smaller size range (783867 aa) than the larger GV enhancins in Cluster I (900 aa). This phylogenetic tree would suggest that the enhancins of LdMNPV, MacoNPV and ORF 150, 152 and 166 of XecnGV may have originated from a common ancestor. In our phylogenetic tree the remainder of the GV enhancins group together in Cluster I and the separation of Clusters I and II is well supported by bootstrap analysis (100 out of 100) (Fig. 4
). Within Cluster I two subgroups were identified and well supported by bootstrap analysis, including TnGV, CfGV and PsunGV enhancins in one group, and HearGV and XecnGV ORF154 (E3) in a second group (Fig. 4
). This is similar to the phylogenetic tree subgrouping from Popham et al. (2001)
and thus may indicate that these GV enhancins share a common ancestor.
Potential baculovirus consensus late promoter motifs, ATAAG or TTAAG, have been found in the upstream sequence of all enhancin genes identified thus far. For example, an ATAAG motif has been identified in the upstream sequence of both LdMNPV enhancins (en1 and en2) (Kuzio et al., 1999) and that of PsunGV enhancin (Roelvink et al., 1995
). Interestingly, two and three TTAAG motifs have been identified in the upstream sequences of enhancins from TnGV and HearGV, respectively (Roelvink et al., 1995
; Hashimoto et al., 1991
). There are also one or two late gene promoters in each of the XecnGV enhancins (Hayakawa et al., 1999
). The existence of the late gene promoter motif(s) indicates that enhancin is likely expressed late in infection. This has been demonstrated for LdMNPV, in which an E1 enhancin-specific 3·5 kb transcript was detected in infected Ld652Y cells at 48 and 72 h pi but not earlier (Bischoff & Slavicek, 1997
). Similarly, in TnGV-infected T. ni larvae, enhancin transcripts were detected at 6 days p.i. but not at 3 days p.i. (Hashimoto et al., 1991
). Our Northern blot analysis showed that in AcMNPV-enMP2 (enhancin expression under control of its native promoter)-infected cells, enhancin gene transcription was detected as a late gene (Fig. 5
). Thus it is likely that in its native system MacoNPV enhancin is expressed as a late gene as well.
The biological activity of GV enhancins in terms of their ability to increase the efficacy of NPV infection of insect larvae has been well documented. Two mechanisms have been suggested for GV enhancin activity; namely, enhancement of virushost midgut cell fusion (Kozuma & Hukuhara, 1994) and degradation of peritrophic matrix proteins by metalloprotease activity (Lepore et al., 1996
; Wang & Granados, 1997
). In LdMNPV, both en1 and en2 genes were shown to contribute to virus potency as deletion of either en1 or en2 resulted in an approximately 2-fold decrease in potency (Popham et al., 2001
). Deletion of both the en1 and en2 gene resulted in a 12-fold decrease of virus potency when compared to wild-type LdMNPV virus (Bischoff & Slavicek, 1997
; Popham et al., 2001
). Our bioassay data with the AcMNPV-enMP2 recombinant expressing MacoNPV-A enhancin under the control of its native promoter shows that this gene can impact NPV infectivity. The AcMNPV-enMP2 recombinant was 4·4-fold more potent than AcMNPV wild-type virus based on LD50 estimates. This is in the same range of infectivity enhancement described for the enhancin gene deletion experiments with LdMNPV (Popham et al., 2001
). Analysis of time-to-mortality data showed similar speed of kill for the wild-type, AcMNPV-E2 and the enhancin recombinant, AcMNPV-enMP2, when the two viruses were tested at biologically similar doses (i.e. LD90 dose). This result is consistent with previous observations for LdMNPV enhancins (Popham et al., 2001
). These authors showed that the ST50 estimates were not significantly different for LdMNPV recombinants in which one or both of the enhancin genes were deleted compared to wild-type virus when tested at LD50 dose rates. However, in the current study, when time-to-mortality was examined at the same absolute dose (32 PIBs per larva) the ST50 for the enhancin recombinant was significantly shorter than for the wild-type virus, 130±3·1 h versus 141±2·9 h p.i., respectively. This observed increase in speed of kill may be the result of a higher effective dose of virus reaching the midgut cells due to the action of MacoNPV enhancin on the peritrophic matrix of the host gut. Thus our results suggest that enhancin functions to increase the amount of initial infection in the host insect midgut rather than increasing the speed of spread of virus infection in the host insect. It would be of interest to determine whether MacoMNPV enhancin, as expressed in the AcMNPV-enMP2 recombinant virus, interacts with the intestinal mucin component of T. ni peritrophic matrix in a fashion similar to that of GV enhancins as demonstrated by Wang & Granados (1997)
. We intend to pursue this line of investigation in in vivo feeding assays with AcMNPV-enMP2 in T. ni larvae and Western blot analysis of PM proteins isolated at various times post-ingestion.
Enhancin genes have been found in several GVs, including PsunGV and HearGV (Roelvink et al., 1995), TnGV (Hashimoto et al., 1991
), CfGV (NCBI database) and XecnGV (Hayakawa et al., 1999
). Interestingly, all the above GVs belong to those categorized as slow GVs in terms of their pathogenicity (Winstanley & O'Reilly, 1999
). Granuloviruses in this group have high LD50 values, with pathogenesis being prolonged and mortality occurring as long as 1020 days after infection. These slow GVs also typically have large genomes (170 kbp or larger) as, for example, in TnGV, HearGV and XecnGV (Winstanley & O'Reilly, 1999
). The LdMNPV (161 046 bp) and MacoNPV (155 060 bp) genomes are the two largest NPV genomes sequenced to date (Kuzio et al., 1999
; Li et al., 2002
), at about 2530 kb larger than other sequenced NPVs. Both LdMNPV and MacoNPV are also slow killing, viruses taking upwards of 10 days to kill the insect at an LD50 dose (Erlandson, 1990
). In the MacoNPV genome several cluster of genes, including enhancin, were found to be homologues of XecnGV genes, indicating some similarity or common ancestry between these baculoviruses. It is possible that baculoviruses with larger genomes have developed different infection strategies to overcome some host-specific barrier, such as a robust host peritrophic matrix structure, and this may have included the incorporation of gene clusters, including possibly enhancin, containing potential virulence factors. Whether there may be complicating fitness costs associated with the replication of larger genomes is open to speculation.
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ACKNOWLEDGEMENTS |
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Received 28 June 2002;
accepted 28 August 2002.