Wageningen University, Laboratory of Virology, Binnenhaven 11, 6709 PD Wageningen, The Netherlands
Correspondence
Just Vlak
just.vlak{at}wur.nl
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ABSTRACT |
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INTRODUCTION |
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Deletions in the SeMNPV genome predominantly occur within the XbaI-A restriction fragment and these deleted genotypes are present in SeMNPV wild-type isolates where they may act as parasitic genotypes (Muñoz et al., 1998). Heldens et al. (1996)
described a SeMNPV mutant with a deletion in XbaI-A of about 25 kb, spanning open reading frames (ORFs) 1441 (IJkel et al., 1999
). This mutant replicated efficiently in cell culture but lacked bioactivity in vivo. Dai et al. (2000)
isolated an SeMNPV recombinant with a deletion of 10·6 kb from nt 18513 to 29106, encompassing ORFs 1528 (Fig. 1
A). This recombinant (SeXD1) was isolated by alternate cloning between Se301 insect cells and S. exigua larvae. A genotypic variant with the same 10·6 kb deletion was shown to exist in the SeMNPV wild-type isolate and seemed preferentially amplified in cell culture, indicating that deletion mutants may have a replicative advantage in cell culture. The recombinant SeXD1, however, was still infectious in vivo by oral ingestion of OBs, but caused no typical liquefaction of the S. exigua larvae, probably due to the deletion of the cathepsin (ORF16) and chitinase (ORF19) genes (Hawtin et al., 1997
). More recently, a spontaneous SeMNPV mutant was generated during passaging of bacmid-derived SeMNPV in Se301 insect cell culture and this mutant lacked virulence in vivo (Pijlman et al., 2002
). The mutant contained a major deletion from nt 20162 to 36398, spanning ORFs 1735 and possibly affecting parts of the promoter region of ORF36. From these preceding studies, we deduced that the gene(s) responsible for the observed loss of virulence in vivo was located within SeMNPV ORFs 2935 (or perhaps 36).
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The spontaneous SeMNPV bacmid deletion mutant (Fig. 1A; Pijlman et al., 2002
) lacked ORFs 1735 and possibly part of the promoter of Se36, as only a sequence 18 bp upstream from the translation start of Se36 was left intact. Thus, it could be that the expression of Se36 was affected, explaining the loss of oral infectivity in our mutant. However, more detailed investigation of the Se36 coding sequence showed that there was a late transcription motif (TAAG) present 12 bp upstream (-12) of the ATG. This suggests that Se36 (pif) may still be functional in the isolated mutant, as the TAAG motif has been retained. The shortest distance between a functional TAAG motif (at position -6) and the translation start site (ATG) in SeMNPV was reported for the ubiquitin gene (Van Strien et al., 1996
).
Using a previously constructed SeMNPV bacmid (Pijlman et al., 2002) and a rapid site-directed mutagenesis protocol in Escherichia coli, known as ET recombination or lambda-red recombination (Muyrers et al., 1999
), progressive deletions within the ORF 2935 region were made. SeMNPV mutants were tested for their ability to infect S. exigua larvae per os by oral feeding and by injection into the haemolymph. Analysis of repair mutants resulted in the identification of a novel baculovirus gene that is required for the per os infection of S. exigua larvae with baculovirus OBs. This gene mapped to SeMNPV ORF35 and belongs to a core set of 30 genes shared by all known baculoviruses (Herniou et al., 2003
).
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METHODS |
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Deletion of ORFs by ET recombination and bacmid DNA preparation.
A series of SeMNPV bacmid mutants with progressive deletions of ORFs 2935 was made by ET recombination as previously described (Pijlman et al., 2002). To obtain genomic stability of the generated deletion mutant bacmids following replication in cell culture, ORFs 1528 were also deleted. These ORFs are responsible for genetic instability but have been shown to be non-essential for oral infectivity of SeMNPV (Dai et al., 2000
). This resulted in bacmids SeBAC
1528/32/34/35 (Fig. 1B
). We also generated a mutant (SeBAC
1535*) similar to the spontaneous bacmid SeMNPV mutant lacking most of the upstream (promoter) sequence of Se36, to investigate whether Se36 was still functional or not. Briefly, PCR products with 50 bp viral flanking overhangs were generated with the Expand long template PCR system (Roche) and with the custom-made primers (Invitrogen) listed in Table 1
. Plasmid pBeloBAC11 (Shizuya et al., 1992
) was used as a template for PCR amplification of the chloramphenicol resistance gene. E. coli DH10B cells harbouring the SeMNPV bacmid SeBAC and the recombination helper plasmid pBAD
(Muyrers et al., 1999
) were induced with L-arabinose and made electrocompetent by subsequent washes with 10 % glycerol (Fig. 1C
). For electrotransformation (200
, 25 µF, 2·3 kV) with a Biorad Genepulser and 2 mm cuvettes (Eurogentec), 0·5 µg PCR product was used. Cells were incubated in a shaker at 37 °C for 1 h and plated on LB agar plates containing 20 µg chloramphenicol ml-1. Bacmid DNA was isolated using an alkali lysis protocol as described previously (Pijlman et al., 2002
).
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Transfection, microinjections and oral infectivity assay.
Se301 cells were seeded in six-well plates (Nunc) with 5x105 cells per well. Transfection was performed with approximately 0·5 µg bacmid DNA using 10 µl Cellfectin (Invitrogen). Supernatant containing BV and polyhedra from infected cells was harvested by centrifugation 14 days post-transfection. The microinjection of BV into fourth instar larvae was performed using a 1·5 ml volume B-D Pen (Becton & Dickinson) and a 28 gauge half-inch NovoFine needle (Novo Nordisk). Ten µl supernatant (or Grace's insect medium for the negative control) was injected into the haemocoel of each larva. The infectivity of recombinant polyhedra was determined with second and fourth instar S. exigua larvae by diet contamination, using at least 106 OBs per larva. For the injection as well as the feeding experiments, the larvae were kept separately in 24-well plates (Nunc) and monitored daily until all larvae had either pupated or died as a result SeMNPV infection. At least 24 larvae were tested per treatment.
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RESULTS |
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Since the original SeMNPV polyhedrin gene was disrupted by the insertion of the bacmid vector, this polyhedrin gene was reintroduced by site-specific transposon-mediated integration (Pijlman et al., 2002). Subsequently, Se301 insect cells were transfected with the recombinant SeMNPV bacmids and OBs were harvested. To check whether all constructed recombinants generated infectious virus, transfection supernatant containing BVs was injected into the haemocoel of fourth instar S. exigua larvae (Table 2
). All larvae injected with BVs from all recombinants died and produced OBs, whereas larvae injected with Grace's medium only (negative control) survived. To test the oral infectivity of the recombinants, OBs were fed to third instar S. exigua larvae by diet contamination. The results showed that SeBACph
1528, SeBACph
1532 and SeBACph
1534 were still infectious per os (Table 2
). Many polyhedra were found in the cadavers, but the larvae did not liquefy as expected as a result of deletion of the cathepsin and chitinase genes (Hawtin et al., 1997
). In contrast, SeBACph
1535 and SeBACph
1535* lacked the ability to infect larvae by oral ingestion of polyhedra. From these results it was concluded that a deletion of ORF35 is responsible for the observed phenotype lacking activity following in vivo infection.
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To investigate whether, in addition to Se35, a deletion of the putative promoter region of Se36 in SeBAC1535* could also have been responsible for a lack of virulence in vivo, Se35 was also reintroduced into SeBAC
1535* (by transposon-mediated integration) generating SeBACph
15-35*rep35 (Fig. 2
), in which the deletion ended at position -18 of the ATG of Se36. Polyhedra from this repair mutant SeBACph
15-35*rep35 also resulted in restoration of oral infectivity, indicating that the 18 bp sequence (including a TAAG motif) upstream of the ATG start site of Se36 serves as an active promoter in the mutant bacmid SeBAC
1535*. Alternatively, Se36 may not be essential for oral infectivity, which would be in contrast to other results (Kikhno et al., 2002
).
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Amino acid sequences of Se35 and its homologues from all 18 completely sequenced baculovirus genomes and the currently unclassified Heliothis zea-1 virus (Hz-1V), were aligned using CLUSTALX (Thompson et al., 1997). Alignment of a selection of Se35 homologues from Helicoverpa armigera SNPV-G4 (a single nucleocapsid NPV), Autographa californica MNPV (a group I MNPV), Cydia pomonella granulovirus (a granulovirus), Culex nigripalpus NPV (a dipteran NPV) and Hz-1V (a more distantly related lepidopteran virus) is shown in Fig. 3
. It can be seen that Se35 has a longer ORF than its homologues and therefore the genuine start of the protein might be at the third methionine, as suggested before. Hydrophobic, putative transmembrane domains in Se35 were identified with the use of TMPRED (Hofmann & Stoffel, 1993
). For Se35, a strong hydrophobic domain was predicted at the N terminus (Fig. 3
), which is conserved in the other Se35 homologues. Using the computer prediction program SIGNALP (Nielsen et al., 1997
), it was found that Se35 (and its homologues) had a predicted signal peptide at the N terminus and a putative cleavage site (indicated with an arrow). The TARGETP program (Emanuelsson et al., 2000
) predicted that Se35 employs the secretory pathway. In the Se35 alignment (Fig. 3
), 11 cysteines were found at conserved positions (indicated with asterisks). These conserved cysteines were also present in all the other homologues (not shown). This indicates that the protein can form multiple disulfide bonds and that it might be heavily folded. No conserved N-glycosylation sites were found in the alignment of Se35.
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DISCUSSION |
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Interestingly, a granulovirus from the potato tuber moth called PhopGV was recently sequenced (GenBank accession no. NC_004062) and appeared to lack exactly the same ORFs as the SeMNPV deletion mutants, namely cathepsin, chitinase, GP37, PTPT-2, EGT, PKIP and ARIF-1. In PhopGV, the gene encoding LEF-1 (ORF66) is adjacent to the Se36 homologue (PhopGV ORF67), as is the case in our SeMNPV ORF 1535 deletion mutant (Fig. 1B). This indicates that this large cluster of non-essential genes is not conserved throughout the family of Baculoviridae. For the utilization of SeMNPV as an expression vector for recombinant proteins, the dispensability of the SeMNPV XbaI-A region spanning ORFs 1535 may be advantageous with regard to its biological safety. Furthermore, by deliberate site-specific deletion of ORFs 1534, the intrinsic genetic instability of the SeMNPV genome in cell culture can be eliminated, while the virus is still infectious per os. Therefore, the SeBACph1534 mutant may be an attractive, biologically active SeMNPV recombinant, which could be efficiently scaled up, both in vitro and in vivo, to be used as a bioinsecticide for pest control. Further analysis of the infectivity and pathobiology of this mutant should be carried out to evaluate its potential for use as a biopesticide.
The results presented here provide genetic and biological evidence that a deletion of SeMNPV ORF35 (Se35) results in the formation of OBs that have lost the ability to infect insects by the oral route. Oral infectivity was rescued by reintroduction of Se35 at a different locus via transposition in the SeMNPV bacmid (Fig. 2). These results also support the hypothesis that Se36 (pif; Kikhno et al., 2002
) was still functional in our mutants and that Se36 transcription is likely to start from the TAAG at position -12. We did not compare transcription levels of Se36 between the various mutants, since this may only have a quantitative but not a qualitative effect on the identification of Se35 as essential gene for oral infectivity. Se35 belongs to the group of so-called baculovirus core genes (Herniou et al., 2001
, 2003
), which are shared by all known baculoviruses. A homologue is also present in the non-assigned Hz-1V (Cheng et al., 2002
). This virus has structural similarities to baculoviruses, but the virions are not occluded. However, Hz-1V is infectious per os. Se35 was shown to be essential for the per os infection of S. exigua, but not necessary for systemic infection of S. exigua larvae by injection into the haemolymph or for succesful virus replication in cell culture. This indicates that only the Se35 protein and not the gene itself is needed for the initial steps of midgut infection by ODVs, either for passing the peritrophic membrane, or for fusion with the midgut epithelial cells, or both.
Together with p74 and Se36 (pif), Se35 is one of the few baculovirus genes that is conserved among all baculoviruses (Herniou et al., 2003) as well as in the distantly related Hz-1V (Cheng et al., 2002
). Moreover, these three conserved proteins are all involved in oral infectivity. Therefore, we predict a highly conserved entry mechanism for baculovirus ODVs and Hz-1V virions in insect midgut cells, with P74, Se36 and Se35 as key components. Hz-1V does not produce BVs, which is in line with the lack of homologues of GP64 or F-protein groups in the predicted Hz-1V ORFs. The Hz-1V virions may therefore be structurally quite similar to baculovirus ODV particles containing a single nucleocapsid (similar to granuloviruses and SNPVs) and spread the infection after cell lysis (Burand, 1998
).
Analogous to the designation of pif to Se36 and its homologues (e.g. AcMNPV ORF119), we propose the name per os infectivity factor 2 (pif-2) for Se35 and its homologues (e.g. AcMNPV ORF22) in other baculoviruses. Kikhno et al. (2002) showed by Western blot analysis that PIF is a structural protein of the ODV envelope and they speculated on a putative interaction between PIF and P74, but now PIF-2 must be considered as well. The overlap of the Se35 (pif-2) 3'UTR and the promoter of the neighbouring Se36 (pif) may indicate that these two genes in SeMNPV have co-evolved and are closely associated, although their homologues in other baculoviruses (including SpliNPV) are located in separate gene clusters.
Although the strong N-terminal hydrophobic domain in Se35 suggests that the gene encodes a protein associated with membranes, it remains to be elucidated whether PIF-2 is indeed an ODV-specific structural protein. The alignments of Se35 (Fig. 3) and Se36 with their homologues demonstrated that both proteins have strong hydrophobic domains at their N termini (Fig. 4
). The Se35 and Se36 N-terminal sequences, which are rich in valines, isoleucines and leucines, are very similar to N-terminal sequences of AcMNPV ODV-E66 (Ac46) and ODV-E25 (Ac94). These latter proteins are conserved among all lepidopteran baculoviruses (Herniou et al., 2003
) and were previously identified as ODV-specific structural proteins. It was shown that their N-terminal hydrophobic domains were sufficient to direct reporter proteins to the nuclear envelope, intranuclear microvesicles and the ODV envelope within baculovirus-infected cells (Hong et al., 1997
). It was shown by N-terminal amino acid sequencing that ODV-E66 and ODV-E25 were uncleaved in the ODV envelope. Although Se35 and Se36 have a predicted cleavage site after the hydrophobic domain, it remains unclear whether cleavage occurs. Hong et al. (1997)
also proposed a model predicting that ODV envelope proteins are incorporated into the endoplasmic reticulum and are subsequently transported to the inner and outer nuclear membrane. In agreement with these findings, an Se36 homologue (Ac119) may be part of the ODV envelope of SpliNPV (Kikhno et al., 2002
). The similar hydrophobic N-terminal amino acid sequences of ODV-E25, ODV-E66, PIF and PIF-2 from AcMNPV and SeMNPV (Fig. 4
) and the involvement of Se35 in oral infectivity may suggest that Se35 (Ac22) also encodes a structural protein associated with ODVs. Further biochemical and immunological studies are required to demonstrate conclusively the location of pif-2.
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ACKNOWLEDGEMENTS |
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Received 30 January 2003;
accepted 2 April 2003.