Identification of pif-2, a third conserved baculovirus gene required for per os infection of insects

Gorben P. Pijlman, Andrea J. P. Pruijssers and Just M. Vlak

Wageningen University, Laboratory of Virology, Binnenhaven 11, 6709 PD Wageningen, The Netherlands

Correspondence
Just Vlak
just.vlak{at}wur.nl


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Infection of cultured insect cells with Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) resulted in the generation of mutants with major genomic deletions. Some of the mutants lacked the ability to infect S. exigua larvae per os. The gene(s) responsible for this phenotype in SeMNPV was mapped within a contiguous sequence encoding ORFs 29–35. In this paper we have shown that SeMNPV ORFs 15–35 (including genes encoding cathepsin, chitinase, GP37, PTPT-2, EGT, PKIP-1 and ARIF-1) are not essential for virus replication in cell culture or by in vivo intrahaemocoelic injection. By site-specific deletion mutagenesis of a full-length infectious clone of SeMNPV (bacmid) using ET recombination in E. coli, a series of SeMNPV bacmid mutants with increasing deletions in ORFs 15–35 was generated. Analyses of these mutants indicated that a deletion of SeMNPV ORF35 (Se35) resulted in loss of oral infectivity of polyhedral occlusion bodies. Reinsertion of ORF35 in SeMNPV bacmids lacking Se35 rescued oral infectivity. We propose the name pif-2 for Se35 and its baculovirus homologues (e.g. Autographa californica MNPV ORF22), by analogy to a different gene recently characterized in Spodoptera littoralis NPV, which was designated per os infectivity factor (pif). Similar to the p74 gene, which encodes an essential structural protein of the occlusion-derived virus envelope, pif and pif-2 belong to a group of 30 genes that are conserved among the Baculoviridae.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) infects the single insect species S. exigua (Smits & Vlak, 1988) and belongs to the group II NPVs. Baculoviruses of this group do not contain a GP64 homologue, but have a functionally homologous F protein as a structural element of the budded virus (BV) (IJkel et al., 2000). BVs are required for the systemic spread of infection throughout the insect, whereas occlusion-derived viruses (ODVs) are involved in the horizontal spread of the virus in insect populations (Blissard & Rohrmann, 1990). Occlusion bodies (OBs) are ingested orally and the alkaline environment of the midgut causes the release of the ODVs. The ODVs first pass the peritrophic membrane and subsequently fuse to the midgut epithelial cells, thereby causing the initial infection (Funk et al., 1997). Large-scale production of SeMNPV for biological control is carried out in vivo using insect larvae, since SeMNPV infection in cell culture leads to the rapid generation and predominance of deletion mutants. These mutants, with deletions up to 25 kb, often lack the ability to infect S. exigua larvae by oral ingestion of OBs (Heldens et al., 1996). Therefore, the genetic engineering of SeMNPV via recombination in cell culture is complicated (Dai et al., 2000) and precludes the in vitro production of biologically active SeMNPV in insect cell bioreactors.

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) 14–41 (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 15–28 (Fig. 1A). 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 17–35 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 29–35 (or perhaps 36).



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Fig. 1. (A) Genomic organization of the hypervariable region in SeMNPV and schematic representation of SeMNPV deletion mutants. The virus lacking ORFs 17–35 is non-infectious per os (Pijlman et al., 2002), whereas the virus with retained oral infectivity lacks ORFs 15–28 (Dai et al., 2000). The region of interest (ORFs 29–35) containing the gene(s) required for oral infectivity is indicated. (B) Schematic overview of SeMNPV deletion mutant bacmids. HindIII and NotI restriction sites are indicated in the full-length bacmid SeBAC. Nucleotide positions according to the complete SeMNPV genome sequence (IJkel et al., 1999) are indicated on either side of the introduced chloramphenicol resistance gene (CmR). (C) ET recombination protocol. The PCR product with a CmR gene and viral flanking sequences of 50 bp was electroporated into arabinose-induced E. coli DH10{beta} cells, which harbour the homologous recombination helper plasmid pBAD{alpha}{beta}{gamma} and the full-length SeMNPV bacmid SeBAC. Recombinant bacmids with deletions from ORFs 15 to 28, 32, 34, 35 or 35* were isolated by selection with kanamycin (Kan) and chloramphenicol (Cm). (D) NotI–HindIII restriction pattern of the full-length SeMNPV bacmid SeBAC and the SeMNPV deletion mutant bacmids. The 26102 bp HindIII–HindIII and 9084 bp NotI–HindIII fragment of SeBAC are indicated with a black asterisk at the left, and the NotI–HindIII fragments (24593, 19692, 18703, 17462 and 17303 bp) with progressive deletions are indicated with white asterisks.

 
Until recently the only baculovirus protein demonstrated to be involved in oral infectivity by ingestion of polyhedra was the ODV-specific P74 (Kuzio et al., 1989), which contains a hydrophobic C terminus involved in protein localization and transmembrane anchoring. None of the other proteins of the ODV envelope, such as ODV-E18, -EC27, -E35, -E25, -E56 and -E66, were proven to participate in the oral infectivity process (Slack et al., 2001). Recently, however, a conserved baculovirus gene encoded by ORF7 of Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) was also found to be required for the oral infection of S. littoralis. This gene, which is homologous to SeMNPV ORF36 (Se36), was designated per os infectivity factor (pif) (Kikhno et al., 2002).

The spontaneous SeMNPV bacmid deletion mutant (Fig. 1A; Pijlman et al., 2002) lacked ORFs 17–35 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 29–35 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).


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Insect cells, insects and virus.
The S. exigua cell line Se301 (Hara et al., 1995) was donated by Dr T. Kawarabata (Institute of Biological Control, Kyushu University, Japan) and was propagated at 27 °C in Grace's supplemented medium (Gibco BRL) containing 10 % foetal calf serum (Gibco BRL). S. exigua larvae were reared on an artificial diet at 27 °C, 70 % humidity and a 16 : 8 h photoperiod. Fourth instar S. exigua larvae were infected by contamination of the artificial diet with 4x105 SeMNPV-US1 (Gelernter & Federici, 1986) polyhedra per larva (Smits & Vlak, 1988). Haemolymph was collected as previously described (IJkel et al., 2000). Infectious BV titres were determined using the endpoint dilution assay (Vlak, 1979).

Deletion of ORFs by ET recombination and bacmid DNA preparation.
A series of SeMNPV bacmid mutants with progressive deletions of ORFs 29–35 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 15–28 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{Delta}15–28/32/34/35 (Fig. 1B). We also generated a mutant (SeBAC{Delta}15–35*) 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{alpha}{beta}{gamma} (Muyrers et al., 1999) were induced with L-arabinose and made electrocompetent by subsequent washes with 10 % glycerol (Fig. 1C). For electrotransformation (200 {Omega}, 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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Table 1. Oligonucleotides used for site-specific genomic deletions by ET recombination and PCR amplification of Se35

For the ET primers, the 50 nt long viral flanking sequence used for homologous recombination (italics) is followed by an AvrII restriction site (underlined). The 3' end of the ET primers is used for PCR amplification of the chloramphenicol resistance gene from pBeloBAC11. The PCR primers for amplification of the coding sequences of Se35 are equipped with HindIII restriction sites (underlined) for cloning purposes.

 
Construction of repair bacmids.
The coding sequence of Se35 plus the putative promoter region (from position -173 relative to the ATG) was amplified by PCR using primer pair DZ 241/DZ 242. HindIII restriction sites (underlined) were included in the primers for cloning purposes (Table 1). The PCR products were first cloned into a pGEM-Teasy vector (Promega), giving pSe35. The plasmid insert was sequenced (Baseclear, The Netherlands) to confirm that no errors had been introduced by PCR. For reintroduction into the SeMNPV bacmid, Se35 was cloned as a HindIII fragment into pFB1Sepol (Pijlman et al., 2002) using standard methods (Sambrook et al., 1989). pFB1Sepol is a derivative of the pFastBAC1 vector (Bac-to-Bac; Invitrogen) and contains a complete SeMNPV polyhedrin gene. The resulting pFastBAC vector pFB1SepolSe35 was used to construct the repair bacmids using the Bac-to-Bac transposition protocol (Invitrogen).

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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification of SeMNPV ORFs required for per os infectivity of S. exigua
From previous studies on SeMNPV performed in our laboratory, we concluded that the genomic region from ORF 29 to 35 contains one or more genes required for oral infectivity (Fig. 1A). To identify this ORF(s), progressive site-specific deletions (Fig. 1B) were made in the full-length SeMNPV bacmid (SeBAC) using homologous ET recombination in E. coli (Fig. 1C). With this method, a sequence of contiguous ORFs from ORF15 to ORF32, -34 or -35 was replaced with a chloramphenicol resistance gene (CmR). We also generated a deletion mutant (SeBAC{Delta}15–35*) with the same deletion as a spontaneous mutant found previously (Pijlman et al., 2002; see Fig. 1A), which lacked oral infectivity. The identity of the deletion mutant bacmids (as outlined in Fig. 1B) was confirmed by their unique NotI–HindIII restriction profile (Fig. 1D).

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{Delta}15–28, SeBACph{Delta}15–32 and SeBACph{Delta}15–34 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{Delta}15–35 and SeBACph{Delta}15–35* 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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Table 2. Feeding and injection experiments with S. exigua larvae

Infections of Se301 insect cells were carried out with SeMNPV-US1 wild type and the various deletion mutant SeMNPV bacmids, which had an introduced polyhedrin gene. Twenty-four early fourth instar larvae were injected with budded virus (BV) containing culture medium. Second and fourth instar larvae (24 per treatment) were fed with polyhedral occlusion bodies (OB). A plus (+) indicates mortality, and a minus (-) indicates survival and pupation of the S. exigua larvae following treatment.

 
Construction and analysis of Se35 repair mutants
To confirm the essential role of Se35 in oral infectivity, the gene was reintroduced (along with the polyhedrin gene) into SeBAC{Delta}15-35 to generate SeBACph{Delta}15-35rep35. After transfection, BV-containing supernatant was injected into fourth instar S. exigua larvae and full mortality and formation of OBs in the cadavers was obtained. OBs harvested from infected cells were then fed to second and fourth instar S. exigua larvae to check oral infectivity. OBs from the transfection with repair mutant SeBACph{Delta}15-35rep35 resulted in restoration (rescue) of oral infectivity, confirming that a deletion of Se35 leads to a baculovirus phenotype lacking infectivity in vivo.

To investigate whether, in addition to Se35, a deletion of the putative promoter region of Se36 in SeBAC{Delta}15–35* could also have been responsible for a lack of virulence in vivo, Se35 was also reintroduced into SeBAC{Delta}15–35* (by transposon-mediated integration) generating SeBACph{Delta}15-35*rep35 (Fig. 2), in which the deletion ended at position -18 of the ATG of Se36. Polyhedra from this repair mutant SeBACph{Delta}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{Delta}15–35*. 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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Fig. 2. Schematic overview of SeMNPV bacmid repair mutants and feeding/injection experiments with S. exigua larvae. SeMNPV ORF35 (Se35) was reintroduced by transposon-mediated integration into the recombinant bacmids along with the polyhedrin (ph) gene. Culture supernatant from infections of Se301 insect cells was injected in 24 early fourth instar larvae, while polyhedral occlusion bodies were fed to second and fourth instar larvae (24 per treatment). A plus (+) indicates mortality, and a minus (-) indicates survival and pupation of the S. exigua larvae following treatment.

 
Computer-assisted analysis of Se35 and its homologues
The DNA sequence encoding Se35 was first investigated for the presence of putative baculovirus early and/or late promoter motifs, such as TATA(A), the consensus early transcription initiation motif ATCA(G/T)T(C/T) (Friesen, 1997) and the essential TAAG motif for late genes (Lu & Miller, 1997). No consensus early or late transcription initiation motifs were found upstream of the first ATG of Se35, but there was a TAAG motif present starting 17 nucleotides before the (third) ATG. This may suggest that Se35 is a late gene and that it might be translated from the third ATG, although none of the first three ATGs in Se35 is in a favourable Kozak context. A putative polyadenylation signal (AATAAA) was detected 22 nucleotides after the stop codon.

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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Fig. 3. Comparison of SeMNPV ORF35 (acc. no. AAF33565) with its homologues HaSNPV-G4 ORF132 (acc. no. AAG53875), AcMNPV ORF22 (acc. no. AAA66652), CpGV ORF48 (acc. no. AAK70708), CuniNPV ORF38 (acc. no. AAK94116) and Hz-1V ORF123 (acc. no. AAN04416). Shading levels indicate amino acid identity/similarity. Conserved cysteine residues are indicated with an asterisk above the sequence. A hydrophobic, putative transmembrane (TM) domain is indicated with a straight line above the sequences. The predicted signal peptide cleavage site is the same for SeMNPV (YDA-HL), AcMNPV (YQA-YL), CpGV (YHA-HQ), CuniNPV (GEA-AV) and Hz-1V (DNS-KY) and is indicated with an arrow. The predicted signal peptide cleavage site for HaSNPV-G4 is slightly different (VLY-RP).

 
Similar N-terminal hydrophobic domains to those found in Se35 were also found for AcMNPV ODV-E66 and ODV-E25, which encode ODV-specific structural proteins directly targeted to the nucleus (Hong et al., 1997). Se36 (pif) also contains such a hydrophobic domain at the N terminus. A comparison of the homologues of these four proteins from AcMNPV and SeMNPV is shown in Fig. 4. All amino acid sequences had a strong hydrophobic domain at the N terminus that was rich in valines, leucines and isoleucines.



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Fig. 4. Comparison of N-terminal sequences of ODV-specific proteins and proteins required for oral infectivity. Amino acid sequences of ODV-E66 (AcMNPV acc. no. AAA66676, SeMNPV acc. no. AAF33587 and AAF33643), ODV-E25 (AcMNPV acc. no. AAA66724, SeMNPV acc. no. AAB88623), PIF (AcMNPV acc. no. AAA66749, SeMNPV acc. no. AAF33566) and the newly identified per os infectivity factor PIF-2 (AcMNPV acc. no. AAA66652, SeMNPV acc. no. AAF33565) were aligned with CLUSTALX. ORF numbers are indicated according to the complete genome sequences (Ayres et al., 1994; IJkel et al., 1999). Se35 is the truncated version of the published sequence (see also Fig. 3). Shaded amino acids indicate the strong hydrophobic domain, which is rich in valines (V), leucines (L) and isoleucines (I).

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this paper, evidence is presented showing that SeMNPV ORF35 (Se35) is essential for oral infectivity and that a region of SeMNPV encompassing ORFs 15–35 is not required for virus replication in cultured insect cells or in vivo after intrahaemocoelic injection. In previous studies involving SeMNPV deletion mutants generated via cell culture or in vivo (Heldens et al., 1996; Dai et al., 2000), it could never be excluded that a minor amount of intact helper virus was still present enabling predominant replication of deletion mutants. With the current strategy using bacmids, pure baculovirus mutants were generated from single-copy bacterial artificial chromosomes maintained in E. coli (Luckow et al., 1993; Pijlman et al., 2002). The deleted region spanning ORFs 15–35 contained genes encoding cathepsin, chitinase, GP37, PTP-2, EGT, PKIP-1 and ARIF-1 (IJkel et al., 1999). Cathepsin (Se16) and chitinase (Se19) are involved in the liquefaction of baculovirus-infected insects, but virus lacking these genes was not less effective in secondary infections either in vivo or in cell culture (Hawtin et al., 1997). GP37 (Se25) may be a chitin-binding protein and is homologous to spindolin and entomopoxvirus fusolin genes, but it appears to be non-essential for baculovirus replication (Cheng et al., 2001). PTP-2 (Se26) is a protein tyrosine phosphatase and is not essential for DNA replication (Li & Miller, 1995). EGT (Se27) encodes a non-essential ecdysteroid UDP-glucosyltransferase that is involved in the moulting of insect larvae (O'Reilly & Miller, 1990). Pkip-1 (Se32) and arif-1 (Se34) are located within the genomic region studied in this paper, containing the genes required for oral infectivity (ORFs 29–35). Pkip-1 is a late gene that has been found to play an essential role in AcMNPV DNA replication by stimulating the activity of the viral protein kinase-1 (Fan et al., 1998), but has now been shown to be non-essential in SeMNPV. A temperature-sensitive mutant, defective in pkip, lacks the ability to form plaques and OBs at non-permissive temperatures (McLachlin et al., 1998), but we found normal OB production with SeMNPV pkip deletion mutants. ARIF-1 has been identified as an early AcMNPV gene product that is involved in remodelling of the actin cytoskeleton of the infected cell (Dreschers et al., 2001), but is also non-essential and not required for oral infectivity.

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 15–35 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 15–35 may be advantageous with regard to its biological safety. Furthermore, by deliberate site-specific deletion of ORFs 15–34, the intrinsic genetic instability of the SeMNPV genome in cell culture can be eliminated, while the virus is still infectious per os. Therefore, the SeBACph15–34 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.


   ACKNOWLEDGEMENTS
 
The authors would like to thank Els C. Roode and Magda Usmany for insect rearing and cell culture maintenance. We acknowledge Sandra W. M. Janssen for help with the infectivity assays. This research was supported by the Technology Foundation STW (grant no. 790-44-730), applied science division of NWO and the technology programme of the Dutch Ministry of Economic Affairs.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 30 January 2003; accepted 2 April 2003.