Báo cáo Y học: Detailed characterization of polydnavirus immunoevasive proteins in an endoparasitoid wasp doc

Detailed characterization of polydnavirus immunoevasive proteins in an endoparasitoid wasp Kohjiro Tanaka, Hitoshi Matsumoto and Yoichi Hayakawa Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan Polydnaviruses are a unique group of insect viruses in terms of their obligate and symbiotic associations with some parasitic wasps. The Cotesia kariyai polydnavirus (CkPDV) replicates only in ovarian calyx cells of C. kariyai female wasps and is injected into the wasp’s host, the armyworm Pseudaletia separata, along with the eggs. A previous study indicated the possibility that one of the CkPDV surface proteins mediates immunoevasion by the wasp from the encapsulation reaction of the host insect’s hemocytes. This protein was named immunoevasive protein (IEP). The pre- sent studies substantially confirmed the previous observation by showing that an anti-IEP IgG neutralizes immunoevasive activity on the wasp eggs. Further, we isolated the IEP homologue (IEP-2) cDNA and IEP (IEP-1) cDNA, sequenced them and found that both are cysteine-rich proteins, each containing epidermal growth factor (EGF)- like repeats. IEP genes were not found to reside in the CkPDV genome, but in the wasp chromosomal DNA. IEPs are synthesized in the female reproductive tract and their expression was detected from 4 days after pupation, 1 day later than expression of the virus capsid proteins. In situ hybridization and immunocytochemistry indicated that the lateral oviduct cells of the reproductive tracts produce IEP-1/ IEP-2 mRNAs and secrete the proteins into the oviduct. These data suggest that the expression pattern and local- ization of IEPs are different from other components of CkPDV virions. Keywords: parasitic wasps; polydnavirus; envelope protein; symbiosis; immunoevasion. Endoparasitoid wasps oviposit into the host insects. During oviposition, the wasps inject several factors from the oviduct and accessory glands. Early studies led to the discovery of a particulate fraction in the lumen of the oviducts of several species of the wasps within the families Ichneumonidae and Braconidae. The particles in many species are now recog- nized as polydnaviruses, a family of viruses distinguished by their multiple superhelical DNA genomes [1]. Polydnavi- ruses also have another distinct character: they exhibit an unusual relationship to two insects, an endoparasitoid wasp and its host. They are symbiotically associated with the wasps where they are integrated into the chromosomal DNA of male and female wasps and are transmitted vertically as an endogenous provirus [2–4]. They replicate only in specialized cells (calyx cells) of the female wasp reproductive tract, and are introduced into the host during oviposition [5–7]. In the parasitized host, they do not replicate but they induce a variety of physiological changes in immunity and development through expression of viral genes [7,8]. Alteration of the wasp’s host physiology contributes to survival of the wasp eggs and larvae. Hence, polydnaviruses are unique in terms of their obligate mutualistic association with their hosts, parasitoid wasps [9]. Although a variety of immunosuppressive phenomena have been reported in parasitized hosts, they are generally divided into two modes, passive and active mechanisms [10]. Passive mechanisms include developing in locations that protect the parasitoid from encapsulation or possessing surface features that prevent the host from recognizing the parasitoid as nonself [11]. Active mechanisms refer to a parasitoid disrupting one or more distinct elements of the host immune system [12]. Several pronounced immunolo- gical changes are concomitant with viral gene expression in parasitized hosts; for example, apoptosis of granulocytes, a major subclass of insect blood cells, is associated with the gene expression of Microplitis demolitor PDV [13]. PDV gene expression can usually be detected from 2–4 h postparasitization even though the expression continues over the period required for the parasitoid progeny to complete its development [14–17]. It is therefore apparent that the PDV-induced immunosuppression occurs through alteration of host hemocytes and correlates with PDV gene expression [10,18]. Once the parasitoid wasps lay eggs in the host insects, the eggs should be targeted by the host immune system because the cellular encapsulation reaction against foreign objects can be very rapid; cellular encapsulations have been observed within 10–15 min in some insects [19,20]. How- ever, even at the stage immediately after oviposition, the wasp eggs are protected against cellular encapsulation, indicating the presence of an early protective mechanism that is not associated with PDV transcripts. A previous study revealed that the protein on the Cotesia kariyai PDV virion might have immunoevasive activity against the host armyworm defence reaction [21]. Therefore, PDVs may contribute to both passive and active mechanisms of the immunoevasion. Correspondence to Y. Hayakawa, Institute of Low Temperature Science, Hokkaido University, Kita-19, Nishi-8, Kita-ku, Sapporo, 060-0819, Japan. Fax: + 81 11 706 7142, Tel.: + 81 11 706 6880, E-mail: hayakawa@orange.lowtem.hokudai.ac.jp Abbreviations: EGF, epidermal growth factor; IEP, immunoevasive protein; PDV, polydnavirus; CkPDV, Cotesia kariyai polydnavirus. (Received 11 December 2001, revised 26 March 2002, accepted 9 April 2002) Eur. J. Biochem. 269, 2557–2566 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02922.x Here, we present further strong evidence that the CkPDV surface protein protects wasp eggs from cellular encapsu- lation, thereby indicating that naming of immunoevasive protein (IEP) for the protein is reasonable. Furthermore, through extensive characterization of IEP, we demonstrated that IEP is not encoded by the PDV genome but by the wasp chromosomal DNA, and expression and localization of IEP are strictly regulated but not concomitant with PDV replication. Because characterization of PDV structural proteins has been quite limited at the molecular level, the present studies focused on one of PDV surface proteins may lead to new insights into the unique relationships between PDVs and their parasitoid wasps. MATERIALS AND METHODS Animals Armyworms P. separata, were reared on an artificial diet at 25 °C±1°C with a photoperiod of 16 h light/8 h dark. Parasitization by C. kariyai was carried out by exposing prospective hosts (day 0 last instar larvae) to female wasps. The endoparasitoid wasp, C. kariyai, was reared on the host P. separata under the same conditions. Adult wasps were maintained with honey. Penultimate instar larvae undergo- ing ecdysis between 2 and 2.5 h after lights on were designated as day 0 last instar larvae [22]. Isolation of C. kariyai PDV capsid proteins and IEPs C. kariyai polydnavirus (CkPDV) particles were purified by using sucrose density gradient centrifugation as described in Hayakawa et al. [23]. Purified CkPDV particles were washed five times in NaCl/P i (8 m M Na 2 HPO 4 ,1.5m M KH 2 PO 4 ,137m M NaCl and 2.7 m M KCl, pH 7.2) by sedimentation and resuspension. After washing, the parti- cles were incubated in NaCl/P i containing 1% Nonidet P-40 on ice for 15 min, centrifuged at 11 000 g for 5 min at 4 °C and the pellet was washed three times with 1% Nonidet P-40 in NaCl/P i [19]. The washed pellet was used for the preparation of anti-(CkPDV capsid protein) IgG. IEPs were isolated from the supernatant after the centrifugation by a reversed-phase HPLC with C 4 column (YMC Co., Japan) [21]. Approximately five major proteins other than IEPs were found in the above supernatant fraction, as analyzed by SDS/PAGE. In vivo encapsulation experiments Fifty female wasps were dissected in Pringle’s saline [24], and the ovaries were collected and incubated with venom for 20 min at 25 °C. Eggs were centrifuged at 100 g for 5 min at room temperature. To examine whether IEPs contribute to avoid host hemocytes encapsulation reaction, intact eggs or eggs preincubated with anti-IEP IgG for 30 min were injected into a day 0 last instar larvae. After 12 h, injected eggs were collected and observed under a microscope. Cloning, sequencing and in vitro expression of IEP cDNA Total ovary RNA was isolated from adult female wasps, by the method of Chomczynski & Sacchi [25]. Polyadenylated mRNA was purified by oligo(dT)-cellulose chromatogra- phy (Amersharm Pharmacia). A cDNA library was con- structed using a ZAP-cDNA Synthesis kit (Stratagene) and a Gigapack in vitro packaging kit (Stratagene) according to the manufacturer’s instructions. Based on the N-terminal sequence of 23 amino-acid residues of IEP determined by Hayakawa & Yazaki [21], two primers were synthesized: 5¢-AAGAATTCATHWSN GTNGARAAYGTN-3¢,5¢-AAGAATTCGGYTTNGTN GCRTANGG-3¢. The DNA fragments corresponding to the 23 amino-acid residues were amplified by PCR using these primers and C. kariyai genome DNA as a template. The PCR amplification reaction was conducted according to the method of Hayakawa & Noguchi [26]. The amplified fragments were subcloned into pBluescript KS(–) (Strata- gene) and sequenced by a Taq dye primer cycle sequencing kit (PerkinElmer). DNA sequencing was performed with an automatic DNA sequencer (model 377, PE Applied Biosystems). A 32 P-labeled N-terminal DNA fragment was used to screen the cDNA library. Radiolabelling was performed using the method of random prime labelling kit, ÔReady-To-GoÕ (Amersharm Pharmacia). The cDNA fragment coding for IEP-1 was cloned into the BamHI/XhoI site in pET32 (Novagen) under a lac promoter and transformed into Escherichia coli, BL21(DE3). The production of the protein containing an additional His 6 tag residues was induced by 0.1 m M isopropyl thio-b- D -galactoside for 12 h at 25 °C. The fusion protein found to be insoluble in the inclusion body was sonicated and directly used as an antigen to prepare anti- (IEP-1) IgG. SDS/PAGE and Western-blot analysis To determine the tissue distribution of IEP proteins, heads, thoraces, abdomens and ovariectomized abdomens were collected from adult male or female wasps into liquid nitrogen, and homogenized in NaCl/P i containing 1% SDS. The homogenates were centrifuged at 17 400 g for 10 min to remove cell debris, and the supernatants were used as samples for SDS/PAGE (10%) after incubating with 80 m M Tris/HCl buffer (pH 8.8) containing 1% SDS and 2.5% 2-mercaptoethanol in boiling water for 5 min. The PAGE gels were developed and stained with Coomassie Brilliant Blue R-250 [27]. Protein bands on SDS/PAGE gel were electrically transferred to a poly(vinylidene difluoride) membrane filter, essentially as described by Burnette [28]. Immunostaining with the anti-(IEP-1) IgG was performed using peroxidase conjugated secondary antibodies according to the method of Hiraoka et al. [29]. Polyclonal anti-(IEP-1) IgG was made by immunizing rabbits with the bacteria- expressed IEP-1 in Freund’s complete adjuvant (TiterMax Gold, CytRx Corporation). Polyclonal anti-(CkPDV capsid protein) IgG was prepared in mice by subcutaneous injection of capsid proteins isolated from CkPDV virions in adjuvant. IgGs in the serums were purified by ammonium sulfate fractionation and Affigel-protein A column chromatogra- phy [30]. To compare the expression of IEP proteins with that of CkPDV capsid proteins after pupation, three female reproductive tracts of each developmental stage were homogenized in NaCl/P i containing 1% SDS. Supernatants after centrifugation at 17 400 g for 5 min were used for SDS/PAGE samples after the treatment described above. 2558 K. Tanaka et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Northern-blot analysis Northern-blot hybridization was performed according to a procedure slightly modified from the method of Hayakawa & Noguchi [26]. Total RNA was extracted from adult male heads and thoraces, abdomens, female heads and thoraces, ovari-ectomized abdomens and reproductive tract ovaries. Twenty microgram aliquots of each RNA preparation were electrophoresed in 1% agarose gel and transferred to a nylon membrane (Hybond N + , Amersharm Pharmacia) according to the manufacturer’s instructions. Hybridization was carried out using a 32 P-labeled IEP-1 cDNA fragment (1088 bp) as a probe in hybridization solution (5 · NaCl/Cit, 5 · Denhardt’s reagent, 50% formamide, 0.1% SDS, 100 lgÆmL )1 of denatured salmon sperm DNA) for 16 h at 42 °C. After hybridization, the membrane were washed with 2 · NaCl/Cit, 0.1% SDS at 60 °Cfor 30 min, followed by 0.1 · NaCl/Cit, 0.1% SDS at 60 °C for 30 min. Immunofluorescence microscopy For immunostaining on sectioned preparations, adult female wasp reproductive tracts fixed with Carnoy’s fixative were embedded in paraffin, and 5-lmthick sections were pretreated with 5% nonfat dry milk, incubated with anti-IEP IgG or anti-(CkPDV capsid protein) IgG for 12 h at 4 °C. Fluorescein isothiocyanate (FITC)-conjugated goat anti-(mouse IgG) IgG (Roche Fig. 1. Nucleotide and predicted amino-acid sequences of IEP-1 and IEP-2 cDNAs. (A) Nucleotide sequences of IEP-1 and -2 cDNAs. Nucleotide numbers are on the right. Bold letters indicate the translation initiation codon and the stop codon. The putative polyadeny- lation signal is underlined. (B) The predicted amino-acid sequences of IEP-1 and IEP-2. Numbers of the amino acids are shown to the rightofeachline.ThesequenceoftheN-ter- minal 25 amino acid residues (IEP-1) deter- mined in the prior study [21] is underlined. The amino acid sequence (10 amino acids) of IEP-2 determined in the present study is also underlined. Potential Asn-glycosylation sites are double underlined. Predicted signal pep- tide sequences are in italic type. Searching various databases for sequence similarity revealed two (in IEP-1) and three (in IEP-2) EGF-like motifs (Cx 0)48 Cx 3)12 Cx 1)70 Cx 1)6 Cx 2 Gax 0)21 Gx 2 Cx; G, often conserved glycine; a, often conserved aromatic acid; x, any residue) dotted underline [40]. Gaps have been introduced for proper alignment of the conserved cysteine residues. Ó FEBS 2002 Polydnavirus immunoevasive proteins (Eur. J. Biochem. 269) 2559 Diagnostics) and rhodamine-conjugated goat anti-(rabbit IgG) IgG (Biosource International, Inc.) were used for detection of the capsid proteins and IEPs, respectively. In situ hybridization analysis In situ hybridization was performed on paraformaldehyde- fixed whole adult female reproductive tracts [31] and paraformaldehyde-fixed sections of the reproductive tracts [32]. Antisense and sense control transcripts were synthes- ized from a IEP-1 cDNA template with digoxigenin (DIG) using a DIG RNA labeling kit (Roche Diagnostics) [33]. For the detection of DIG-labeled RNA, 5-bromo-4-chloro- 3-indolylphosphate and nitroblue tetrazolium salt were used as substrates for alkaline phosphatase conjugated with anti- DIG IgG. Fig. 2. Immunodetective analyses of C. kariyai polydnavirus and immunoevasive activity of IEPs. (A) Left, immunogold staining of CkPDVs on the wasp egg with anti-IEP IgG. Right, control run stained with IgG from nonimmunized rabbit. (B) Encapsulated egg which was treated with anti-IEP IgG prior to injection and recovered from the armyworm after 12 h (C) Intact egg which was dissected from wasp ovary was injected, and recovered from the armyworm after 12 h (D) After being thoroughly washed (insert), the egg was coated with purified IEPs prior to injection and recovered from the armyworm after 12 h. Note that the egg encapsulated by hemocytes are melanized as indicated with arrows. More than 90% of anti-IEP IgG-treated eggs were encapsulated as shown here. The intact egg and the egg with IEPs coated are attached on muscle and fat body, respectively. 2560 K. Tanaka et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Genomic southern-blot analysis CkPDV genome DNA was extracted by the method of Yamanaka et al. [34]. For preparing wasp chromosomal DNA, male and female pupae were frozen by liquid nitrogen and powdered by using a motor-driven pestle. Chromosomal DNA was extracted with DNAzol (Gibco-BRL) according to the instruction manual. The extracted DNA was restricted by various restriction enzymes, electrophoresed in 1% agarose gel and was transferred to nylon membrane (Hybond N + , Amersharm Pharmacia). The transferred membrane was probed with 32 P-labeled IEP-1 cDNA (1088 bp) in hybridization solution (5 · NaCl/Cit, 5· Den- hardt’s reagent, 0.1% SDS, 100 lgÆmL )1 of denatured salmon sperm DNA) for 16 h at 60 °C. After hybridization, themembranewerewashedwith2 · NaCl/Cit, 0.1% SDS at 60 °C for 30 min, followed by 0.1 · NaCl/Cit, 0.1% SDS at 60 °C for 30 min. Microscopic observation Eggs embedded in LR-Gold (London Resin, Surrey, UK) were thin-sectioned with glass knives and placed on Formvar-coated nickel grids (100 mesh). Specimens were rinsed with NaCl/P i , treated with 10% fetal bovine serum, in NaCl/P i for 1 h, and incubated for 2 h at room temperature with anti-IEP IgG or preimmune IgG (1 : 500) with 10% fetal bovine serum in NaCl/P i .Afterathoroughwashingin NaCl/P i , the specimens were incubated for 1 h at room temperature in goat anti-(rabbit IgG) IgG conjugated to colloidal gold particles (15 nm, British Biocell International Ltd) (1 : 100 dilution), with 10% fetal bovine serum. The grids were washed with NaCl/P i and distilled water, and then dried. Finally, they were stained with uranyl acetate. Ovaries were dissected from the second and third days after pupation and fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4 °C. Postfixation was per- formed in 1% aqueous OsO 4 . The tissue was embedded in Epon 812 (TAAB Laboratories Equipment Ltd, England) after dehydration. Thin sections were cut on an Ultracut microtome (Reichert-Jung, Germany). For electron micros- copy, thin sections were briefly stained in 2% aqueous uranyl acetate and 0.1% lead citrate. Micrographs were taken with a JEM-1200EX (Jeol Ltd, Japan) electron microscope. RESULTS Finding of IEP homolog To elucidate a primary structure of IEP, cDNAs encoding IEP were cloned by a combination of PCR and cDNA library screening. The PCR-derived cDNA fragment for the amino terminal peptide was used to screen cDNA library in kZAP II. Five positive clones were isolated with inserts of similar size (1.1 kbp). Complete sequencing of the inserts in these clones revealed that four of them contain the same 278 residue ORF whose N-terminus in the deduced amino-acid sequence is identical with that of IEP as shown in Fig. 1A. One of five clones contained a slightly different ORF, whose deduced amino-acid sequence was a 272-residue sequence 85% identical with IEP, thereby suggesting that these are homologs. The former and latter proteins were named IEP-1 and IEP-2, respectively. The amino-acid sequences of IEP-1 and IEP-2 include 26- and 29-residue predicted signal peptides, respectively, and a mature protein region rich in cysteine residues (about 12%). The mature protein region consists of a tandem repeat of the six cysteine residues with a spacing similar to other EGF-like motifs as shown in Fig. 1B. Further, three potential sites for Asn-glycosylation were found in the region. To produce anti-IEP IgG, the IEP-1 cDNA, with an N-terminal His 6 tag, was expressed in E. coli for use as an antigen. This antibody detects IEP-1 and IEP-2 well by immunoblot analysis. IEP is an CkPDV surface protein with an immunoevasive activity Immunoelectron microscopic observation suggests IEPs are present on the surface of the C. kariyai polydnavirus (CkPDV) particle (Fig. 2A). Furthermore, as it also showed Fig. 3. Tissue distribution of the IEP proteins and corresponding mRNA. (A) Western-blot analyses were performed with anti-IEP IgG. Immunostaining of male and female wasp tissue extract with anti-IEP IgG. m.h., male heads; m.t., male thoraces; m.a., male abdomens; ov., ovaries; f.h., female heads; f.t., female thoraces; f.a., female abdomens. (B) Northern-blot analysis of IEP mRNA. Twenty micrograms total RNA prepared from each tissue were separated on a 1% agarose gel. 32 P-Labeled IEP-1 cDNA fragment was used as a hybridization probe. Actin mRNA was shown below. m.h. & t, male heads and thoraces; m.a., male abdomens; f.h. & t., female heads and thoraces; f.a. (n), ovariectomized female abdomens; ov., ovaries. Ó FEBS 2002 Polydnavirus immunoevasive proteins (Eur. J. Biochem. 269) 2561 that the CkPDV particles with IEPs coated and IEPs themselves are attached to the surface of the parasitic wasp egg, it was of interest to determine whether IEPs contribute to avoiding host recognition of eggs as foreign. If IEPs serve as an immunoevasive mediator, wasp eggs coated with IEPs should not be encapsulated by hemocytes of the wasp’s host. In order to examine this possibility, intact and anti-(IEP-1) IgG-pretreated wasp eggs were injected into last instar larvae of the armyworm Pseudaletia separata, andthenthe surfaces of both types of the eggs were observed for 12 h after injection. As shown in Fig. 2B–D, eggs pretreated with anti-(IEP-1) IgG were clearly encapsulated (Fig. 2B), but intact eggs and the eggs with IEPs coated were protected from the encapsulation (Fig. 2C,D), thereby indicating that the presence of IEPs on the eggs is essential for the wasps to evade encapsulation by host hemocytes. IEP mRNA expression initiates 1 day after CkPDV starts to express Because IEPs are present on the surface of the PDV virions, it is interesting to examine whether IEP expres- sion occurs in the reproductive tracts of female wasps and the expression is synchronized with the replication of CkPDV virions. Immunoblotting showed that IEP pro- teins were present in the reproductive tracts of adult female wasps but not in other tissues of female wasps or any tissue of male wasps (Fig. 3A). Results of northern- blot analyses are consistent with those of the immuno- blotting; IEP-1 mRNAs are only expressed in the ovary (Fig. 3B). To obtain an overview of IEP and CkPDV capsid protein expression in female wasps after pupation, protein samples extracted from each developmental stage of three female reproductive tracts were probed with anti-(IEP-1) IgG or anti-(CkPDV capsid protein) IgG. The western-blots show that capsid proteins could be detected from day three (Fig. 4A), while IEP from day four (Fig. 4B). To deny the possibility that this difference in the both expression timing may be due to differences in the qualities of both antibodies, the ovary extracts prepared from second to fourth day after pupation were analyzed by a reversed phase HPLC. The results are consistent with those of the western-blots; the IEP protein peak is visible from fourth day after pupation, while the capsid protein peak is detectable from the third day after pupation (data not shown). Furthermore, Fig. 4. Expression of IEPs and CkPDV capsid proteins after pupation. For both SDS/PAGE and western-blot analyses, protein samples prepared from three female reproductive tractswereappliedontoeachlane. (A) Immunostaining with anti-(CkPDV capsid protein) IgG. (B) Immunostaining with anti-IEP IgG. Lane 1–6, reproductive tracts collected from pupae at 1st, 2nd, 3rd, 4th, 5th and 6th day after pupation. Lane 7, repro- ductive tracts collected from 1st day of adult female wasp. (C) Electron microscopic obser- vations of CkPDV particles in the nuclei of the ovarian calyx cells of 2nd (2nd) and 3rd (3rd) day female wasps after pupation. Note that CkPDV particles are produced in the calyx cell nucleus and become visible from the 3rd day (indicated with white arrow heads) when the capsid proteins can be detected. Fig. 5. Localization of C. kariyai IEPs and capsid proteins. Immunodetective analyses of IEPs (A) and capsid proteins (B) in the same section of female reproductive tract. White arrows indicate the calyx cells cross-reacted with the antibody [anti-(CkPDV capsid protein) IgG]. Note that only anti-(CkPDV capsid protein) IgGs cross-reacted with the calyx cells, although both antibodies [anti-IEP IgG and anti-(CkPDV capsid protein) IgG] cross-reacted with the calyx fluid regions. (C) In situ hybridization of whole female reproductive tracts to localize expression of IEP mRNA using a digoxigenin labeled antisense IEP-1 mRNA. (D) Control in situ hybridization of reproductive tracts using the sense probe. Note that IEP-1 mRNA is expressed in the bluish region of the lateral oviducts shown in (C). Dark bluish staining of the venom glands seems to be nonspecific. (E) In situ hybridization of sections of female reproductive tracts using the antisense probe. (F) Control run using the sense probe. Note that IEP-1 mRNA is not transcribed in the calyx cells but abundantly transcribed in the lateral oviduct cells. 2562 K. Tanaka et al. (Eur. J. Biochem. 269) Ó FEBS 2002 electron microscopy was conducted to examine at which stage CkPDV virions first appear in the calyx cells. As shown in Fig. 4C, the virions are not detectable in the calyx cells at second day after pupation, but are visible in the nucleus on the calyx cells at third day after pupation. Therefore, the capsid protein expression and the virus particle formation initiates one day before the IEP expression. Ó FEBS 2002 Polydnavirus immunoevasive proteins (Eur. J. Biochem. 269) 2563 IEP expresses in the oviduct cells of wasp reproductive tract It has been reported that the PDV replication is detected only in the ovarian calyx cells of female wasps [5,7]. In fact, we observed CkPDV virions in the calyx cells of C. kariyai female reproductive tracts (K. Tanaka, H. Matsumoto & Y. Hayakawa, unpublished result). Furthermore, we con- firmed that capsid proteins of CkPDV are present in the calyx cells (Fig. 5A). To examine whether IEPs are also synthesized in the calyx cells, immunocytochemical and in situ hybridization analyses were carried out. Unexpect- edly, the calyx cells were not immunoreactive but the inside of the oviduct was strongly immunoreactive (Fig. 5B). A whole-mount in situ hybridization confirmed the immuno- chemical observation that IEP expression is localized in the lateral oviduct end region adjacent to the calyx cells (Fig. 5C). A sectioned in situ hybridization clearly visualized the lateral oviduct cells expressing IEP-1 mRNAs (Fig. 5E). These results were interpreted to indicate that, although IEPs are present on the PDV virions, IEP genes and their expression are independent from the PDV gene expression or the PDV replication. IEP genes are present only in the host chromosomal DNA To assess the location of genes encoding IEP, PDV genomic DNA and wasp chromosomal DNA were tested on a southern-blot using the IEP-1 cDNA fragment as a probe. Although no significant hybridization signal was detected in the viral DNA, the probe hybridized with the chromosomal DNAs from both male and female wasps (Fig. 6). These results clearly demonstrate that the IEP genes do not reside in the virus genomic DNA but in the wasp chromosomal DNA. In the light of these data including in situ hybridiza- tion and genomic-Southern blot analyses, it is possible to assume that, although IEPs attach to the CkPDV virions and function as immunoevasive surface proteins of CkPDVs, the site and pattern of the IEP gene expressions are completely different from those of the other PDV component genes. DISCUSSION Polydnaviruses (PDVs) are obligate symbionts with certain parasitic wasps in the families Ichneumonidae and Bracon- idae. PDVs replicate only in ovarian calyx cells of the female wasps. Despite the absence of replication in the parasitized hosts, PDVs induce an array of physiological alterations that are likely essential for survival of the wasp’s progeny. Of particular importance are the immunosuppressive effects PDVs have on the hosts. A previous study revealed that Cotesia kariyai PDV (CkPDV) possesses surface features that prevent the wasp’s host from recognizing the parasitoid as nonself [21]. Based on the observation that glass capillaries precoated with one of the CkPDV surface proteins were not encapsulated, we thought that this protein with a molecular weight of approximately 50 kDa may serve as an immunoevasive mediator, and thereby named it immunoevasive protein (IEP). In the present study, we confirmed the immunoevasive activity of IEP for wasp eggs by a strong evidence that wasp eggs neutralized with anti- IEP IgG were extensively encapsulated (Fig. 2). Although the molecular mechanism by which IEP protects wasp eggs from being encapsulated is not totally resolved, we speculate that the armyworm (wasp’s host) hemolymph proteins having antigenic similarities may be responsible for a molecular disguise [21]. CkPDVs replicate from integrated proviral DNA only in the calyx cells located at the junction of the ovary and oviduct of the female wasps, as seen in Campoletis sonorensis PDV [35]. Therefore, we expected that IEP transcripts should be present in the calyx cells, even though IEPs are not encoded by the PDV genome but by the wasp chromosomal DNA. However, IEP-1 mRNAs and proteins were observed only in the oviduct cells and inside of the oviduct, respectively, instead of the calyx cells. Further, the expression of IEP proteins is not synchronized with that of CkPDV capsid proteins; IEP proteins were observed approximately one day after the initiation of the capsid protein expression in the pupal stage (Fig. 4). These data suggest that characteristics of IEPs are a typical for PDV structural proteins. Reports on PDV structural proteins are quite limited but the PDV envelope-like and surface proteins have been character- ized. Both the proteins, p44 in Campoletis sonorensis PDV [36] and Crp32 in Cotesia rubecula PDV [37], are encoded by the wasp genome, and expressed in calyx cells. Deng et al. suggested the possibility of the evolutionary transfer of p44 gene from the viral genome to the host wasp genome as seen in mitochondrial genes [36]. Crp32 protects Cotesia rubecula eggs from the host defense by attaching to the surface of the eggs and PDV virions [37]. Fig. 6. Localization of the IEP gene. Genomic southern-blot analysis was performed using 32 P-labeled IEP-1 cDNA as a probe. Chromoso- mal DNAs (each 10 lg) extracted from female or male pupa, and genomic DNA (5 lg) purified CkPDV, were applied onto each well after treating with a restriction enzyme (EcoRI, MspIandXhoI). 2564 K. Tanaka et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Therefore, Crp32 and IEPs have a similar function but no sequence exists among two proteins. An EGF-related gene family has been found in the Microplitis demolitor PDV genome [38]. Each of these gene products contains a single EGF-like motif and there is no other significant amino-acid sequence homology of these proteins except within the EGF-like motif. Other PDV gene family containing the cysteine motifs structurally analogous to the motif of conotoxins have also been reported [39]. However, both these and the above EGF-related genes expressed in parasitized insects. Because we could not observe any expression of the IEP transcripts in the parasitized host insects, it is thought that the reported cysteine-rich PDV genes are completely different from the IEP genes. At present, we do not have direct evidence that shows that IEP is one of the CkPDV virion components, such as envelope proteins. Of course, it is possible that IEPs are ovarian proteins of host wasps that may attach to various surfaces such as those of polydnaviruses and eggs. However, it is worth emphasizing that IEPs strongly attach to the surface of the CkPDV particle and appear to behave like a viral envelope protein. Further functional and structural studies of IEPs should lead to a better understanding of the evolutional relationship between PDVs and the host wasps, and also clarify the mechanism by which parasitoid wasps escape from the host defence reactions. ACKNOWLEDGEMENTS We thank Dr Bruce A. Webb (University of Kentucky) for critical comments on the manuscript. We also thanks for Dr Megumi Moriya for technical assistance with the electron microscopic studies. This work was supported by Program for Promotion of Basic Research Activities for Innovative Biosciences (Japan). REFERENCES 1. Stoltz, D.B., Krell, P.J., Summers, M.D. & Vinson, S.B. 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(A) Left, immunogold staining of CkPDVs on the wasp. Stettler, P., Heiniger, D., Schumperli, D. & Lanzrein, B. (1996) Polydnavirus DNA of the braconid wasp Chelonus inanitus is integrated in the wasp s genome and excised only in later pupal and adult