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:
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. (1984)
Polydnaviridae – a proposed family of insect viruses with seg-
mented, double-stranded, circular DNA genomes. Intervirology
21, 1–4.
2. Fleming, J.G.W. & Summers, M.D. (1991) Polydnavirus DNA is
integrated in the DNA of its parasitoid wasp host. Proc. Natl
Acad.Sci.USA88, 9770–9774.
3. Xu, D. & Stoltz, D.B. (1991) Evidence for chromosomal location
of polydnavirus DNA in the ichneumonid parasitoid Hyposoter
fugitives. J. Virol. 65, 6693–6704.
4. Gruber, A., 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 stages of the female. J. Gen. Virol. 77, 2873–2879.
5. Stoltz, D.B. (1993) The polydnavirus life cycle. In Parasites and
Pathogens of Insects, Vol. 1 (Beckage, N.E., Thompson, S.N. &
Federici, B.A., eds), pp. 23–57. Academic Press, New York.
6. Summers, M.D. & Dib-Hajj, S.D. (1995) Polydnavirus-facilitated
endoparasite protection against host immune defenses. Proc. Natl
Acad.Sci.USA92, 29–36.
7. Webb, B.A. (1998) Polydnavirus biology, genome structure, and

evolution. In The Insect Viruses (Miller, L.K. & Ball, L.A., eds),
pp. 105–139. Plenum Press, New York.
8. Fleming, J.G.W. & Krell, P.J. (1993) Polydnavirus genome
organization. In Parasites and Pathogens of Insects,Vol.1
(Beckage, N.E., Thompson, S.N. & Federici, B.A., eds), pp. 189–
225.AcademicPress,NewYork.
9. Stoltz, D.B. (1986) Interaction between parasitoid-derived prod-
ucts and host insects: an overview. J. Insect Physiol. 32, 347–350.
10. Strand, M.R. & Pech, L.L. (1995) Immunological basis for com-
patibility in parasitoid-host relationship. Ann. Rev. Entomol. 40,
31–56.
11. Davies, D.H. & Vinson, S.B. (1986) Passive evasion by eggs of the
braconid parasitoid Cardiochiles nigriceps from encapsulation in
vitro by haemocytes of host Heliothis virescens. J. Insect Physiol.
32, 1003–1010.
12. Hayakawa, Y. (1994) Cellular immunosuppressive protein in the
plasma of parasitized insect larvae. J. Biol. Chem. 269, 14536–
14540.
13. Strand, M.R. & Pech, L.L. (1995) Microplitis demolitor poly-
dnavirus induces apoptosis of a specific hemocyte morphotype in
Pseudoplusia includens. J. Gen. Virol. 76, 283–291.
14. Fleming, J.G.W., Blissard, G.W. & Summers, M.D. (1983)
Expression of Campoletis sonorensis virus in the parasitized host,
Heliothis virescens. J. Virol. 48, 74–78.
15. Stoltz, D.B., Guzo, D., Belland, E.R., Lucarotti, C.J. &
Mackinnon, E.A. (1988) Venom promotes uncoating in vitro and
persistence in vivo of DNA from a braconid polydnavirus. J. Gen.
Virol. 69, 903–907.
16. Blissard, G.W., Theilmann, D.A. & Summers, M.D. (1989) Seg-
ment W of Campoletis sonorensis virus: expression, gene products,

and organization. Virology 169, 78–89.
17. Strand, M.R., Mckenzie, D.I., Grassl, V., Dover, B.A. & Aiken,
J.M. (1992) Persistence and expression of Microplitis demolitor
polydnavirus in Pseudoplusia includens. J. Gen. Virol. 73,
1627–1635.
18. Li, X. & Webb, B.A. (1994) Apparent functional role for a
cysteine-rich polydnavirus protein in suppression of the insect
cellular immune response. J. Virol. 68, 7482–7489.
19. Webb, B.A. & Luckhart, S. (1994) Evidence for an early
immunosuppressive role for related Campoletis sonorensis venom
and ovarian protein in Heliothis virescens. Arch. Insect Biochem.
Physiol. 26, 147–163.
20. Ratcliffe, N.A. (1993) Cellular defense responses of insect: unre-
solved problems. In Parasites and Pathogens of Insects,Vol.1
(Beckage, N.E., Thompson, S.N. & Federici, B.A., eds), pp. 267–
304.AcademicPress,NewYork.
21. Hayakawa, Y. & Yazaki, K. (1997) Envelope protein of parasitic
wasp symbiont virus, polydnavirus, protects the wasp eggs from
cellular immune reactions by the host insect. Eur. J. Biochem. 246,
820–826.
22. Hayakawa, Y. (1990) Juvenile hormone esterase activity repressive
factor in the plasma of parasitized insect larvae. J. Biol. Chem. 265,
10813–10816.
23. Hayakawa, Y., Yazaki, K., Yamanaka, A. & Tanaka, T. (1994)
Expression of polydnavirus genes from the parasitoid wasp Cotesia
kariyai in two noctuid hosts. Insect Mol. Biol. 3, 97–103.
24. Pringle, J.W.S. (1938) Proprioception in insects. J. Exp. Biol. 15,
101–103.
25. Chomczynski, P. & Sacchi, N. (1987) Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform

extraction. Anal. Biochem. 152, 156–159.
26. Hayakawa, Y. & Noguchi, H. (1998) Growth-blocking peptide
expressed in the insect nervous system: cloning and functional
characterization. Eur. J. Biochem. 253, 810–816.
27. Laemmli, U.K. (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227, 680–685.
28. Burnette, W.N. (1981) Western blotting: electrophoretic transfer
of proteins from sodium dodecyl sulfate-polyacrylamide gels to
unmodified nitrocellulose and radiographic detection with anti-
body and radioiodinated protein A. Anal. Biochem. 112, 195–203.
29. Hiraoka, T., Hayakawa, Y. & Downer, R.G.H. (1995)
Immunocytochemical localization of trehalase inhibitor in some
insect species. Cell Tissue Res. 279, 465–468.
Ó FEBS 2002 Polydnavirus immunoevasive proteins (Eur. J. Biochem. 269) 2565
30. Harlow, E. & Lane, D. (1988) Antibodies: a Laboratory Manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
31. Tauz, D. & Pfeifle, C. (1989) A non-radioactive in situ
hybridization method for the localization of specific RNAs in
Drosophila embryos reveals translational control of the segmen-
tation gene Hunchback. Chromosoma 98, 81–85.
32. Wilcox, J.N. (1983) Fundamental principles of in situ hybridiza-
tion. J. Histochem. Cytochem. 41, 1725–1733.
33. Hayakawa, Y., Ohnishi, A., Mizoguchi, A. & Yamashika, C.
(2000) Distribution of growth-blocking peptide in the insect cen-
tral nervous tissue. Cell Tissue Res. 300, 459–464.
34. Yamanaka, A., Hayakawa, Y., Noda, H., Nakashima, N. &
Watanabe, H. (1996) Characterization of polydnavirus-encoded
mRNA in parasitized armyworm larvae. Insect Biochem. Mol.
Biol. 26, 529–536.
35. Norton, W.N. & Vinson, S.B. (1983) Correlating the initiation of

virus replication with a specific pupal developmental phase of an
ichneumonid parasitoid. Cell Tissue Res. 231, 387–398.
36. Deng, L., Stoltz, D.B. & Webb, B.A. (2000) A gene encoding
polydnavirus structural polypeptide is not encapsidated. Virology
269, 440–450.
37. Asgari, S., Theopold, U., Wellby, C. & Schmidt, O. (1998) A
protein with protective properties against the cellular defense
reactions in insects. Proc. Natl Acad. Sci. USA 95, 3690–3695.
38. Strand, M.R., Witherell, A.R. & Trudeau, D. (1997) Two
Microplitis demolitor polydnavirus mRNAs expressed in hemo-
cytes of Pseudoplusia includens contain a common cysteine-rich
domain. J. Virol. 71, 2146–2156.
39. Dib-Hajj, S.D., Webb, B.A. & Summers, M.D. (1993) Structure
and evolutionary implication of a ı
`
cysteine-richıˆ Campoletis
sonorensis polydnavirus gene family. Proc. Natl Acad. Sci. USA
90, 3765–3769.
40. Ancsin, J.B. & Kisilevsky, R. (1996) Laminin interactions
important for basement membrane assembly are promoted by zinc
and implicate laminin zinc finger-like sequeneces. J. Biol. Chem.
271, 6845–6851.
2566 K. Tanaka et al. (Eur. J. Biochem. 269) Ó FEBS 2002