Morphological and Genomic Characterization of the Polydnavirus Associated with the Parasitoid Wasp Glyptapanteles Indiensis (Hymenoptera: Braconidae)

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Journal of General Virology (2003), 84, 2051–2060 DOI 10.1099/vir.0.19234-0

Morphological and genomic characterization of the polydnavirus associated with the parasitoid wasp Glyptapanteles indiensis (Hymenoptera: Braconidae)

Y. P. Chen and D. E. Gundersen-Rindal

Correspondence USDA-ARS Insect Biocontrol Laboratory, Beltsville, MD 20705, USA Dawn Gundersen-Rindal [email protected] Glyptapanteles indiensis polydnavirus (GiPDV) is essential for successful parasitization of the larval stage of the lepidopteran Lymantria dispar (gypsy moth) by the endoparasitic wasp Glyptapanteles indiensis. This virus has not been characterized previously. Ultrastructural studies of GiPDV showed that virions had a rod-like or rectangular form and each contained as many as ten nucleocapsids enclosed by a single unit membrane envelope. Field inversion gel electrophoresis (FIGE) analysis of the virus genomic DNA revealed that GiPDV had a segmented genome composed of 13 dsDNA segments, ranging in size from approximately 11 kb to more than 30 kb. Four genomic segments were present in higher molar concentration than the others. Further characterization of the GiPDV genome yielded several cDNA clones which derived from GiPDV-speciﬁc mRNAs, and Northern blot analysis conﬁrmed expression of isolated cDNA clones in the parasitized host. Each was present on more than one GiPDV genomic DNA segment, suggesting the existence of related sequences among DNA segments. It has been proposed previously that in polydnavirus systems, genome segmentation, hypermolar ratio segments and segment nesting may function to increase the copy number of essential genes and to increase the levels of gene expression in the absence of virus replication. The present data Received 17 March 2003 support this notion and suggest that GiPDV morphology and genomic organization may be Accepted 3 April 2003 intrinsically linked to the function and evolutionary strategies of the virus.

INTRODUCTION The PDVs are recognized as two genera: the ichnoviruses found exclusively in parasitoid wasps of the Ichneumo- Polydnaviruses (PDVs) are complex insect viruses charac- nidae, and the Bracoviruses found only in parasitoid wasps terized by segmented double-stranded circular DNA of the Braconidae. Ichnovirus and Bracovirus differ from genomes that vary in size and molar ratio (Stoltz et al., each other in morphology, host range and molecular 1984). Viral DNA is integrated into the wasp genome as characteristics (Stoltz & Whitfield, 1992). Ichnoviruses provirus and transmitted vertically through the germ-line characteristically contain one lenticular nucleocapsid per (Stoltz et al., 1984, 1986; Fleming & Summers, 1986, 1991; virion surrounded by two unit membranes, whereas Fleming, 1992; Fleming & Krell, 1993; Stoltz, 1993). PDVs bracoviruses characteristically contain one or more rod- replicate only in the nucleus of calyx epithelial cells of the shaped nucleocapsids per virion surrounded by a single wasp ovaries during the pupal stage, and are stored in the unit membrane envelope. It is thought that Ichnoviruses lumen of the oviduct (Norton & Vinson, 1983; Theilmann are released into the ovary lumen by budding from calyx & Summers, 1986). During oviposition, PDVs are injected cells, while bracoviruses, in contrast, are believed to be with eggs into the host, and enter different host tissues. released through lysis of the calyx cells. Within the host tissues, PDVs do not replicate, but host- specific viral genes are expressed. PDV gene products The PDV genome structure is complex and poorly under- function to disrupt the host immune system as well as host stood. Only a few PDV genomes have been described in development, which appear to be essential for successful detail. Estimates of PDV genome sizes have been compli- parasitoid development (Stoltz, 1993; Lawrence & Lanzrein, cated by the characteristic presence of unequal molar ratios 1993, 1994; Lavine & Beckage, 1995; Strand & Pech, 1995). of genomic segments, comigrating DNA segments of equal size, and direct terminal repeats (Fleming, 1992; Fleming & Krell, 1993; Stoltz, 1993). Comparisons of various PDV The GenBank accession numbers of the sequences reported are genomes showed that the DNA segment number and molar AF414845 and AF414846. ratio and the total genome size appeared to be specific to

0001-9234 Printed in Great Britain 2051 Y. P. Chen and D. E. Gundersen-Rindal the PDVs of each individual PDV-containing wasp species parasitization. After a single oviposition was observed, each parasi- (Krell, 1991; Stoltz, 1993; Fleming & Krell, 1993). Descrip- tized larva was removed from the Petri dish and reared in a plastic tions of ichnovirus genomes have been largely based on cup containing high wheat-germ diet for 24 h prior to RNA extraction. studies of PDVs isolated from the parasitoid wasps Virus purification and viral DNA isolation. Virus was purified Campoletis sonorensis (Krell et al., 1982), Hyposoter exiguae from the calyx fluid of female wasps. Female wasps were anaesthe- (Krell & Stoltz, 1980) and Diadegma terebrans (Krell, tized in 75 % ethanol for a few seconds and then rinsed in sterile 1987). Of these ichnoviruses, the C. sonorensis polydnavirus H2O. Each pair of ovaries was dissected in a 15 ml drop of cold PBS (CsPDV) genome has been by far the most systematically and punctured individually with forceps, causing the calyx fluid to diffuse into the PBS drop. The calyx fluid was filtered through a characterized, and appears to contain 28 DNA segments 0?45 mm filter to remove eggs and cellular debris by the methods of ranging in size from 5?5 to 21 kbp. Genome analyses of Beckage et al. (1994). The resulting viral fluid was transferred into a bracoviruses have been less complete than those described 1?5 ml microcentrifuge tube and immediately processed for viral for Ichnoviruses. The available information on bracovirus DNA isolation and electron microscopy of viral ultrastructure. genomes comes mainly from descriptions of the PDVs Electron microscopy of viral particles. For negative staining, associated with the parasitoids Cardiochiles nigriceps GiPDV virus particles in filtered calyx fluid were absorbed onto a (Varricchio et al., 1999), Chelonus inanitus (Albrecht et al., 400 mesh Formvar-coated nickel grid for 2 s, quickly washed in dis- 1994; Johner & Lanzrein, 2002; Wyder et al., 2002) and tilled water, stained with 4 % uranyl acetate for 1 min, and then Microplitis demolitor (Strand et al., 1992). In general, the viewed in an H-7000 Hitachi electron microscope at 75 kV. For thin genome segments of bracoviruses tend to be fewer in sections, embedded ovaries dissected from female G. indiensis were number but larger in size than those of the ichnoviruses, fixed in 3 % glutaraldehyde/0?05 M sodium cacodylate buffer and exhibit a relaxed open circular topology (Albrecht et al., (pH 7?0) for 2 h at room temperature and placed in a 4 uC refri- gerator overnight. After washing in sodium cacodylate buffer six 1994; Webb, 1998). For example, characterization of the times within 1 h, ovaries were post-fixed in 2 % buffered osmium bracovirus of C. inanitus showed that its genome consisted tetroxide for 2 h, dehydrated in graded ethanol solutions and infil- of 10 different segments ranging in size from 7 to 31 kbp, trated with Spurr’s low-viscosity embedding resin. Ovaries were and that individual DNA segments appeared to be singly sectioned on a Riechert/AO Ultracut microtome with a Diatome encapsidated (Albrecht et al., 1994). diamond knife. Silver sections of the ovaries were mounted on 200- mesh Ni grids, stained with 4 % uranyl acetate and 3 % lead citrate, The braconid endoparasitic wasp Glyptapanteles indiensis and observed in an H-7000 Hitachi electron microscope at 75 kV. parasitizes the larval stage of the gypsy moth, Lymantria Viral DNA isolation and field inversion gel electrophoresis dispar. Previous studies on G. indiensis polydnavirus (FIGE). Filter-purified viral fluids were incubated in an equal (GiPDV) in our laboratory demonstrated that GiPDV volume of extraction buffer (4 % sarcosyl, 1 % SDS, 50 mM EDTA, could be integrated not only as a provirus within the 10 mM Tris, 0?2 M NaCl, 20 mg RNase ml21,50mg proteinase 2 parasitoid wasp genome, but also in vitro within the Kml 1)at50uC for 2 h, followed by two extractions with ultrapure chromosomal DNA of cells derived from the natural host buffer-saturated phenol (Invitrogen). DNA was precipitated in L. dispar (Gundersen-Rindal & Dougherty, 2000). More 2 vols of cold ethanol and centrifuged at 15 000 r.p.m. for 30 min. The viral DNA pellet was resuspended in TE buffer and stored at recently, a GiPDV putative protein tyrosine phosphatase 4 uC. The purified viral DNA was analysed by FIGE, an electro- gene believed to be associated with gene regulation during phoretic method used to resolve DNAs in the 10–150 kb range. immune response was shown to be differentially expressed Undigested viral DNA (2 mg) and a molecular size standard (DIG- in various tissues of the parasitized host (Chen et al., 2003). labelled linear l DNA digested with HindIII) were loaded onto a Although information on the in vitro and in vivo properties 0?8 % (w/v) pulse field certified agarose gel (Bio-Rad) in 0?56 TBE of GiPDV has been gathered, fundamental knowledge buffer (50 mM Tris/borate, 1 mM EDTA, pH 8?3). Gel electrophoresis was performed at 4 uC for 2624 h using a 0?1–0?4 s switch pertaining to GiPDV virion morphology and genomic time ramp with 180 V forward voltage and 120 V reverse voltage for organization remains undescribed. To fill this gap, the each 24 h run. The gels were stained with ethidium bromide for morphological features, genomic organization and mole- 15 min, destained with sterile H2O for 30 min, and visualized using cular features of GiPDV were investigated and are reported a UV transilluminator. here. The isolation of cDNA clones representing GiPDV cDNA library construction and screening. Total RNA was iso- sequences from mRNA isolated from parasitized host and lated from parasitized L. dispar larvae 24 h post-parasitization. confirmation of the expression of cDNA clones in the Insects were frozen in liquid nitrogen and ground to a fine powder. parasitized host are described. Further, mapping of these RNA was extracted with TRIzol reagent (Invitrogen) according to cDNA fragments to the GiPDV genome is shown. the manufacturer’s instructions. RNA samples were resuspended in nuclease-free water in the presence of ribonuclease inhibitor (Invitrogen). The RNA quality was verified by formaldehyde gel METHODS electrophoresis, and mRNA was isolated from total RNA using the Messagemaker Reagent Assembly kit (Life Technologies) following Insect rearing. The parasitoid wasp was reared from larvae of the the manufacturer’s instructions. The concentration of mRNA was host insect, L. dispar. Adult wasps were fed with 30 % honey water. measured using a spectrophotometer. cDNA libraries were prepared Both G. indiensis and its host were maintained at 26 uC, 50 % rela- using the Superscript Choice System for cDNA Synthesis (Invitrogen) tive humidity and a 16 h light : 8 h dark photoperiod according to following the manufacturer’s instructions. mRNA was converted into the methodology established by Bell et al. (1981). To obtain parasi- size-fractionated, EcoRI-adapted cDNA. The cDNA was ligated into tized larvae, individual L. dispar larvae were exposed to a single the EcoRI-digested lZIPLOX expression vector (Life Technologies). G. indiensis female within a 35610 mm Petri dish to ensure 100 % The ligated vector-cDNAs were packaged in Gigapack III Gold

2052 Journal of General Virology 84 Glyptapanteles indiensis polydnavirus

Packaging Extract (Stratagene). The packaged cDNA-containing Genome mapping of cDNAs. Southern hybridization was per- phage were introduced into Y1090 (ZL) E. coli that had been grown formed to map the two isolated cDNA fragments to a specific overnight in LB medium with 10 mM MgSO4 and 0?2 % maltose. region(s) of the GiPDV genome. FIGE-separated viral genomic DNA The transformants were plated on LB-agar and incubated overnight segments were transferred from the gel to nylon membrane at 37 uC. The cDNA libraries were titrated and amplified, and (Hybond-N+; Amersham Pharmacia) and UV cross-linked for ana- approximately 56104 p.f.u. of library were plated onto ten large LB lysis. cDNA clones were DIG-labelled using the DIG DNA labelling plates. The plaques were transferred to nitrocellulose membrane kit (Roche) according to the manufacturer’s instructions. Mem- (Roche) and the phage DNAs were denatured, neutralized and UV branes with separated GiPDV genomic segments were hybridized cross-linked to membrane according to standard procedures. The with a DIG-labelled cDNA probe in DIG Easy Hybridization solu- GiPDV genomic DNA used as a probe was radiolabelled to high tion (Roche) for 16 h at 48 uC. After hybridization, the membranes specific activity with the Random Primers DNA Labelling System were washed twice in low stringency wash solution (26 SSC, 0?5% (Invitrogen) following the manufacturer’s instructions. Hybridization SDS) at room temperature for 5 min and washed twice in high screening of cDNA libraries using the 32P-labelled GiPDV probe was stringency wash solution (0?16 SSC, 0?5 % SDS) at 48 uC for carried out in the hybridization solution (66 SSPE, 0?5 % SDS, 56 15 min. After the washes, anti-DIG antibody was used to bind to Denhardt’s solution, 100 mg denatured fragment salmon sperm the DIG-labelled probe and the chemiluminescent substrate, CSPD, DNA ml21)at65uC for 16 h. Membranes were washed once in was used to visualize antibody binding. The banding pattern and lower stringency solution (16 SSPE, 0?1 % SDS) for 15 min at signal were detected on X-ray film (Amersham) after a 30 min expo- room temperature and twice in medium stringency solution (0?56 sure. The same membrane was probed with two different cDNA SSPE, 0?1 % SDS) at 48 uC for 15 min. After air-drying, membranes probes individually. For repeated hybridization of the same blot, the were exposed to Hyperfilm (Amersham Pharmacia) at 280 uC over- nylon membranes were washed in stripping solution (0?2 M NaOH, night. Positive plaques were identified and subjected to a second 0?1 % SDS) for 30 min at 37 uC to remove the probe. round of screening. Two positive clones confirmed by secondary screening were picked with a Pasteur pipette and resuspended in 500 ml of SM buffer (50 mM Tris/HCl, pH 7?9, 100 mM NaCl, 10 mM MgSO4,0?01 % gelatin) and stored at 4 uC with 20 mlof RESULTS chloroform. Conversion of recombinant phage into plasmid (in vivo excision) was done by infecting E. coli DH10B(ZIP), according to Electron micrographs of GiPDV the manufacturer’s instructions (Life Technologies). Plasmid DNA containing the cDNA insert was purified for each using a Plasmid Thin section analysis of the calyx region in the lumen of Miniprep kit (Bio-Rad). the G. indiensis oviduct revealed the presence of large quantities of viral particles (Fig. 1a). Variable densities DNA sequence analysis and nucleotide sequence accession of viral particles were seen among different calyx cells numbers. Sequencing reaction of isolated cDNA clones was carried (Fig. 1b). Each viral particle contained, at most, ten out with the ABI PRISM Big Dye Terminator Cycle Sequence kit (Applied Biosystems) using T7 and SP6 promoter-specific primers, nucleocapsids enveloped by a single unit membrane, with followed by primer walking. The nucleotide sequences of the cDNA an average of six nucleocapsids per virion (Fig. 1c). The size clones were determined using an ABI PRISM 310 Genetic Analyser and shape of virions were variable, with a mean length of (Applied Biosystems). Sequence fragments were edited and assembled 273±34?24 nm, ranging from 175 to 350 nm, and a mean into contiguous regions using the SeqManII sequence assembling width of 162±19?32 nm, ranging from 140 to 190 nm software of the DNASTAR package. Sequence homology searches were (n=80) (Fig. 2a). done using BLAST (Altschul et al., 1990) and searched against the GenBank database. Sequences have been deposited in GenBank with A negative stain of GiPDV particles present in calyx fluid accession numbers AF414845 and AF414846, respectively. showed the nucleocapsids were rod-like or rectangular in Northern blot analysis. Expression of isolated cDNA clones in shape. The electron-dense cores were probably DNA the parasitized host was confirmed by Northern blot analysis. The genome with their associated proteins (Fig. 1d). The sizes RNA samples isolated from parasitized L. dispar larvae 24 h post- of nucleocapsids were slightly variable, with a mean length parasitization and nonparasitized L. dispar larvae were resolved on of 58±5?21 nm, ranging from 46 to 66 nm, and a mean denaturing formaldehyde/1 % agarose gel and blotted onto nylon width of 38±6?37 nm, ranging from 34 to 52 nm (n=60) membrane. DIG-labelled RNA probes complementary to two isolated (Fig. 2b). cDNA clones were synthesized using the DIG RNA labelling kit (Roche). The membrane was prehybridized in prehybridization solution (50 % formamide, 5 % blocking reagent, 56 SSC, 0?1% sarcosyl, 0?1 % SDS) at 52 uC for 2 h, followed by hybridization Electrophoretic profiles of GiPDV genomic DNA with DIG-labelled RNA probes overnight. After hybridization, the GiPDV FIGE hybridization patterns showed that GiPDV membrane was washed twice in low stringency wash solution (26 SSC, 0?1 % SDS) at room temperature for 5 min and washed comprised multiple DNA segments, similar to other twice in high stringency wash solution (0?16 SSC, 0?1 % SDS) at previously described polydnaviruses, and 13 visible bands 52 uC for 15 min. The hybridization signals were detected with anti- were recognized ranging in size from approximately 11 kb digoxigenin–AP Fab fragments (Roche) and visualized with chemi- to more than 30 kb (Fig. 3). The various DNA segments luminescence substrate CDP-Star, ready-to-use (Roche). The same were present in non-equimolar ratios, as indicated by membrane was probed with each DIG-labelled RNA probe (1?1 and differing band intensities. Four segments (D, F, K and M) 1?8) and a DIG-labelled b-actin cDNA probe, individually, under identical conditions. For repeated hybridization to the same were present in higher molar concentration than the other Northern blot, the nylon membranes were washed in stripping segments (Fig. 3). Assuming that each DNA segment was solution (50 % formamide, 50 mM Tris/HCl, pH 7?5, 5 % SDS) for unique, and that there were no co-migrating DNA segments, 26 45 min at 60 uC to remove the probe. GiPDV genomic size was estimated to be 250 kb. http://vir.sgmjournals.org 2053 Y. P. Chen and D. E. Gundersen-Rindal

Fig. 1. Electron micrographs of G. indiensis polydnavirus. (a) Thin section showing calyx region in the lumen of the G. indiensis oviduct. Large numbers of viral particles were seen in calyx epithelial cells. (b) Thin section showing separate desmosomes joining two calyx cells. Different densities of viral particles between the calyx cells may show different levels or stages of replication occurring in the two cells. (c) Thin section showing virus particles within the calyx ﬂuid. Each virion consists of 5 to 10 nucleocapsids enclosed by a single membrane envelope. Bar marker represents 0?5 mm. (d) Negative staining of liberated GiPDV nucleocapsids (35 nm wide646 nm long). Electron micrograph: Charlie Murphy and Christopher Pooley.

Screening of parasitized host cDNA libraries sequences of cDNA 1?1 and cDNA 1?8 are presented in and isolation of expressed GiPDV-specific Fig. 4 and Fig. 5. Clone 1?1 contained a single large open cDNA clones reading frame (ORF) encoding 325 amino acids. Clone 1?8 contained one large ORF comprising 494 amino acids. To further characterize the complexity of the GiPDV BLAST searches of these sequences did not yield any signi- genome, cDNA was synthesized and cloned from mRNA ficant homology to nucleotide or amino acid sequences in isolated from parasitized L. dispar larvae 24 h after GenBank. G. indiensis parasitization. After screening the library with GiPDV, two positive cDNA clones, which derived from GiPDV-specific mRNA expressed in parasitized hosts, were Northern blot analysis of cDNA clones identified, isolated and sequenced. The first cDNA clone, cDNA 1?1, contained a 1?1 kb insert, whereas the second The expression of cDNA 1?1 and cDNA 1?8 in parasitized cDNA clone, cDNA 1?8, contained a 1?8 kb insert. The host was confirmed by Northern hybridization. Using

2054 Journal of General Virology 84 Glyptapanteles indiensis polydnavirus

Fig. 2. (a) Distribution of GiPDV virion length (nm) (n=80). (b) Distribution of GiPDV nucleocapsid length (n=60).

DIG-labelled RNA complementary to cDNA 1?1asa hybridization probe, one transcript of 1?1 kb was identified. Using DIG-labelled RNA complementary to cDNA 1?8 as a hybridization probe, one major transcript of 1?8 kb and two minor transcripts of 1?7 kb and 2?6 kb were Fig. 3. FIGE profile of GiPDV genomic DNA. Undigested identified. No hybridization signal was detected with RNA GiPDV genomic DNA and DIG-labelled linear l DNA markers extracted from non-parasitized gypsy moth host (Fig. 6). digested with HindIII were loaded into a 0?8 % pulse field certified agarose gel. FIGE was performed at 4 ˚C. Major DNA Mapping of isolated cDNA clones to the GiPDV segments are indicated by upper-case letters. Fragment sizes genome (kb) are labelled. In order to determine which GiPDV genomic DNA segments encoded these two expressed GiPDV-specific mRNAs, cDNA 1?1 and cDNA1?8 were DIG-labelled and observed by transmission electron microscopy and the used as probes to hybridize individually to an identical morphological characteristics were similar to those of Southern blot containing size-fractionated genomic DNA. other described bracoviruses. The calyx fluid of G. indiensis Mapping studies indicated that both cDNAs hybridized females contained large quantities of GiPDV viral particles. to more than one genomic DNA segment and different The GiPDV virions were ovoid in form and contained cDNAs were present on the same genome segment. cDNA electron-dense nucleocapsids enclosed by a single unit 1?1 probe hybridized to four viral DNA segments (C, D, E, membrane envelope. Compared with previously character- K) and cDNA 1?8 probe hybridized to two viral DNA ized bracoviruses, virions of GiPDV seemed to contain segments (J, K), as shown in Fig. 7. more numerous nucleocapsids; as many as six to ten nucleocapsids in a single virion were frequently observed. Variation in the number of nucleocapsids that appeared in DISCUSSION the virions may have been due to the different angles at which the virions were observed under electron micro- Characteristics of GiPDV morphology scopy. Compared with those of other bracoviruses, GiPDV Polydnaviruses have been found to be mutualistically asso- nucleocapsids were fairly large, with a mean length of ciated with some hymenopteran endoparasites in the families 58±5?21 nm, ranging from 46 to 66 nm, and a mean width Braconidae and Ichneumonidae and have been character- of 38±6?37 nm, ranging from 34 to 52 nm. The largest ized in approximately 40 species of wasps (Fleming, 1992). nucleocapsids reported to date were found in the PDV The morphology of the braconid polydnavirus GiPDV was associated with Chelonus texanus, with each PDV having a http://vir.sgmjournals.org 2055 Y. P. Chen and D. E. Gundersen-Rindal

Fig. 4. Nucleotide sequence and deduced translated amino acids of GiPDV cDNA 1?1. The putative translation initiation site is in bold and indicated by an arrow. Stop codon is bold and indicated by an asterisk.

diameter of 35 to 40 nm and a length of 50 to 100 nm (Stoltz nucleocapsids were species-speciﬁc among the bracoviruses & Vinson, 1979). The smallest nucleocapsids were seen in and were positively related to the size of DNA segments the PDV associated with Cardiochiles nigriceps, with a enclosed. It was reported that nucleocapsids of bracovirus diameter of 25 to 50 nm and a length of 10 nm (Stoltz & contained only one DNA segment and DNA molecules Vinson, 1979). As a general rule, the number and size of were singly encapsidated, as indicated by the release of

2056 Journal of General Virology 84 Glyptapanteles indiensis polydnavirus

Fig. 5. Nucleotide sequence and deduced translated amino acids of GiPDV cDNA 1?8. The putative translation initiation site is in bold and indicated by an arrow. Stop codon is bold and indicated by an asterisk. http://vir.sgmjournals.org 2057 Y. P. Chen and D. E. Gundersen-Rindal

(a) cDNA 1.1 cDNA 1.8

1 23 456

23.1 kb

2661 bp 1821 bp 1517 bp 1049 bp 9.4 kb 575 bp

483 bp 6.6 kb

(b) 1 23 4 5 b-actin

Fig. 7. Localization of cDNAs 1?1 and 1?8 within the GiPDV Fig. 6. Northern blot analysis of cDNA 1?1 and cDNA 1?8. genome. DIG-labelled l DNA/HindIII (lane 2; fragment kb sizes (a) Total RNA was extracted from parasitized and non-parasitized labelled in lane 1) and GiPDV genomic DNA (lane 3) were L. dispar. The RNA samples were resolved on a denaturing separated by FIGE and blotted to nylon membrane. The formaldehyde 1 % agarose gel, blotted to nylon membrane and Southern-blotted GiPDV genomic DNA was probed individually individually hybridized to the DIG-labelled RNA probes comple- with cDNA 1?1 (lane 4) and cDNA 1?8 (lane 5) to map the mentary to cDNA 1?1 and cDNA 1?8. The mobility and sizes two isolated cDNA fragments to specific regions of the GiPDV (bp) of DIG-labelled RNA molecular mass marker I are indi- genome. cDNA 1?1 hybridized to four genomic DNA segments cated. (b) Labelled insect b-actin was hybridized to the same (C, D, E and K) and cDNA 1?8 hybridized to two genomic membranes to show that uniform quantities of RNA were DNA segments (J and K). loaded. Lanes 1 and 4, DIG-labelled RNA molecular mass marker. Lanes 2 and 5, RNA extracted from parasitized host. Lanes 3 and 6, RNA extracted from non-parasitized host. ichnovirus, C. sonorensis polydnavirus (CsPDV), containing 28 DNA segments ranging in size from 5?5 to 21 kbp, GiPDV comprised fewer but larger DNA segments. Con- individual closed circular DNA segments from the nucleo- sistent with previous findings for other ichno- and braco- capsids after osmotic shock (Albrecht et al., 1994). viruses, GiPDV consisted of multiple DNA segments with variable sizes and molar ratios (Theilmann & Summers, Characteristics of the GiPDV genome 1987, 1988; Summers & Dib-Hajj, 1995; Cui & Webb, 1997; Strand et al., 1997; Webb & Cui, 1998, Varricchio et al., Bracoviruses appear to comprise fewer but larger DNA 1999). Four segments (D, F, K and M) were present in higher molecules than ichnoviruses, and exhibit a relaxed open molar concentration than other segments, as indicated by circular topology (Albrecht et al., 1994; Webb, 1998). a higher band intensity. The higher molar concentration Electrophoretic profiles of genomic DNA showed that the of DNA segments indicated the presence of multiple GiPDV genome contained 13 segments ranging in size DNA segments of similar size with either homologous or from 11 kb to more than 30 kb. The aggregated GiPDV polymorphic sequences. genome size was approximately 250 kb. The visible GiPDV segments may represent distinctive genomic molecules or Identification of cDNA clones encoding GiPDV- the same genomic molecules in different conformations. specific mRNA and expression of cDNA clones Since supercoiled, relaxed circular and linear forms of in the parasitized host viral DNA were very difficult to differentiate, the potent- ial increase in sequence complexity associated with co- Construction and screening of a cDNA library allowed migration of DNA segments of the same size or the same identification of cDNA clones deriving from GiPDV- DNA segment present in more than one conformation was specific mRNA expressed in parasitized host 24 h post- not included in the size estimate for the GiPDV genome in parasitization. Both cDNA clones contained a single ORF. this study. The ongoing GiPDV sequencing project in our Analysis of amino acid sequences indicated that isolated laboratory will provide a better and more precise character- transcripts have no significant homology to other ichno- ization of genome size and organization in the future. virus or bracovirus gene families (Webb & Cui, 1998). Compared with the most systematically characterized Northern blot analysis confirmed the expression of cDNA

2058 Journal of General Virology 84 Glyptapanteles indiensis polydnavirus

1?1 and cDNA 1?8 in the parasitized host, with cDNA 1?1 REFERENCES expressed as a single 1?1 kb transcript and cDNA 1?8 expressed as one major 1?8 kb transcript, with minor 1?7 Albrecht, U., Wyler, T., Pfister-Wilhelm, R., Gruber, A., Stettler, P., Heiniger, P., Kurt, E., Schu¨ mperli, D. & Lanzrein, B. (1994). and 2?6 kb transcripts also detected in the parasitized Polydnavirus of the parasitic wasp Chelonus inanitus (Braconidae): host. Whether the minor transcripts resulted from alter- characterization, genome organization and time point of replication. native splicing of the same precursor transcript or were J Gen Virol 75, 3353–3363. derived from independent genes is unknown. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403–410. Southern hybridization showed that cDNA 1?1 resided Beckage, N. E., Tan, F. F., Schleifer, K. W., Lane, R. 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(2003). sequence homologies noted for GiPDV. The features of Quantitative expression analysis of a Glyptapanteles indiensis genome segmentation, hypermolar segment ratios and polydnavirus protein tyrosine phosphatase gene in its natural lepidopteran host, Lymantria dispar. Insect Mol Biol 12, 271–280. sequence homology have been suggested to be involved in Cui, L. & Webb, B. A. (1997). increasing the copy number of essential genes and the Homologous sequences in the Campo- letis sonorensis polydnavirus genome are implicated in replication levels of gene expression in the absence of virus replica- and nesting of the W segment family. J Virol 71, 8504–8513. tion (Xu & Stoltz, 1993; Cui & Webb, 1997). Homologous Fleming, J. G. W. (1992). Polydnaviruses: mutualists and pathogens. viral genes residing on different DNA segments could be Annu Rev Entomol 37, 401–425. transcribed simultaneously or separately to exert additive Fleming, J. G. W. & Krell, P. J. (1993). 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Proc Natl Acad Sci segments. USA88, 9770–9774. Gundersen-Rindal, D. & Dougherty, E. M. (2000). Evidence for The morphological features and genomic organization of integration of Glyptapanteles indiensis PDV DNA into the chromo- the bracovirus of GiPDV are unique, although characteristic some of Lymantria dispar in vitro. Virus Res 66, 27–37. of braconid PDVs in general. Isolation of cDNAs encoding Johner, A. & Lanzrein, B. (2002). Characterization of two genes of GiPDV-specific mRNA expressed in the parasitized host, the polydnavirus of Chelonus inanitus and their stage-specific expression in the host Spodoptera littoralis. J Gen Virol 83, 1075–1085. as well as mapping of these cDNAs to multiple GiPDV genomic DNA segments, supported the notion that GiPDV Krell, P. J. (1987). Polydnavirus replication and tissue organization of infected cells in the parasitic wasp Diadegma terebrans. Can morphology and genomic organization are intrinsically J Microbiol 33, 176–190. linked to the function and evolutionary strategies of the Krell, P. J. (1991). The polydnaviruses, multipartite DNA viruses virus. This study enhances knowledge about this virus from parasitic hymenoptera. In Viruses of Invertebrates, pp. 141–177. and provides a basis for future functional characterization Edited by E. Kurstak. New York: Marcel Dekker. of GiPDV. Krell, P. J. & Stoltz, D. B. (1980). Virus-like particles in the ovary of an ichneumonid wasp. Purification and preliminary characterization. Virology 101, 408–418. Krell, P. J., Summers, M. D. & Vinson, S. B. (1982). Virus with ACKNOWLEDGEMENTS multipartite superhelical DNA genome from the ichneumonid parasitoid Campoletis sonorensis. J Virol 43, 859–870. We gratefully acknowledge the financial support of USDA-NRI grant 99353028522. We express our appreciation to C. Murphy and Lavine, M. D. & Beckage, N. E. (1995). Polydnaviruses: potent C. Pooley of the USDA Electron Microscopy Unit, Beltsville, MD, mediators of host insect immune dysfunction. Parasitol Today 11, for their help in electron microscopy. We thank M. Shapiro of the 368–378. USDA Insect Biocontrol Laboratory, Beltsville, MD, for providing Lawrence, P. O. & Lanzrein, B. (1993). Hormonal interactions L. dispar larvae, P. B. Taylor of the USDA Beneficial Insect Introduc- between insect endoparasites and their host insects. In Parasites and tion Laboratory, Newark, DE, for rearing G. indiensis wasps, and Yan Pathogens of Insects, pp. 59–86. Edited by N. E. Beckage, S. N. Zhao for critical review of an early draft of this manuscript. Thompson & B. A. Federici. San Diego: Academic Press. http://vir.sgmjournals.org 2059 Y. P. Chen and D. E. Gundersen-Rindal

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