USDA-ARS Insect Biocontrol Laboratory, Beltsville, MD 20705, USA
Correspondence
Dawn Gundersen-Rindal
gundersd{at}ba.ars.usda.gov
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ABSTRACT |
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The GenBank accession numbers of the sequences reported are AF414845 and AF414846.
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INTRODUCTION |
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The PDVs are recognized as two genera: the ichnoviruses found exclusively in parasitoid wasps of the Ichneumonidae, and the Bracoviruses found only in parasitoid wasps of the Braconidae. Ichnovirus and Bracovirus differ from each other in morphology, host range and molecular characteristics (Stoltz & Whitfield, 1992). Ichnoviruses characteristically contain one lenticular nucleocapsid per virion surrounded by two unit membranes, whereas bracoviruses characteristically contain one or more rod-shaped nucleocapsids per virion surrounded by a single unit membrane envelope. It is thought that Ichnoviruses are released into the ovary lumen by budding from calyx cells, while bracoviruses, in contrast, are believed to be released through lysis of the calyx cells.
The PDV genome structure is complex and poorly understood. Only a few PDV genomes have been described in detail. Estimates of PDV genome sizes have been complicated by the characteristic presence of unequal molar ratios of genomic segments, comigrating DNA segments of equal size, and direct terminal repeats (Fleming, 1992; Fleming & Krell, 1993
; Stoltz, 1993
). Comparisons of various PDV genomes showed that the DNA segment number and molar ratio and the total genome size appeared to be specific to the PDVs of each individual PDV-containing wasp species (Krell, 1991
; Stoltz, 1993
; Fleming & Krell, 1993
). Descriptions of ichnovirus genomes have been largely based on studies of PDVs isolated from the parasitoid wasps Campoletis sonorensis (Krell et al., 1982
), Hyposoter exiguae (Krell & Stoltz, 1980
) and Diadegma terebrans (Krell, 1987
). Of these ichnoviruses, the C. sonorensis polydnavirus (CsPDV) genome has been by far the most systematically characterized, and appears to contain 28 DNA segments ranging in size from 5·5 to 21 kbp. Genome analyses of bracoviruses have been less complete than those described for Ichnoviruses. The available information on bracovirus genomes comes mainly from descriptions of the PDVs associated with the parasitoids Cardiochiles nigriceps (Varricchio et al., 1999
), Chelonus inanitus (Albrecht et al., 1994
; Johner & Lanzrein, 2002
; Wyder et al., 2002
) and Microplitis demolitor (Strand et al., 1992
). In general, the genome segments of bracoviruses tend to be fewer in number but larger in size than those of the ichnoviruses, and exhibit a relaxed open circular topology (Albrecht et al., 1994
; Webb, 1998
). For example, characterization of the bracovirus of C. inanitus showed that its genome consisted of 10 different segments ranging in size from 7 to 31 kbp, and that individual DNA segments appeared to be singly encapsidated (Albrecht et al., 1994
).
The braconid endoparasitic wasp Glyptapanteles indiensis parasitizes the larval stage of the gypsy moth, Lymantria dispar. Previous studies on G. indiensis polydnavirus (GiPDV) in our laboratory demonstrated that GiPDV could be integrated not only as a provirus within the parasitoid wasp genome, but also in vitro within the chromosomal DNA of cells derived from the natural host L. dispar (Gundersen-Rindal & Dougherty, 2000). More recently, a GiPDV putative protein tyrosine phosphatase gene believed to be associated with gene regulation during immune response was shown to be differentially expressed in various tissues of the parasitized host (Chen et al., 2003
). Although information on the in vitro and in vivo properties of GiPDV has been gathered, fundamental knowledge pertaining to GiPDV virion morphology and genomic organization remains undescribed. To fill this gap, the morphological features, genomic organization and molecular features of GiPDV were investigated and are reported here. The isolation of cDNA clones representing GiPDV sequences from mRNA isolated from parasitized host and confirmation of the expression of cDNA clones in the parasitized host are described. Further, mapping of these cDNA fragments to the GiPDV genome is shown.
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METHODS |
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Virus purification and viral DNA isolation.
Virus was purified from the calyx fluid of female wasps. Female wasps were anaesthetized in 75 % ethanol for a few seconds and then rinsed in sterile H2O. Each pair of ovaries was dissected in a 15 µl drop of cold PBS and punctured individually with forceps, causing the calyx fluid to diffuse into the PBS drop. The calyx fluid was filtered through a 0·45 µm filter to remove eggs and cellular debris by the methods of Beckage et al. (1994). The resulting viral fluid was transferred into a 1·5 ml microcentrifuge tube and immediately processed for viral DNA isolation and electron microscopy of viral ultrastructure.
Electron microscopy of viral particles.
For negative staining, GiPDV virus particles in filtered calyx fluid were absorbed onto a 400 mesh Formvar-coated nickel grid for 2 s, quickly washed in distilled water, stained with 4 % uranyl acetate for 1 min, and then viewed in an H-7000 Hitachi electron microscope at 75 kV. For thin sections, embedded ovaries dissected from female G. indiensis were fixed in 3 % glutaraldehyde/0·05 M sodium cacodylate buffer (pH 7·0) for 2 h at room temperature and placed in a 4 °C refrigerator overnight. After washing in sodium cacodylate buffer six times within 1 h, ovaries were post-fixed in 2 % buffered osmium tetroxide for 2 h, dehydrated in graded ethanol solutions and infiltrated with Spurr's low-viscosity embedding resin. Ovaries were sectioned on a Riechert/AO Ultracut microtome with a Diatome diamond knife. Silver sections of the ovaries were mounted on 200-mesh Ni grids, stained with 4 % uranyl acetate and 3 % lead citrate, and observed in an H-7000 Hitachi electron microscope at 75 kV.
Viral DNA isolation and field inversion gel electrophoresis (FIGE).
Filter-purified viral fluids were incubated in an equal volume of extraction buffer (4 % sarcosyl, 1 % SDS, 50 mM EDTA, 10 mM Tris, 0·2 M NaCl, 20 µg RNase ml-1, 50 µg proteinase K ml-1) at 50 °C for 2 h, followed by two extractions with ultrapure buffer-saturated phenol (Invitrogen). DNA was precipitated in 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 4 °C. The purified viral DNA was analysed by FIGE, an electrophoretic method used to resolve DNAs in the 10150 kb range. Undigested viral DNA (2 µg) and a molecular size standard (DIG-labelled linear DNA digested with HindIII) were loaded onto a 0·8 % (w/v) pulse field certified agarose gel (Bio-Rad) in 0·5x TBE buffer (50 mM Tris/borate, 1 mM EDTA, pH 8·3). Gel electrophoresis was performed at 4 °C for 2x24 h using a 0·10·4 s switch time ramp with 180 V forward voltage and 120 V reverse voltage for each 24 h run. The gels were stained with ethidium bromide for 15 min, destained with sterile H2O for 30 min, and visualized using a UV transilluminator.
cDNA library construction and screening.
Total RNA was isolated from parasitized L. dispar larvae 24 h post-parasitization. Insects were frozen in liquid nitrogen and ground to a fine powder. RNA was extracted with TRIzol reagent (Invitrogen) according to 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 electrophoresis, and mRNA was isolated from total RNA using the Messagemaker Reagent Assembly kit (Life Technologies) following the manufacturer's instructions. The concentration of mRNA was measured using a spectrophotometer. cDNA libraries were prepared using the Superscript Choice System for cDNA Synthesis (Invitrogen) following the manufacturer's instructions. mRNA was converted into size-fractionated, EcoRI-adapted cDNA. The cDNA was ligated into the EcoRI-digested ZIPLOX expression vector (Life Technologies). The ligated vector-cDNAs were packaged in Gigapack III Gold Packaging Extract (Stratagene). The packaged cDNA-containing phage were introduced into Y1090 (ZL) E. coli that had been grown overnight in LB medium with 10 mM MgSO4 and 0·2 % maltose. The transformants were plated on LB-agar and incubated overnight at 37 °C. The cDNA libraries were titrated and amplified, and approximately 5x104 p.f.u. of library were plated onto ten large LB plates. The plaques were transferred to nitrocellulose membrane (Roche) and the phage DNAs were denatured, neutralized and UV cross-linked to membrane according to standard procedures. The GiPDV genomic DNA used as a probe was radiolabelled to high specific activity with the Random Primers DNA Labelling System (Invitrogen) following the manufacturer's instructions. Hybridization screening of cDNA libraries using the 32P-labelled GiPDV probe was carried out in the hybridization solution (6x SSPE, 0·5 % SDS, 5x Denhardt's solution, 100 µg denatured fragment salmon sperm DNA ml-1) at 65 °C for 16 h. Membranes were washed once in lower stringency solution (1x SSPE, 0·1 % SDS) for 15 min at room temperature and twice in medium stringency solution (0·5x SSPE, 0·1 % SDS) at 48 °C for 15 min. After air-drying, membranes were exposed to Hyperfilm (Amersham Pharmacia) at -80 °C overnight. Positive plaques were identified and subjected to a second round of screening. Two positive clones confirmed by secondary screening were picked with a Pasteur pipette and resuspended in 500 µl of SM buffer (50 mM Tris/HCl, pH 7·9, 100 mM NaCl, 10 mM MgSO4, 0·01 % gelatin) and stored at 4 °C with 20 µl of chloroform. Conversion of recombinant phage into plasmid (in vivo excision) was done by infecting E. coli DH10B(ZIP), according to the manufacturer's instructions (Life Technologies). Plasmid DNA containing the cDNA insert was purified for each using a Plasmid Miniprep kit (Bio-Rad).
DNA sequence analysis and nucleotide sequence accession numbers.
Sequencing reaction of isolated cDNA clones was carried out with the ABI PRISM Big Dye Terminator Cycle Sequence kit (Applied Biosystems) using T7 and SP6 promoter-specific primers, followed by primer walking. The nucleotide sequences of the cDNA clones were determined using an ABI PRISM 310 Genetic Analyser (Applied Biosystems). Sequence fragments were edited and assembled into contiguous regions using the SeqManII sequence assembling software of the DNASTAR package. Sequence homology searches were done using BLAST (Altschul et al., 1990) and searched against the GenBank database. Sequences have been deposited in GenBank with accession numbers AF414845 and AF414846, respectively.
Northern blot analysis.
Expression of isolated cDNA clones in the parasitized host was confirmed by Northern blot analysis. The RNA samples isolated from parasitized L. dispar larvae 24 h post-parasitization and nonparasitized L. dispar larvae were resolved on denaturing formaldehyde/1 % agarose gel and blotted onto nylon membrane. DIG-labelled RNA probes complementary to two isolated cDNA clones were synthesized using the DIG RNA labelling kit (Roche). The membrane was prehybridized in prehybridization solution (50 % formamide, 5 % blocking reagent, 5x SSC, 0·1 % sarcosyl, 0·1 % SDS) at 52 °C for 2 h, followed by hybridization with DIG-labelled RNA probes overnight. After hybridization, the membrane was washed twice in low stringency wash solution (2x SSC, 0·1 % SDS) at room temperature for 5 min and washed twice in high stringency wash solution (0·1x SSC, 0·1 % SDS) at 52 °C for 15 min. The hybridization signals were detected with anti-digoxigeninAP Fab fragments (Roche) and visualized with chemiluminescence substrate CDP-Star, ready-to-use (Roche). The same membrane was probed with each DIG-labelled RNA probe (1·1 and 1·8) and a DIG-labelled -actin cDNA probe, individually, under identical conditions. For repeated hybridization to the same Northern blot, the nylon membranes were washed in stripping solution (50 % formamide, 50 mM Tris/HCl, pH 7·5, 5 % SDS) for 2x 45 min at 60 °C to remove the probe.
Genome mapping of cDNAs.
Southern hybridization was performed to map the two isolated cDNA fragments to a specific region(s) of the GiPDV genome. FIGE-separated viral genomic DNA segments were transferred from the gel to nylon membrane (Hybond-N+; Amersham Pharmacia) and UV cross-linked for analysis. cDNA clones were DIG-labelled using the DIG DNA labelling kit (Roche) according to the manufacturer's instructions. Membranes with separated GiPDV genomic segments were hybridized with a DIG-labelled cDNA probe in DIG Easy Hybridization solution (Roche) for 16 h at 48 °C. After hybridization, the membranes were washed twice in low stringency wash solution (2x SSC, 0·5 % SDS) at room temperature for 5 min and washed twice in high stringency wash solution (0·1x SSC, 0·5 % SDS) at 48 °C for 15 min. After the washes, anti-DIG antibody was used to bind to the DIG-labelled probe and the chemiluminescent substrate, CSPD, was used to visualize antibody binding. The banding pattern and signal were detected on X-ray film (Amersham) after a 30 min exposure. The same membrane was probed with two different cDNA probes individually. For repeated hybridization of the same blot, the nylon membranes were washed in stripping solution (0·2 M NaOH, 0·1 % SDS) for 30 min at 37 °C to remove the probe.
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RESULTS |
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Electrophoretic profiles of GiPDV genomic DNA
GiPDV FIGE hybridization patterns showed that GiPDV comprised multiple DNA segments, similar to other previously described polydnaviruses, and 13 visible bands were recognized ranging in size from approximately 11 kb to more than 30 kb (Fig. 3). The various DNA segments were present in non-equimolar ratios, as indicated by differing band intensities. Four segments (D, F, K and M) were present in higher molar concentration than the other segments (Fig. 3
). Assuming that each DNA segment was unique, and that there were no co-migrating DNA segments, GiPDV genomic size was estimated to be 250 kb.
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DISCUSSION |
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Characteristics of the GiPDV genome
Bracoviruses appear to comprise fewer but larger DNA molecules than ichnoviruses, and exhibit a relaxed open circular topology (Albrecht et al., 1994; Webb, 1998
). Electrophoretic profiles of genomic DNA showed that the GiPDV genome contained 13 segments ranging in size from 11 kb to more than 30 kb. The aggregated GiPDV genome size was approximately 250 kb. The visible GiPDV segments may represent distinctive genomic molecules or the same genomic molecules in different conformations. Since supercoiled, relaxed circular and linear forms of viral DNA were very difficult to differentiate, the potential increase in sequence complexity associated with co-migration of DNA segments of the same size or the same DNA segment present in more than one conformation was not included in the size estimate for the GiPDV genome in this study. The ongoing GiPDV sequencing project in our laboratory will provide a better and more precise characterization of genome size and organization in the future. Compared with the most systematically characterized 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. Consistent with previous findings for other ichno- and bracoviruses, GiPDV consisted of multiple DNA segments with variable sizes and molar ratios (Theilmann & Summers, 1987
, 1988
; Summers & Dib-Hajj, 1995
; Cui & Webb, 1997
; Strand et al., 1997
; Webb & Cui, 1998
, Varricchio et al., 1999
). Four segments (D, F, K and M) were present in higher molar concentration than other segments, as indicated by a higher band intensity. The higher molar concentration of DNA segments indicated the presence of multiple DNA segments of similar size with either homologous or polymorphic sequences.
Identification of cDNA clones encoding GiPDV-specific mRNA and expression of cDNA clones in the parasitized host
Construction and screening of a cDNA library allowed identification of cDNA clones deriving from GiPDV-specific mRNA expressed in parasitized host 24 h post-parasitization. Both cDNA clones contained a single ORF. Analysis of amino acid sequences indicated that isolated transcripts have no significant homology to other ichnovirus or bracovirus gene families (Webb & Cui, 1998). Northern blot analysis confirmed the expression of cDNA 1·1 and cDNA 1·8 in the parasitized host, with cDNA 1·1 expressed as a single 1·1 kb transcript and cDNA 1·8 expressed as one major 1·8 kb transcript, with minor 1·7 and 2·6 kb transcripts also detected in the parasitized host. Whether the minor transcripts resulted from alternative splicing of the same precursor transcript or were derived from independent genes is unknown.
Southern hybridization showed that cDNA 1·1 resided on four separate viral DNA segments, while cDNA 1·8 apparently resided on two viral DNA segments, indicating multiple gene loci within GiPDV. The multiple gene loci suggested the probability of homologous sequences or related genes among different GiPDV DNA segments, as is characteristic of other described polydnaviruses (Theilmann & Summers, 1988; Cui & Webb, 1997
, 1998
; Strand et al., 1997
; Volkoff et al., 1999
). These are the first internal sequence homologies noted for GiPDV. The features of genome segmentation, hypermolar segment ratios and sequence homology have been suggested to be involved in increasing the copy number of essential genes and the levels of gene expression in the absence of virus replication (Xu & Stoltz, 1993
; Cui & Webb, 1997
). Homologous viral genes residing on different DNA segments could be transcribed simultaneously or separately to exert additive or complementary functions in host regulation. Webb & Cui (1998)
reported that abundantly expressed genes are often associated with nested DNA segments, while genes that are expressed at a lower level are not generally present on nested DNA segments. The multiple loci among GiPDV genome segments suggested that cDNA 1·1- and 1·8-encoded GiPDV-specific genes represent related genes of importance in the parasitized host, members of gene families, and are probably not associated with nested segments.
The morphological features and genomic organization of the bracovirus of GiPDV are unique, although characteristic of braconid PDVs in general. Isolation of cDNAs encoding GiPDV-specific mRNA expressed in the parasitized host, as well as mapping of these cDNAs to multiple GiPDV genomic DNA segments, supported the notion that GiPDV morphology and genomic organization are intrinsically linked to the function and evolutionary strategies of the virus. This study enhances knowledge about this virus and provides a basis for future functional characterization of GiPDV.
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ACKNOWLEDGEMENTS |
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Received 17 March 2003;
accepted 3 April 2003.