Isolation and characterization of a Cotesia rubecula bracovirus gene expressed in the lepidopteran...

Isolation and characterization of a Cotesiarubecula bracovirus gene expressed in thelepidopteran Pieris rapae

Richard Glatz,13 Otto Schmidt1 and Sassan Asgari2

Correspondence

Sassan Asgari

[email protected]

1Insect Molecular Biology, University of Adelaide, Waite Campus, Glen Osmond, SA 5064,Australia

2Department of Zoology and Entomology, School of Life Sciences, University of Queensland,St Lucia, QLD 4072, Australia

Received 23 May 2004

Accepted 16 June 2004

Polydnaviruses are endogenous particles that are crucial for the survival of endoparasitoid

wasps, providing active suppression of the immune function of the lepidopteran host in which

wasp larvae develop. The Cotesia rubecula bracovirus (CrBV) is unique in that only four gene

products are detected in larval host (Pieris rapae) tissues and expression of CrBV genes is

transient, occurring between 4 and 12 h post-parasitization. Two of the four genes, CrV1 and

CrV3, have been characterized. CrV1 is a secreted glycoprotein that has been implicated in

depolymerization of the actin cytoskeleton of host haemocytes, leading to haemocyte inactivation;

CrV3 is a multimeric C-type lectin that shares homology with insect immune lectins. Here, a

third CrBV-specific gene is described, CrV2, which is expressed in larval P. rapae tissues. CrV2,

which is transcribed in haemocytes and fat body cells, has an ORF of 963 bp that produces a

glycoprotein of approximately 40 kDa. CrV2 is secreted into haemolymph and appears to be

internalized by host haemocytes. CrV2 has a coiled-coil region predicted at its C-terminus, which

may be involved in the formation of putative CrV2 trimers that are detected in haemolymph of

parasitized host larvae.

INTRODUCTION

Endogenous polydnaviruses (PDVs) are produced exclu-sively within the ovaries of certain hymenopteran endo-parasitoids that belong to the ichneumonoid familiesBraconidae and Ichneumonidae (Stoltz & Vinson, 1979).Based on differences in host range and morphology, PDVsare subdivided into bracoviruses and ichnoviruses (Stoltz& Whitfield, 1992). Particles may contain one or severaldouble-stranded circular DNAmolecules (or segments) andare injected into the haemocoel of a host at oviposition,together with the parasitoid egg and other maternal proteinsecretions. Most of the proteins encoded by encapsidatedgenes are expressed within the parasitized host and functionto suppress the host immune response [reviewed by Webb(1998); Kroemer & Webb (2004)]. Polydnaviruses are alsoknown to interfere with the host endocrine system, causingdevelopmental arrest and other physiological disorders[reviewed by Beckage (1998); Beckage & Gelman (2004)].The PDVs are anomalous in that encapsidated circular

DNA segments do not appear to encode PDV structural orreplicative proteins, and new PDV particles or DNA are notproduced in the parasitized host’s cells (Stoltz & Vinson,1979). Instead, particle-associated DNA segments areknown to originate from wasp chromosomal DNA andare transferred from one generation to the next, in theirintegrated form, in Mendelian fashion (Stoltz et al., 1986).Particles enter many host cell types, including haemocytesand fat body cells (Strand et al., 1992; Harwood & Beckage,1994), and virus transcripts are detected in the first fewhours following parasitization. Transcripts are generatedeither transiently (Asgari et al., 1996) or persistently (Strandet al., 1992) during parasitism.

The Cotesia rubecula bracovirus (CrBV) is unique as itappears to generate only four gene products, production ofwhich in the larval host (Pieris rapae) is highly transient,from 4 to 12 h post-parasitization (h.p.p.) (Asgari et al.,1996). By contrast, the Campoletis sonorensis ichnovirus(CsIV) is estimated to express over 35 genes that belong toseveral gene families (Turnbull & Webb, 2002) and expres-sion of at least some genes continues throughout develop-ment of the parasitoid larva (Webb, 1998). Transientgene expression is associated with reversible inactivationof the cellular immune system, which is considered to bean advanced evolutionary adaptation that restores the

3Present address: CSIRO Health Sciences and Nutrition, Therapeuticand Diagnostic Technologies, PO Box 10041 Adelaide BC, Australia.

The GenBank/EMBL/DDBJ accession number for the sequencereported in this paper is AY631272.

0008-0307 G 2004 SGM Printed in Great Britain 2873

Journal of General Virology (2004), 85, 2873–2882 DOI 10.1099/vir.0.80307-0

parasitized host’s ability to defend the developing parasitoidagainst pathogens or other parasitoids.

Asgari et al. (1996, 1997) cloned and sequenced CrV1-encoding DNA from CrBV, demonstrating that CrV1 is anencapsidated gene that is expressed as a single transcriptin parasitized host haemocytes. CrV1 is secreted frominfected cells into serum interacting with the surface ofhaemocytes (Asgari et al., 1997). Although the exact modeof action is unknown, the presence of depolymerized actinin CrV1-treated haemocytes suggests that CrV1 interactionwith the cell surface leads to depolymerization of cyto-plasmic actin structural components (Asgari et al., 1997).Without functional actin filaments, haemocytes are unableto undergo the rearrangements of the cytoskeleton that arerequired for immune-related spreading and phagocytosisreactions (Rosales et al., 1994; Yano et al., 1994; Strand &Pech, 1995; Asgari et al., 1997). Homologues of CrV1 havebeen found to occur in six Cotesia species and the matchingphylogenetic trees created by analysis of wasp 16S rRNA andNADH1 genes also matched that produced by analysis ofCrV1 homologue sequences (Whitfield, 2000), suggestingthat PDVs were not acquired independently among Cotesiaspecies, but co-evolved with the hymenopteran parasitoid(Whitfield & Asgari, 2003).

More recently, a second CrBV gene (CrV3) was character-ized as a C-type lectin (CTL) (Glatz et al., 2003). CrV3 hashomologues in bracoviruses that are associated with Cotesiaruficrus and Cotesia karyai (Teramoto & Tanaka, 2003) andthis group of CTLs forms a unique CTL family. Interestingly,the CrV3 homologues are related more closely to inverte-brate CTLs, which have been implicated in humoralimmune defence of such animals (Haq et al., 1996; Saitoet al., 1997; Arai et al., 1998; Kakiuchi et al., 2002; Yu &Kanost, 2003), than to known viral lectins. CrV3 formsmultimeric structures in the haemolymph, composed ofmonomers (occurring as two glycoforms for CrV3) thateach contain a single carbohydrate-binding domain,features that are shared with invertebrate immune CTLs(Kilpatrick, 2002; Glatz et al., 2003).

In this study, we report the isolation and characterization ofCrV2, the third of four expressed CrBV genes to be isolated.Like CrV1, CrV2 has a coiled-coil domain and is found inoligomeric form in the haemolymph of parasitized larvae,where it is taken up by host haemocytes.

METHODS

Insect cultures. The parasitoid C. rubecula and its host weremaintained at 25 uC on a 14 : 10 h (light : dark) photoperiod. P.rapae larvae were reared on cabbage plants. Adult C. rubecula waspswere fed with honey–water solution.

CrBV and genomic DNA isolation. Virus purification and geno-mic DNA extraction were carried out as described previously (Glatzet al., 2003).

Southern and Northern blot hybridization. DNA samples wererun on a 1% agarose gel and transferred to a nylon membrane asdescribed by Sambrook et al. (1989). RNA was isolated fromunparasitized and 6 h-parasitized P. rapae caterpillars accordingto Chomczynski & Sacchi (1987). RNA samples were run on 1%agarose gels under denaturing conditions using formaldehyde andtransferred to nylon membranes as described by Sambrook et al.(1989). Probes were prepared as described by the manufacturer(Ready-To-Go DNA labelling beads; Amersham Biosciences).Slot-blots were carried out by using a Bio-Dot SF microfiltrationapparatus (Bio-Rad), according to the manufacturer’s instructions.

5§ amplification of CrV2 cDNA (5§ RACE). Partial CrV2 cDNAwas extended in the 59 direction by using the 59 RACE system forrapid amplification of cDNA ends (Life Technologies). PCR productobtained from 59 RACE was ligated into the pGEM-T Easy vector asdescribed by the manufacturer (Promega). The insert was sequencedby using M13 forward and reverse primers.

Computer analysis. Sequences were compared against thosecontained in GenBank by using a nucleotide BLAST search, accessedvia the National Centre for Biotechnology Information website(www.ncbi.nlm.nih.gov/blast). All CrV2 protein analysis tools wereaccessed through the ExPASy molecular biology server (http://us.expasy.org/tools).

RT-PCR. Primers specific to the CrV2 ORF (Fig. 1) were used inRT-PCR analysis of RNA from unparasitized and 6 h-parasitizedP. rapae larvae by using AMV reverse transcriptase (AMV-RT;Promega). XhoI and HindIII restriction sites were added to primersequences (underlined) to provide sites for direct ligation of thefragment into the pQE30 expression vector (Qiagen). Primer CrV2-R (59-GCGCAAGCTTTTAGGGATGATCTCGAGC-39) was usedin the RT reaction, followed by PCR using primers CrV2-R andCrV2-F (59-CGCGGCATGCCCGTTGCAAGACAGAAG-39).

Collection of protein samples and Western blotting.Haemolymph, haemocyte and fat body cells were collected and pre-pared as described previously (Glatz et al., 2003). Protein sampleswere electrophoresed on denaturing 12% SDS–polyacrylamide gels,as described by Laemmli (1970). Standard conditions were denatur-ing; however, non-denaturing conditions were sometimes used,whereby SDS was absent from gels, sample and running buffers.Proteins were either stained within gels by using Coomassie brilliantblue (Sigma) or transferred to a nitrocellulose membrane as

Fig. 1. DNA nucleotide and deduced amino acid sequence for CrV2 from the Cotesia rubecula bracovirus. The putativetransmembrane signal peptide is boxed and contains the methionine start codon as the first amino acid in the signal. Thepredicted signal peptide cleavage point is denoted by an arrowhead. Single amino acid [serine (S) and threonine (T)] residuesidentified as putative O-glycosylation sites are underlined; nucleotides representing putative N-glycosylation sites are double-underlined. The stop codon is indicated by an asterisk and the polyadenylation signal is in bold type. CrV2-F and CrV2-Rprimer binding sites are underlined and are located at the 59 and 39 ends, respectively, of the gene. The original 450 bp cDNAfragment (obtained by screening a cDNA library) is underlined by dots and the CloneC-F and CloneC-R primers that wereused to amplify 290 bp of the cDNA fragment are wavy underlined. Note that CloneC-R and CrV2-R share 1 bp (primernucleotide C) at position 943.

2874 Journal of General Virology 85

R. Glatz, O. Schmidt and S. Asgari

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Cotesia rubecula bracovirus gene expression

described by Sambrook et al. (1989). Before obtaining anti-CrV2,blots were probed with either a 1 : 10 000 dilution of peroxidase-conjugated Helix pomatia lectin (HPL, 50 ng ml21; Sigma) or ananti-polyhistidine mAb (clone His-1; Sigma).

Expression and purification of bacterial CrV2. Primers CrV2-Fand CrV2-R were designed to amplify the ORF of the CrV2 gene,excluding a putative signal sequence corresponding to the first 20 aaof the protein (see Fig. 1 and RT-PCR for CrV2). These primerswere used in PCR of pGEM-T Easy vector (Promega) containingthe CrV2 ORF to obtain the required fragment for ligation to thepQE30 bacterial expression vector (Qiagen). Production of bacterialCrV2 (containing six additional, vector-derived histidine residues)was induced by addition of 1 mM IPTG to the bacterial suspensionfor 2 h at 37 uC. The resulting fusion protein was largely containedin the insoluble fraction and was purified under denaturing con-ditions as described by the manufacturer (Qiagen). Samples weredialysed overnight against TBS (0?15 M NaCl, 0?01 M Tris, pH 8?0)at 4 uC.

Anti-CrV2 polyclonal antibody production. Purified bacterialCrV2 was visualized on preparative 12% SDS–acrylamide gels bystaining with water-dissolved Coomassie brilliant blue (Sigma).CrV2 protein bands were excised from the gel and used for immuni-zation of two rabbits as described previously (Glatz et al., 2003). Theantiserum was used at a dilution of 1 : 5000. Bound anti-CrV2 wasthen visualized by alkaline phosphatase-labelled secondary anti-rabbit antibody (1 : 10 000).

Fluorescent labelling of CrV2 associated with P. rapaehaemocytes. Unparasitized and 24 h-parasitized larvae were bledinto PBS that was saturated with phenylthiourea before centrifuga-tion at 2300 g for 5 min. The pellet was then resuspended gently inPBS before transfer to multiwell slides. Time was allowed for settlingbefore fixing cells with 4% paraformaldehyde in PBS. Anti-CrV2antiserum and fluorescein isothiocyanate (FITC)-conjugated anti-rabbit secondary antibody were used to visualize CrV2 associatedwith haemocytes, as described previously (Asgari et al., 1996).

RESULTS

CrBV genes are expressed transiently within a few hours ofinfection of host tissues (Asgari et al., 1996). When totalCrBV genomic DNA was used to probe total RNA fromparasitized P. rapae caterpillars, four abundant virus trans-cripts were detected 4 h.p.p. (Asgari et al., 1996). Theresulting signal became stronger at 6 h, to the extent that itwas overexpressed compared with the amount of transcriptat 4 and 12 h. Although additional virus transcripts maybe expressed at low levels that are masked by the strongsignals at 6 h after parasitization, the four detected trans-cripts are nevertheless referred to as CrV1–4 by decreasingsize. CrV1 and CrV3 were previously isolated and identifiedby screening a cDNA library made from 6 h-parasitizedlarvae by using total CrBV DNA as a probe (Asgari et al.,1996; Glatz et al., 2003). A similar approach was employedhere to isolate the CrBV gene encoding CrV2 and to investi-gate its possible role in the host–parasitoid interaction.

Isolation and characterization of CrV2

Several screens of the cDNA library led to the isolation of anapproximately 450 bp cDNA (CloneC) that encoded part ofa putative CrBV gene and included a poly(A) tail (Fig. 1).

The fragment was cloned and sequenced, allowing primersto be designed to amplify 290 bp of the 59 end (CloneC-Fand CloneC-R; Fig. 1). To confirm the cDNA fragment asparticle-derived, the amplified fragment was then used as aprobe in both a Southern blot of CrBV DNA (Fig. 2a) anda Northern blot of RNA from naı̈ve and 6 h-parasitizedlarvae (Fig. 2b). Hybridization occurred to a CrBV restric-tion fragment of >15 kbp and to a parasitism-specifictranscript of approximately 1?2 kbp. These data, and the factthat the same probe bound to genomic DNA from femalewasps but not to genomic DNA from P. rapae (data notshown), indicate that the cDNA originated from particlesthat were introduced to the larvae at oviposition. Basedon the transcript size being intermediate between CrV1(1?4 kbp) and CrV3 (1?1 kbp), matching the transcript sizeof CrV2-encoding mRNA, we designated the newly isolatedgene as CrV2. Binding of the cDNA to a single RNA band onthe Northern blot revealed that CrV2 shows no significanthomology to other CrBV-related genes within the 290 bpprobe. Slot-blot analysis of RNA isolated from haemocytesand fat body cells from 6 h-parasitized larvae showed thatthere was no significant difference in the amount of CrV2transcripts detected in the two samples (Fig. 2d). In orderto measure RNA loading within slots, a fragment of 18SrRNA was used from P. rapae as a control.

59 RACE was employed to extend the cDNA at the 59 endand complete the ORF. This method produced a fragmentof approximately 850 bp (data not shown), which wassubsequently sequenced. Sequence data from the 59 RACEfragment were combined with those from the 450 bp cDNAclone to assemble the total CrV2 ORF of 960 bp and flank-ing sequences (Fig. 1). A methionine codon (ATG) at thebeginning of the ORF was identified as the only possiblecodon with a suitable nucleotide sequence environmentfor functional initiation (Cavener & Ray, 1991). Computeranalyses of the deduced amino acid sequence revealed aputative signal peptide encompassing the first 20 aa of theprotein, with a cleavage point predicted at the end of thesignal peptide (Fig. 1), indicating that the CrV2 protein isprobably secreted from infected cells.

Four putative N- and six putative O-glycosylation sites werepredicted in the ORF, as well as a polyadenylation signalapproximately 120 bp downstream of the stop codon(Fig. 1). These data were used to generate primers specificto theCrV2ORF (CrV2-F and CrV2-R; Fig. 1). Comparisonof RT-PCR and genomic DNA PCR products that weregenerated by utilizing these primers revealed no sequencedifferences, indicating that no introns are present in thegenomic CrV2 DNA. The CrV2 nucleotide and deducedamino acid sequences were compared against knownsequences in GenBank; however, no significant homologywas detected.

CrV2, without the signal peptide, was expressed in Escheri-chia coli and produced an approximately 40 kDa fusionprotein, following induction with IPTG (Fig. 2c). Computeranalyses predicted a molecular mass of 33?7 kDa and a pI of



8?94 for secreted CrV2. Purified recombinant CrV2 wasused to immunize rabbits for production of polyclonalanti-CrV2 antibodies. Western blot analysis of serum fromnon-parasitized and 6 h-parasitized P. rapae larvae, probedwith anti-CrV2, allowed visualization of the 37 kDa CrV2only in parasitized larvae (Fig. 3a). Previous data showedthe presence of a parasitism-specific glycoprotein in thehaemolymph of P. rapae larvae, the production of which wasinitiated at approximately 6 h.p.p. (Asgari, 1997; Fig. 3b).By using anti-CrV2 to probe serum proteins from 6 h-parasitized larvae, it was determined that the previouslyunidentified glycoprotein is CrV2 (Fig. 3b). These dataconfirm that CrV2 is a secreted glycoprotein and, further,that it contains N-acetyl-D-galactosamine residues at itsO-glycosylation sites, as it was previously detected byGalNAc-specific HPL (Asgari, 1997). Western blot analysesof larval serum, haemocytes and fat body cells at variouspoints after parasitization showed that CrV2 was presentin each sample at 6 h.p.p., reached a maximum level atabout 24 h.p.p. and was declining at 48 h.p.p. (Fig. 3c).These data are consistent with secretion of CrV2 intocell-free haemolymph from CrBV-infected haemocytes andfat body cells.

CrV2 forms trimers

When cell-free haemolymph proteins were analysed undernon-denaturing conditions, a putative CrV2 trimer ofapproximately 98 kDa was detected in parasitized larvae(Fig. 4a). Coiled-coil regions are commonly associated withthe multimerization of proteins (Alber, 1992). COILS soft-ware predicted the presence of a coiled-coil region near theC-terminus of CrV2, comprised of aa 266–313 (Fig. 4b).Analysis of the coiled-coil region with MULTICOIL softwarepredicted a high probability of trimer formation, due tothe presence of the C-terminal coiled-coil region (data notshown).

Relative expression of CrV2 and CrV3

During an earlier CrV3-related study, anecdotal evidencepointed to large differences in the amount of CrV2 andCrV3 transcripts and protein detected in parasitized larvae.Previous data also indicated that CrV3 transcript levels aregenerally lower than transcript levels from the other threemajor CrBV genes (Asgari et al., 1996). Therefore, severalexperiments were undertaken in order to directly comparerelative levels of transcription and protein production in

Fig. 2. Confirmation of CrBV as the origin of a 290 bp (CrV2-related) cDNA fragment obtained by screening a 6 h-parasitized P. rapae larval cDNA library and purification of recombinant CrV2 protein. (a) Southern blot analysis of total CrBVDNA (CrBV) digested with EcoRI, using the [32P]-labelled 290 bp cDNA fragment as a probe. The probe hybridized to arestriction fragment of >15 kbp. The corresponding ethidium bromide-stained DNA is shown on the left. (b) Northern blothybridization of the [32P]-labelled 290 bp cDNA fragment to total RNA extracted from non-parasitized (N) and 6 h-parasitized(P) P. rapae larvae. Hybridization was to a parasitism-specific transcript of approximately 1250 bp. Approximately 20 mg RNAwas loaded in each lane (see loading control at bottom). (c) Western blot analysis (12% SDS-PAGE; anti-polyhistidine,1 : 10000) of non-induced (T0) and 2 h post-induced (T2) recombinant M15 bacterial cells (containing CrV2 fusion proteinlacking the signal peptide) and the recombinant protein, C, purified from insoluble proteins taken from induced cells. Theinduced purified protein (arrowhead) was used for production of anti-CrV2 antibodies. (d) Slot-blot analysis of RNA (2 mg)isolated from haemocytes and fat body of 6 h-parasitized larvae probed with CrV2 cDNA and P. rapae 18S rRNA genefragment to measure RNA loading.



cell-free haemolymph, haemocytes and fat bodies of para-sitized larvae. Previous work showed that each of the fourCrBV genes expressed in parasitized larvae was expressedtransiently (with maximum expression near 6 h.p.p.) andthat the up- and downregulation of each gene occurred oversimilar periods (Asgari et al., 1996). Thus, 6 h-parasitizedlarvae were used for the comparison. Slot-blot analysis wasused to compare relative CrV2 and CrV3 transcript levels

contained in RNA extracted from naı̈ve and parasitizedP. rapae larvae (Fig. 5a). Probing similar amounts of RNAwith CrV2 and CrV3 probes indicated that CrV2 transcriptmade up a significantly greater proportion of total trans-cripts than CrV3 transcript. Indeed, RNA probed with 32P-labelled CrV2 produced a visible signal in just a few hoursof autoradiograph exposure, whereas the same amount ofRNA probed with 32P-labelled CrV3 required nearly 3 days

Fig. 3. Expression and secretion of CrV2. The position of CrV2 is indicated by an arrowhead in each blot. (a) Western blotanalysis (12% SDS-PAGE; anti-CrV2 antiserum, 1 : 5000) of cell-free haemolymph from non-parasitized (N) and 6 h-parasitized (P) P. rapae larvae. (b) A parasitism-specific glycoprotein (arrowhead) detected at 6 h.p.p. on Western blotscontaining proteins extracted from serum from naı̈ve (N) and parasitized P. rapae larvae and probed with a GalNAc-specificHPL (1 : 10000). Anti-CrV2 antibodies (1 : 5000) confirmed that the secreted glycoprotein is CrV2 (P6). (c) Western blotanalyses (12% SDS-PAGE; anti-CrV2 antiserum, 1 : 5000) of P. rapae cell-free haemolymph, haemocytes and fat body cells.Proteins were collected at various time points (h) after parasitization and are indicated above the blots. Total haemocytes or fatbody cells were pooled from three larvae at each time point, from which 20 ml was loaded per lane. Similarly, for haemolymphsamples, 5 ml cell-free haemolymph from each pool was loaded per lane. Protein source is shown below blots. For eachprotein source, expression is detected at 6 h.p.p., large amounts of CrV2 are still present at 24 h.p.p. and CrV2 levels are indecline at 48 h.p.p.



autoradiograph exposure to produce a visible signal(Fig. 5a).

Western blot analyses of similar amounts of protein fromhaemocytes, serum and fat bodies, probed with both anti-CrV2 and anti-CrV3, indicated that this difference ismaintained at the protein level (Fig. 5b). In the comparativeanalyses shown, CrV2 was detected in large amounts in eachsample, while CrV3 was not detected under conditions ofthe same amount of total loaded protein (Fig. 5b). Thesedata indicate that CrV2 protein is present in significantlyhigher amounts than CrV3 in haemocytes, cell-freehaemolymph and fat bodies of parasitized larvae. As thisreflects the relative amounts of transcripts, this differenceis regulated at the transcriptional level. The biologicalsignificance of this differential expression is not known. Thedifference may reflect a function different from that ofCrV3, in that CrV2 (like CrV1) targets haemocytes, whichinternalize the protein in a systemic fashion and thereforerequire large amounts of active protein in the serum. Incontrast, CrV3 appears to function in cell-free haemolymphand may possibly be recycled after binding to a targetmolecule. Alternatively, the target molecule of CrV3 maybe present in small amounts, e.g. an immune-induced formof a molecule that contains an unusual glycosylation orproteolytic cleavage site.

Immunofluorescence detection of CrV2 inhaemocytes

Most of the PDV genes characterized so far targethaemocytes of parasitized larvae, e.g. CrV1 (Asgari et al.,

1996, 1997), EP1 fromCotesia congregata bracovirus (CcBV)(Beckage &Kanost, 1993; Beckage et al., 1994), VHv1.1 fromCampoletis sonorensis ichnovirus (Dib-Hajj et al., 1993) andEGF-like gene products from Microplitis demolitor braco-virus (Strand et al., 1997; Trudeau et al., 2000). To investi-gate whether haemocytes are targeted by CrV2, haemocyteswere isolated from larvae at different times post-parasitizationand tested for CrV2 presence by staining with FITC-linkedsecondary antibody. The maximum amount of stainingoccurred at 24 h.p.p. (Fig. 6a). At this point, much of theCrV2 appeared to be localized within the haemocytes inlarge endosomes (Fig. 6b). Most CrV2 protein is foundin cell-free haemolymph at this stage, which suggests thatCrV2 is taken up by haemocytes, similarly to CrV1. CrBV-related proteins remain present in the haemolymph forseveral days after parasitization, although transcripts areproduced only transiently (Asgari et al., 1996). However, alow, persistent level of CrBV expression that was notdetected by Northern blotting cannot be ruled out. A com-prehensive, real-time, RT-PCR quantification approach isrequired to confirm expression levels.

DISCUSSION

The relatively small number of four CrBV genes that areexpressed in the host provides the opportunity to elucidatethe role of CrBV in parasitization in a comprehensivemanner, compared with other PDV systems where a largenumber of genes are expressed by PDV-infected host cells(Kroemer & Webb, 2004). The overall role of PDVs thatencode active immune suppressors is widely accepted.

Fig. 4. Trimer formation and coiled-coil structure of CrV2. (a) Trimerization of CrV2. Non-denaturing Western blot analysis(12% PAGE; anti-CrV2 antiserum, 1 : 5000) of protein extracts from cell-free haemolymph from 6 h-parasitized (P) and naı̈ve(N) Pieris rapae larvae. A putative CrV2 trimer of approximately 98 kDa was detected (arrowhead). (b) Deduced CrV2 aminoacid sequence was analysed by COILS software. A putative coiled-coil region is predicted near the C-terminus of the protein(aa 266–313).



Nearly all of the characterized genes that have been reportedso far are known to target host haemocytes; however, otherfunctions, such as alteration of developmental regulationand behaviour, are also known (Beckage & Gelman, 2004).From our investigations, it appears that the main role ofCrBV is immune suppression of the larval host haemocytes.CrV1 is known to be taken up by host haemocytes, causinginactivation of F-actin and reducing the ability of infectedhaemocytes to carry out immune-related tasks, such asphagocytosis and spreading (Asgari et al., 1996, 1997). Thus,CrV1 acts as a haemocyte-specific toxin, although itstransient expression means that cells recover after a fewdays. Interestingly, a CrV1 homologue was identified inCcBV, but differed in that expression was strong for 72 hand continued at low levels throughout development ofthe wasp larvae (Le et al., 2003). It is believed that the CrV1homologue affects haemocytes bymediating apoptosis (unlikefor CrBV), perhaps a function of continuous expression.However, the level of complexity of the CcBV system pre-cludes linking apoptosis to a single gene for the time being.

The function of CrV2 is, as yet, undetermined. The largeamount of CrV2 in haemolymph and haemocytes at24 h.p.p., and low levels of CrV2 transcripts in haemocytes

at the same time point, indicate that haemocytes internalizethe protein. In such a scenario, haemocytes and fat bodycells would secrete CrV2 into the serum, from wherehaemocytes acquire CrV2, as is the case for CrV1 (Asgariet al., 1996, 1997). Further studies are required to determinethe molecular interactions between CrV2 and haemocytesin vitro, in both the presence and absence of other CrBVproteins. Injection of active recombinant CrV2 into naı̈velarvae may also reveal the effects of CrV2 on haemocytesand whether P. rapae haemolymph proteins are requiredfor haemocyte uptake. As CrV2 is similar to CrV1 in terms ofmonomer size, expression levels, presence of a coiled-coilregion and formation of small oligomers, together with thefact that most characterized class II PDV genes (thoseexpressed in the host caterpillar; Theilmann & Summers,1988) appear to target haemocytes, may further indicatethat the function of CrV2 is similar to CrV1. It is possiblethat CrV2 enhances or complements the activity of CrV1by targeting a distinct haemocyte type.

CrV3 is also implicated in immune disruption in that it isrelated closely to invertebrate immune proteins. Apart fromCrV3 homologues that are expressed by other Cotesia-associated bracoviruses (Teramoto & Tanaka, 2003), the

Fig. 5. Transcription of CrV2. (a) Northernslot-blot comparing relative levels of CrV2

and CrV3 transcripts in total RNA from un-and 6 h-parasitized P. rapae larvae. Probesconsisted of 32P-labelled CrV2 and CrV3

fragments to measure virus transcripts and a32P-labelled P. rapae 18S rRNA gene frag-ment. Putative amounts of loaded RNA areshown at the top; the source of RNA (naı̈veversus parasitized larvae) is shown at thebottom. Autoradiograph exposure period isshown on the left; probe type on the right.CrV2 transcripts comprise a significantlygreater proportion of total transcripts thanCrV3 transcripts. (b) Comparison of relativeCrV2 and CrV3 protein levels in haemocytes(H), cell-free haemolymph (C) and fat body(F) from 6 h-parasitized P. rapae larvae. Theamount of each protein sample was cali-brated, allowing direct comparison of CrV2and CrV3 levels within a given sample. Theprobe used for each analysis is shownbeneath the blot. CrV2 appears to beexpressed at a significantly higher level ineach sample than CrV3.



closest relatives to CrV3 were insect CTLs that are secretedinto cell-free haemolymph on induction by foreign elicitors,such as lipopolysaccharide on bacterial surfaces. TheseCTLs act as immune molecules by binding to specific sugarmoieties associated with foreign surfaces, rendering themvisible to the immune system and facilitating their removalfrom circulation. It seems probable that the unusual regula-tion of the CrV3 protein is associated with its role inimmune suppression, as opposed to immune protection.

The P. rapae–CrBV–C. rubecula system represents a uniqueopportunity to develop a comprehensive model of immune-suppressive activity carried out by CrBV and thus to gleanmore general information relating to virus-related manipu-lation of host physiology. Genes such as CrV1 and CrV3,whose homologues occur in a range of Cotesia-relatedbracoviruses, also raise interesting questions about theorigin of PDVs and their genes. Evolutionary studies willexplore the origin of this relationship further by targetingthe ancestral PDV forms and the way PDVs have apparentlydriven the successful radiation of certain ichneumonoidendoparasitoids.

ACKNOWLEDGEMENTS

This work was funded by an Australian Research Council fellowshipand a University of Adelaide Small Grant Scheme to S. A. and anAustralian Postgraduate Award to R. G.

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Phase-contrast UV illumination(a)

(b)

Fig. 6. Visualization of CrV2 in haemocytesfrom 24 h-parasitized P. rapae larvae, usingantibodies against recombinant CrV2and FITC-conjugated secondary antibodies.(a) Fluorescent labelling of CrV2 associatedwith haemocytes from naı̈ve and 24 h-parasitized P. rapae larvae. Backgroundlabelling was low (see unparasitized control),indicating that most staining results fromlabelled CrV2 detected in infected haemo-cytes. (b) Magnified view of several infectedhaemocytes showing CrV2, which is app-arently internalized in large granular-shapestructures that are possibly lysosomes(arrowheads), in contrast to earlier stages ofinfection where staining is diffuse over thecytoplasm (not shown).



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Isolation and characterization of a Cotesia rubecula bracovirus gene expressed in the lepidopteran...

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