1 Insect Molecular Biology, University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia
2 Department of Zoology and Entomology, School of Life Sciences, University of Queensland, St Lucia, QLD 4072, Australia
Correspondence
Sassan Asgari
s.asgari{at}uq.edu.au
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY631272.
Present address: CSIRO Health Sciences and Nutrition, Therapeutic and Diagnostic Technologies, PO Box 10041 Adelaide BC, Australia.
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INTRODUCTION |
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The Cotesia rubecula bracovirus (CrBV) is unique as it appears to generate only four gene products, production of which 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 to several gene families (Turnbull & Webb, 2002
) and expression of at least some genes continues throughout development of the parasitoid larva (Webb, 1998
). Transient gene expression is associated with reversible inactivation of the cellular immune system, which is considered to be an advanced evolutionary adaptation that restores the parasitized host's ability to defend the developing parasitoid against pathogens or other parasitoids.
Asgari et al. (1996, 1997)
cloned and sequenced CrV1-encoding DNA from CrBV, demonstrating that CrV1 is an encapsidated gene that is expressed as a single transcript in parasitized host haemocytes. CrV1 is secreted from infected cells into serum interacting with the surface of haemocytes (Asgari et al., 1997
). Although the exact mode of action is unknown, the presence of depolymerized actin in CrV1-treated haemocytes suggests that CrV1 interaction with the cell surface leads to depolymerization of cytoplasmic actin structural components (Asgari et al., 1997
). Without functional actin filaments, haemocytes are unable to undergo the rearrangements of the cytoskeleton that are required for immune-related spreading and phagocytosis reactions (Rosales et al., 1994
; Yano et al., 1994
; Strand & Pech, 1995
; Asgari et al., 1997
). Homologues of CrV1 have been found to occur in six Cotesia species and the matching phylogenetic trees created by analysis of wasp 16S rRNA and NADH1 genes also matched that produced by analysis of CrV1 homologue sequences (Whitfield, 2000
), suggesting that PDVs were not acquired independently among Cotesia species, but co-evolved with the hymenopteran parasitoid (Whitfield & Asgari, 2003
).
More recently, a second CrBV gene (CrV3) was characterized as a C-type lectin (CTL) (Glatz et al., 2003). CrV3 has homologues in bracoviruses that are associated with Cotesia ruficrus and Cotesia karyai (Teramoto & Tanaka, 2003
) and this group of CTLs forms a unique CTL family. Interestingly, the CrV3 homologues are related more closely to invertebrate CTLs, which have been implicated in humoral immune defence of such animals (Haq et al., 1996
; Saito et al., 1997
; Arai et al., 1998
; Kakiuchi et al., 2002
; Yu & Kanost, 2003
), than to known viral lectins. CrV3 forms multimeric structures in the haemolymph, composed of monomers (occurring as two glycoforms for CrV3) that each 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 of CrV2, the third of four expressed CrBV genes to be isolated. Like CrV1, CrV2 has a coiled-coil domain and is found in oligomeric form in the haemolymph of parasitized larvae, where it is taken up by host haemocytes.
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METHODS |
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CrBV and genomic DNA isolation.
Virus purification and genomic DNA extraction were carried out as described previously (Glatz et al., 2003).
Southern and Northern blot hybridization.
DNA samples were run on a 1 % agarose gel and transferred to a nylon membrane as described by Sambrook et al. (1989). RNA was isolated from unparasitized and 6 h-parasitized P. rapae caterpillars according to Chomczynski & Sacchi (1987)
. RNA samples were run on 1 % agarose gels under denaturing conditions using formaldehyde and transferred 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 microfiltration apparatus (Bio-Rad), according to the manufacturer's instructions.
5' amplification of CrV2 cDNA (5' RACE).
Partial CrV2 cDNA was extended in the 5' direction by using the 5' RACE system for rapid amplification of cDNA ends (Life Technologies). PCR product obtained from 5' RACE was ligated into the pGEM-T Easy vector as described by the manufacturer (Promega). The insert was sequenced by using M13 forward and reverse primers.
Computer analysis.
Sequences were compared against those contained in GenBank by using a nucleotide BLAST search, accessed via the National Centre for Biotechnology Information website (www.ncbi.nlm.nih.gov/blast). All CrV2 protein analysis tools were accessed through the ExPASy molecular biology server (http://us.expasy.org/tools).
RT-PCR.
Primers specific to the CrV2 ORF (Fig. 1) were used in RT-PCR analysis of RNA from unparasitized and 6 h-parasitized P. rapae larvae by using AMV reverse transcriptase (AMV-RT; Promega). XhoI and HindIII restriction sites were added to primer sequences (underlined) to provide sites for direct ligation of the fragment into the pQE30 expression vector (Qiagen). Primer CrV2-R (5'-GCGCAAGCTTTTAGGGATGATCTCGAGC-3') was used in the RT reaction, followed by PCR using primers CrV2-R and CrV2-F (5'-CGCGGCATGCCCGTTGCAAGACAGAAG-3').
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Expression and purification of bacterial CrV2.
Primers CrV2-F and CrV2-R were designed to amplify the ORF of the CrV2 gene, excluding a putative signal sequence corresponding to the first 20 aa of the protein (see Fig. 1 and RT-PCR for CrV2). These primers were used in PCR of pGEM-T Easy vector (Promega) containing the CrV2 ORF to obtain the required fragment for ligation to the pQE30 bacterial expression vector (Qiagen). Production of bacterial CrV2 (containing six additional, vector-derived histidine residues) was induced by addition of 1 mM IPTG to the bacterial suspension for 2 h at 37 °C. The resulting fusion protein was largely contained in the insoluble fraction and was purified under denaturing conditions as described by the manufacturer (Qiagen). Samples were dialysed overnight against TBS (0·15 M NaCl, 0·01 M Tris, pH 8·0) at 4 °C.
Anti-CrV2 polyclonal antibody production.
Purified bacterial CrV2 was visualized on preparative 12 % SDSacrylamide gels by staining with water-dissolved Coomassie brilliant blue (Sigma). CrV2 protein bands were excised from the gel and used for immunization of two rabbits as described previously (Glatz et al., 2003). The antiserum was used at a dilution of 1 : 5000. Bound anti-CrV2 was then visualized by alkaline phosphatase-labelled secondary anti-rabbit antibody (1 : 10 000).
Fluorescent labelling of CrV2 associated with P. rapae haemocytes.
Unparasitized and 24 h-parasitized larvae were bled into PBS that was saturated with phenylthiourea before centrifugation at 2300 g for 5 min. The pellet was then resuspended gently in PBS before transfer to multiwell slides. Time was allowed for settling before fixing cells with 4 % paraformaldehyde in PBS. Anti-CrV2 antiserum and fluorescein isothiocyanate (FITC)-conjugated anti-rabbit secondary antibody were used to visualize CrV2 associated with haemocytes, as described previously (Asgari et al., 1996).
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RESULTS |
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Isolation and characterization of CrV2
Several screens of the cDNA library led to the isolation of an approximately 450 bp cDNA (CloneC) that encoded part of a putative CrBV gene and included a poly(A) tail (Fig. 1). The fragment was cloned and sequenced, allowing primers to be designed to amplify 290 bp of the 5' end (CloneC-F and CloneC-R; Fig. 1
). To confirm the cDNA fragment as particle-derived, the amplified fragment was then used as a probe in both a Southern blot of CrBV DNA (Fig. 2a
) and a Northern blot of RNA from naïve and 6 h-parasitized larvae (Fig. 2b
). Hybridization occurred to a CrBV restriction fragment of >15 kbp and to a parasitism-specific transcript of approximately 1·2 kbp. These data, and the fact that the same probe bound to genomic DNA from female wasps but not to genomic DNA from P. rapae (data not shown), indicate that the cDNA originated from particles that were introduced to the larvae at oviposition. Based on the transcript size being intermediate between CrV1 (1·4 kbp) and CrV3 (1·1 kbp), matching the transcript size of CrV2-encoding mRNA, we designated the newly isolated gene as CrV2. Binding of the cDNA to a single RNA band on the Northern blot revealed that CrV2 shows no significant homology to other CrBV-related genes within the 290 bp probe. Slot-blot analysis of RNA isolated from haemocytes and fat body cells from 6 h-parasitized larvae showed that there was no significant difference in the amount of CrV2 transcripts detected in the two samples (Fig. 2d
). In order to measure RNA loading within slots, a fragment of 18S rRNA was used from P. rapae as a control.
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Four putative N- and six putative O-glycosylation sites were predicted in the ORF, as well as a polyadenylation signal approximately 120 bp downstream of the stop codon (Fig. 1). These data were used to generate primers specific to the CrV2 ORF (CrV2-F and CrV2-R; Fig. 1
). Comparison of RT-PCR and genomic DNA PCR products that were generated by utilizing these primers revealed no sequence differences, indicating that no introns are present in the genomic CrV2 DNA. The CrV2 nucleotide and deduced amino acid sequences were compared against known sequences in GenBank; however, no significant homology was detected.
CrV2, without the signal peptide, was expressed in Escherichia coli and produced an approximately 40 kDa fusion protein, following induction with IPTG (Fig. 2c). Computer analyses predicted a molecular mass of 33·7 kDa and a pI of 8·94 for secreted CrV2. Purified recombinant CrV2 was used to immunize rabbits for production of polyclonal anti-CrV2 antibodies. Western blot analysis of serum from non-parasitized and 6 h-parasitized P. rapae larvae, probed with anti-CrV2, allowed visualization of the 37 kDa CrV2 only in parasitized larvae (Fig. 3a
). Previous data showed the presence of a parasitism-specific glycoprotein in the haemolymph of P. rapae larvae, the production of which was initiated 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 previously unidentified glycoprotein is CrV2 (Fig. 3b
). These data confirm that CrV2 is a secreted glycoprotein and, further, that it contains N-acetyl-D-galactosamine residues at its O-glycosylation sites, as it was previously detected by GalNAc-specific HPL (Asgari, 1997
). Western blot analyses of larval serum, haemocytes and fat body cells at various points after parasitization showed that CrV2 was present in each sample at 6 h.p.p., reached a maximum level at about 24 h.p.p. and was declining at 48 h.p.p. (Fig. 3c
). These data are consistent with secretion of CrV2 into cell-free haemolymph from CrBV-infected haemocytes and fat body cells.
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Immunofluorescence detection of CrV2 in haemocytes
Most of the PDV genes characterized so far target haemocytes of parasitized larvae, e.g. CrV1 (Asgari et al., 1996, 1997
), EP1 from Cotesia congregata bracovirus (CcBV) (Beckage & Kanost, 1993
; Beckage et al., 1994
), VHv1.1 from Campoletis sonorensis ichnovirus (Dib-Hajj et al., 1993
) and EGF-like gene products from Microplitis demolitor bracovirus (Strand et al., 1997
; Trudeau et al., 2000
). To investigate whether haemocytes are targeted by CrV2, haemocytes were isolated from larvae at different times post-parasitization and tested for CrV2 presence by staining with FITC-linked secondary antibody. The maximum amount of staining occurred at 24 h.p.p. (Fig. 6a
). At this point, much of the CrV2 appeared to be localized within the haemocytes in large endosomes (Fig. 6b
). Most CrV2 protein is found in cell-free haemolymph at this stage, which suggests that CrV2 is taken up by haemocytes, similarly to CrV1. CrBV-related proteins remain present in the haemolymph for several days after parasitization, although transcripts are produced only transiently (Asgari et al., 1996
). However, a low, persistent level of CrBV expression that was not detected by Northern blotting cannot be ruled out. A comprehensive, real-time, RT-PCR quantification approach is required to confirm expression levels.
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DISCUSSION |
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The function of CrV2 is, as yet, undetermined. The large amount of CrV2 in haemolymph and haemocytes at 24 h.p.p., and low levels of CrV2 transcripts in haemocytes at the same time point, indicate that haemocytes internalize the protein. In such a scenario, haemocytes and fat body cells would secrete CrV2 into the serum, from where haemocytes acquire CrV2, as is the case for CrV1 (Asgari et al., 1996, 1997
). Further studies are required to determine the molecular interactions between CrV2 and haemocytes in vitro, in both the presence and absence of other CrBV proteins. Injection of active recombinant CrV2 into naïve larvae may also reveal the effects of CrV2 on haemocytes and whether P. rapae haemolymph proteins are required for haemocyte uptake. As CrV2 is similar to CrV1 in terms of monomer size, expression levels, presence of a coiled-coil region and formation of small oligomers, together with the fact that most characterized class II PDV genes (those expressed in the host caterpillar; Theilmann & Summers, 1988
) appear to target haemocytes, may further indicate that the function of CrV2 is similar to CrV1. It is possible that CrV2 enhances or complements the activity of CrV1 by targeting a distinct haemocyte type.
CrV3 is also implicated in immune disruption in that it is related closely to invertebrate immune proteins. Apart from CrV3 homologues that are expressed by other Cotesia-associated bracoviruses (Teramoto & Tanaka, 2003), the closest relatives to CrV3 were insect CTLs that are secreted into cell-free haemolymph on induction by foreign elicitors, such as lipopolysaccharide on bacterial surfaces. These CTLs act as immune molecules by binding to specific sugar moieties associated with foreign surfaces, rendering them visible to the immune system and facilitating their removal from circulation. It seems probable that the unusual regulation of the CrV3 protein is associated with its role in immune suppression, as opposed to immune protection.
The P. rapaeCrBVC. rubecula system represents a unique opportunity to develop a comprehensive model of immune-suppressive activity carried out by CrBV and thus to glean more general information relating to virus-related manipulation of host physiology. Genes such as CrV1 and CrV3, whose homologues occur in a range of Cotesia-related bracoviruses, also raise interesting questions about the origin of PDVs and their genes. Evolutionary studies will explore the origin of this relationship further by targeting the ancestral PDV forms and the way PDVs have apparently driven the successful radiation of certain ichneumonoid endoparasitoids.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Arai, T., Kawasaki, K., Kubo, T. & Natori, S. (1998). Cloning of cDNA for regenectin, a humoral C-type lectin of Periplaneta americana, and expression of the regenectin gene during leg regeneration. Insect Biochem Mol Biol 28, 987994.[CrossRef][Medline]
Asgari, S. (1997). Cotesia rubecula polydnavirus-specific gene expression in the host Pieris rapae. PhD thesis, University of Adelaide, Australia.
Asgari, S., Hellers, M. & Schmidt, O. (1996). Host haemocyte inactivation by an insect parasitoid: transient expression of a polydnavirus gene. J Gen Virol 77, 26532662.[Abstract]
Asgari, S., Schmidt, O. & Theopold, U. (1997). A polydnavirus-encoded protein of an endoparasitoid wasp is an immune suppressor. J Gen Virol 78, 30613070.[Abstract]
Beckage, N. E. (1998). Parasitoids and polydnaviruses. Bioscience 48, 305311.
Beckage, N. E. & Gelman, D. B. (2004). Wasp parasitoid disruption of host development: implications for new biologically based strategies for insect control. Annu Rev Entomol 49, 299330.[CrossRef][Medline]
Beckage, N. E. & Kanost, M. R. (1993). Effects of parasitism by the braconid wasp Cotesia congregata on host hemolymph proteins of the tobacco hornworm, Manduca sexta. Insect Biochem Mol Biol 23, 643653.[CrossRef][Medline]
Beckage, N. E., Tan, F. F., Schleifer, K. W., Lane, R. D. & Cherubin, L. L. (1994). Characterization and biological effects of Cotesia congregata polydnavirus on host larvae of the tobacco hornworm, Manduca sexta. Arch Insect Biochem Physiol 26, 165195.
Cavener, D. R. & Ray, S. C. (1991). Eukaryotic start and stop translation sites. Nucleic Acids Res 19, 31853192.[Abstract]
Chomczynski, P. & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanatephenolchloroform extraction. Anal Biochem 162, 156159.[CrossRef][Medline]
Dib-Hajj, S. D., Webb, B. A. & Summers, M. D. (1993). Structure and evolutionary implications of a "cysteine-rich" Campoletis sonorensis polydnavirus gene family. Proc Natl Acad Sci U S A 90, 37653769.[Abstract]
Glatz, R., Schmidt, O. & Asgari, S. (2003). Characterization of a novel protein with homology to C-type lectins expressed by the Cotesia rubecula bracovirus in larvae of the lepidopteran host, Pieris rapae. J Biol Chem 278, 1974319750.
Haq, S., Kubo, T., Kurata, S., Kobayashi, A. & Natori, S. (1996). Purification, characterization, and cDNA cloning of a galactose-specific lectin from Drosophila melanogaster. J Biol Chem 271, 2021320218.
Harwood, S. H. & Beckage, N. E. (1994). Purification and characterization of an early-expressed polydnavirus-induced protein from the hemolymph of Manduca sexta larvae parasitized by Cotesia congregata. Insect Biochem Mol Biol 24, 685698.[CrossRef]
Kakiuchi, M., Okino, N., Sueyoshi, N., Ichinose, S., Omori, A., Kawabata, S., Yamaguchi, K. & Ito, M. (2002). Purification, characterization, and cDNA cloning of -N-acetylgalactosamine-specific lectin from starfish, Asterina pectinifera. Glycobiology 12, 8594.
Kilpatrick, D. C. (2002). Animal lectins: a historical introduction and overview. Biochim Biophys Acta 1572, 187197.[Medline]
Kroemer, J. A. & Webb, B. A. (2004). Polydnavirus genes and genomes: emerging gene families and new insights into polydnavirus replication. Annu Rev Entomol 49, 431456.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Le, N. T., Asgari, S., Amaya, K., Tan, F. F. & Beckage, N. E. (2003). Persistence and expression of Cotesia congregata polydnavirus in host larvae of the tobacco hornworm, Manduca sexta. J Insect Physiol 49, 533543.[CrossRef][Medline]
Rosales, C., Jones, S. L., McCourt, D. & Brown, E. J. (1994). Bromophenacyl bromide binding to the actin-bundling protein l-plastin inhibits inositol trisphosphate-independent increase in Ca2+ in human neutrophils. Proc Natl Acad Sci U S A 91, 35343538.[Abstract]
Saito, T., Hatada, M., Iwanaga, S. & Kawabata, S. (1997). A newly identified horseshoe crab lectin with binding specificity to O-antigen of bacterial lipopolysaccharides. J Biol Chem 272, 3070330708.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Stoltz, D. B. & Vinson, S. B. (1979). Viruses and parasitism in insects. Adv Virus Res 24, 125171.[Medline]
Stoltz, D. B. & Whitfield, J. B. (1992). Viruses and virus-like entities in the parasitic Hymenoptera. J Hymenopt Res 1, 125139.
Stoltz, D. B., Guzo, D. & Cook, D. (1986). Studies on polydnavirus transmission. Virology 155, 120131.[Medline]
Strand, M. R. & Pech, L. L. (1995). Microplitis demolitor polydnavirus induces apoptosis of a specific haemocyte morphotype in Pseudoplusia includens. J Gen Virol 76, 283291.[Abstract]
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, 16271635.[Abstract]
Strand, M. R., Witherell, R. A. & Trudeau, D. (1997). Two Microplitis demolitor polydnavirus mRNAs expressed in hemocytes of Pseudoplusia includens contain a common cysteine-rich domain. J Virol 71, 21462156.[Abstract]
Teramoto, T. & Tanaka, T. (2003). Similar polydnavirus genes of two parasitoids, Cotesia kariyai and Cotesia ruficrus, of the host Pseudaletia separata. J Insect Physiol 49, 463471.[CrossRef][Medline]
Theilmann, D. A. & Summers, M. D. (1988). Identification and comparison of Campoletis sonorensis virus transcripts expressed from four genomic segments in the insect hosts Campoletis sonorensis and Heliothis virescens. Virology 167, 329341.[CrossRef][Medline]
Trudeau, D., Witherell, R. A. & Strand, M. R. (2000). Characterization of two novel Microplitis demolitor polydnavirus mRNAs expressed in Pseudoplusia includens haemocytes. J Gen Virol 81, 30493058.
Turnbull, M. & Webb, B. (2002). Perspectives on polydnavirus origins and evolution. Adv Virus Res 58, 203254.[Medline]
Webb, B. A. (1998). Polydnavirus biology, genome structure, and evolution. In The Insect Viruses, pp. 105139. Edited by L. K. Miller & L. A. Ball. New York: Plenum.
Whitfield, J. B. (2000). Phylogeny of microgastroid braconid wasps, and what it tells us about polydnavirus evolution. In Hymenoptera: Evolution, Biodiversity and Biological Control, pp. 97105. Edited by A. D. Austin & M. Dowton. Melbourne, Australia: CSIRO.
Whitfield, J. B. & Asgari, S. (2003). Virus or not? Phylogenetics of polydnaviruses and their wasp carriers. J Insect Physiol 49, 397405.[CrossRef][Medline]
Yano, Y., Kambayashi, J., Shiba, E., Sakon, M., Oiki, E., Fukuda, K., Kawasaki, T. & Mori, T. (1994). The role of protein phosphorylation and cytoskeletal reorganization in microparticle formation from the platelet plasma membrane. Biochem J 299, 303308.[Medline]
Yu, X.-Q. & Kanost, M. R. (2003). Manduca sexta lipopolysaccharide-specific immulectin-2 protects larvae from bacterial infection. Dev Comp Immunol 27, 189196.[CrossRef][Medline]
Received 23 May 2004;
accepted 16 June 2004.