Characterization of two genes of Cotesia vestalis ... - Semantic Scholar
Journal of General Virology (2007), 88, 3317–3322
Short Communication
DOI 10.1099/vir.0.82999-0
Characterization of two genes of Cotesia vestalis polydnavirus and their expression patterns in the host Plutella xylostella Ya-Feng Chen, Min Shi, Fang Huang and Xue-xin Chen
Correspondence
Institute of Insect Sciences, Zhejiang University, 268 Kaixuan Road, Hangzhou 310029, PR China
Xue-xin Chen [email protected]
Received 15 March 2007 Accepted 23 August 2007
Cotesia vestalis is an endoparasitoid of larval stages of Plutella xylostella, the diamondback moth. For successful parasitization, this parasitoid injects a polydnavirus into its host during oviposition. Here we isolated two genes, which we named CvBV1 and CvBV2. CvBV1 was located on segment CvBV-S5 with a size of 790 bp, while CvBV2 was located on segment CvBV-S51 with a size of 459 bp. A gene copy of CvBV2 was found on segment CvBV-S48, which we name CvBV2’. Gene duplication occurred in both genes, tandem gene duplication for CvBV1 and segmental duplication for CvBV2. Gene transcripts of the two genes were detected in hosts as early as 0.5 h post-parasitization (p.p.) and continued to be detected for six days, and tissue-specific expression patterns showed that they could be detected in the haemolymph and brain at 2 h p.p., suggesting that they could participate in early protection of parasitoid eggs from host cellular encapsulation.
Polydnavirus (PDV) is a unique group of insect viruses because of their obligate and symbiotic associations with parasitic wasps in the families Braconidae and Ichneumonidae, as Bracovirus (BV) and Ichnovirus (IV) genera, respectively (Kroemer & Webb, 2004). They are so named because their genome is polydisperse, containing a series of different circular DNAs (Turnbull & Webb, 2002). The endoparasitoid, Cotesia vestalis (Hymenoptera: Braconidae), employs multiple strategies of active and passive immune-suppression in overcoming the defences of its host, Plutella xylostella (Lepidoptera: Plutellidae). We have demonstrated previously that the venom from C. vestalis has a limited effect on haemocytes, whereas calyx fluid from C. vestalis may play a major role in the suppression of the host immune system and the venom probably synergizes the effect of calyx fluid or C. vestalis polydnavirus (CvBV) (Yu et al., 2007). Most of the CvBV genome has been sequenced using the plasmid capture system (PCS), although the segments over 30 kb do not amplify due to PCS system restrictions (Choi et al., 2005). CvBV shares some genetic similarity with the Cotesia congregata bracovirus (CcBV), the first BV genome to be fully sequenced (Espagne et al., 2004). Here we isolated two CvBV genes and their genomic organization and expression patterns were investigated. The GenBank accession numbers of the sequences reported in this paper are EF467277 and EF467278. Sequences of the primers used in this study are available with the online version of this paper.
0008-2999 G 2007 SGM
Printed in Great Britain
The parasitoid species of the current study is commonly known as Cotesia plutellae (Kurdjumov) in the literature, but now is referred to as C. vestalis (Haliday) (Shaw, 2003). The cDNA library was prepared with the SMART cDNA Library Construction Kit following the manufacturer’s protocols (Clontech), using 2 mg of total RNA isolated from parasitized P. xylostella 6 h post-parasitization (p.p.). PDV virions and viral DNA were collected from 200–300 2-day-old female wasps as previously described by Beckage et al. (1994) and Chen & Gundersen-Rindal (2003). CvBV probe was prepared by double-digesting the viral DNA with BamHI and HindIII and labelling it with 32P using Random Primer DNA Labelling Kit Ver. 2.0 (TaKaRa) according to the manufacturer’s instructions. Library screening was carried out by the method described by Chen & Gundersen-Rindal (2003). Two positive clones confirmed by secondary screening were converted to pTriplEx2 by infecting E. coli BM25.8. These two clones were sequenced and named CvBV1 and CvBV2 (GenBank accession numbers are EF467277 and EF467278). BLAST searches of these two sequences indicated that CvBV1 and CvBV2 were located on segments CvBV-S5 (14.5 kb) and CvBV-S51 (17.5 kb) of the CvBV genome, respectively (Fig. 1a). CvBV2 has a gene copy on segment CvBV-S48 (15.4 kb) with 3 bp nucleotide differences in the 39 untranslated region (UTR) and we refer to it as CvBV2’. Highly homologous sequences characterized the two genes on their respective segments, segment CvBV-S5 and CvBVS11 for CvBV1, and segment CvBV-S51 and CvBV-S48 for CvBV2 (Fig. 1a). Homologous sequences of CvBV1 were
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Fig. 1. (a) Location of CvBV1, CvBV2 and their homologous sequences on CvBV segments. Boxes represent exon sequences and their homologous sequences. Numbers above boxes indicate percentage similarity. Dark shaded regions indicate UTRs. GenBank accession numbers: DQ075357 for CvBV-S5, DQ075360 for CvBV-S11, EF067330 for CvBV-S48 and EF067332 for CvBV-S51. (b, c) Nucleotide and deduced amino acid sequences of CvBV1 and CvBV2. Potential polyadenylation signals are in bold characters. The most likely cleavage sites of putative signal peptides are underlined in the figure. The sole potential O-glycosylation site of CvBV 1 is underlined and in italics. Three potential N-glycosylation sites of CvBV2 are underlined and in bold characters. The positions of the single intron present in the genomic sequence of CvBV1 and CvBV2 are denoted with asterisks in the cDNA sequence. Sequences of the introns present in the genomic fragment coding for CvBV1 and CvBV2 are flanked by the last coding triplet of exon1 and the first triplet of exon2.
CvBV1-s5 favoured the hypothesis of tandem gene duplication since both genes clustered on the same segment, whereas CvBV2/CvBV2’ and CvBV-HP5107/ CvBV-HP4805 depicted segmental duplication. Gene duplication along with genome segmentation, segment nesting and non-equimolar segment ratios are different strategies adopted by the virus to increase the copy number of essential genes and the levels of gene expression in the absence of virus replication (Kroemer & Webb, 2004; Webb & Cui, 1998). The current results revealed a high degree of similarity between segments CvBV-S51 and CvBV-S48, and between segments CvBV-S5 and CvBVS11. Despite the close resemblances, Choi et al. (2005) made it clear that segments CvBV-S51 and CvBV-S48, CvBV-S5 and CvBV-S11 are all different segments with different digestion patterns.
predicted to encode proteins using Fgenesh (Salamov & Solovyev, 2000; Solovyev & Salamov, 1999) and were named CvBV1-s5 and CvBV1-s11, respectively (Fig. 1a). Gene duplication has been previously reported in BVs and IVs and categorized as follows: (1) tandem gene duplication, (2) segmental duplication or (3) unresolved between the two hypotheses (Friedman & Hughes, 2006; Hilgarth & Webb, 2002; Provost et al., 2004). The pair of CvBV1 and 3318
CvBV1 had a size of 790 bp and was composed of two exons and an intron, which was consistent with the prediction in GenBank. In the 39 UTR, two potential polyadenylation signals (Beaudoing et al., 2000) were observed (Fig. 1b). CvBV2 had a size of 459 bp and was similarly composed of two exons and an intron, but the first exon was not consistent with the prediction in GenBank. There were three potential polyadenylation signals in the 39 UTR (Fig. 1c). The complicated genome organization and gene expression patterns make PDV gene prediction difficult, so different parameters may be adopted for PDV genes expressed in different hosts (hymenopteran and lepidopteran). In order to improve PDV gene prediction, more comprehensive PDV gene transcription, processing and expression models are needed (Gundersen-Rindal & Pedroni, 2006). In the current study gene prediction was carried out using Fgenesh (Solovyev & Salamov, 1999; Salamov & Solovyev, 2000) with human, Drosophila melanogaster, honey bee and Brugia malayi (parasitic nematode) settings. Most settings correctly predicted the nature of CvBV1, except for the Drosophila Journal of General Virology 88
Two Cotesia vestalis polydnavirus genes
settings, whereas CvBV2 could only be predicted correctly using the Brugia malayi settings. Brugia malayi, a mosquito-borne nematode parasite and a cause of lymphatic filariasis in humans (Ottesen et al., 1997), has evolved specific measures to counter host immune defences–strategies quite similar to those employed by PDVs, which is a possible reason for the correct prediction by this species. The open reading frame (ORF) of CvBV1 comprises 97 aa encoding a protein with a predicted molecular mass of 10.9 kDa (Fig. 1b). Computer analyses of the deduced amino acid sequence suggested that the protein could have a signal peptide (Nielsen et al., 1997), indicating that CvBV1 protein was probably secreted from CvBV-infected cells. The most likely cleavage sites of putative signal peptides were between positions 24 and 25 (SGS-SP) or
between positions 31 and 32 (AYA-KP). A potential Oglycosylation site at position 94 in the deduced protein was predicted by NetOGlyc 3.1 (http://www.cbs.dtu.dk/services/NetOGlyc/). The search for conserved domains using Blocks (Henikoff & Henikoff, 1994) indicated significant similarity to a staphylococcal enterotoxin, which has been shown to induce apoptosis in thymocytes, which are a kind of immune cell (Lin et al., 1999). The ORF of CvBV2 encoded 91 amino acids with a predicted molecular mass of 10.0 kDa (Fig. 1c). The predicted protein also seemed to have a N-terminal signal peptide and was proposed to be localized in the lysosome (Nakai & Horton, 1999). The predicted cleavage sites of the putative signal peptides were between positions 18 and 19 (GTS-WF) or between positions 30 and 31 (VDA-LP) (Nielsen et al., 1997). There were three potential N-glycosylation sites predicted by NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc/).
Fig. 2. Sequence comparison of CvBV1 and CvBV2 with homologous hypothetical proteins found in CcBV, CvBV and GiBV. GenBank accession numbers: YP_184861.1 for CcBV_23.3, ABK63302.1 for CvBV-HP1002, AAZ04271.1 for CvBVHP401, ABK63336.1 for CvBV-HP3701, YP_184904.1 for CcBV_33.6, YP_184802.1 for CcBV_9.3, ABK57021.1 for GIP_L1_00350, ABK57038.1 for GIP_L1_00520, AAZ04270.1 for CvBV-HP302, YP_184803.1 for CcBV_9.4, YP_184902.1 for CcBV_33.4, ABK63358.1 for CvBV-HP4805, ABK63370.1 for CvBV-HP5107, ABK57050.1 for GIP_L1_00640, and ABK57047.1 for GIP_L1_00610. http://vir.sgmjournals.org
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Fig. 3. (a) Phylogenetic analysis of CvBV1 and CvBV2 proteins. CcBV_9.4 and GIP_L1_00640 were used as the outgroup for the CvBV1 and CvBV2 tree, respectively. (b) Temporal and tissuespecific expression patterns of CvBV1 and CvBV2 genes in the parasitized host assessed by RT-PCR. Non-quantitative RT-PCR of the two genes was performed using specific primers based on each identified ORF and total RNAs from P. xylostella larvae parasitized by C. vestalis were isolated at various times p.p. (nonparasitized and 0.5, 2, 4, 6, 9, 12 h, 1, 2, 4, and 6 days p.p.) and different tissues. haem, Haemolymph; bra, brain; mid, midgut; viral, direct PCR product using viral DNA as template; neg, negative control (H2O).
The search for conserved domains using Blocks (Henikoff & Henikoff, 1994) indicated significant similarity to a hydrolase of the glycosyl hydrolase 92 family. Many studies have indicated that lysosomes and lysosomal enzymes, including some hydrolases, are involved in apoptosis (Kroemer & Jaattela, 2005; Tardy et al., 2006). Apoptosis is ubiquitously observed in parasitized hosts. A number of characterized genes of PDV appear to induce apoptosis of haemocytes (Lapointe et al., 2005; Le et al., 2003; Strand & Pech, 1995; Strand et al., 1997), which are involved in cellular immunity of insects and take part in phagocytosis, encapsulation and nodule formation (Lavine & Strand, 2002). Homologues of CvBV1 and CvBV2 were found among CvBV, CcBV and Glyptapanteles indiensis polydnavirus (GiBV) by BLASTP searches with an E-value cut-off of 1028 (Fig. 2). CvBV1 and CcBV_23.3 are the same size (97 aa) 3320
and show a high degree of similarity (68 %), indicating that CcBV_23.3 might be an equivalent gene in CcBV. CvBV2 shares 57 % similarity with CvBV-HP5107 and CvBVHP4805 over 84 aa. The CLUSTAL_X alignment shown in Fig. 2 revealed that there were two conserved domains on sequences of homologues for both genes, with one domain located on the N-terminal region and the other located on a non-secretive region. The peptide sequence functions like a postal address on an envelope by targeting proteins for secretion or for transfer to specific organelles for further processing (Choo et al., 2005); therefore we suggest that these homologues are transferred to the same location and share similar functions. Several other hypothetical proteins from CvBV, CcBV, GiBV or Cotesia kariyai polydnavirus (CkBV) only contained the conserved N-terminal domains, indicating that they might be transferred to the same destination but had different functions. For phylogenetic analysis, neighbour-joining trees were generated using PAUP4 (Fig. 3a). The result revealed that CvBV1 is closely related to CcBV_23.3, CvBV-HP302, GIP_L1_00350 and GIP_L1_00520, and CvBV2 is closely related to CvBVHP4805 and CvBV-HP5107 (Fig. 3a). These homologous genes were among CvBV, CcBV and GiBV, suggesting that these BVs are closely related. Transmission of PDVs is exclusively vertical and no replication occurs while inside the host (Stoltz, 1990; Stoltz et al., 1986), so genetic changes occur solely in the female wasp. Therefore, evolution of the polydnavirus is expected to parallel that of the wasp (Friedman & Hughes, 2006). Studies of BV phylogeny indicate that all BV-containing wasps share a common ancestor (Whitfield, 1997). The available fossildating techniques reveal that the ancestor of this lineage lived approximately 73.7±10 million years ago (Whitfield, 2002). Phylogenetic relationships among homologues of CvBV1 and CvBV2 reconfirm that the closer the relationship among the wasps, the more genetically similar are their polydnaviruses. As an advanced step, examination of temporal and tissuespecific expression patterns of CvBV1 and CvBV2 were carried out using non-quantitative RT-PCR with genespecific RT-PCR primers (Fig. 3b; Supplementary Table S1, available with the online version of this paper). Total RNAs from parasitized P. xylostella larvae at various time points and different tissues were treated with RNase-free DNase (TaKaRa) to eliminate DNA contamination and were used as templates in non-quantitative RT-PCR, which was performed according to the manufacturer’s protocol using an RT-PCR kit (QIAGEN). The absence of contaminating DNA in RNA samples was verified by running PCR directly without reverse transcription. The b-tubulin gene of P. xylostella was used as an internal control. Both CvBV1 and CvBV2 were detected in hosts as early as 0.5 h p.p. and continued to be detected for 6 days, which was similar to gene GiPDV 1.1 of GiBV (Chen et al., 2003). The early protein (EP) genes, which many researchers have shown to be early expressed PDV genes, are also detected within 30 min in the host Manduca sexta, and EPs accumulate to Journal of General Virology 88
Two Cotesia vestalis polydnavirus genes
comprise over 10 % of the total haemolymph proteins by 24 h p.p. (Beckage et al., 1987; Harwood et al., 1994). Tissue-specific expression patterns of the two genes showed that they could be detected in haemolymph and brain at 2 h p.p. and later they could be detected in the midgut. These findings suggest that these two genes could be involved in early protection of parasitoid eggs from host cellular encapsulation. For CvBV1 and CvBV2, further research will focus on obtaining purified native proteins and determining their mode of action.
an ankyrin repeat gene of the parasitoid Glyptapanteles indiensis polydnavirus in the parasitized host. J Gen Virol 87, 311–322. Harwood, S. H., Grosovsky, A. J., Cowles, E. A., Davis, J. W. & Beckage, N. E. (1994). An abundantly expressed hemolymph
glycoprotein isolated from newly parasitized Manduca sexta larvae is a polydnavirus gene product. Virology 205, 381–392. Henikoff, S. & Henikoff, J. G. (1994). Protein family classification
based on searching a database of blocks. Genomics 19, 97–107. Hilgarth, R. S. & Webb, B. A. (2002). Characterization of Campoletis
sonorensis ichnovirus segment I genes as members of the repeat element gene family. J Gen Virol 83, 2393–2402. Kroemer, G. & Jaattela, M. (2005). Lysosomes and autophagy in cell
Acknowledgements We are grateful to Dr M. R. Strand (University of Georgia) for his help at the initial stage of this research and his critical review of an early draft of the manuscript. We also thank Dr R. V. Glatz (SARDI, Australia) for his suggestions on our experiments and Dr M. Sharkey (University of Kentucky) for improving the English text. Funding for this study was provided jointly by 973 Program (2006CB102005), Key Program of Natural Science Foundation of Zhejiang Province (Z306031), the National Science Funds For Distinguished Young Scholars (Grant No. 30625006), Program for New Century Excellent Talents in University (NCET-04-0521), Innovation Research Team Program of the Ministry of Education of China (IRT0355), and the China Postdoctoral Science Foundation (20060400322).
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Journal of General Virology 88
Short Communication
DOI 10.1099/vir.0.82999-0
Characterization of two genes of Cotesia vestalis polydnavirus and their expression patterns in the host Plutella xylostella Ya-Feng Chen, Min Shi, Fang Huang and Xue-xin Chen
Correspondence
Institute of Insect Sciences, Zhejiang University, 268 Kaixuan Road, Hangzhou 310029, PR China
Xue-xin Chen [email protected]
Received 15 March 2007 Accepted 23 August 2007
Cotesia vestalis is an endoparasitoid of larval stages of Plutella xylostella, the diamondback moth. For successful parasitization, this parasitoid injects a polydnavirus into its host during oviposition. Here we isolated two genes, which we named CvBV1 and CvBV2. CvBV1 was located on segment CvBV-S5 with a size of 790 bp, while CvBV2 was located on segment CvBV-S51 with a size of 459 bp. A gene copy of CvBV2 was found on segment CvBV-S48, which we name CvBV2’. Gene duplication occurred in both genes, tandem gene duplication for CvBV1 and segmental duplication for CvBV2. Gene transcripts of the two genes were detected in hosts as early as 0.5 h post-parasitization (p.p.) and continued to be detected for six days, and tissue-specific expression patterns showed that they could be detected in the haemolymph and brain at 2 h p.p., suggesting that they could participate in early protection of parasitoid eggs from host cellular encapsulation.
Polydnavirus (PDV) is a unique group of insect viruses because of their obligate and symbiotic associations with parasitic wasps in the families Braconidae and Ichneumonidae, as Bracovirus (BV) and Ichnovirus (IV) genera, respectively (Kroemer & Webb, 2004). They are so named because their genome is polydisperse, containing a series of different circular DNAs (Turnbull & Webb, 2002). The endoparasitoid, Cotesia vestalis (Hymenoptera: Braconidae), employs multiple strategies of active and passive immune-suppression in overcoming the defences of its host, Plutella xylostella (Lepidoptera: Plutellidae). We have demonstrated previously that the venom from C. vestalis has a limited effect on haemocytes, whereas calyx fluid from C. vestalis may play a major role in the suppression of the host immune system and the venom probably synergizes the effect of calyx fluid or C. vestalis polydnavirus (CvBV) (Yu et al., 2007). Most of the CvBV genome has been sequenced using the plasmid capture system (PCS), although the segments over 30 kb do not amplify due to PCS system restrictions (Choi et al., 2005). CvBV shares some genetic similarity with the Cotesia congregata bracovirus (CcBV), the first BV genome to be fully sequenced (Espagne et al., 2004). Here we isolated two CvBV genes and their genomic organization and expression patterns were investigated. The GenBank accession numbers of the sequences reported in this paper are EF467277 and EF467278. Sequences of the primers used in this study are available with the online version of this paper.
0008-2999 G 2007 SGM
Printed in Great Britain
The parasitoid species of the current study is commonly known as Cotesia plutellae (Kurdjumov) in the literature, but now is referred to as C. vestalis (Haliday) (Shaw, 2003). The cDNA library was prepared with the SMART cDNA Library Construction Kit following the manufacturer’s protocols (Clontech), using 2 mg of total RNA isolated from parasitized P. xylostella 6 h post-parasitization (p.p.). PDV virions and viral DNA were collected from 200–300 2-day-old female wasps as previously described by Beckage et al. (1994) and Chen & Gundersen-Rindal (2003). CvBV probe was prepared by double-digesting the viral DNA with BamHI and HindIII and labelling it with 32P using Random Primer DNA Labelling Kit Ver. 2.0 (TaKaRa) according to the manufacturer’s instructions. Library screening was carried out by the method described by Chen & Gundersen-Rindal (2003). Two positive clones confirmed by secondary screening were converted to pTriplEx2 by infecting E. coli BM25.8. These two clones were sequenced and named CvBV1 and CvBV2 (GenBank accession numbers are EF467277 and EF467278). BLAST searches of these two sequences indicated that CvBV1 and CvBV2 were located on segments CvBV-S5 (14.5 kb) and CvBV-S51 (17.5 kb) of the CvBV genome, respectively (Fig. 1a). CvBV2 has a gene copy on segment CvBV-S48 (15.4 kb) with 3 bp nucleotide differences in the 39 untranslated region (UTR) and we refer to it as CvBV2’. Highly homologous sequences characterized the two genes on their respective segments, segment CvBV-S5 and CvBVS11 for CvBV1, and segment CvBV-S51 and CvBV-S48 for CvBV2 (Fig. 1a). Homologous sequences of CvBV1 were
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Fig. 1. (a) Location of CvBV1, CvBV2 and their homologous sequences on CvBV segments. Boxes represent exon sequences and their homologous sequences. Numbers above boxes indicate percentage similarity. Dark shaded regions indicate UTRs. GenBank accession numbers: DQ075357 for CvBV-S5, DQ075360 for CvBV-S11, EF067330 for CvBV-S48 and EF067332 for CvBV-S51. (b, c) Nucleotide and deduced amino acid sequences of CvBV1 and CvBV2. Potential polyadenylation signals are in bold characters. The most likely cleavage sites of putative signal peptides are underlined in the figure. The sole potential O-glycosylation site of CvBV 1 is underlined and in italics. Three potential N-glycosylation sites of CvBV2 are underlined and in bold characters. The positions of the single intron present in the genomic sequence of CvBV1 and CvBV2 are denoted with asterisks in the cDNA sequence. Sequences of the introns present in the genomic fragment coding for CvBV1 and CvBV2 are flanked by the last coding triplet of exon1 and the first triplet of exon2.
CvBV1-s5 favoured the hypothesis of tandem gene duplication since both genes clustered on the same segment, whereas CvBV2/CvBV2’ and CvBV-HP5107/ CvBV-HP4805 depicted segmental duplication. Gene duplication along with genome segmentation, segment nesting and non-equimolar segment ratios are different strategies adopted by the virus to increase the copy number of essential genes and the levels of gene expression in the absence of virus replication (Kroemer & Webb, 2004; Webb & Cui, 1998). The current results revealed a high degree of similarity between segments CvBV-S51 and CvBV-S48, and between segments CvBV-S5 and CvBVS11. Despite the close resemblances, Choi et al. (2005) made it clear that segments CvBV-S51 and CvBV-S48, CvBV-S5 and CvBV-S11 are all different segments with different digestion patterns.
predicted to encode proteins using Fgenesh (Salamov & Solovyev, 2000; Solovyev & Salamov, 1999) and were named CvBV1-s5 and CvBV1-s11, respectively (Fig. 1a). Gene duplication has been previously reported in BVs and IVs and categorized as follows: (1) tandem gene duplication, (2) segmental duplication or (3) unresolved between the two hypotheses (Friedman & Hughes, 2006; Hilgarth & Webb, 2002; Provost et al., 2004). The pair of CvBV1 and 3318
CvBV1 had a size of 790 bp and was composed of two exons and an intron, which was consistent with the prediction in GenBank. In the 39 UTR, two potential polyadenylation signals (Beaudoing et al., 2000) were observed (Fig. 1b). CvBV2 had a size of 459 bp and was similarly composed of two exons and an intron, but the first exon was not consistent with the prediction in GenBank. There were three potential polyadenylation signals in the 39 UTR (Fig. 1c). The complicated genome organization and gene expression patterns make PDV gene prediction difficult, so different parameters may be adopted for PDV genes expressed in different hosts (hymenopteran and lepidopteran). In order to improve PDV gene prediction, more comprehensive PDV gene transcription, processing and expression models are needed (Gundersen-Rindal & Pedroni, 2006). In the current study gene prediction was carried out using Fgenesh (Solovyev & Salamov, 1999; Salamov & Solovyev, 2000) with human, Drosophila melanogaster, honey bee and Brugia malayi (parasitic nematode) settings. Most settings correctly predicted the nature of CvBV1, except for the Drosophila Journal of General Virology 88
Two Cotesia vestalis polydnavirus genes
settings, whereas CvBV2 could only be predicted correctly using the Brugia malayi settings. Brugia malayi, a mosquito-borne nematode parasite and a cause of lymphatic filariasis in humans (Ottesen et al., 1997), has evolved specific measures to counter host immune defences–strategies quite similar to those employed by PDVs, which is a possible reason for the correct prediction by this species. The open reading frame (ORF) of CvBV1 comprises 97 aa encoding a protein with a predicted molecular mass of 10.9 kDa (Fig. 1b). Computer analyses of the deduced amino acid sequence suggested that the protein could have a signal peptide (Nielsen et al., 1997), indicating that CvBV1 protein was probably secreted from CvBV-infected cells. The most likely cleavage sites of putative signal peptides were between positions 24 and 25 (SGS-SP) or
between positions 31 and 32 (AYA-KP). A potential Oglycosylation site at position 94 in the deduced protein was predicted by NetOGlyc 3.1 (http://www.cbs.dtu.dk/services/NetOGlyc/). The search for conserved domains using Blocks (Henikoff & Henikoff, 1994) indicated significant similarity to a staphylococcal enterotoxin, which has been shown to induce apoptosis in thymocytes, which are a kind of immune cell (Lin et al., 1999). The ORF of CvBV2 encoded 91 amino acids with a predicted molecular mass of 10.0 kDa (Fig. 1c). The predicted protein also seemed to have a N-terminal signal peptide and was proposed to be localized in the lysosome (Nakai & Horton, 1999). The predicted cleavage sites of the putative signal peptides were between positions 18 and 19 (GTS-WF) or between positions 30 and 31 (VDA-LP) (Nielsen et al., 1997). There were three potential N-glycosylation sites predicted by NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc/).
Fig. 2. Sequence comparison of CvBV1 and CvBV2 with homologous hypothetical proteins found in CcBV, CvBV and GiBV. GenBank accession numbers: YP_184861.1 for CcBV_23.3, ABK63302.1 for CvBV-HP1002, AAZ04271.1 for CvBVHP401, ABK63336.1 for CvBV-HP3701, YP_184904.1 for CcBV_33.6, YP_184802.1 for CcBV_9.3, ABK57021.1 for GIP_L1_00350, ABK57038.1 for GIP_L1_00520, AAZ04270.1 for CvBV-HP302, YP_184803.1 for CcBV_9.4, YP_184902.1 for CcBV_33.4, ABK63358.1 for CvBV-HP4805, ABK63370.1 for CvBV-HP5107, ABK57050.1 for GIP_L1_00640, and ABK57047.1 for GIP_L1_00610. http://vir.sgmjournals.org
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Fig. 3. (a) Phylogenetic analysis of CvBV1 and CvBV2 proteins. CcBV_9.4 and GIP_L1_00640 were used as the outgroup for the CvBV1 and CvBV2 tree, respectively. (b) Temporal and tissuespecific expression patterns of CvBV1 and CvBV2 genes in the parasitized host assessed by RT-PCR. Non-quantitative RT-PCR of the two genes was performed using specific primers based on each identified ORF and total RNAs from P. xylostella larvae parasitized by C. vestalis were isolated at various times p.p. (nonparasitized and 0.5, 2, 4, 6, 9, 12 h, 1, 2, 4, and 6 days p.p.) and different tissues. haem, Haemolymph; bra, brain; mid, midgut; viral, direct PCR product using viral DNA as template; neg, negative control (H2O).
The search for conserved domains using Blocks (Henikoff & Henikoff, 1994) indicated significant similarity to a hydrolase of the glycosyl hydrolase 92 family. Many studies have indicated that lysosomes and lysosomal enzymes, including some hydrolases, are involved in apoptosis (Kroemer & Jaattela, 2005; Tardy et al., 2006). Apoptosis is ubiquitously observed in parasitized hosts. A number of characterized genes of PDV appear to induce apoptosis of haemocytes (Lapointe et al., 2005; Le et al., 2003; Strand & Pech, 1995; Strand et al., 1997), which are involved in cellular immunity of insects and take part in phagocytosis, encapsulation and nodule formation (Lavine & Strand, 2002). Homologues of CvBV1 and CvBV2 were found among CvBV, CcBV and Glyptapanteles indiensis polydnavirus (GiBV) by BLASTP searches with an E-value cut-off of 1028 (Fig. 2). CvBV1 and CcBV_23.3 are the same size (97 aa) 3320
and show a high degree of similarity (68 %), indicating that CcBV_23.3 might be an equivalent gene in CcBV. CvBV2 shares 57 % similarity with CvBV-HP5107 and CvBVHP4805 over 84 aa. The CLUSTAL_X alignment shown in Fig. 2 revealed that there were two conserved domains on sequences of homologues for both genes, with one domain located on the N-terminal region and the other located on a non-secretive region. The peptide sequence functions like a postal address on an envelope by targeting proteins for secretion or for transfer to specific organelles for further processing (Choo et al., 2005); therefore we suggest that these homologues are transferred to the same location and share similar functions. Several other hypothetical proteins from CvBV, CcBV, GiBV or Cotesia kariyai polydnavirus (CkBV) only contained the conserved N-terminal domains, indicating that they might be transferred to the same destination but had different functions. For phylogenetic analysis, neighbour-joining trees were generated using PAUP4 (Fig. 3a). The result revealed that CvBV1 is closely related to CcBV_23.3, CvBV-HP302, GIP_L1_00350 and GIP_L1_00520, and CvBV2 is closely related to CvBVHP4805 and CvBV-HP5107 (Fig. 3a). These homologous genes were among CvBV, CcBV and GiBV, suggesting that these BVs are closely related. Transmission of PDVs is exclusively vertical and no replication occurs while inside the host (Stoltz, 1990; Stoltz et al., 1986), so genetic changes occur solely in the female wasp. Therefore, evolution of the polydnavirus is expected to parallel that of the wasp (Friedman & Hughes, 2006). Studies of BV phylogeny indicate that all BV-containing wasps share a common ancestor (Whitfield, 1997). The available fossildating techniques reveal that the ancestor of this lineage lived approximately 73.7±10 million years ago (Whitfield, 2002). Phylogenetic relationships among homologues of CvBV1 and CvBV2 reconfirm that the closer the relationship among the wasps, the more genetically similar are their polydnaviruses. As an advanced step, examination of temporal and tissuespecific expression patterns of CvBV1 and CvBV2 were carried out using non-quantitative RT-PCR with genespecific RT-PCR primers (Fig. 3b; Supplementary Table S1, available with the online version of this paper). Total RNAs from parasitized P. xylostella larvae at various time points and different tissues were treated with RNase-free DNase (TaKaRa) to eliminate DNA contamination and were used as templates in non-quantitative RT-PCR, which was performed according to the manufacturer’s protocol using an RT-PCR kit (QIAGEN). The absence of contaminating DNA in RNA samples was verified by running PCR directly without reverse transcription. The b-tubulin gene of P. xylostella was used as an internal control. Both CvBV1 and CvBV2 were detected in hosts as early as 0.5 h p.p. and continued to be detected for 6 days, which was similar to gene GiPDV 1.1 of GiBV (Chen et al., 2003). The early protein (EP) genes, which many researchers have shown to be early expressed PDV genes, are also detected within 30 min in the host Manduca sexta, and EPs accumulate to Journal of General Virology 88
Two Cotesia vestalis polydnavirus genes
comprise over 10 % of the total haemolymph proteins by 24 h p.p. (Beckage et al., 1987; Harwood et al., 1994). Tissue-specific expression patterns of the two genes showed that they could be detected in haemolymph and brain at 2 h p.p. and later they could be detected in the midgut. These findings suggest that these two genes could be involved in early protection of parasitoid eggs from host cellular encapsulation. For CvBV1 and CvBV2, further research will focus on obtaining purified native proteins and determining their mode of action.
an ankyrin repeat gene of the parasitoid Glyptapanteles indiensis polydnavirus in the parasitized host. J Gen Virol 87, 311–322. Harwood, S. H., Grosovsky, A. J., Cowles, E. A., Davis, J. W. & Beckage, N. E. (1994). An abundantly expressed hemolymph
glycoprotein isolated from newly parasitized Manduca sexta larvae is a polydnavirus gene product. Virology 205, 381–392. Henikoff, S. & Henikoff, J. G. (1994). Protein family classification
based on searching a database of blocks. Genomics 19, 97–107. Hilgarth, R. S. & Webb, B. A. (2002). Characterization of Campoletis
sonorensis ichnovirus segment I genes as members of the repeat element gene family. J Gen Virol 83, 2393–2402. Kroemer, G. & Jaattela, M. (2005). Lysosomes and autophagy in cell
Acknowledgements We are grateful to Dr M. R. Strand (University of Georgia) for his help at the initial stage of this research and his critical review of an early draft of the manuscript. We also thank Dr R. V. Glatz (SARDI, Australia) for his suggestions on our experiments and Dr M. Sharkey (University of Kentucky) for improving the English text. Funding for this study was provided jointly by 973 Program (2006CB102005), Key Program of Natural Science Foundation of Zhejiang Province (Z306031), the National Science Funds For Distinguished Young Scholars (Grant No. 30625006), Program for New Century Excellent Talents in University (NCET-04-0521), Innovation Research Team Program of the Ministry of Education of China (IRT0355), and the China Postdoctoral Science Foundation (20060400322).
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