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B-like gene family in polydnaviruses associated with wasps belonging to different Braconid subfamilies
1 Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, Università della Basilicata, Potenza, Italy
2 Istituto di Genetica e Biofisica, CNR, Napoli, Italy
3 Institut de Recherche sur la Biologie de l'Insecte, CNRS, Université de Tours, Faculté des Sciences, Tours, France
4 Dipartimento di Biologia Strutturale e Funzionale, Università dell'Insubria, Varese, Italy
5 Dipartimento di Entomologia e Zoologia Agraria ‘F. Silvestri’, Università di Napoli ‘Federico II’, Via Università 100, 80055 Portici (NA), Italy
Correspondence
Francesco Pennacchio
f.pennacchio{at}unina.it
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ABSTRACT |
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B-like proteins in the BVs associated with Cotesia congregata (CcBV) and Toxoneuron nigriceps (TnBV) was analysed. PDV-encoded IB-like proteins (ANK) are similar to insect and mammalian IB, an inhibitor of the transcription factor nuclear factor B (NF-B), but display shorter ankyrin domains and lack the regulatory domains for signal-mediated degradation and turnover. Phylogenetic analysis of ANK proteins indicates that those of IVs and BVs are closely related, even though these two taxa are believed to lack a common ancestor. Starting from a few hours after parasitization, the transcripts of BV ank genes were detected, at different levels, in several host tissues. The structure of the predicted proteins suggests that they may stably bind NF-B/Rel transcription factors of the tumour necrosis factor (TNF)/Toll immune pathway. Accordingly, after bacterial challenge of Heliothis virescens host larvae parasitized by T. nigriceps, NF-B immunoreactive material failed to enter the nucleus of host haemocytes and fat body cells. Moreover, transfection experiments in human HeLa cells demonstrated that a TnBV ank1 gene product reduced the efficiency of the TNF-
-induced expression of a reporter gene under NF-B transcriptional control. Altogether, these results suggest strongly that TnBV ANK proteins cause retention of NF-B/Rel factors in the cytoplasm and may thus contribute to suppression of the immune response in parasitized host larvae.
Present address: Institut de Génétique et Microbiologie, Université Paris Sud, Bat. 400, 91405 Orsay cedex, France. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Pennacchio & Strand, 2006). These host regulation factors include venom and ovarian secretions, which, in many parasitoid species of lepidopteran larvae, contain viruses of the family Polydnaviridae (Webb & Strand, 2005). These viruses are associated with a large number of species of ichneumonid and braconid wasps and are classified in the genera Ichnovirus and Bracovirus, respectively (Webb et al., 2000).
The phylogeny of braconid wasps indicates that species associated with bracoviruses (BVs) form a monophyletic group (Whitfield, 2002). In spite of this, only a few genes shared by BVs associated with wasp species in different braconid subfamilies have been identified so far. These include members of a large gene family encoding protein tyrosine phosphatases (PTPs), for which we reported an analysis comparing representatives of two BVs, namely those associated with the microgastrine Cotesia congregata and the cardiochiline Toxoneuron nigriceps (Provost et al., 2004). Here, a similar comparative characterization of a second gene family found in the BVs associated with these two parasitoid species is presented. The putative proteins, consisting mostly of ankyrin repeats, display significant sequence similarity (approx. 50 %) with members of the IB protein family, which act as inhibitors of NF-B signalling pathways in insects and vertebrates (Silverman & Maniatis, 2001), but lack the regulatory domains controlling their signal-induced degradation. Similar viral ankyrin genes (ank) have been described recently in Microplitis demolitor (Hymenoptera, Braconidae, Microgastrinae) bracovirus (MdBV) (Thoetkiattikul et al., 2005) and Campoletis sonorensis (Hymenoptera, Ichneumonidae, Campopleginae) ichnovirus (Kroemer & Webb, 2005), which, along with sequences from other PDVs deposited recently in GenBank (Choi et al., 2005), are included in our phylogenetic analysis.
In Drosophila, the IB protein Cactus regulates multiple cellular responses activated by the nuclear import of a set of NF-B/Rel proteins, which control embryonic dorsoventral patterning (Bergmann et al., 1996; Roth et al., 1991) and antimicrobial response (De Gregorio et al., 2001; Hoffmann, 2003). The presence of sequences in TnBV and CcBV genomes encoding truncated IB-like viral ANK proteins suggests that they may play a role in the impairment of the host immune response, as occurs with MdBV (Thoetkiattikul et al., 2005) and African swine fever virus (ASFV), a large DNA virus of vertebrates (Revilla et al., 1998).
In this paper, the ank gene family in TnBV and CcBV genomes was characterized, the transcription profile in parasitized host tissues was analysed and phylogenetic analysis using the amino acid sequences of ANK proteins encoded by different PDVs was performed. Finally, a functional analysis was undertaken to assess whether the TnBV viral ANK protein interfered with the NF-B/Rel-mediated immune response.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Pennacchio et al., 1998).
Viral DNA extraction and sequencing.
DNA was extracted from purified virus particles (Beckage et al., 1994; Falabella et al., 2003). CcBV genome segments were sequenced by using a shotgun strategy (Espagne et al., 2004). HindIII and EcoRI fragments of the TnBV genome were cloned into the plasmid pGEM-3Z by standard procedures (Sambrook et al., 1989) and sequenced at the TIGEM-IGB Sequencing Core Service, Naples, Italy.
RNA extraction and purification.
Host larvae were staged in developmentally synchronous cohorts, which were either parasitized or used as healthy controls. Tissues were homogenized in ice-cold extraction buffer. Total RNA was extracted with Tri reagent (Sigma) and polyA+ mRNAs were enriched by using Dynabeads Oligo dT (Dynal). Alternatively, RNA extraction was carried out by using an RNeasy kit (Qiagen). The quality of the samples was verified by 1 % agarose-gel electrophoresis and ethidium bromide staining. To eliminate trace amounts of viral DNA present in the total RNA, polyA+ mRNAs were purified by using an Oligotex kit (Qiagen) or treated with RQ1 RNase-free DNase (Promega), according to the manufacturer's instructions.
Isolation of cDNAs.
PolyA+ mRNAs obtained from haemocytes of mature H. virescens larvae 24 and 48 h after T. nigriceps oviposition were used to produce a lambda ZAP-cDNA library, containing clones packaged in pBluescript phagemids (Stratagene). Colony hybridization filters were prepared and screened by standard methods (Pennacchio & Strand, 2006), using the genomic clones 93H3 (4727 bp) and 139RI (5673 bp) as probes for TnBVank1 and TnBVank3, respectively, which were 5' end-labelled to high specific activity with 32P.
CcBV ank cDNAs were isolated by RT-PCR from mRNA extracted from the fat body and haemocytes of M. sexta fifth-instar larvae, 24 h after parasitization, by using Omniscript reverse transcriptase (Qiagen) and high-fidelity Ex Taq (TaKaRa). The following primers were designed from the annotated genomic sequence to amplify the whole GENESCAN-predicted coding DNA sequences (CDS): CcBVank1 – 5'-CAGTCGTTTTAAGCACCGAAA (start 52 bp before the ATG codon), 5'-CCTGTTCACCAATTCCTGGA (end 33 bp after the stop codon); CcBVank2 – 5'-GCCTGTAAGCTTCGTCTGCT (start 44 bp before the ATG codon), 5'-CCTTGAGTGGTGTTAGCTTGC (end 39 bp after the stop codon); CcBVank3 – 5'-ATGGCGCAAAACAATATCATA (start at the ATG codon), 5'-TTAGAATCCACCTGAACCGC (end at the stop codon); CcBVank4 – 5'-ATGGAAGTGCCAAATTTTGTAA (start at the ATG codon), 5'-TTACACTAGATCGGTGTTTTCG (end at the stop codon); CcBVank5 – 5'-ATGGGATCCAACAAGTTTCTTC (start at the ATG codon), 5'-TCATTCAGAACATTTTCGCTTA (260 bp after the stop codon); CcBVank6 – 5'-TAACATCATTGGCATGTTTACATG (start 37 bp before the ATG of a first predicted exon); and CcBVank6int – 5'-TCTTGACATGCTGAGCGCCAGGAG (start 7 bp before the ATG of a second predicted exon), 5'-CCGGTGCCAAGTGCGAACCTTAGA (end 33 bp after the stop codon). CcBVank6int, but not CcBVank6, allowed the successful amplification of the cDNA, indicating that the first intron and exon predicted by GENESCAN are not present in the cDNA.
Southern blot mapping.
Undigested TnBV DNA samples (5 µg) or samples digested with EcoRI or HindIII restriction enzymes were separated on 1 % agarose gels, transferred to a nitrocellulose membrane and hybridized with [-32P]dCTP-labelled probes, according to Sambrook et al. (1989). The isolated cDNAs or genomic sequences of TnBV ank genes were used as probes.
Northern blot analysis and RT-PCR.
RNA samples were run on 1.4 % agarose gels under denaturing conditions using 2.2 % formaldehyde, transferred to nylon membranes and hybridized with [-32P]dCTP-labelled probes, according to standard methods (Sambrook et al., 1989). The cDNAs or genomic clones of TnBV ank genes were used as probes.
The expression of TnBVank2, which did not give any hybridization signal in Northern blot experiments, was further analysed by RT-PCR. In this case, total RNA samples of 5 µg were used for cDNA first-strand synthesis (SuperScript First-Strand Synthesis system for RT-PCR; Gibco). PCR was carried out, using as template one-fifth of the reverse transcription product and the TnBVank2 primers 5'-CTTACCGTCAACGAACAACCA and 5'-TAGGAGCTCGATCATGTACTC.
The PCR program included a denaturation step (95 °C for 2 min), followed by 30 amplification cycles (denaturation, 94 °C for 30 s; annealing, 57 °C for 30 s; extension, 72 °C for 60 s) and a final extension step (72 °C for 10 min).
Total mRNA purified from parasitized M. sexta larvae was reverse-transcribed (Omniscript reverse transcriptase; Qiagen) and RT multiplex PCRs (Qiagen) were performed according to the manufacturer's instructions. The six CcBV ank products were amplified in two different reactions, using single-strand cDNA obtained from 10 ng mRNA and the following primers: reaction 1 – CcBVank1 (5'-GTGGCCATCGGAAAACTTTA and 5'-GTTGTGCTGCGGTTTCTTG), CcBVank3 (5'-TCGAGTTCATGGAATCCGTA and 5'-TCCATCATTCTTTGGTCACG) and CcBVank6 (5'-CACCGGCCTTTCTCAGTATC and 5'-CGGGTATTAATATCGCTCTGTCC); and reaction 2 – CcBVank2 (5'-GTCCGGCAACTTCGTGTTT and 5'-GACACTTCACATTGAGCACCA), CcBVank4 (5'-ACATTGCCGGACAAATGAGT and 5'-TCAAATGGCGAAAGGTTTTT) and CcBVank5 (5'-AACAGTGCACTCATTTCATCG and 5'-TTCAGAACATTTTCGCTTAGCA).
PCRs were also performed on 10 ng mRNA (without reverse transcription) to exclude viral DNA contamination. The multiplex PCR program included a denaturation step (95 °C for 15 min), followed by 30 amplification cycles (denaturation, 94 °C for 30 s; annealing, 55 or 57 °C for 90 s; extension, 72 °C for 90 s) and a final extension step (72 °C for 10 min).
Phylogenetic analysis.
Vertebrate and invertebrate proteins containing ankyrin repeats were retrieved by BLASTP analysis. They were then aligned with ankyrin repeats of BV proteins by using CLUSTAL_X (Thompson et al., 1997). The alignment was improved by visual assessment using MACCLADE (Maddison & Maddison, 2000). Distance and parsimony analyses were performed by using the program PAUP4 (Swofford et al., 1996). Trees were checked by bootstrap analysis (Felsenstein, 1985). Maximum-likelihood analysis was performed by using TREE-PUZZLE 5.1 (Schmidt & von Haeseler, 2003).
Immunocytochemistry.
H. virescens final (fifth)-instar larvae parasitized by T. nigriceps and synchronous non-parasitized controls were punctured 12 h after parasitization with a thin needle previously dipped in a concentrated culture of Micrococcus luteus. Haemocyte and fat body samples were collected 1 h after bacterial infection.
Fat body cryosections (10 µm) and haemocytes were processed for immunocytochemistry as described elsewhere (de Eguileor et al., 2004; Ferrarese et al., 2005). The primary antibodies used were raised against the Rel domain of Drosophila Relish (kindly provided by D. Hultmark and S. Stoven, Umeå University, Umeå, Sweden) or human NF-B (NF-B p65, H-286; Santa Cruz Biotechnology) (dilution 1 : 20). The secondary antibody, conjugated with fluorescein isothiocyanate, was used at 1 : 200 dilution (Jackson ImmunoResearch Laboratories). The cell nuclei were revealed by DAPI (4,6-diamidino-2-phenylindole) staining (Russell et al., 1975).
Slides were examined with a confocal laser microscope (laser 568 nm; MRC 1024, Bio-Rad) and images were recorded with a DeltaVision microscope (DeltaVision Real-time System; Applied Precision, Elcomind). For each experimental condition, several randomly selected microscopic fields were considered for counting the total number of cells and those showing the immune signal in the nucleus. Data were compared by 
2 analysis using SYSTAT software.
Cell culture.
The human HeLa (human cervix epithelioid carcinoma) cell line was maintained in Dulbecco's modified Eagle's medium supplemented with 10 % fetal calf serum, penicillin (100 U ml–1) and streptomycin (100 µg ml–1), and kept at 37 °C in a humidified 5 % CO2 atmosphere.
Cell transfection and luciferase assay.
HeLa cells were co-transfected by using FuGENE6 reagent (Roche) according to the manufacturer's instructions, with 1 µg of the reporter pIgk-Luc plasmid (kindly provided by G. Courtois, Hôpital Saint-Louis, Paris, France), which contained the luciferase reporter gene downstream of three NF-B-binding sites (Fusco et al., 2004), 20 ng pRLSV40 reference plasmid (Promega), and increasing amounts (300 ng to 1 µg) of the pCMVTag-3C plasmid (Stratagene), into which the viral gene TnBVank1 was fused in frame upstream of a myc Tag (pANK-1), or 1 µg empty plasmid (mock). By 48 h after transfection, cells were divided in two groups, one of which was treated with tumour necrosis factor alpha (TNF-) (20 ng ml–1 for 6 h) (Promega). Subsequently, both stimulated and unstimulated cells were lysed and luciferase activity was determined by using the Dual-Luciferase Reporter Assay system (Promega) and a Turner TD2420 luminometer. Experimental data were expressed as a ratio of the luciferase activity recorded in cells stimulated with TNF- and those under basal conditions. Expression of the viral TnBVANK1 protein in transfected HeLa cells was confirmed by Western blot analysis of cell extract using anti-myc antibody. Each experiment was carried out in triplicate. Data were analysed by one-way analysis of variance (ANOVA) and Tukey's multiple comparisons using SYSTAT software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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B-like genes in TnBV and CcBV genomes).
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, is shown in Fig. 2. Because the CcBV genome has been sequenced and annotated, based on the predictions of the GENESCAN program and using specific primers flanking the CDS, CcBV ank cDNAs were generated by RT-PCR and sequences of the encoded proteins were predicted (Fig. 2). The sequences of four amplification products corresponded to those of CcBVank1–4 in the annotated genome sequence. The CcBVank5 cDNA contained a sequence predicted to be a small intron; a stop codon is present in the first half of the cDNA, probably due to a deletion determining a frameshift (see Fig. 2). The structure of CcBVank6 is very similar to that of CcBVank1–4, as the presence of an upstream exon predicted by GENESCAN was not confirmed experimentally (see Methods). Altogether, these results indicated that CcBV ank genes have a simple structure with no introns.
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), a situation that is similar to that described for the CcBV EP1 gene, which has two transcripts that differ in the length of the 3' UTR (Harwood et al., 1994).
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The structure of BV ANK proteins predicted from isolated cDNAs is very similar (Fig. 2
). The viral proteins are made up almost entirely of an ankyrin domain comprising four repeats, which show similarity with repeats 3–6 of Cactus and IB (Fig. 2). Interestingly, these viral ANK proteins do not contain the N-terminal IKK target motif that mediates the signal-induced degradation of Cactus or the C-terminal PEST domain, present in the Cactus/IB proteins, which is involved in rapid protein turnover (Rogers et al., 1986). The CcBVANK5 protein would have a structure similar to that of other ANK proteins, but the stop codon results in a very short putative product, lacking most of the ankyrin-repeat regions (Fig. 2) and, thus, having probably lost the functional activity of the original ANK protein.
Phylogenetic analysis of polydnavirus and related ank genes
To analyse the relationship between different PDV-encoded ANK proteins and to explore the possible origin of their coding genes, sequences displaying significant similarities with BV ANK proteins were retrieved by BLASTP analysis. An alignment of the most closely related proteins, improved by using MACCLADE, is shown in Fig. 3. In addition to BV and IV proteins, the alignment included Cactus proteins from insects, human IB and NF-B proteins, a vertebrate virus protein (ASFV IB-like) and ankyrin repeat-containing molecules with non-immune functions (gankyrin, cyclin kinase inhibitor). The phylogenetic relationship between ankyrin repeats was analysed by using the methods of distance, parsimony and maximum likelihood (Fig. 4). PDV ANK proteins show a significant level of similarity with Cactus from Drosophila, much higher than that shown with a truncated IB encoded by ASFV, which acts as an NF-B inhibitor (Revilla et al., 1998). However, due to the high divergence of viral sequences, it was not possible to demonstrate by phylogenetic analysis that BV genes originated from a cactus gene and to reconstruct a complete filiation of bracovirus genes. The most interesting result of the phylogenetic analysis was the unexpected finding that BV and IV ank gene products constitute a monophyletic group. This group was obtained with all methods used and supported by highly significant bootstrap values (Fig. 4). Furthermore, this result was supported strongly by the lack of the insertion in the ank3 region present in Cactus proteins from Drosophila and other insects and by the occurrence of conserved signatures (see Fig. 3 below the alignment), including a Y-WLC motif present specifically in most BV and IV ANK proteins. Thus, ank genes are not only present both in BVs and IVs, but they are derived from a common gene source.
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). TnBVank3 was also an ‘early gene’, which produced multiple hybridization signals (Fig. 5b) consistent with the different sizes of the isolated cDNAs (see above). As indicated above, TnBVank2 expression was not detected by either Northern blot or RT-PCR analyses (data not shown).
A study of the tissue-specific transcription profile over time was undertaken for CcBV ank genes by using a multiplex PCR approach (Fig. 6). Whilst no amplification was obtained from mRNA samples before retrotranscription, which indicated the absence of viral DNA contamination in the samples, a product of the expected size was obtained for each gene using mRNAs extracted from most of the analysed tissues (Fig. 6). The general picture is a widespread, but heterogeneous, expression of CcBV ank genes in different host tissues. Even though multiplex PCR is only a semi-quantitative method, the amounts of PCR product obtained for different genes in the same amplification run can be compared. The different amounts of PCR product obtained for different genes in different host tissues indicate the occurrence of variations in the mRNA levels. For example, the ratio of CcBVank2 to CcBVank5 transcription levels was considerably higher in the fat body than in the haemocytes, where the amplification product for CcBVank2 was barely detectable. Conversely, the ratio of expression levels of CcBVank6 to those of CcBVank1 was comparatively higher in the haemocytes than in the fat body.
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B-related factors in parasitized host cellsB/Rel factors was altered in H. virescens larvae parasitized by T. nigriceps. In Drosophila, septic injury by Gram-positive bacteria and fungi activates the Toll pathway and elicits the translocation into the nucleus of NF-B/Rel factors, which induce, in the fat body and haemocytes, the transcription of genes encoding antimicrobial peptides (Hoffmann & Reichhart, 2002; Hultmark, 2003; Lamberty et al., 1999; Lavine & Strand, 2002). As this pathway is fully conserved in mosquitoes (Christophides et al., 2002) and probably in lepidopterans, it was considered as a reference model for our in vivo experiments.
Parasitized and control larvae were challenged with haemocoelic infections of a Gram-positive bacterium. Following infection, the subcellular localization of NF-B/Rel in the fat body and haemocytes was assessed by using an antibody raised against the conserved Rel domain of Drosophila Relish. In control fat body cells of non-parasitized larvae, a positive signal was found in the nuclei of 6.7 % of observed cells (n=265) (Fig. 7, np/b–) and this value increased significantly to 63 % (n=110) after infection [2=139.8; degrees of freedom (df)=1; P<0.0001] (Fig. 7, np/b+). In parasitized host larvae, the signal was localized in the nucleus of 11.6 % of fat body cells (n=215) and this percentage did not increase significantly after immune challenge (n=180; 17.7 %) (Fig. 7, p/b+). Similar results were obtained with haemocytes, where the percentage of cells showing nuclear localization of the NF-B signal increased significantly from 19 % (n=110) to 68 % (n=150) (2=60.9; df=1; P<0.0001) after infection of non-parasitized larvae, but not in the haemocytes of parasitized larvae (p/b–, 15.3 %, n=130; p/b+, 15.0 %, n=117) (Fig. 7). Comparable results were obtained by using an antibody reacting against the Rel domain of human NF-B (NF-B p65, H-286; Santa Cruz Biotechnology) (data not shown). Collectively, these experiments indicate that the nuclear import of NF-B/Rel immunoreactive proteins is disrupted in H. virescens larvae parasitized by T. nigriceps.
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B-like genes on TNF signalling in HeLa cellsB proteins. These virally encoded proteins would behave as dominant inhibitors, insensitive to host signalling pathways that regulate Cactus/IB phosphorylation and degradation in response to immune challenge, as demonstrated for a similarly truncated IB-like protein of a vertebrate virus (Revilla et al., 1998).
As the TNF pathway of mammals and the Toll pathway of Drosophila are functionally conserved in the use of Cactus/IB and NF-B/Rel molecules, the impact of BV ANK proteins on NF-B transcriptional control was assessed in human HeLa cells. The effect of a transfected plasmid (pANK1) encoding TnBVANK1 protein on the efficiency of the TNF--mediated induction of NF-B-driven expression of a reporter gene was measured. The reporter construct contained the luciferase gene downstream of three NF-B-binding sites. The rate of luciferase activation by TNF- treatment was measured 48 h after co-transfection of HeLa cells with the reporter plasmid and increasing amounts of the plasmid driving TnBVANK1 production. The rate of activation was influenced significantly by the presence of pANK1 (F=10360.36; P<0.0001), which, at the highest dose, induced up to a nearly threefold reduction of luciferase activation (Fig. 8).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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B inhibitor, a protein conserved from Drosophila to mammals (Espagne et al., 2004; Webb & Strand, 2005). Similar genes were characterized recently in MdBV, a bracovirus associated with M. demolitor, a parasitoid species in the subfamily Microgastrinae (Thoetkiattikul et al., 2005), and in IVs (Kroemer & Webb, 2004, 2005; Webb & Strand, 2005). Thus, ank genes, along with cysteine-motif genes, are apparently the only genes shared by BVs and IVs (Espagne et al., 2004; Kroemer & Webb, 2004; Webb & Strand, 2005; Webb et al., 2006). Moreover, BV- and IV-encoded ANK proteins have the same structure, and the phylogenetic analysis reported here indicates that they are derived from a common gene source. This finding would not be predicted a priori by the hypothesis that the two PDV genera originated and evolved independently in the braconid and ichneumonid lineages (Webb & Strand, 2005; Whitfield, 2002). A possible explanation is that the BV and IV ank genes may originate from independent acquisitions of wasp cactus genes, as suggested for other PDV genes, such as PTPs, cystatins and vinnexins (Espagne et al., 2005; Provost et al., 2004; Turnbull et al., 2005). Indeed, as ichneumonid and braconid wasps are related phylogenetically, their endogenous Cactus may share common features, such as the Y-WLC motif found in IB-like proteins of PDVs. However, it should be noted that this signature is not found in the Apis mellifera sequence and that alternative interpretations are possible, such as the acquisition of a common ank viral gene from a third viral partner or a previously unrecognized phylogenetic link between the ancestor viruses, which became integrated independently in the ichneumonid and braconid lineages. This issue could be clarified further by cloning cactus homologues from PDV-carrying wasps.
Structure and function of isolated ank genes
The PDV-encoded IB-like proteins are all characterized by the presence of ankyrin domains, which may enable binding to NF-B, but they lack the regulatory motifs necessary for signal-mediated degradation (Ghosh & Karin, 2002; Ghosh et al., 1998; Webb & Strand, 2005) and for control of protein turnover (C-terminal PEST domain) (Rogers et al., 1986). These molecular features of the viral ankyrin gene products suggest strongly that they may disrupt cell pathways by binding host NF-B molecules irreversibly. This hypothesis is supported by recent functional analysis of MdBV ANK proteins N5 and H4, the latter being related closely to CcBVANK6, which inhibit NF-B activation and suppress the insect immune response (Thoetkiattikul et al., 2005). The strong reduction of NF-B-driven luciferase expression in mammalian cells co-transfected with TnBVank1 extends the results obtained for MdBV, showing that bracovirus ANK proteins act as suppressors of NF-B. In addition, our results show that TnBVANK1 is active in an evolutionarily distant heterologous system.
Moreover, it has been shown for the first time that NF-B/Rel immunoreactive proteins are actually sequestered in the cytoplasm of the cells of parasitized larvae. ANK proteins are most probably involved in this sequestration, in which they may be aided by other factors such as ovarian proteins, venom components and other PDV products.
In parasitized lepidopteran larvae, which are unable to encapsulate foreign invaders, the PDV-induced disruption of NF-B signalling probably affects both the humoral and cellular immune responses. The negative impact of NF-B signalling disruption on antimicrobial response, which has been reported in several lepidopteran host–parasitoid associations (Shelby & Webb, 1999; Shelby et al., 1998), could be interpreted as a trade-off associated with an efficient suppressive strategy of the host cellular immune response. In Drosophila, microarray studies have shown the upregulation in parasitized larvae of several genes under NF-B control (Wertheim et al., 2005). Moreover, the Toll pathway is known to be implicated both in the transcription of genes encoding antimicrobial peptides and in the formation of the capsule against a parasite, by regulating haemocyte proliferation (Qiu et al., 1998), as well as in the activation of the phenoloxidase cascade (Ligoxygakis et al., 2002). The possible conservation of these pathways in lepidopteran hosts may well account for a key role of PDV-encoded ANK proteins in the suppression of the immune response.
TnBV ANK proteins might also be involved in the induction of apoptosis observed in parasitized host cells (Ferrarese et al., 2005) by disrupting the synthesis of anti-apoptotic factors and complementing the possible activity in vivo of TnBV1 gene product, which elicits apoptosis in insect cell lines (Lapointe et al., 2005). This is suggested by the fact that, in mammals, a caspase-mediated cleavage of the IB N-terminal region produces a truncated product, which suppresses NF-B nuclear translocation and sensitizes cells to death by blocking NF-B-dependent synthesis of anti-apoptotic factors (Reuther et al., 1999).
Duplication and diversity of ank genes
The diversity of ank genes may reflect the redundancy or the specialization of their functions. Some BV ank genes, such as CcBVank2 and CcBVank6, display some tissue specificity in their quantitative level of expression, as reported also for IV vankyrin genes (Kroemer & Webb, 2005), which might be a consequence of the specialization of their function. Different ANK proteins might be active in specific tissues in order to target different Rel transcription factors, which, in mammals, have cell lineage-specific functions (Mason et al., 2004) and might differ in their binding preference, as suggested in the case of MdBV (Thoetkiattikul et al., 2005). To support this hypothesis, it would be necessary to isolate the Rel proteins targeted in the lepidopteran hosts.
However, the widespread expression of ank genes and the fact that some gene-family members are probably pseudogenes are in contrast to the hypothesis above and strongly indicate a redundant function of the encoded proteins. Moreover, the facts that TnBVANK1 is active in human HeLa cells and that, in MdBV, H4 and N5 bind to both Dif and Relish do not suggest a highly specific interaction with the target molecules (Thoetkiattikul et al., 2005). The redundancy of PDV genes probably reflects the evolutionary process of adaptive radiation and host shift by parasitoid wasps. The capacity of facing the immune challenge in a new host environment is undoubtedly one of the major functional constraints limiting the successful colonization of a new ecological niche. Thus, the genes involved in the suppression of the immune response probably play a major role in this adaptive process and are expected to have a remarkable degree of plasticity to cope with the great diversity of available new hosts. Interestingly, a recent evolutionary model (Francino, 2005) proposes that the origin of new gene functions does not require that all of the different gene copies generated must acquire a specialized function. This model postulates that the evolution of a new function may start with the amplification of an existing gene with some level of pre-adaptation for that function, followed by a period of competitive evolution among gene copies, resulting in the preservation of the most effective variant and the ‘pseudogenization’ and eventual loss of the rest. Thus, this model may account for the existence of gene families, for the occurrence of pseudogenes and for the high divergence of BV ank genes, which may even lose the conserved ANK domains, as in the case of a gene, CcBV_26.5, located downstream of CcBVank6. This gene encodes a protein that is similar to CcBVANK6, but lacks any identified ankyrin repeats (see Fig. 3).
Divergent proteins, pseudogenes and proteins with eroded conserved domains are also observed in BV PTPs (Provost et al., 2004). The study of the molecular targets of products encoded by these gene families in different hosts will probably shed some light on the evolutionary processes that have led to this genetic diversification and will provide the opportunity to assess whether the proposed ‘adaptive-radiation model’ (Francino, 2005) offers a new general framework for a better understanding of the evolution of PDV gene families.
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ACKNOWLEDGEMENTS |
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B-like proteins, Jérome Lesobre (Institut de Recherche sur la Biologie de l'Insecte, Université de Tours) for his contribution to sequencing of the CcBV cDNAs, Genoveffa Ciancio, Elisa Teresa Caprioli, Giandonato Caggianese (Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali – Università della Basilicata) and Cindy Ménoret (Institut de Recherche sur la Biologie de l'Insecte, Université de Tours) for their technical assistance and for insect rearing, and Maria Guarino for editorial assistance. |
REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 19 June 2006;
accepted 4 September 2006.
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-strands, cylinders for 
50 % amino acid similarity; amino acids highlighted in black, 
0.01).