Institute of Cell Biology, University of Berne, Baltzerstrasse 4, CH-3012 Bern, Switzerland1
Author for correspondence: Beatrice Lanzrein. Fax +41 31 631 46 16. e-mail beatrice.lanzrein{at}izb.unibe.ch
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Abstract |
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Introduction |
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We are working with the bracovirus of the egglarval parasitoid Chelonus inanitus (CiV) and have shown that its genome consists of at least 10 segments with sizes of between 7 and 31 kbp which appear to be singly encapsidated (Albrecht et al., 1994 ). For a 12 kbp segment (CiV12) integration into the wasps genome has been demonstrated as well as stage-specific excision and rejoining of flanking regions (Gruber et al., 1996
). Transcriptional activity has been analysed for three segments (CiV12, CiV14 and CiV16.8) and was seen to increase in the later phase of parasitization (Johner et al., 1999
). Here we show the complete sequences of four CiV segments (CiV12, CiV14, CiV14.5 and CiV16.8) and their analysis. We demonstrate that proviral CiV12 and CiV14 are flanked by other viral segments in the wasps genome and that CiV14 is nested in a larger segment. We also show the sequence of the integration/excision site of CiV14 and compare it with that of CiV12 and that of a Cotesia congregata bracovirus segment. Furthermore, we demonstrate that excision of CiV14 and CiV12 sets in simultaneously at a particular stage of pupaladult development.
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Methods |
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DNA isolation and sequencing.
Calyx DNA was collected and isolated as described in Gruber et al. (1996) . Cloning and mapping of entire viral segments is described in Albrecht et al. (1994)
. Plasmid subcloning was done into pBluescript KS+ or pSP64 or pSP65 vectors. For sequencing of large subclones the GPS-1 Genome Priming System (New England Biolabs) was used according to the manufacturers protocol. GPS-1 is a Tn7 transposon-based in vitro system which uses TnsABC transposase to insert a transprimer randomly into the DNA target. Plasmid DNA was prepared using Wizard Plus SV Minipreps (Promega). Sequencing reactions were performed using the Thermo Sequenase sequencing kit (Amersham) with IRD800-labelled primers. Automatic sequencing was carried out on a Gene ReadIR 4200 (Licor). Each sequence was determined at least three times and more determinations were done in case of ambiguities.
Sequence analysis.
DNA sequence data were analysed using the GCG suite (Wisconsin Package version 10.1, Genetic Computer Group, Madison, WI, USA). Genes were predicted using Fgenesh 1.0 with Drosophila settings (Solovyev & Salamov, 1999 ; http://genomic.sanger.ac.uk/gf/gf.html). Sequence comparisons were made using BLAST 2 (Tatusova & Madden, 1999
; http://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html) and dot plot sequence comparisons were generated with GCG with a sliding window of 20 nt and a stringency of 16 nt. Searches for motifs within predicted proteins were performed using the Prosite (Hofmann et al., 1999
; http://www.expasy.ch/tools/scnpsit1.html) and Blocks databases (Henikoff & Henikoff, 1994
; http://www.blocks.fhcrc.org). N-terminal signal and transmembrane domains were screened using SignalP (Nielsen et al., 1997
; http://www.cbs.dtu.dk/services/SignalP) and Tmpred (Hofmann & Stoffel, 1993
; http://www.ch.embnet.org/software) and localization of predicted proteins was analysed by PSORT (Nakai & Horton, 1999
; http://psort.nibb.ac.jp). Potential N- and O-glycosylation sites were predicted by Prosite (Hofmann et al., 1999
) and NetOGlyc 2.0 (Hansen et al., 1998
; http://www.cbs.dtu.dk/services/NetOGlyc), respectively. Kyte and Doolittle hydrophobicity plots were generated using ProtScale (ExPasy, http://www.expasy.ch/cgi-bin/protscale.pI).
Analysis of the CiV14 integration/excision site and time-point of excision.
A male C. inanitus genomic library (Gruber et al., 1996 ) was screened with the 1300 bp HindIII fragment of clone 2A6 from segment CiV14 (Figs 1
and 3) with methods described in Sambrook et al. (1989)
. DNA from male or female pupae of stages 2 to 4 (for designation of pupal stages see Albrecht et al., 1994
) was isolated as described in Gruber et al. (1996)
. PCR reactions were carried out in a volume of 50 µl with 100 ng of template DNA, 1 U of Taq polymerase, 0·2 µM primers (14LL/14RR or 14LR/14RL, see Fig. 3 and accession nos AJ278677 and AJ319653; 12LL/12RR or 12LR/12RL and accession nos Z58828 and Z58832) and 100 µM of each dNTP (Qiagen Taq PCR core kit) on a Mastercycler gradient (Eppendorf). The denaturation temperature was 95 °C, annealing was done at 55 °C (CiV14) or 60 °C (CiV12), each step lasting 1 min, and synthesis was at 72 °C for 2 min. Aliquots of 10 µl were taken from the reaction after 30 cycles and electrophoresed in MetaPhor agarose (FMC Bio Products) gels (35 g/l) in TAE buffer according to Sambrook et al. (1989)
. PCR clones were generated to investigate the frequency of the ATA and TAC sequence type variants of CiV14. The PCR product obtained with primers 14LL/14RR (circular DNA, see Fig. 3) and 10 ng of calyx DNA as template was polished with T4 polymerase and ligated into a SmaI-cut pBluescript vector. The PCR product obtained with primers 14LR/14RL (rejoined DNA, see Fig. 3) and 1 µg of DNA from adult female wasps as template was treated in the same way. PCR reactions of 30 cycles were carried out in a volume of 50 µl with 1 U Taq polymerase (Qiagen), 0·2 µM primers and 0·2 mM of each dNTP. The denaturation temperature was 95 °C, annealing was done at 55 °C, each step lasting 1 min, and synthesis was at 72 °C for 2 min.
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Results |
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Discussion |
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Hybridization of calyx DNA with probes specific for the flanking regions of CiV12 and CiV14 indicated that both are flanked by other viral segments (Fig. 6a), suggesting that proviral CiV segments are integrated in tandem arrays in the wasps genome. We cannot show physical linkage to non-viral wasp genomic sequences so far as we have not yet analysed segments at the edge of the proviral cluster. Estimation of the approximate size of the flanking segments by comparison with the size of identified segments and contour length data (Albrecht et al., 1994
) suggests that CiV12 is flanked by a ca. 9 kbp and 20 kbp segment and CiV14 by a ca. 25 kbp and 31 kbp segment. In the bracovirus of C. congregata the EP1 circle is flanked on one side by another viral circle and on the other side by wasp DNA (Savary et al., 1997
). In contrast, segments B and W of the ichnovirus of C. sonorensis have been shown to be flanked by wasp DNA on both sides (Fleming & Summers, 1991
; Cui & Webb, 1997
).
Sequence analyses and gene prediction
Comparisons of CiV nucleotide sequences with sequences in databases did not reveal any significant similarities. Analysis for repeats showed that inverted repeats with identities between 77% and 92%, lengths of 26100 bp and AT contents between 33% and 73% were found on all four sequenced segments outside of the predicted genes. Palindromic structures of comparable sizes have been reported to act as origins of replication in baculoviruses (Pearson et al., 1992 ; Ahrens et al., 1995
; Kool et al., 1995
) and it is thus conceivable that certain AT-rich palindromic structures in CiV might serve the same function. A large palindromic structure was also found in the EP1 circle of the C. congregata bracovirus (Savary et al., 1997
).
All four sequenced segments are predicted to contain genes, namely one on CiV14.5 and CiV16.8, two on CiV12 and three on CiV14 (Fig. 1 and Table 1
). The predicted proteins consist of 80549 amino acids and have some potential O- and/or N-glycosylation sites. With the exception of CiV14g3, experimental information has been obtained to substantiate the existence of these genes. For CiV14g1, CiV14g2 and CiV12g2 the cDNA has been cloned (A. Johner, D. Kojic and B. Lanzrein, unpublished). For CiV12g1, CiV14.5g1 and CiV16.8g1 primers have been designed and quantitative reverse transcription PCR analyses have shown that they are all transcribed in a stage-specific manner (S. Zumbach, D. Kojic and B. Lanzrein, unpublished). The accuracy of the predictions was as follows. The percentage of nucleotides correctly predicted as coding was 68% for CiV14g1, 56% for CiV14g2 and 100% for CiV12g2. The percentage of false positives at the nucleotide level was 7% for CiV14g1, 67% for CiV14g2 and 0% for CiV12g2.
Comparison of the predicted proteins with databases did not reveal any significant similarities to known proteins. Nor did we find motifs such as cystein-rich motifs observed in some genes of the ichnovirus of C. sonorensis (Dib-Hajj et al., 1993 ; Cui & Webb, 1996
) and the bracovirus of M. demolitor (Strand et al., 1997
; Trudeau et al., 2000
). Thus, the identified and predicted genes and proteins from the four CiV segments appear to be unrelated to proteins reported for other bracoviruses (Harwood et al., 1994
; Asgari et al., 1996
; Yamanaka et al., 1996
; Strand et al., 1997
; Varricchio et al., 1999
; Trudeau et al., 2000
) or ichnoviruses (Cui & Webb, 1996
; Cui et al., 1997
; Deng & Webb, 1999
; Béliveau et al., 2000
). All these bracoviruses and ichnoviruses are from larval parasitoids and many of the identified proteins have been shown to be involved in abrogation of the immune response of the host. In the egglarval parasitoid C. inanitus abrogation of the hosts immune system appears to be different from that of larval parasitoids (Stettler et al., 1998
) and thus the predicted CiV proteins found here may be involved in other aspects of host regulation.
Integration/excision site and time-point of excision
Analysis of the integration/excision site of CiV14 and comparison to that of CiV12 (Gruber et al., 1996 ) revealed great similarities. On both termini of proviral CiV12 and CiV14 as well as in the excised circular molecule and the rejoined DNA a very similar repeat of 14 bp was found (Fig. 4
). For both CiV12 and CiV14 two sequence variants (ATA, TAC) were seen in the excised segment and the rejoined DNA and a model illustrating where the terminal repeats might recombine to yield the two variants of CiV12 and CiV14 is presented in Fig. 4(c)
. For the EP1 bracovirus segment of C. congregata rejoining of DNA after excision of the viral DNA was also observed and in this case one base pair is lost during the excision process, possibly at the position where DNA strand exchange occurs (Savary et al., 1997
). The direct repeats of the EP1 segment have a length of 24 bp (5') and 22 bp (3') and contain a DNA motif that resembles a Hin recombinase recognition site (Savary et al., 1997
). Searches for this motif in the CiV12 and CiV14 repeats revealed weak similarity (Fig. 5
) and the existence of nucleotides which would prevent Hin binding in vitro according to Feng et al. (1994)
. The existence of direct repeats on the termini of proviral segments appears to be a general feature of polydnaviruses, but the size of the repeats is very variable. For segment W of the C. sonorensis ichnovirus 1185 bp repeats with 100% identity were found (Cui & Webb, 1997
) and for segment B 59 bp repeats with 83% identity (Fleming & Summers, 1991
). Up to now rejoined DNA after excision of viral DNA has not been documented in ichnoviruses. It is not known whether this indicates a difference in the excision process between ichnoviruses and bracoviruses and has to do with the fact that the ichnoviral segments analysed to date are flanked by wasp DNA (Fleming & Summers, 1991
; Cui & Webb, 1997
) while the bracoviral segments appear to be clustered.
Excision of viral DNA appears to be restricted to females and sets in at a very precise time-point of pupaladult development for both CiV12 and CiV14 (Fig. 7; Gruber et al., 1996
). The appearance of excised viral DNA was also seen to be stage-dependent in the ichnovirus of C. sonorensis (Norton & Vinson, 1983
; Webb & Summers, 1992
) and the bracovirus of C. congregata (Savary et al., 1999
). This bracovirus was also excised in diploid, but not haploid males (Savary et al., 1999
). The absence of excised viral molecules in the C. inanitus males (Fig. 7
; Gruber et al., 1996
) might thus indicate that our C. inanitus colony contains no diploid males or that diploid males do not occur in this species. Extrachromosomal viral DNA was found also in males of the braconid Cotesia melanoscela (Stoltz et al., 1986
) and the ichneumonids C. sonorensis (Fleming & Summers, 1986
; Cui & Webb, 1997
) and Hyposoter fugitivus (Xu & Stoltz, 1991
), but it is not known whether this has to do with the existence of diploid males also in these species.
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Acknowledgments |
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Footnotes |
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References |
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Received 19 July 2001;
accepted 27 September 2001.