Department of Biochemistry, University of Kentucky, Lexington, KY 40536, USA1
Department of Entomology, University of Kentucky, Lexington, KY 40546, USA2
Author for correspondence: Bruce Webb. Fax +1 859 323 1120. e-mail bawebb{at}pop.uky.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CsIV gene expression has been studied in some detail with RNA blot hybridizations. Viral genes may be expressed in the wasp during virus replication (class I), in the parasitized lepidopteran host (class II), or in both hosts (class III) (Theilmann & Summers, 1988 ). The published studies have emphasized characterization of genes that are expressed in parasitized lepidopteran hosts (class II) in order to identify those viral genes that may alter their physiology. Two distinct viral gene families have been studied in CsIV: a cysteine-rich gene family with five identified members, which is expressed only in parasitized lepidopteran larvae (Dib-Hajj et al., 1993
; Cui & Webb, 1996
), and a second putative gene family, the repeat element (rep) gene family, which has been proposed based on cross-hybridization of a sequenced rep gene, BHv 0.9, to several mRNAs on Northern blots and on sequence similarity among viral DNA segments (Theilmann & Summers, 1988
). BHv 0.9 has a class II gene expression pattern, although some of the cross-hybridizing mRNAs show class III expression (Theilmann & Summers, 1988
). The proposed rep gene family is distinguished by an imperfectly conserved 540 bp repeat sequence (Theilmann & Summers, 1987
). This rep sequence hybridized to the majority of CsIV segments at reduced stringencies, suggestive of either a widely distributed gene family or a conserved viral DNA structural element. The segment B rep gene and three other expressed rep sequences were identified on viral segments H and O1 by Theilmann & Summers (1988)
. The putative H and O1 rep genes contain multiple 540 bp rep sequences, while BHv 0.9 has a single copy of the repeat sequence (Theilmann & Summers, 1987
). Because the 540 bp repeat sequence cross-hybridized with most, possibly all, CsIV segments, Theilmann & Summers (1988)
hypothesized that the rep genes may represent a gene family having many members. Whether or not the cross-hybridizing sequences were expressed genes, potentially expressed genes or non-functional pseudogenes was not determined. In the present study, we describe the isolation and sequence of segment I, and identify three new members of the rep gene family. Characterization of these genes suggests that the rep genes have evolved in lineages of single-repeat-containing genes and multiple-repeat-containing genes. Preliminary characterization of the genes indicates that they are functional but expressed at different levels, even though they are found on the same viral segment.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation of the segment I clone.
Probes were labelled by the random primer method (Feinberg & Vogelstein, 1983 ) with [32P]dATP using the Prime-a-Gene labelling system (Promega). Thirteen cDNA clones that hybridized to the rep sequence, HC1185 (Theilmann & Summers, 1987
, 1988
), were isolated by screening a
gt11 parasitized H. virescens cDNA library under low-stringency conditions (30% formamide, 6x SSC, 5x Denhardt's solution, 0·5% SDS at 42 °C, with two washes of 20 min in 2x SSC, 0·5% SDS at 42 °C), followed by two washes of 0·1x SSC, 0·5% SDS at 65 °C for 20 min with autoradiography at -80 °C overnight (Sambrook et al., 1989
). Hybridizing phage plaques were amplified by PCR, and then subcloned into pBluescript II KS(-) (Stratagene), as previously described (Cui & Webb, 1996
). Five clones representing different insert sizes were sequenced. To isolate genomic clones encoding the expressed rep transcripts, the largest cDNA clone of 1·6 kbp was used to screen CsIV genomic libraries prepared by digesting CsIV genomic DNA with XhoI, SphI, BamHI and SacI, and ligating inserts into compatibly digested pZero-1.0 vector (Invitrogen) (L. Cui, unpublished data). Hybridization of cDNA clones to viral DNA was carried out under high stringency conditions (50% formamide, otherwise as above, followed by two washes in 0·1x SSC, 0·5% SDS at 65 °C for 20 min) with autoradiography at -80 °C overnight.
Sequencing of segment I.
For sequencing, segment I subclones were constructed by digesting SphI35, a positive segment I clone identified by hybridization, with XhoI, BamHI, SacI and EcoRI individually and cloning fragments into pBluescript II KS (Stratagene). Nested unidirectional deletion subclones were also constructed using the Erase-a-Base System (Promega) after linearizing 10 µg of SphI35 with SpeI and KpnI. Some site-specific primers were designed and used to complete the segment I sequence.
Identification of segment I rep genes by rapid amplification of cDNA ends (RACE).
Rep sequences were identified by homology to BHv 0.9, O1 and H rep sequences from segments B, O1 and H (Theilmann & Summers, 1987 , 1988
) with gene expression confirmed by amplification and sequencing of the 3' end of the segment I rep transcripts. Gene-specific primers were designed to the three putative segment I rep genes: I 1.1 (5' ATG GAT TCC CCG GTC AAA G 3', I1.1a-SphI35, nt 38963914), I 1.2 (5' ATG GGA TCA TTG ACT AGT G 3', I1.2a-SphI35, nt 70627080) and I 0.9 (5' ATG GAG TCC CTG GGC GAA GG 3', I 0.9a-SphI35, nt 26952714). First-strand cDNA synthesis used an oligo(dT) primer [5' CCA GTG AGC AGG TGA CGA GGA CTC GAG CTC AAG C(T)17] (Frohman, 1994
), and 4 µg total RNA isolated from parasitized H. virescens larvae, non-parasitized H. virescens larvae and C. sonorensis. Reverse transcription reactions of 1x cDNA synthesis buffer [250 mM TrisHCl, pH 8·3, 375 mM KCl, 15 mM MgCl2, 200 µM of each dNTP, 10 mM DDT, 50 pmol oligo(dT) and 200 units of Superscript II (Gibco)] in 20 µl were incubated at 42 °C for 50 min. The amplification of the 3' end of segment I rep genes followed a procedure modified from Frohman (1994)
. One µl of the reverse transcription mixture was added to a 50 µl PCR reaction containing 1x PCR buffer [10 mM TrisHCl, pH 9·0, 50 mM KCl], 1·5 mM MgCl2, 50 µM dNTP, 25 pmol of oligo(dT) and 25 pmol of a segment I rep gene-specific primer, and amplified through five amplification cycles of 94 °C for 1 min, 61 °C for 1 min and 72 °C for 1·5 min, followed by 25 amplification cycles of 94 °C for 1 min, 56 °C for 1 min and 72 °C for 1·5 min, in a Perkin-Elmer/Cetus 480 DNA Thermal Cycler. Segment I rep gene PCR reactions were analysed by electrophoresis of 8 µl of each reaction on a 1% agarose gel in 1x TAE. To detect segment I transcripts in C. sonorensis and I 0.9 transcripts in parasitized H. virescens, a second round of amplification was required. The initial amplification was diluted 1:100 in water, and 1 µl of this dilution used in a second amplification reaction with a nested gene-specific primer: I 1.1 (5' TTA GAA GAG GAC GTA TGC CCA 3', I1.1b-SphI35, nt 43734393), I 1.2 (5' ATG CGA GAT ATT GGC TAC GTC GT 3', I1.2b-SphI35, nt 75827604) and I 0.9 (5' GCG AAG AAG ACG ACA CTT TC 3', I0.9b-SphI35, nt 28502869) with oligo(dT) as the reverse primer and amplification reactions and conditions as described above. The 3' RACE amplimers corresponding to the predicted sizes of the transcripts were cloned into pGEM-T Easy (Promega) and sequenced to confirm amplification of segment I rep gene transcripts. Control amplifications used a segment I genomic DNA template with negative controls lacking template (water only) in primer combinations as described above.
The 5' ends of the segment I rep cDNAs were isolated by 5' RACE (5' RACE system, Gibco BRL). Individual segment I rep gene primers were used in the reverse transcription reactions with 3 µg of total H. virescens RNA [I 1.1 (5' CAT GGT CAA TGT GTT TAC AAA TTC 3'; I1.1c-SphI35, nt 30693048), I 1.2 (5' GTC CTC TCG TTC GTA GG 3', I1.2c-SphI35, nt 72077190) and I 0.9 (5' GTT TTT CTT TGA AAG TGT CG 3', I0.9c-SphI35, nt 28792860)]. PCR amplification of each segment I rep gene reverse transcription product was carried out with specific primers for each rep gene [I 1.1 (5' GTA GCT CGA CTT CCC CAA GGA TTG 3', I1.1d-SphI35, nt 42324209), I 1.2 (5' GCG TAC AGA CCA TGA GAA CAT 3' I1.2d-SphI35, nt 71687148) and I 0.9 (5' TAG TCC TCG ATG TTC CTT CGC 3', I0.9d-SphI35, nt 27282708)] in conjunction with the anchor primer (5' GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG 3') (Gibco BRL), following the manufacturers instructions. PCR conditions were 30 cycles of 94 °C for 1 min, 56 °C for 1 min and 72 °C for 1 min. The 5' RACE-amplified products were analysed on a 1% agaraose gel in 1x TAE, and then cloned into pGEM-T Easy (Promega) and sequenced.
DNA sequence determination and analysis.
DNA sequencing reactions routinely used the ABI Prism dRhodamine or Big Dye Terminator Cycle Sequencing Ready Reaction kits with the sequence visualized on ABI 377 or ABI 310 DNA sequencers (PE Applied Biosystems). Sequence data were assembled with the Lasergene sequence analysis software (DNASTAR). ORF analysis employed Gene Construction Kit 2 (Textco) and the Baylor College of Medicine Gene Finder Find genes H program set on Drosophila (http://dot.imgen.bcm.tmc.edu:9331/gene-finder/gf.html). The segment I rep cDNA sequences were analysed for protein motifs and cellular localization signals using the PSORT prediction program (http://psort.nibb.ac.jp/). Nucleotide and peptide sequence similarity searches utilized BLAST search programs (Altschul et al., 1997 ) with DNA alignments utilizing the University of Wisconsin Genetics Computer Group DNA analysis software for the VAX computer (release 7.2) and protein alignments used the Clustal X alignment program (Thompson et al., 1997
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Segment I rep gene structure
The I 0.9 transcript is 937 bp excluding the poly(A) tract (Fig. 2b). This gene encodes one copy of the 546 bp sequence characteristic of the rep gene family. The I 0.9 ORF encodes a predicted protein size of 28·6 kDa having a 56 amino acid sequence upstream of the conserved repeat. This transcript has a 170 bp 5' UTR with a consensus TATA box 27 bp upstream of its 5' RACE terminus. The 3' UTR is 51 bp with a polyadenylation signal (AATAAA) 15 bp upstream of the poly(A) tract.
ORF II encodes the I 1.1 transcript of 1069 bp excluding the poly(A) tract (Fig. 2b). This gene contains a 531 bp sequence with repeat sequence homology and a 195 bp coding region upstream of the repeat sequence. The ORF in this transcript encodes a predicted protein of 28·9 kDa. The 5' UTR of this transcript is at least 268 bp, with a putative TATA box 26 bp upstream of its terminus and a 75 bp 3' UTR having a consensus polyadenylation signal (AATAAA) 13 bp upstream of the 3' poly(A) tract.
The third segment I rep gene, I 1.2, encodes a transcript 1241 bp in length excluding the poly(A) tract (Fig. 2b). This gene contains two copies of the repeat sequence in a 525 bp tandem repeat. A 48 bp coding region is present upstream of the repeat sequences resulting in a predicted protein of 43·6 kDa. The 525 bp tandem repeats have 89% nucleotide sequence similarity. The 5' UTR of the I 1.2 transcript is at least 88 bp, with a putative TATA box present 25 bp upstream of the transcript's 5' terminus. A 26 bp 3' UTR contains a potential polyadenylation signal (AATAAA) 15 bp upstream of the 3' poly(A) tract.
Consensus translation initiation sequences (PuNNATGPu) (Kozak, 1983 ) flank the first methionine codon of each segment I rep gene ORF described above. The N termini of these predicted proteins do not encode a consensus signal peptide (von Heijne, 1986
), suggesting that the proteins encoded by these ORFs are not secreted.
Expression of the segment I gene
To evaluate segment I rep gene expression, RTPCR was performed at selected time-points after parasitization with primers specific for each of the segment I rep genes. Consistent with the expression of other CsIV genes, transcripts from I 0.9, I 1.1 and I 1.2 were detected 3 h after oviposition and throughout 8 days of parasitization (Fig. 3). The I 1.1 and I 1.2 rep genes were readily amplified from parasitized H. virescens mRNA, but two rounds of amplification were required to detect I 0.9 rep gene transcripts. To determine if segment I rep genes are transcribed in C. sonorensis, RNAs were extracted from adult, stage I, stage II, stage III and stage IV C. sonorensis ovaries for RTPCR analyses (Webb & Summers, 1992
). RNA was also isolated from ovariectomized females and male C. sonorensis. Transcripts were detected in all stages of ovarian development, in males, and in ovariectomized females (Fig. 3
). Negative controls using the segment I clone (SphI35) showed that a DNA template would not produce an amplicon in these reactions.
|
|
The BHv 0.9 predicted protein (Theilmann & Summers, 1988 ) and the three predicted segment I rep proteins were compared at the amino acid level. I 0.9, I 1.1 and BHv 0.9 had 56 amino acids located at the N terminus with an identity ranging from 5853%. The I 1.2 gene lacks 40 amino acids in the non-repeat portion of the N terminus relative to the BHv 0.9, I 0.9 and I 1.1 genes, with the rest of the predicted peptides encoded by the rep sequence. Within the repeat sequences, the four predicted proteins had an average amino acid identity of 42%. The two I 1.2 repeats were most similar, having amino acid identities of 79%. Alignments of the proteins encoded by BHv 0.9, I 0.9, I 1.1 and I 1.2 revealed two amino acid sequence motifs conserved within the repeat coding portion of these genes (Fig. 5
). A 16 amino acid conserved sequence of F-N---IEVEY-Y-RE was encoded by the N-terminal portion of the repeat, while the 19 amino acid consensus sequence D-CP--HFHH--P-H---W was present near the C terminus. Two additional motifs were observed in comparisons of the predicted proteins of the non-repeat coding portion of the single-repeat rep genes, BHv 0.9, I 0.9 and I 1.1 (Fig. 5
). A 13 amino acid consensus sequence, TP-YFSTRQ--LP, and a 10 amino acid consensus sequence, FR-F-RAMWP, appeared in the BHv 0.9, I 1·0 and I 0.9 genes near the N terminus. The second motif was at the boundary of the 540 bp repeat sequence. In the I 1.2 gene, the two sequences present outside the repeat region were absent (Fig. 5
).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The three segment I rep genes are expressed suggesting that most, possibly all, rep sequences are part of expressed genes. Previous studies have suggested that the rep genes have two expression patterns. The BHv 0.9 rep gene is expressed only in parasitized larvae making it a class II CsIV gene (Theilmann & Summers, 1988 ). Two other rep sequences from segments H and O1 hybridize to mRNAs isolated from parasitized lepidopteran larvae and C. sonorensis adult female ovaries, suggesting that the putative segment H and O1 rep genes are class III CsIV genes. Detection of segment I rep gene transcripts in both parasitized H. virescens larvae and C. sonorensis confirm that rep gene family members belong to both class II and class III CsIV genes. This result is in contrast with the CsIV cysteine-motif gene family, which is only expressed in parasitized H. virescens (Blissard et al., 1987
; Cui & Webb, 1996
). The temporal expression in parasitized Lepidoptera of the segment I genes is identical to that of the BHv 0.9 rep gene, with transcripts detected as early as 2 h post-parasitization (Theilmann & Summers, 1988
). The segment I rep genes may be expressed at different levels in parasitized insects, since two rounds of PCR amplification were required to detect the I 0.9 transcripts while one was sufficient for detection of the I 1.1 and I 1.2 rep gene transcripts. Quantitative studies are required to confirm this difference in transcript level, but different levels of expression may suggest that the virus has functional genes that are transcriptionally inactive but available for activation by genetic rearrangement. Alternatively, these genes may be differentially expressed in a tissue- or developmental stage-dependent manner, although there is no experimental evidence to support this hypothesis.
Expression of the BHv 0.9 rep gene in parasitized tissues has not been investigated, as prior studies utilized homogenates of whole larvae and evaluated expression in Northern blots. In this study, segment I rep gene transcripts were detected by RTPCR in all tissues assayed, suggesting that rep genes are expressed ubiquitously in parasitized tissues, albeit at low levels for some genes and some tissues. Segment I rep gene expression was also detected in C. sonorensis in tissues that do and do not support virus replication. Expression of PDV genes in tissues that do not support virus replication has been previously reported by Johner et al. (1999) who detected PDV gene expression in Chelonus inanitus males by RTPCR, although virus replication was not detected.
The known rep sequences have an overall nucleotide identity of 50%, with some areas more highly conserved (up to 90% identical). Segment I has two rep genes with single repeat sequences and one with two copies of the rep sequence in tandem. The two single-repeat segment I rep genes, I 0.9 and I 1.1, have a 56 amino acid N terminus upstream of the repeat sequence. This organization is similar to that of the BHv 0.9 rep gene. Interestingly, the single-repeat segment I genes are more similar to the BHv 0.9 repeat than to the I 1.2 rep gene, which has two copies of the rep sequence. These two genes, I 0.9 and I 1.1, have a 74% nucleotide identity, suggesting that gene duplication has occurred to produce these two genes, whereas the I 1.2 gene, which contains two rep sequences in tandem, has an origin more divergent than the repeat gene on segment B.
CsIV segment O1 may have multiple rep genes, as two regions of the segment hybridize to the 540 bp rep sequence and to transcripts on Northern blots (Theilmann & Summers, 1988 ). Theilmann & Summers (1988)
also showed that many CsIV segments, possibly all, hybridize to repeat sequence probes in genomic Southern blots. They also identified multiple repeat sequences in tandem arrays on segment H (five tandem repeats) and O1 (three tandem repeats). Interestingly, the I 1.2 tandem repeats are more similar to the sequences from segments H and O1 that exist in tandem arrays than to the rep sequences from genes encoding a single repeat (BHv 0.9, I 1.1 and I 0.9). This suggests that the I 1.2 rep gene may have arisen from other multiple-repeat-sequence-containing rep genes rather than from duplication of a single repeat sequence on segment I. Thus, two types of rep genes seem to exist and may represent two major lineages within this gene family. One lineage is comprised of rep genes encoding a single repeat sequence. The second lineage is comprised of those genes that have repeat sequences in tandem array. Segment I may be unusual in that it encodes both single-repeat-containing and multiple-repeat-containing rep genes.
Northern blot analysis of mRNAs from H. virescens larvae parasitized by C. sonorensis detects at least 10 viral mRNAs that hybridize to CsIV DNA (Blissard et al., 1986 ). The number of transcripts expressed in parasitized H. virescens larvae is probably greater because of co-migrating transcripts that would not be differentiated by this method. Four of the expressed genes in parasitized H. virescens belong to the cysteine-motif gene family (Blissard et al., 1987
; Dib-Hajj et al., 1993
; Cui & Webb, 1996
). These genes are highly expressed, with gene amplification occurring through an intramolecular recombination process known as segment nesting (Cui & Webb, 1998
). With the three rep genes reported here, there are four rep genes now known to be expressed in parasitized larvae, giving a total of seven identified CsIV genes. This number is likely to rise as rep sequences on segments H and O1 also hybridize to transcripts in parasitized lepidopteran larvae.
The number of CsIV genes (class I and class II) expressed in C. sonorensis has not been studied in detail. One CsIV-encoded nucleocapsid gene, p12, has been isolated (Deng & Webb, 1999 ). Presently, at least six rep genes appear to be expressed in C. sonorensis: the three identified segment I rep genes, two O1 repeat-hybridizing transcripts and one H repeat-hybridizing transcript. Segment B is also predicted to contain a class I gene that does not belong to the rep gene family, but this gene has not yet been fully isolated (Theilmann & Summers, 1988
). The p12 gene is expressed only in tissues that support CsIV replication, with expression coincident with virus replication (Deng & Webb, 1999
). Segment I rep gene expression appears to differ from that of the p12 gene, as expression is detected prior to virus replication and in non-replicative tissues. However, the RTPCR technique is more sensitive than Northern analysis previously used to assay for CsIV gene expression, so it is conceivable that our understanding of CsIV transcriptional regulation may be revised as this technique is applied to analyses of additional viral genes.
Although rep gene expression may differ in a host- and tissue-specific manner, similar portions of the rep proteins are conserved among all known rep genes. Two conserved amino acid motifs are evident within the 180 amino acid repeat. One motif is located near the N terminus of the repeat (F-N---IEVEY-Y-RE). The second motif is encoded near the repeat's C terminus (D-CP--HFHH--P-H---W). The presence of these conserved amino acid motifs in the segment B and segment I rep genes suggests that these regions are important for function of these proteins.
All the CsIV rep proteins lack secretion signals suggesting that these proteins act intracellularly. However, more directed study of the function of this gene family is required to provide an understanding of their roles in parasitization and/or virus replication. The identification and analysis of three members of the rep gene family from segment I confirm that the rep sequences do constitute a CsIV gene family. Studies on the levels of expression of these rep genes should be undertaken to determine the possible role and biological significance of these rep genes in this system. The evidence that rep genes are a large gene family suggests they may have an important, albeit presently unknown, function in the CsIV life cycle (Theilmann & Summers, 1987 , 1988
).
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389-3402.
Asgari, S., Hellers, M. & Schmidt, O. (1996). Host haemocyte inactivation by an insect parasitoid: transient expression of a polydnavirus gene. Journal of General Virology 77, 2653-2662.[Abstract]
Blissard, G. W., Fleming, J. G. W., Vinson, S. B. & Summers, M. D. (1986). Campoletis sonorensis virus: expression in Heliothis virescens and identification of expressed sequences. Journal of Insect Physiology 32, 351-359.
Blissard, G. W., Smith, O. P. & Summers, M. D. (1987). Two related viral genes are located on a single superhelical DNA segment of the multipartite Campoletis sonorensis virus genome. Virology 160, 120-134.[Medline]
Cui, L. & Webb, B. A. (1996). Isolation and characterization of a member of the cysteine-rich gene family from Campoletis sonorensis polydnavirus. Journal of General Virology 77, 797-809.[Abstract]
Cui, L. & Webb, B. A. (1997). Homologous sequences in the Campoletis sonorensis polydnavirus genome are implicated in replication and nesting of the W segment family. Journal of Virology 71, 8504-8513.[Abstract]
Cui, L. & Webb, B. A. (1998). Relationships between polydnavirus genomes and viral gene expression. Journal of Insect Physiology 44, 785-793.[Medline]
Deng, L. & Webb, B. A. (1999). Cloning and expression of a gene encoding a Campoletis sonorensis polydnavirus structural protein. Archives of Insect Biochemistry and Physiology 40, 30-40.[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. Proceedings of the National Academy of Sciences, USA 90, 3765-3769.[Abstract]
Edson, K. M., Stoltz, D. B. & Vinson, S. B. (1981). Virus in a parasitoid wasp: suppression of the cellular immune response in the parasitoids host. Science 211, 582-583.[Medline]
Feinberg, A. P. & Vogelstein, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Analytical Biochemistry 132, 6-13.[Medline]
Fleming, J. A. (1992). Polydnaviruses: mutualists and pathogens. Annual Review of Entomology 37, 401-425.[Medline]
Fleming, J. A. & Summers, M. D. (1986). Campoletis sonorensis endoparasitic wasps contain forms of C. sonorensis virus DNA suggestive of integrated and extrachromosomal polydnavirus DNAs. Journal of Virology 60, 552-562.
Fleming, J. G. & Summers, M. D. (1991). Polydnavirus DNA is integrated in the DNA of its parasitoid wasp host. Proceedings of the National Academy of Sciences, USA 88, 9770-9774.[Abstract]
Frohman, M. A. (1994). Cloning PCR Products. In The Polymerase Chain Reaction , pp. 14-37. Edited by K. B. Mullis, F. Ferre & R. A. Gibbs. Boston:Birkhauser.
Johner, A., Stettler, P., Gruber, A. & Lanzrein, B. (1999). Presence of polydnavirus transcripts in an egglarval parasitoid and its lepidopterous host. Journal of General Virology 80, 1847-1854.[Abstract]
Kozak, M. (1983). Comparison of initiation of protein synthesis in prokaryotes, eukaryotes, and organelles. Microbiological Reviews 47, 1-45.
Krell, P. J., Summers, M. D. & Vinson, S. D. (1982). A virus with a multipartite superhelical DNA genome from the ichneumonid parasitoid Campoletis sonorensis. Journal of Virology 43, 859-870.
Lavine, M. D. & Beckage, N. E. (1995). Polydnaviruses: potent mediators of host insect immune dysfunction. Parasitology Today 11, 368-378.[Medline]
Norton, W. N. & Vinson, S. B. (1983). Correlating the initiation of virus replication with a specific pupal developmental phase of an ichneumonid parasitoid. Cell and Tissue Research 231, 387-398.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Shelby, K. S. & Webb, B. A. (1999). Polydnavirus-mediated suppression of insect immunity. Journal of Insect Physiology 45, 507-514.[Medline]
Stoltz, D. B. (1993). The polydnavirus life-cycle. In Parasites and Pathogens of Insects , pp. 167-187. Edited by N. E. Beckage, B. A. Federici & S. N. Thompson. San Diego, CA:Academic Press.
Stoltz, D. B., Guzo, D. & Cook, D. (1986). Studies on polydnavirus transmission. Virology 21, 120-131.
Strand, M. R. & Pech, L. L. (1995). Immunological basis for compatibility in parasitoidhost relationships. Annual Review of Entomology 40, 31-56.[Medline]
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. Journal of General Virology 73, 1627-1635.[Abstract]
Theilmann, D. A. & Summers, M. D. (1987). Physical analysis of the Campoletis sonorensis virus multipartite genome and identification of a family of tandemly repeated elements. Journal of Virology 61, 2589-2598.
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, 329-341.[Medline]
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 24, 4876-4882.
von Heijne, G. (1986). A new method for predicting signal sequence cleavage sites. Nucleic Acids Research 14, 4683-4690.[Abstract]
Webb, B. A. & Summers, M. D. (1992). Stimulation of polydnavirus replication by 20-hydroxyecdysone. Experientia 48, 1018-1022.[Medline]
Webb, B. A., Beckage, N. E., Hayakawa, Y., Krell, P. J., Lanzrein, B., Stoltz, D. B., Strand, M. R. & Summers, M. D. (2000). Polydnaviridae. In Virus Taxonomy. Seventh Report of the International Committee on Taxonomy of Viruses , pp. 253-260. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. San Diego:Academic Press.
Received 10 January 2002;
accepted 11 June 2002.