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Two Microplitis demolitor polydnavirus mRNAs expressed in hemocytes of Pseudoplusia includens contain a common cysteine-rich domain.

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JOURNAL OFVIROLOGY, 0022-538X/97/$04.0010

Mar. 1997, p. 2146–2156 Vol. 71, No. 3

Copyrightq1997, American Society for Microbiology

Two

Microplitis demolitor

Polydnavirus mRNAs Expressed

in Hemocytes of

Pseudoplusia includens

Contain a

Common Cysteine-Rich Domain

MICHAEL R. STRAND,* R. ANDREWS WITHERELL,ANDDOMINIQUE TRUDEAU

Department of Entomology, University of Wisconsin—Madison, Madison, Wisconsin 53706

Received 9 September 1996/Accepted 18 November 1996

Microplitis demolitoris a polydnavirus-carrying wasp that parasitizes the larval stage ofPseudoplusia

inclu-dens. A previous study indicated that M. demolitor polydnavirus (MdPDV) infects primarily hemocytes in parasitized hosts. Thereafter, several alterations that compromise the immune response ofP. includenstoward the developing parasitoid occur in hemocytes. In this study, we identified two MdPDV mRNAs (1.0 and 1.5 kb) expressed inP. includenshemocytes that have homology to the viral genomic clone pMd-2. Corresponding 1.0-and 1.5-kb cDNA clones (MdPi455 1.0-and MdPi59) were isolated from an MdPDV-infected hemocyte cDNA library. Nucleotide sequence analysis of the cDNA clones confirmed that the 1.5- and 1.0-kb mRNAs have significant regions of homology. Sequence alignment revealed that the gene, OMd1.0, encoding the 1.0-kb mRNA is present in pMd-2. This gene contains two introns and three exons that agree with the sequence for MdPi455. In contrast, the 1.5-kb mRNA is likely encoded by a related gene located on the same MdPDV genomic DNA as is OMd1.0. The predicted peptide sequences for the 1.0- and 1.5-kb transcripts contain a cysteine-rich region at their 5* ends that have some similarity with epidermal growth factor-like motifs. Hybridization studies revealed that both mRNAs are expressed in granular cells and plasmatocytes, the primary classes of hemocytes involved in defense againstM. demolitorand other parasites.

Certain parasitic wasps in the families Ichneumonidae and Braconidae carry polydnaviruses (PDVs), a family of viruses characterized by double-stranded DNA genomes that are het-erologous in size (11, 28–30). Most PDV-carrying wasps par-asitize the larval stage of Lepidoptera. PDVs replicate in fe-male wasps in a region of the ovary called the calyx. Virions are stored in the lumen of the oviducts with the resulting suspen-sion of virus and protein called calyx fluid. When a wasp ovi-posits, she injects a quantity of calyx fluid, venom, and one or more eggs into the hemocoel of the host. Virus enters different host tissues, with transcription in the apparent absence of rep-lication occurring over the period required for the wasp’s prog-eny to complete development. PDVs appear to be transmitted vertically as proviruses through the germ line of the wasp (11, 29). Despite the absence of replication, however, PDVs induce an array of physiological alterations in parasitized hosts that appear to be essential for survival of the wasp’s progeny. Of particular importance are the immunosuppressive effects PDVs have on hosts (34). In the absence of virus, parasitoid progeny are often encapsulated and killed by host blood cells (hemo-cytes), whereas in the presence of virus they are not.

Microplitis demolitoris a braconid wasp that parasitizes the

larval stage of the mothPseudoplusia includens(Lepidoptera: Noctuidae). The genome ofMicroplitis demolitorPDV (MdPDV) consists of at least 13 circularized DNA segments, ranging in size from 4 to over 30 kb (33). Viral expression is detectable by 4 h postparasitization or postinjection of MdPDV intoP.

in-cludenslarvae and continues over the 7-day period required for

completion of development by the immature wasp (33, 34). Although MdPDV-specific mRNAs are detected in several host tissues, hemocytes are the primary site of infection (32, 33). Injection of MdPDV alone intoP. includensblocks

encap-sulation of an array of foreign targets, includingM. demolitor

eggs (31). In contrast,M. demolitoreggs are encapsulated at 24 to 30 h postinjection in the absence of MdPDV.

P. includenshemocytes can be divided into several subclasses

based on morphology and antigenic properties (23, 36). En-capsulation is mediated by two subclasses of cells, granular cells that initiate capsule formation by opsonizing the surface of the foreign target and plasmatocytes that attach to the opsonized surface and to one another, forming a multicellular sheath around the target (24). We previously demonstrated that plasmatocytes and granular cells exhibit different alter-ations after injection of MdPDV into hosts or infection of cells in vitro. Granular cells undergo apoptosis, while plasmatocytes lose the ability to adhere to foreign surfaces (31, 35, 36). These alterations did not occur in vitro when cells were cultured in medium conditioned by virus-infected hemocytes or plasma from parasitized hosts or when cells were infected with MdPDV pretreated with psoralen and UV (31, 35). Collec-tively, these results suggest that immunosuppression is due to direct infection of hemocytes by transcriptionally active virus. Northern blot analysis indicated that approximately six size classes of MdPDV mRNAs are expressed in P. includens he-mocytes within 24 h of parasitization or injection of virus (32). In the present study, we examined two MdPDV mRNAs ex-pressed in hemocytes ofP. includens. We found that an abun-dantly expressed 1.5-kb mRNA is partially homologous to a 1.0-kb mRNA. Nucleotide sequence analysis of the associated cDNA clones confirmed that the 1.5- and 1.0-kb mRNAs have significant regions of homology but are likely encoded by dif-ferent genes located on a single MdPDV DNA. Hybridization studies revealed that both mRNAs are expressed in granular cells and plasmatocytes ofP. includens.

MATERIALS AND METHODS

Insects.M. demolitorandP. includenswere reared at 278C with a 16-h-light– 8-h-dark photoperiod as described previously (31). Moths were fed a 20%

su-* Corresponding author. Mailing address: Department of Entomol-ogy, 237 Russell Labs, University of Wisconsin—Madison, Madison, WI 53706. Fax: (608) 262-3322. E-mail: mstrand@calshp.cals.wisc.edu.

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crose solution.M. demolitorwas reared at 278C by parasitizing 12-h-old fourth-stadiumP. includenslarvae. All parasitized larvae used in experiments were parasitized individually. For experiments where hemocytes were collected, 24- to 36-h-old fifth-instarP. includenslarvae were injected with 0.05 wasp equivalents of calyx fluid plus venom, a dose within the physiological range of virus injected byM. demolitorfemales at oviposition (31). Although venom itself does not affect host development or hemocyte behavior, its presence synergizes the effects of MdPDV and other PDVs carried by braconid wasps (29, 32). For in vitro experiments, hemocytes were collected from 24- to 36-h-old fifth-instarP. inclu-denslarvae, as described by Pech et al. (23). Time points were calculated in hours from the time a wasp oviposited into a host or was injected with calyx fluid plus venom.

Calyx fluid and virus.Calyx fluid was collected from wasps by established methods, with the quantities of calyx fluid and MdPDV used in experiments expressed in wasp equivalents (31). MdPDV was purified on sucrose gradients, and MdPDV DNAs were isolated by previously established methods (32, 33). Briefly, calyx fluid or gradient-purified MdPDV was diluted in physiological saline and treated with proteinase K (0.1 mg/ml)–25 mM EDTA–2% (vol/vol) Sarkosyl for 1 h at 428C. MdPDV DNAs were extracted with 1 volume of phenol–1 volume of ether and ethanol precipitated in the presence of 0.25 M NaCl. Viral DNA extracted directly from calyx fluid is only minimally contami-nated with wasp genomic DNA; therefore, identical results were obtained with either purified virus or calyx fluid (32).

DNA and RNA isolations fromP. includens.Nucleic acids were extracted from

P. includenshemocytes by homogenizing cells in an equal volume of lysis buffer (0.5% sodium dodecyl sulfate [SDS], 0.20 M NaCl, 25 mM EDTA, pH 8.0) and phenol-chloroform (1:1). P. includenshemocytes were collected by bleeding larvae from a proleg into anticoagulant buffer (98 mM NaOH, 0.19 M NaCl, 1.7 mM EDTA, and 41 mM citric acid, pH 4.5). Cells were resuspended in Ex-cell 400 medium (JRH Scientific, Lenexa, Kans.) and pelleted at 4003g(23). Total DNA and RNA was isolated as described previously (33), while polyadenylated mRNAs were isolated by oligo(dT)-cellulose chromatography (25). Purified DNA was stored at 48C, and RNA was stored at2808C.

DNA cloning and sequencing.HindIII andEcoRI fragments of the MdPDV genome were cloned into plasmid pGEM 3Z by standard procedures (25). This plasmid library was screened by colony hybridization using 32P-labeled

first-strand cDNA probes synthesized from total mRNA isolated fromP. includens

hemocytes at 18 h postparasitization. Approximately 10mg of RNA was reverse transcribed in the presence of 0.5 mM (each) dATP, dGTP, and dTTP, 100mCi (400 Ci/nmol) of [a-32P]dCTP (Amersham), 1mg of oligo(dT)

12–18, and

first-strand cDNA synthesized with Moloney murine leukemia virus reverse transcrip-tase for 1 h at 378C. Selected cloned fragments which hybridized to cDNA probes were physically mapped.

Double-stranded cDNAs were synthesized from polyadenylated mRNA iso-lated fromP. includenshemocytes at 18 h postinfection with calyx fluid plus venom. First-strand cDNAs were synthesized with Moloney murine leukemia virus reverse transcriptase as described above. Second-strand cDNA synthesis was accomplished by incubating the cDNA-RNA hybrids in a 100-ml reaction mixture containing 40 mM (each) dATP, dGTP, dTTP, and dCTP and DNA polymerase I. Reactions were terminated by the addition of 20 mM EDTA. Double-stranded cDNAs were extracted with phenol-chloroform, followed by ethanol precipitation in the presence of 2.5 M ammonium acetate. Double-stranded cDNAs were cloned by either blunt-end ligation into theSmaI site of PGEM 3Z or Uni-Zap Lambda arms (Stratagene). PGEM 3Z clones were propagated inEscherichia coliDH5a, while Uni-Zap clones were propagated in XL1-Blue cells. Recombinant plasmids or plaques containing cDNA inserts homologous to MdPDV genomic DNAs were identified by colony-plaque hy-bridization. Specific MdPDV genomic fragments were gel purified and labeled to high specific activity by random priming in the presence of [32P]dCTP (3,000

Ci/nmol; Amersham). Then colony hybridization filters were prepared and screened by standard methods (25). Filters were exposed to autoradiographic film overnight. Positive cDNA clones were analyzed by restriction enzyme di-gestion and Southern blotting. Dideoxy DNA sequencing reactions of selected clones were performed by the dideoxy-chain termination method using either Sequenase (United States Biochemical Corp.) or Abi Prism cycle (Perkin-Elmer) sequencing kits. Sequence assembly and analysis were performed by using the Genetics Computer Group package (7).

Gel electrophoresis and blot hybridization.For Southern and Northern hy-bridizations,32

P-labeled DNA probes were prepared by random priming using [32

P]dCTP. Synthetic oligonucleotide probes were labeled by addition of a ho-mopolymeric tail of [a-32

P]dATP using terminal deoxynucleotidyl transferase. To identify MdPDV DNA sequences expressed in P. includens,32

P-labeled cDNAs were hybridized to Southern blots of plasmid DNAs from theHindIII library of MdPDV DNA. For Southern blots, undigested or restriction enzyme-digested viral DNAs (1.0mg/lane) were size fractionated on 1 to 0.4% agarose gels and transferred to nylon in 103SSC (13SSC is 0.15 M NaCl plus 0.015 M sodium citrate) by standard methods (24). Blots were prehybridized for 4 h at 428C in hybridization buffer (53SSC, 0.1% SDS, 103Denhardt’s solution, and 50% formamide).32P-labeled cDNAs or viral genomic DNAs were added to the

hybridization buffer at a concentration of 106cpm/ml and hybridized for 12 to

36 h at 428C. Blots were washed under high-stringency conditions in 0.13SSC– 1.0% SDS for 1 h at 688C and autoradiographed. For Northern blots, total RNA

(2.0mg/lane) from hemocytes was collected from parasitized or unparasitizedP. includenslarvae, size fractionated on 6% formaldehyde–0.9% agarose gels, and transferred to nylon in 103SSC (25). The conditions for hybridizations using

32

P-labeled viral DNAs or cDNAs as probes were the same as described above for Southern blots. Northern blots were washed under conditions of high strin-gency in 0.13SSC–0.9% SDS at 688C for 1 h. The conditions for hybridizations using oligonucleotide probes were those of Lee et al. (20). Northern blots were prehybridized for 6 h in 1 M sodium phosphate (pH 7.2)–7.5% SDS–1% bovine serum albumin–50 mM EDTA. Probe was added to filters at 53106

cpm/ml and hybridized for 18 h at 488C, 58C below the melting temperature of the probe. Filters were washed at high stringency in 0.23SSC–0.9% SDS at 388C for 45 min.

In situ hybridizations. In situ hybridization studies were conducted with digoxigenin-labeled cDNAs or oligonucleotide probes. cDNAs were labeled by random priming using digoxigenin-dUTP and a commercially available kit (Genius; Boehringer Mannheim). Oligonucleotide probes were labeled by homo-polymeric tailing using digoxigenin-dATP. The probe concentration was deter-mined by slot blot analysis against a known standard according to the manu-facturer’s instructions. Hybridization was carried out by established methods (32, 38). Hemocytes from normal, parasitized, or calyx fluid-injected larvae were placed into 96-well culture plates containing 70ml of Ex-cell 400 medium at a density of 2.03104

cells per well. Cells were allowed to settle for 15 to 30 min and then fixed for 20 min with an equal volume of 10% formalin–50 mM EDTA in phosphate-buffered saline (PBS). Cells were permeabilized with PBS plus 0.1% Tween 20 with subsequent incubation of cells in 0.1mg of proteinase K per ml for 2 min. The cells in each well were prehybridized in hybridization buffer for 3 h at 448C followed by addition of digoxigenin-labeled probe at a concentration of 100 ng/ml for 24 h. Hybridized cells were washed for 6 h (1 h per wash) in PBS plus 0.1% Tween 20. A positive hybridization signal was detected by using an anti-digoxigenin Fab fragment conjugated to alkaline phosphatase at a 1:2,000 dilution for 1 h at room temperature. Reactions were stopped by rinsing cells in PBS. Normal hemocytes at a density of 2.03104

cells per well were also infected in vitro by addition of 0.10 wasp equivalents of gradient-purified MdPDV plus venom per well (32, 35). At 12 h postinfection, hemocytes were fixed and pro-cessed as described above. As controls, hemocytes infected in vitro or collected from parasitized larvae were pretreated with RNase before hybridization or were processed with the probe or secondary antibody omitted from the protocol. Additional controls included incubating hemocytes with venom only or infecting hemocytes in vitro with MdPDV and conducting in situ hybridization experi-ments using pMd-8 as a probe. This 2.1-kb MdPDV genomic clone maps to MdPDV DNA L but does not hybridize to any mRNA expressed in parasitized larvae (41a).

Nucleotide sequence accession numbers.The sequence data reported here appear in the GenBank database under accession no. U76033 and U76034.

RESULTS

Screening and mapping of expressed MdPDV sequences.A number of cloned restriction fragments containing expressed sequences were identified by screening the MdPDV genomic library with 32P-labeled cDNA probes. For this study, one positively hybridizingHindIII fragment of 5.2 kb, designated pMd-2, was selected for further study. To identify MdPDV DNAs with homology to pMd-2, this clone was physically mapped and hybridized under conditions of high stringency to Southern blots of undigested and HindIII-digested MdPDV DNAs. pMd-2 hybridized strongly to MdPDV DNA O and moderately to DNAs K and I (Fig. 1). Electrophoretic and electron microscopic studies indicated that MdPDV DNAs O, K, and I are approximately 32.1, 16.0, and 14.4 kb in size, respectively (33) (unpublished observations). pMd-2 also hy-bridized to sevenHindIII fragments (8.2, 7.5, 7.0, 5.2, 3.5, 3.2, and 1.6 kb) (Fig. 1).

To determine whether any MdPDV mRNAs expressed in

P. includenshemocytes have homology with pMd-2, Northern

blot studies were conducted with hemocyte RNA fromP.

in-cludens larvae 2 to 96 h after injection with calyx fluid plus

venom. pMd-2 hybridized strongly to two mRNAs of approx-imately 1.5 and 1.0 kb at 12 and 24 h (Fig. 2). A weaker hybridization signal to a 0.4-kb mRNA was also detected at 12 h postinjection. Upon longer exposure of autoradiograms, hybridization to the 1.5- and 1.0-kb mRNAs was also detected at 96 h. In contrast, pMd-2 did not hybridize to any mRNAs from unparasitized larvae or from larvae 2 h postparasitization.

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Mapping cDNA clones to MdPDV DNAs.To identify, map, and sequence the 1.5- and 1.0-kb MdPDV mRNAs, cDNAs were synthesized and cloned from polyadenylated mRNA iso-lated from hemocytes fromP. includens larvae 18 h after in-jection with calyx fluid plus venom. cDNA clones with homol-ogy to the 1.5- and 1.0-kb MdPDV mRNAs were identified by colony hybridization using pMd-2 as the hybridization probe. Seven cDNA clones with inserts of$500 bp were identified, and two (MdPi59 and MdPi455) containing inserts of approx-imately 1.5 and 1.0 kb, respectively, were selected for further study. Since pMd-2 hybridized to multiple MdPDV DNAs (O, K, and I), the MdPi59 and MdPi455 cDNAs could have ho-mology to one or more circles of the MdPDV genome. To identify which viral DNAs have homology to these cDNAs, each was labeled with [32P]dCTP and hybridized to undigested andHindIII-digested MdPDV DNAs. Both cDNAs hybridized specifically to MdPDV DNA O (Fig. 3). Hybridization to other MdPDV DNAs was not detected even after prolonged auto-radiographic exposure. However, both cDNAs also hybridized to five (8.2, 7.0, 5.2, 3.5, and 3.2 kb) of the seven Hin dIII-cleaved MdPDV DNA bands that hybridized with pMd-2 (Fig. 3). This suggested that the 1.5- and 1.0-kb mRNAs are en-coded either by multiple copies of a common gene or by re-lated genes on MdPDV DNA O.

Sequencing of cDNA clones.MdPi455 was 964 bp in length and included a 66-bp poly(A) sequence (Fig. 4A). This cDNA contained two potential translation initiation codons in frame with one another. The sequence preceding the first ATG was in good agreement with the consensus for translation initiation (17). A 226-amino-acid open reading frame (ORF) followed the first ATG. Sequence analysis of this ORF revealed a cys-teine-rich region of 34 amino acids from nucleotides 190 to 289 and an imperfectly repeated element arranged in tandem array beginning at position 439. MdPi59 was 1,490 bp in length, with a single initiation codon at its 59end and an ORF of 316 amino acids (Fig. 4B). The same cysteine-rich domain and 106-nucle-otide direct repeat present in MdPi455 were present in the ORF of MdPi59.

[image:3.612.151.468.84.407.2]

Comparison of these cDNAs also revealed differences in sequence (Fig. 4). MdPi59 began at nucleotide 22 of MdPi455, a position 2 bp downstream of the first initiation codon in MdPi455, which strongly suggested that this cDNA was trun-cated. The two cDNAs were thereafter homologous until nu-cleotide 515 of MdPi455, where a single nunu-cleotide substitu-tion occurred. Thirteen other single-base differences occurred between the two cDNAs through nucleotide 731 of MdPi455. This included an A-to-G substitution in the putative stop codon of MdPi455, which resulted in an extended ORF for

FIG. 1. Analysis of the 5.2-kb MdPDV genomic clone pMd-2 by physical mapping and Southern blotting. (A) Physical map of the 5.2-kbHindIII insert. Restriction sites are indicated above the line, while numbers below the line represent relative distances (in kilobases) from the left terminal restriction site. (B) Hybridization of pMd-2 with uncut DNAs from MdPDV. Undigested MdPDV DNAs were separated on 0.4% agarose gels (left lane), transferred to nylon, and probed with the insert from pMd-2 that had been gel purified and radiolabeled. Filters were washed under conditions of high stringency and exposed to autoradiographic film at2808C for 2 h. The positions of DNAs O, K, and I are shown on the left. (C) Hybridization of pMd-2 withHindIII-digested MdPDV DNAs. Size markers (in kilobases) are shown on the left. Digested MdPDV DNAs were separated on 1% agarose gels (left lane) and analyzed as described for panel B.

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MdPi59. The sequences of the two cDNAs diverged signifi-cantly downstream of nucleotide 735 of MdPi455. This resulted in the ORF of MdPi455 containing three direct repeats and the ORF of MdPi59 containing five. The 39untranslated regions of the two cDNAs differed significantly from one another.

Sequence analysis.By using the first ATG in each cDNA as the translation initiation site, the ORFs of MdPi455 and MdPi59 encoded proteins with predicted molecular masses of 27.1 and 35.2 kDa, respectively. The predicted protein from MdPi455 began with a strongly hydrophobic region of 12 amino acids, typical of a eukaryotic signal sequence. The pre-dicted protein from MdPi59 lacked this signal sequence. Com-parisons with sequences in GenBank/EMBL and SwissProt databases revealed that the cysteine-rich region in both cDNAs has some similarity with epidermal growth factor (EGF)-like motifs present in several proteins (Fig. 4). These cysteine-rich domains are characterized by the spacing of 6 cysteine residues within a 30- to 60-amino-acid domain (1). The homologous domain of MdPi455 and MdPi59 contained the requisite six cysteine residues and spacing similar to that of other EGF-like motifs (Fig. 5). A potential N-linked glycosylation site (N-X-T) was also present in both cDNAs immediately upstream of the cysteine-rich region, as observed for other proteins containing EGF-like motifs (41). A hydrophobic stretch at residues 274 to 316 of MdPi59 served as a putative membrane anchor se-quence. No hydrophobic domain was present at the C terminus of MdPi455. Searches of available databases did not reveal any significant overall homology between the tandem repeats present in MdPi455 and MdPi59 and other proteins.

Genomic sequencing.Preliminary studies indicated that the 59 ends of MdPi455 and MdPi59 map to thePvuI/EcoRI re-striction fragment of pMd-2. This fragment and adjoining up-stream and downup-stream regions, representing a total of 2.6 kb, of pMd-2 were therefore sequenced and aligned to MdPi455 and MdPi59 (Fig. 6). pMd-2 aligned identically with MdPi455 but not MdPi59, indicating that this genomic clone encodes the gene, designated OMd1.0, for the 1.0-kb mRNA. OMd1.0 con-tained three putative TATA boxes and a CAAT box located 39, 130, 149, and 179 nucleotides upstream of the 59 end of MdPi455. OMd1.0 consists of two introns with a single ORF of

216 amino acids that agrees with the predicted amino acid sequence for MdPi455. As expected, pMd-2 aligned with the 59 end of MdPi59 but did not align with the 39end of this cDNA. Temporal expression of MdPDV mRNAs.To determine whether MdPi59 and MdPi455 represented the 1.5- and 1.0-kb mRNAs detected on Northern blots by pMd-2, cDNA-specific probes were hybridized to hemocyte RNA from P. includens

larvae injected with calyx fluid plus venom (Fig. 7). A 271-bp

KpnI/PstI fragment (KP-271) from the 39untranslated region of MdPi59 was used as an MdPi59-specific probe. This restric-tion fragment has no sequence identity with MdPi455. Since only short regions at the 59and 39ends of MdPi455 differ from those of MdPi59, oligonucleotide (TTAGTTTACTTATCAA CTAGG) 819-R was used as an MdPi455-specific probe. This oligonucleotide corresponds to positions 819 to 839 of MdPi455 on the reverse strand. Northern blots were also probed with the entire insert from MdPi59 and MdPi455.

KP-271 hybridized strongly to a single mRNA of 1.5 kb at 12 h postinjection (Fig. 7). A weaker hybridization signal was also detected at 24 h. Upon long exposure of autoradiograms, hybridization to the 1.5-kb mRNA was also detected at 2 and 96 h postinjection; however, no hybridization signal was de-tected in unparasitized P. includens. Oligonucleotide 819-R hybridized strongly to a single mRNA of approximately 1.0 kb at 12 h postinjection, with weaker hybridization signals de-tected at 24 and 96 h (Fig. 7). No mRNAs were dede-tected at 2 h postinjection or in unparasitizedP. includensafter prolonged autoradiographic exposure with this probe. Full-length inserts from MdPi455 and MdPi59 hybridized to both the 1.5- and 1.0-kb mRNAs but did not hybridize to any mRNAs from unparasitized hosts (Fig. 7).

Cross-hybridization to the 1.5- and 1.0-kb mRNAs by MdPi455 and MdPi59 is consistent with the high level of ho-mology for these cDNAs. Hybridization with KP-271 and 819-R indicated, however, that MdPi59 and MdPi455 repre-sent the 1.5- and 1.0-kb MdPDV mRNAs, respectively,

[image:4.612.120.237.70.240.2]

ex-FIG. 2. Identification of MdPDV mRNAs fromP. includenshemocytes with homology to pMd-2. The insert from pMd-2 was gel purified, radiolabeled, and hybridized to Northern blots of total hemocyte RNA (2mg/lane) from unpara-sitizedP. includenslarvae (up) and larvae injected 2, 12, 24, and 96 h previously with 0.05 wasp equivalents of calyx fluid plus venom. Size markers (in kilobases) are shown on the left. Blots were washed under conditions of high stringency and exposed to autoradiographic film by using an intensifying screen at2808C for 3 days.

FIG. 3. Identification of MdPDV DNAs homologous to the cDNA clones MdPi59 and MdPi455. (A) Hybridization of MdPi59 and MdPi455 with uncut DNAs from MdPDV. Undigested MdPDV DNAs were separated on 0.4% agarose gels (left lane), transferred to nylon, and probed with the insert from pMd-2 that had been gel purified and radiolabeled. Filters were washed under conditions of high stringency and exposed to autoradiographic film at2808C for 2 h. The position of DNA O is shown on the left. (B) Hybridization of MdPi59 and MdPi455 withHindIII-digested MdPDV DNAs. Size markers (in kilobases) are shown on the left. Digested MdPDV DNAs were separated on 1% agarose gels (left lane) and analyzed as described for panel A. Southern blots were processed and analyzed as described in the legend to Fig. 1.

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[image:5.612.63.551.86.666.2]

FIG. 4. Nucleotide sequences of the MdPi455 (A) and MdPi59 (B) cDNAs (GenBank accession no. U76033 and U76034). The predicted amino acid sequence for each protein is shown below the corresponding codons. Selected restriction sites are indicated. Potential initiation codons and polyadenylation signals are underlined and in bold type. The putative signal sequence in MdPi455 is in italics, while the EGF-like motifs and upstream N-glycosylation sites on both cDNAs are underlined. Each direct repeat in the central region of each cDNA is bounded by arrows. The asterisks above nucleotides 731 of MdPi455 and 710 of MdPi59 indicate where the two sequences significantly diverge.

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pressed inP. includenshemocytes. Although mRNAs may have 39poly(A) tracts of variable length, the sizes of MdPi59 (1,490 nucleotides) and MdPi455 (961 nucleotides) correspond closely to the size of the mRNAs detected on Northern blots. We conclude, therefore, that MdPi59 and MdPi455 extend to near the 59ends of the corresponding 1.5- and 1.0-kb mRNAs.

In situ hybridization. With digoxigenin-labeled probes, in situ hybridization studies indicated that both the 1.5- and 1.0-kb MdPDV mRNAs were expressed in a large percentage of the hemocytes collected from parasitizedP. includenslarvae (Fig. 8 and 9A). Approximately 6% of hemocytes exhibited both cytoplasmic and nuclear hybridization signals when col-lected from larvae 2 h postparasitization. The proportion of positive cells increased to more than 75% at 24 h postparasiti-zation and then declined. No hybridipostparasiti-zation signal was detected when probes were hybridized to hemocytes from unparasitized larvae (data not presented).

Since granular cells and plasmatocytes do not spread after infection with MdPDV (31, 35, 36), it is difficult to unambig-uously identify hemocyte classes when cells are infected in vivo. Thus, to assess whether the 1.5- and 1.0-kb MdPDV mRNAs were expressed in these hemocyte types, we purified each mor-photype by gradient centrifugation and infected the cells with MdPDV in vitro. Both plasmatocytes and granular cells

exhib-FIG. 5. Cysteine number and spacing consensus for the cysteine-rich do-mains of MdPi455 and MdPi59 (455/59) and three recognized classes of EGF-like motifs (1), EGF-EGF-like, pancreatic secretory trypsin inhibitor (PSTI)-EGF-like, and laminin-like motifs. X represents any amino acid.

FIG. 6. Alignment of nucleotide sequences between pMd-2 (Md-2) and MdPi455 (455). Three putative TATA boxes and a CAAT box in pMd-2 are underlined, while the putative initiation codons, stop codons, and polyadenylation signals in MdPi455 are underlined and in bold type. Introns identified in pMd-2 are shown in lowercase type, while periods are used in MdPi455 for alignment purposes. A vertical line between nucleotides indicates identity.

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ited nuclear and cytoplasmic hybridization signals when probed with digoxigenin-labeled KP-271 and 819-R (Fig. 8 and 9). In control experiments, no hybridization signal was de-tected in hemocytes from unparasitized larvae or hemocytes cultured in the presence of venom only (Fig. 9E). In addition, no hybridization signal was detected when virus-infected he-mocytes were pretreated with RNase, when the probe was omitted, or when the antidigoxigenin antibody was omitted from the hybridization protocol (data not presented). How-ever, when hemocytes were probed in situ with pMd-8, a viral

genomic clone that has no homology with MdPDV mRNAs expressed in parasitized hosts (41a), only a nuclear hybridiza-tion signal was detected (Fig. 9F).

DISCUSSION

PDVs are associated with several thousand species of para-sitic wasps and are known to play an essential role in the successful development of these insects in their lepidopteran hosts. All PDV-carrying wasps occur in two families of

Hyme-FIG. 7. Identification of MdPDV mRNAs fromP. includenshemocytes homologous with MdPi59 and MdPi455. (A) Physical maps of MdPi59 and MdPi455. A

KpnI/PstI fragment (KP-271) from MdPi59 and an oligonucleotide (819-R) designed from MdPi455 were radiolabeled and used as hybridization probes to Northern blots ofP. includenshemocyte RNA. (B) Probes were hybridized to Northern blots of total hemocyte RNA (2mg/lane) from unparasitizedP. includenslarvae (up) and larvae injected 2, 12, 24, and 96 h previously with 0.05 wasp equivalents of calyx fluid plus venom. KP-271 was hybridized to the blot on the left, and 819-R was hybridized to the blot on the right. Size markers (in kilobases) are shown on the left. Blots were washed under conditions of high stringency and exposed to autoradiographic film by using an intensifying screen at2808C for 4 days. (C) Inserts from MdPi59 (59) and MdPi455 (455) were gel purified, radiolabeled, and hybridized to Northern blots of total hemocyte RNA (2mg/lane) from unparasitizedP. includenslarvae (up) and larvae injected 12 h previously with calyx fluid plus venom (p). mRNAs were analyzed as described for panel B. The positions of the 1.5- and 1.0-kb mRNAs are indicated by labeled arrows.

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noptera, Ichneumonidae and Braconidae, and their associated viruses are divided into the ichnovirus and bracovirus groups. Ichnoviruses and bracoviruses appear to have a common life cycle and induce similar alterations in parasitized hosts (11, 30, 34). In particular, viruses from both groups encode genes tran-scribed in the wasp’s host and play essential roles in compro-mising host cellular defense responses toward the developing parasitoid (6, 13, 19, 21, 31). However, ichnoviruses and bra-coviruses differ significantly in morphology, and too few genes have been characterized to assess whether the viruses in these two groups disrupt host defenses by similar or unrelated mech-anisms (11, 30). Currently, more information about the phys-ical organization and transcriptional activity of ichnoviruses than of bracoviruses is available. Therefore, our goal has been to develop a better understanding of bracoviruses through the study ofM. demolitor, MdPDV, andP. includens.

A previous study indicated that MdPDV induces several alterations in granular cells and plasmatocytes, the two hemo-cyte morphotypes identified to be essential for normal capsule formation by P. includens(24). The results of in vitro experi-ments further suggest that both the inhibition of spreading by plasmatocytes and apoptosis of granular cells require direct infection of hemocytes by MdPDV and subsequent gene ex-pression (32, 35). In this study, we isolated cDNAs homologous to two MdPDV-encoded transcripts expressed inP. includens

hemocytes. Physical mapping initially suggested that the mRNAs homologous to MdPi455 and MdPi59 could arise from alter-native splicing of a single transcription unit. However, hybrid-ization of these cDNAs to several HindIII restriction frag-ments of MdPDV genomic DNA (Fig. 3) suggested that these mRNAs could also be transcribed from related genes located on MdPDV DNA O.

Sequence alignment confirmed that the genomic clone pMd-2 encodes the gene for the 1.0-kb mRNA (OMd1.0) but not the 1.5-kb mRNA. Although pMd-2 cross-hybridizes among three viral DNAs (O, K, and I), MdPi455 and MdPi59 hybridized only to MdPDV DNA O. We currently hypothesize that OMd1.0 and the unidentified gene(s) encoding the 1.5-kb mRNA are located on a single MdPDV DNA and that the hybridization observed between pMd-2 and other viral DNAs is likely due to flanking sequences. It is important to note, however, that MdPDV and other PDVs currently cannot be propagated outside of their wasp hosts. Since these studies were not conducted with clonal isolates of virus, it is possible that the cross-hybridization observed in Fig. 3 could be due to the existence of restriction fragment length polymorphisms. Genetic heterogeneity in MdPDV could also explain in part the sequence variation between MdPi455 and MdPi59. We assume that our culture ofM. demolitoris relatively homoge-neous, given that no wasps from elsewhere have been intro-duced into this culture for at least 8 years. We also did not observe any differences in hybridization patterns when these cDNAs were hybridized to MdPDV genomic DNA isolated from individual wasps versus pooled samples as presented in Fig. 3 (30a). This suggests that the cross-hybridization observed in Fig. 3 is not due to genetic heterogeneity in M. demolitor. However, nucleocapsid sizes for MdPDV do vary within sam-ples of virus collected from individual wasps (32a), leading to the possibility that the MdPDV DNAs packaged in individual virions vary. Variation in particle size is widespread among PDVs, but workers in this field will not be able to characterize the putative genetic heterogeneity between virions until meth-ods are developed to propagate these viruses outside of their wasp hosts.

The similarity in sequence between the 1.0- and 1.5-kb mRNAs suggests that the associated genes on MdPDV DNA O have a common ancestry, possibly evolving from a recent gene duplication event. Our results also suggest that the 1.0-and 1.5-kb mRNAs represent a gene family targeted for ex-pression in parasitized hosts. The transcripts described here have no significant homology to the small number of other genes expressed in hosts by ichnoviruses (4, 8, 40) or bracovi-ruses (2, 14). However, the presence of a putative gene family in MdPDV is consistent with observations for the ichnovirus

Campoletis sonorensisPDV (CsPDV), where at least two gene

families have been identified on the basis of nucleotide se-quence similarity and immunological cross-reactivity (4, 8, 40, 42). The sequence similarity observed between the cDNAs described here is higher than the imperfect homology (68 to 88%) reported for candidate gene family members on, for example, CsPDV DNA W (8). This may reflect differences between these species as to when putative gene duplication

FIG. 8. Mean percentages6standard deviations ofP. includenshemocytes hybridizing with digoxigenin-labeled KP-271 (MdPi59) and 819-R (MdPi455) when infected in vivo (A) and in vitro (B) with MdPDV. (A) Probes were hybridized to hemocytes collected at selected intervals (2 to 72 h postparasitiza-tion) from parasitized larvae. Each datum point is the mean percentage of cells exhibiting a hybridization signal from three different larvae. (B) Mean percent-ages6standard deviations of granular cells and plasmatocytes hybridizing with KP-271 and 819-R when infected with purified MdPDV plus venom in vitro. Hemocyte types were isolated by gradient centrifugation, infected with 0.10 wasp equivalents of gradient-purified MdPDV plus venom, and hybridized with probes at 6 h postinfection. Each datum point is the mean percentage of cells exhibiting a hybridization signal from three independent collections of hemocytes.

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[image:9.612.79.535.46.650.2]

FIG. 9. In situ hybridization of digoxigenin-labeled KP-271 (MdPi59) and 819-R (MdPi455) toP. includenshemocytes. (A) Hemocytes from aP. includenslarva at 12 h postparasitization, probed with KP-271 (scale bar, 35mm). Note that hemocytes collected from parasitized larvae at this time are rounded and unspread. (B through F) Hemocytes infected in vitro with MdPDV plus venom. Granular cells and plasmatocytes collected from normal larvae assume a spread morphology when cultured in vitro. Cells infected in vitro with MdPDV remain spread for approximately 20 h postinfection but thereafter apoptose or disattach from culture plates (35). (B) Granular cells at 6 h after infection with MdPDV plus venom, probed with 819-R (scale bar, 15mm). (C) Plasmatocytes at 6 h postinfection, probed with KP-271 (scale bar, 15mm). (D) Granular cells at 6 h postinfection probed with KP-271 (scale bar, 10mm). (E) Granular cells at 6 h after incubation with 0.10 wasp equivalents of venom only, probed with KP-271 (scale bar, 10mm). Note the lack of hybridization signal in either the cytoplasm or nucleus (arrow). (F) Hemocytes infected with MdPDV plus venom and probed with the MdPDV genomic clone pMd-8 (scale bar, 15mm). Note the nuclear hybridization signal (arrow) and absence of a cytoplasmic hybridization signal.

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events occurred or in the importance of the conserved features within these gene families relative to their function in parasit-ism.

Currently, we can only speculate as to why PDVs might encode families of genes targeted for expression in the wasp’s host. One possibility is that gene duplication permits expansion and specialization of gene function. This could be important for expression of transcripts at specific times during parasitism or in specific tissues of the host. For example, Dib-Hajj et al. (8) hypothesized that variation in cysteine-rich regions of the WHv 1.0/1.6 gene family of CsPDV could facilitate targeting of these gene products to specific host cells. Another possibility, however, is that gene families in PDVs reflect an evolutionary outcome of confronting multiple host environments. Although the factors defining the host ranges of parasitoids are poorly understood, it is clear from the literature that very few species of parasitoids are truly host specific (3, 37). While some PDV-carrying wasps parasitize only one or two species of hosts, most regularly encounter and successfully parasitize several species of Lepidoptera. For example,M. demolitorparasitizes at min-imum eight species of moths (26, 30a), whereasC. sonorensis

parasitizes at least 20 (18). The presence of gene families in MdPDV and CsPDV may thus reflect a requirement for cialization that facilitates survival of the wasp in different spe-cies of hosts. If so, we would predict that the relative size of gene families in PDVs varies with the host range size of the associated parasitoid.

Previous studies indicated that two of the mRNAs expressed in hemocytes from parasitizedP. includenswere approximately 1.5 and 1.0 kb (32). The results of the current study indicate that the highest levels of expression for both transcripts occur between 12 and 24 h postparasitization, followed by a decline in expression. This pattern parallels the physiological changes that occur in hemocytes after infection by MdPDV (31, 35). By 24 h postinfection, nearly all plasmatocytes are incapable of spreading on foreign surfaces, while the highest levels of apo-ptosis by granular cells also occur at 24 h. Given that (i) granular cells comprise 60 to 70% of the hemocytes in circu-lation (23) and (ii) MdPDV does not appear to replicate inP.

includens(33), the significant loss of this cell type is consistent

with the reduced levels of expression of the 1.5- and 1.0-kb mRNAs observed after 24 h during the current study.

The most distinguishing structural feature of the transcripts described here is the presence of a cysteine-rich domain at the N terminus. This domain resembles the EGF-like motifs de-scribed for other proteins. Three classes of EGF-like motifs are distinguished on the basis of the number and spacing of cysteine residues within 30- to 60-amino-acid domains (1). Proteins containing these motifs are highly variable outside of these regions. Members of the first class of EGF-like motif contain six cysteine residues with spacing akin to the cysteine-rich domains of EGF (10, 12) and developmental regulators such as the Notch protein of Drosophila melanogaster and

Lin-12 from Caenorhabditis elegans (45). This class also

in-cludes proteins such as thrombomodulin (9), mouse coagula-tion factor IX (43), the human LDL-receptor-related protein (15), and the cysteine-rich coat proteins inGiardia duodenalis

(5). Members of the second class also have six cysteine residues spaced similarly to the active domain of the Kazal family of pancreatic trypsin inhibitors (44). Members of the third class occur in basement membrane proteins such as laminin and contain eight cysteine residues (1, 16, 22). The cysteine-rich domains of the 1.5- and 1.0-kb mRNAs encoded by MdPDV resemble the first class of EGF-like motifs. All EGF-like pro-teins are either secreted, membrane bound, or membrane as-sociated and are involved in specific protein-protein

interac-tions (27, 39). Results to be presented elsewhere indicate that both mRNAs described here are translated and that the re-sulting proteins localize to the plasma membrane and cyto-plasm of MdPDV-infected hemocytes. Functional studies cur-rently in progress should clarify whether the proteins encoded by MdPDV are involved in disruption of the cellular immune response of insects.

ACKNOWLEDGMENTS

We thank J. Johnson and K. Kadash for assistance in sequencing selected clones. We also thank D. McKenzie and P. Friesen for com-ments and suggestions on the manuscript.

This research was supported in part by Public Health Service grant AI32617 from the National Institute of Allergy and Infectious Diseases and the USDA Hatch program to M.R.S. D.T. was a recipient of predoctoral fellowships from the Natural Sciences and Engineering Research Council and from FCAR (Canada).

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Figure

FIG. 1. Analysis of the 5.2-kb MdPDV genomic clone pMd-2 by physical mapping and Southern blotting
FIG. 3. Identification of MdPDV DNAs homologous to the cDNA clonesMdPi59 and MdPi455. (A) Hybridization of MdPi59 and MdPi455 with uncut
FIG. 4. Nucleotide sequences of the MdPi455 (A) and MdPi59 (B) cDNAs (GenBank accession no
FIG. 5. Cysteine number and spacing consensus for the cysteine-rich do-mains of MdPi455 and MdPi59 (455/59) and three recognized classes of EGF-
+4

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