Analysis of gene transcription and relative abundance of the cys-motif gene family from Campoletis sonorensis ichnovirus (CsIV) and further characterization of the most abundant cys-motif protein, WHv1.6.

The cys-motif gene family associated with Campoletis sonorensis ichnovirus contains 10 members, WHv1.6, WHv1.0, VHv1.1, VHv1.4, AHv1.0, A'Hv0.8, FHv1.4, LHv2.8, UHv0.8, and UHv0.8a. The results of this study indicated that, within the encapsidated virion, WHv1.6 is the most abundant cys-motif gene, while the combined AHv genes are the least abundant. During parasitization of Heliothis virescens by Campoletis sonorenis, WHv1.6 transcripts were the mostly highly expressed, while the combined UHv genes had the lowest expression. Further proteomic analysis of WHv1.6 showed that it accumulates at high levels in parasitized plasma by 6 h, and is detectable in the haemocytes, fat body, malpighian tubules, nerve cord and epidermis by 2 days after parasitization. Localization experiments led us to conclude that WHv1.6 interacts with the cell membrane along with other organelles within a virus-infected cell and prevents immunocytes from spreading or adhering to a foreign surface. Similarly to VHv1.4 and VHv1.1, WHv1.6 is able to inhibit the translation of haemocyte and Malpighian tubule RNAs. Our results showed that the expression of cys-motif genes during parasitization is related to the gene copy number of each gene within the encapsidated virion and may also be dependent upon cis-regulatory element activity in different target tissues. In addition, WHv1.6 plays a major role in inhibiting the cellular encapsulation response by H. virescens.

Invited review: the role of caterpillars in mare reproductive loss syndrome: a model for environmental causes of abortion.

A new abortigenic disease, now known as mare reproductive loss syndrome (MRLS), significantly affected the horse industry in the Ohio River Valley of the United States in late April and early May of 2001 and 2002. In 2001, approximately 25% of all pregnant mares aborted within several weeks (over 3,000 mares lost pregnancies), and abortion rates exceeded 60% on some farms. Mare reproductive loss syndrome struck hard and without warning, it was caused by something in the environment, it was not transmitted between animals, and it was not associated with any known abortigenic agent or disease. These experiments demonstrated that horses will inadvertently consume Eastern tent caterpillars (ETC) when the insects are present in the pasture or other feedstuffs, and MRLS-type abortions were induced in experimental animals (mares and pigs) by mixing ETC with the feed of the animals. Eastern tent caterpillars are hirsute (hairy) caterpillars, and the only part of the caterpillar that caused MRLS abortions was the cuticle. The experiments revealed that the setae (hairs) embed into the submucosa of the alimentary tract creating microgranulomatous lesions. It is hypothesized that the alimentary tract lesions allow bacteria from the alimentary tract of the mare, principally streptococci, actinobacilli, and to a lesser extent enterococci, to invade the circulatory system of the mare. The bacteria then establish infections in tissues where the immune surveillance of the mare is reduced, such as the fetus and placenta. Fetal and placental fluid bacterial infections lead to fetal death and abortion characteristic of MRLS. Inadvertent ingestion of ETC by pregnant mares causes MRLS. Currently the only known means to prevent MRLS is to avoid exposure of horses, particularly pregnant mares, to ETC and probably most hirsute caterpillars.

The Campoletis sonorensis ichnovirus vankyrin protein P-vank-1 inhibits apoptosis in insect Sf9 cells.

The Campoletis sonorensis ichnovirus (CsIV) vankyrin genes encode proteins containing truncated ankyrin repeat domains with sequence homology to the inhibitory domains of NF-kappaB transcription factor inhibitors, IkappaBs. The CsIV vankyrin proteins are thought to be involved in the suppression of NF-kappaB activity during immune response and/or developmental events in the parasitized host. Here we report that when P-vank-1 was expressed stably from Sf9 cells, prolonged survival of these cells was observed after baculovirus infection, UV irradiation, and treatment with the apoptosis-inducing chemical camptothecin compared to untransformed Sf9 cells. Furthermore, P-vank-1 inhibited nuclear and internucleosomal degradation and caspase activity after induction of apoptosis in Sf9 cells stably expressing P-vank-1. This is the first report of a polydnavirus protein with anti-apoptotic function.

A teratocyte gene from a parasitic wasp that is associated with inhibition of insect growth and development inhibits host protein synthesis.

After parasitization, some wasps induce hosts prematurely to initiate metamorphic development that is then suspended in a postwandering, prepupal state. Following egression of the parasite larva, the host remains in this developmentally arrested state until death. Teratocytes, cells released at egg hatch from extra-embryonic serosal membranes of some wasp parasites, inhibit growth and development when injected into host larvae independent of other parasite factors (e.g. venom, polydnavirus). Synthesis of some developmentally regulated, abundantly expressed Heliothis virescens host proteins is inhibited in hosts parasitized by Microplitis croceipes and by teratocyte injection. A cDNA encoding a 13.9 kDa protein (TSP14) that inhibited protein synthesis, growth and development was isolated from a protein fraction secreted by teratocytes. TSP14 appears to be responsible, in part, for the teratocyte-mediated inhibition of host growth and development. Interestingly, this cDNA encoded a cysteine-rich amino acid motif similar to that described from Campoletis sonorensis polydnavirus, a mutualistic virus that enables wasp parasitization of lepidopteran larvae. Moreover, TSP14 inhibited protein synthesis in a dose-dependent manner in rabbit reticulocyte lysate and wheat germ extract translation systems. We hypothesize that some wasp parasites inhibit translation as a general means to regulate and redirect lepidopteran host physiology to support endoparasite development.

Two Hyposoter didmator ichnovirus genes expressed in the lepidopteran host encode secreted or membrane-associated serine and threonine rich proteins in segments that may be nested.

We present in this work two novel Hyposoter didymator ichnovirus genes expressed in parasitized Spodoptera larvae. These genes, named HdCorfS6 and HdGorfP30, are unrelated and present in two different genome segments, possibly nested, SH-C and SH-G respectively. HdCorfS6 encodes a predicted transmembrane protein, putatively glycosylated. HdCorfS6 transcripts appear to be abundant in lepidopteran host hemocytes compared to the other tissues analyzed. The second gene described, HdGorfP30, is well expressed in hemocytes, but also in other tissues, such as the fat body, nervous system and epidermis. This gene is peculiar since it presents 17 perfectly conserved repeated sequences arranged in tandem arrays. Each of these repeats contains 58% of serine and threonine residues and therefore several potential sites for glycosylation. This mucin-like protein, predicted as highly glycosylated, could be involved in host immune suppression.

Evidence for a conserved polydnavirus gene family: ichnovirus homologs of the CsIV repeat element genes.

In Campoletis sonorensis Ichnovirus (CsIV), the repeat element genes constitute a gene family of 28 members. In the present work, we document the presence of members of this gene family in two additional ichnoviruses, Hyposoter didymator Ichnovirus (HdIV) and Tranosema rostrale Ichnovirus (TrIV). Two repeat element genes, representing at least one functional gene, were identified in TrIV, whereas HdIV was found to contain at least three such genes. In both HdIV and TrIV, the known repeat element genes are encoded on single genome segments, with hybridization studies suggesting the presence of other, related but as yet uncharacterized genes. The HdIV and TrIV repeat element genes are all transcribed in infected caterpillars, although differences exist among genes in levels and in tissue specificity of expression. A heuristic tree was generated indicating that the repeat element genes are more similar within a species of wasp than between species, with TrIV genes being more closely related to the CsIV than to the HdIV genes. These results suggest that the most significant duplication, divergence, and expansion of the repeat element genes occurred after speciation. The finding that repeat element genes form an interspecific family within the genus Ichnovirus supports the view that the proteins they encode play an important role in ichnovirus biology.

Expression and characterization of a novel teratocyte protein of the braconid, Microplitis croceipes (cresson).

Microplitis croceipes wasps overcome host immunity by inducing changes in host physiology using factors derived from the embryo and/or larva. Teratocytes of some parasitic wasps circulate in the host hemolymph after egg hatch and synthesize proteins (TSPs), some of which are secreted to alter host physiology in support of endoparasitoid development. TSPs appear to alter host physiology, at least in part, by inhibiting synthesis of certain proteins. M. croceipes teratocytes synthesize a 13.9 kDa protein (TSP14), which inhibits synthesis of host proteins that are linked to larval growth and development. A cDNA encoding TSP14 was generated by RT-PCR from teratocyte RNA, and cloned into yeast expression vectors to produce sufficient recombinant protein for functional analyses. RecTSP14 was produced using the yeast expression system at a concentration of more than 300 micrograms/L. The recTSP14 inhibited in vitro translation of larval Heliothis virescens RNA, with the activity sensitive to boiling, protein concentration, incubation time, and storage temperatures. Although recTSP14 inhibited translation of some cellular RNAs in vitro, the in vivo incorporation of [35S]-methionine into proteins of selected insect and mammalian cell lines was not inhibited. These findings suggest that recTSP14 entry is cell type-specific and required to inhibit synthesis of target protein(s).

G2/M arrest caused by actin disruption is a manifestation of the cell size checkpoint in fission yeast.

In budding yeast, actin disruption prevents nuclear division. This has been explained as activation of a morphogenesis checkpoint monitoring the integrity of the actin cytoskeleton. The checkpoint operates through inhibitory tyrosine phosphorylation of Cdc28, the budding yeast Cdc2 homolog. Wild-type Schizosaccharomyces pombe cells also arrest before mitosis after actin depolymerization. Oversized cells, however, enter mitosis uninhibited. We carried out a careful analysis of the kinetics of mitotic initiation after actin disruption in undersized and oversized cells. We show that an inability to reach the mitotic size threshold explains the arrest in smaller cells. Among the regulators that control the level of the inhibitory Cdc2-Tyr15 phosphorylation, the Cdc25 protein tyrosine phosphatase is required to link cell size monitoring to mitotic control. This represents a novel function of the Cdc25 phosphatase. Furthermore, we demonstrate that this cell size-monitoring system fulfills the formal criteria of a cell cycle checkpoint.

Parasitism-linked block of host plasma melanization.

When parasitized by the Ichneumonid parasitoid Campoletis sonorensis, larvae of the Noctuid moth, Heliothis virescens, are unable to mount an effective immune response against parasitoid eggs. Defensive melanization of plasma and cellular encapsulation of parasite eggs are dramatically inhibited by infection with the symbiotic immunosuppressive C. sonorensis ichnovirus (CsIV). This study demonstrates that the CsIV-mediated inhibition of melanization is associated with reduction in the enzymatic activity and protein titer of key enzymes in the melanization pathway, phenoloxidase, dopachrome isomerase, and DOPA decarboxylase. Inhibition of the synthesis of key melanization enzymes leads to reductions in the melanization substrates l-dihydroxyphenylalanine, N-acetyldopamine, and N-beta-alanyl dopamine from millimolar to nanomolar levels in parasitized larvae. By contrast, concentration of a precursor catecholamine, dopamine, rises fourfold in these larvae. Thus in CsIV-infected larvae, enzymatic deficiencies in the melanization pathway lead to reduced concentrations of specific enzyme substrates, causing failure of melanization in parasitized insects.

A role for myosin-I in actin assembly through interactions with Vrp1p, Bee1p, and the Arp2/3 complex.

Type I myosins are highly conserved actin-based molecular motors that localize to the actin-rich cortex and participate in motility functions such as endocytosis, polarized morphogenesis, and cell migration. The COOH-terminal tail of yeast myosin-I proteins, Myo3p and Myo5p, contains an Src homology domain 3 (SH3) followed by an acidic domain. The myosin-I SH3 domain interacted with both Bee1p and Vrp1p, yeast homologues of human WASP and WIP, adapter proteins that link actin assembly and signaling molecules. The myosin-I acidic domain interacted with Arp2/3 complex subunits, Arc40p and Arc19p, and showed both sequence similarity and genetic redundancy with the COOH-terminal acidic domain of Bee1p (Las17p), which controls Arp2/3-mediated actin nucleation. These findings suggest that myosin-I proteins may participate in a diverse set of motility functions through a role in actin assembly.

A gene encoding a polydnavirus structural polypeptide is not encapsidated.

Polydnaviruses are symbiotic viruses associated with some parasitic Hymenoptera that are vertically transmitted as proviruses within wasp genomes. To study this symbiotic association a gene encoding an abundant Campoletis sonorensis polydnavirus virion protein was characterized. This gene is not encapsidated but resides in the wasp genome where it is expressed only during virus replication. Immunolocalization studies detected the encoded 44-kDa protein only in oviduct tissue with ultrastructural studies detecting epitopes between or on virion envelopes. Expression and localization of the 44-kDa protein are consistent with its being a viral structural protein but localization of the gene only within the wasp genome is atypical, raising the possibility that this protein is adventitiously packaged during virion assembly. To address this possibility, quantitative dot blot and genomic Southern blot hybridizations were performed to determine whether the copy number of the p44 gene increased disproportionately during replication, as would be expected for a gene encoding a virion protein. The copy number of the p44 gene increases in tissues supporting virus replication but is unchanged in other tissues, suggesting that this gene is amplified in replicative cells. The data indicate that genes encoding polydnavirus virion proteins may be distributed between wasp and encapsidated viral genomes.

Cloning and expression of a gene encoding a Campoletis sonorensis polydnavirus structural protein.

Polydnaviruses are the only known group of mutualistic viruses. They are required for successful parasitization in many braconid and ichneumonid parasitoids. The intimacy of this mutualistic association is indicated by the integration and vertical transmission of polydnaviruses in wasp genomes and by their asymptomatic, developmentally regulated replication. The evolution of this mutualism raises several interesting issues that require a better understanding of the viral genome and viral replication. To develop probes for virus replication and morphogenesis, we have begun to characterize several viral structural proteins. A 699 bp cDNA encoding the p12 viral structural protein was cloned and sequenced. The p12 gene localizes to viral segment Y and encodes a predicted protein of 92 amino acids that does not encode a signal peptide and is unrelated to known peptide or nucleic acid sequences. The p12 mRNA is detected at the onset of virus replication. mRNA titers increase with increasing rates of virus replication. Polyclonal antisera raised against histidine-tagged p12 protein expressed in bacteria reacted specifically with the p12 polypeptide in Western blots of CsPDV virions. The p12 polypeptide was not detected in non-replicative wasp or lepidopteran tissues by Western blot analyses but was readily detected in protein extracts of wasp ovaries. The data indicate that the p12 gene is a viral gene encoding a virion protein and provides a specific probe for virus replication that will be useful for studying the evolution of this group of mutualistic viruses.

Polydnavirus-mediated suppression of insect immunity.

Polydnaviruses are symbiotic proviruses of some ichneumonid and braconid wasps that modify the physiology, growth and development of host lepidopteran larvae. Polydnavirus infection targets neuroendocrine and immune systems, altering behavior, stunting growth, and immobilizing immune responses to wasp eggs and larvae. Polydnavirus-mediated disruption of cellular and humoral immunity renders parasitized lepidopteran larvae suitable for development of wasp larvae as well as more susceptible to opportunistic infections. Evidence from the Campoletis sonorensis polydnavirus system indicates that the unique genomic organization of polydnaviruses may have evolved to amplify the synthesis of immunosuppressive viral proteins. Immunosuppressive viruses have been essential to elucidating vertebrate immunity. Polydnaviruses have similar potential to clarify insect immune responses and may also provide novel insights into the role of insect immunity in shaping polydnavirus genomes.

Polydnavirus-mediated inhibition of lysozyme gene expression and the antibacterial response.

Parasitism of lepidopteran host larvae by hymenopteran parasitoids impairs the cellular immune response via expression of polydnavirus genes. Encapsulation of parasitoid eggs is thereby prevented. Parasitized insects are susceptible to opportunistic infections, suggesting that additional components of the immune system are affected. Insects normally respond to infection by inducing the synthesis of an array of antibacterial factors, including cecropins and lysozyme via a NFkappaB/lkappaB-like signal transduction pathway. To characterize the effects of PDVs on the antibacterial immune response, plasma antibacterial activities were assayed in H. virescens larvae infected with the C. sonorensis PDV. Plasma lysozyme activity in Heliothis virescens was reduced in parasitized and PDV-infected larvae after immune challenge. To examine the regulation of lysozyme after CsPDV injection, the Heliothis virescens lysozyme cDNA was cloned. In contrast to plasma lysozyme activity, the 1.1 kb lysozyme mRNA was induced in fat body and haemocytes by known elicitors. The data suggest that CsPDV, like some other viruses, regulates host cell gene expression at the level of translation. We propose that the immunodeficiencies caused by CsPDV injection are caused, in part, by the targeted translational inhibition of specific humoral immune response transcripts.

Relationships between polydnavirus genomes and viral gene expression.

Polydnavirus genomes and viral gene functions are atypical for viruses. Polydnaviruses are the only group of viruses with segmented DNA genomes and have an unusual obligate mutualistic association with parasitic Hymenoptera, in which the virus is required for survival of the wasp host and vice versa. The virus replicates asymptomatically in the wasp host but severely disrupts lepidopteran host physiology in the absence of viral DNA replication. It is not surprising then that viral gene expression is divergent in its two insect hosts and that differences in viral gene expression are linked to these divergent functions. Some viral genes are expressed only in the wasp host while other viral genes are expressed only in the lepidopteran host and are presumed to be involved in the disruption of host physiological systems. Our laboratory has described the expression and regulation of a family of viral genes implicated in suppressing the lepidopteran immune system, the cys-motif genes. In conjunction with these studies we have described the physical organization of additional viral gene segments. We have cloned, mapped and begun the sequence analysis of selected viral DNA segments. We have noted that some viral DNA segments are nested and that nested viral DNA segments encode the abundantly expressed, secreted cys-motif genes. Conversely, other viral segments are not nested, encode less abundantly expressed genes and may be targeted intra-cellularly. These results suggest that nesting of segments in polydnavirus genomes may be linked to the levels of gene expression. By extension, the unique, segmented organization of polydnavirus genomes may be associated, in part, with the requirement for divergent levels of viral gene expression in lepidopteran hosts in the absence of viral DNA replication.

Homologous sequences in the Campoletis sonorensis polydnavirus genome are implicated in replication and nesting of the W segment family.

Polydnaviruses (PDVs) are double-stranded DNA viruses with segmented genomes that replicate only in the oviducts of some species of parasitic wasps and are required for the successful parasitization of lepidopteran insects. PDV DNA segments are integrated in the genomes of their associated wasp hosts, and some are nested; i.e., smaller segments are produced from and largely colinear with larger segments. To determine the internal structure of nested viral segments, the first complete nucleotide sequence of a PDV genome segment and its integration locus was determined. By restriction mapping, Southern blot, and sequence analyses, we demonstrated that the Campoletis sonorensis PDV segment W is integrated into wasp genomic DNA. DNA sequence analysis revealed that proviral segment W terminates in two 1,185-bp direct long terminal repeats (LTRs) in the wasp chromosome, while only one LTR copy is present in the extrachromosomal (viral) W. The results suggest that terminal direct repeats are a general feature of PDV DNA segment integration but that the homology and size of the repeats can vary extensively. Segment W contains 12 imperfect direct repeats of six different types between 89 bp and 1.9 kbp with 65 to 90% homology. The orientation and structure of the repeats suggest that W itself may have arisen through sequence duplication and subsequent divergence. Mapping, hybridization, and sequence analyses of cloned R and M demonstrated that these segments are nested within segment W and that internal imperfect direct repeats of one type are implicated in the homologous intramolecular recombination events that generate segments R and M. Interestingly, segment nesting differentially increases the copy number of genes encoded by segment W, suggesting that the unusual genomic organization of PDVs may be directly linked to the unique functions of this virus in its obligate mutualistic association with parasitic wasps.

Expression, assembly, and proteolytic processing of Helminthosporium victoriae 190S totivirus capsid protein in insect cells.

The dsRNA genome (5.2 kbp) of Helminthosporium victoriae 190S totivirus (Hv190SV) consists of two large overlapping open reading frames (ORFs). The 5' proximal ORF codes for the capsid protein (CP) and the 3' ORF codes for an RNA-dependent RNA polymerase. Although the capsid of Hv190SV is encoded by a single gene, it is composed of two major closely related polypeptides, either p88 and p83 or p88 and p78. Whereas p88 and p83 are phosphoproteins, p78 is nonphosphorylated. Expression of the CP ORF in insect cells generated both p78 and p88 which assembled into virus-like particles. The finding that p78, p83, and p88 share a common N-terminal amino acid sequence is consistent with the determination that N-terminal, but not C-terminal, CP deletions were incompetent for assembly. Evidence was obtained that p78 is derived from p88 via proteolytic cleavage at the C-terminus. Proteolytic processing may play a regulatory role in the virus life cycle since it leads to dephosphorylation of CP and a subsequent decrease in virion transcriptional activity.

Promoter analysis of a cysteine-rich Campoletis sonorensis polydnavirus gene.

Promoter activity of the Campoletis sonorensis polydnavirus (CsPDV) WHv1.6 gene was analysed by transient transfection assays in insect cell culture using constructs expressing the CAT gene. Deletions of the WHv1.6 gene promoter were used to define promoter regions important for expression. Progressive deletion of the regions upstream of the TATA box reduced the promoter activity, whereas deletions eliminating the TATA box abolished promoter activity. Cis-activating elements were detected up to 1 kb upstream of the WHv1.6 transcription initiation site (TIS). Promoter elements increasing transcription were detected between -444 and -550 bp and between -831 and -1035 bp relative to the TIS. Analysis of the 3' flanking sequences of the WHv1.6 gene indicated that the polyadenylation signals were the only important elements affecting expression in the constructs. Comparison of promoter regions of four cysteine-rich CsPDV genes revealed homologous sequences that may be important for transcriptional regulation of polydnavirus gene expression in parasitized Heliothis virescens larvae.

Purification and analysis of a polydnavirus gene product expressed using a poly-histidine baculovirus vector.

The VHv1.1 polydnavirus gene has been implicated in suppressing the encapsulation response in parasitized insects [Li and Webb (1994) J. Virol. 68, 7482-7489]. In order to characterize this gene product and to further our analysis of its immunosuppressive function, we expressed the VHv1.1 using a custom-designed C-terminal poly-histidine baculovirus vector which allows for high expression and single-step purification of the protein. The 34 kDa VHv1.1 protein was expressed in baculovirus-infected cell cultures and in H. virescens larvae. Highly enriched preparations of the secreted VHv1.1 protein were obtained after affinity chromatography using a NTA-(Ni2+) resin. Characterization with purified preparations of the VHv1.1 protein established that the protein is N-glycosylated, containing glycogroups which are PNGase F-sensitive but Endo H-resistant. The recombinant VHv1.1 protein bound to hemocytes in vitro and in vivo and was endocytosed in a manner similar to the native protein produced in CsPDV-infected larvae.

Polydnavirus infection inhibits translation of specific growth-associated host proteins.

The wasp Campoletis sonorensis injects a polydnavirus (CsPDV) along with its egg during parasitization of Heliothis virescens larvae. CsPDV protects the wasp egg and larvae by selectively disabling the host's cellular immune response, and by altering host physiology, growth, and development. Among the changes in host physiology brought about by CsPDV infection is a rapid, and specific decline in the translation of fat body mRNAs encoding selected major plasma proteins. Translational inhibition of the synthesis of all storage protein monomers, p82 (Riboflavin binding hexamer), and p74/p76 (arylphorin), occurs upon infection with CsPDV. Moreover, the prewandering peak of the plasma enzyme juvenile hormone esterase (JHE) was blocked by CsPDV injection. Northern blotting of fat body mRNA demonstrated that transcript levels of storage proteins were not affected by infection. Plasma titers of the iron binding proteins transferrin (p72) and ferritin (p24/26), and of the plasma juvenile hormone binding protein (p25) were not changed by CsPDV infection. That storage protein and JHE synthesis are translationally suppressed, while the synthesis of other plasma proteins continues apace, suggests that CsPDV infection may lead to translational discrimination among available mRNAs in CsPDV infected fat bodies. The effect of this translational discrimination is to shunt host resources away from larval growth and adult development, which presumably makes them available to the developing endoparasitoid.