Polydnaviridae - a proposed family of insect viruses with segmented, double-stranded, circular DNA genomes.
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Two related viral genes are located on a single superhelical DNA segment of the multipartite Campoletis sonorensis virus genome.
Campoletis sonorensis virus (CsV) (Polydnaviridae) is a large eucaryotic DNA virus with a structurally complex genome consisting of 28 or more superhelical (SH) DNA segments. Little is known of the relationship between different SH DNAs, but some SH DNAs do cross hybridize, indicating a relatedness between certain SH DNAs. In a previous study of viral expression in parasitized Heliothis virescens larvae, several partially homologous viral mRNAs were also identified (G. W. Blissard, S. B. Vinson, and M. D. Summers, 1986, J. Virol. 57, 318-327). To study the organization of the viral genome and the relationship between two partially homologous viral mRNAs, we analyzed cDNA clones and a cloned SH DNA segment of the CsV genome. Of the two CsV mRNAs examined (1.6 and 1.0 kb), both mRNAs were abundant at 48 hr after parasitization and the 1.6-kb mRNA was detected as early as 2 hr after parasitization. Nucleotide sequence analyses of cDNA clones representing the two partially homologous CsV mRNAs (1.6 and 1.0 kb) show that the two CsV mRNAs share five regions of imperfect homology (68 to 88%) which include a large part of each mRNA. These data indicate that the two mRNAs are transcribed from two separate but closely related CsV genes. Comparison of predicted amino acid sequences shows that the two related viral genes encode proteins with divergent amino acid sequences. Northern and Southern hybridization analyses using cloned cDNAs as probes showed that one CsV mRNA (1.6 kb) is homologous to CsV SH DNAs W, R, and M, while the other mRNA (1.0 kb) shows strong homology only to SH DNA W. By cloning and Southern hybridization mapping of the 15.8-kbp SH DNA W, we demonstrate that the genes for the related 1.6- and 1.0-kb mRNAs are located on different regions of this single 15.8-kbp SH DNA. Alignment of nucleotide sequences from a cloned viral genomic DNA and a cDNA demonstrates that the CsV gene encoding the 1.6-kb mRNA is a spliced gene containing at least two introns. Conservation of splice junctions between the two mRNAs suggests that the 1.0-kb mRNA is also spliced. These data represent the detailed analysis of two closely related CsV mRNAs abundantly expressed in parasitized H. virescens larvae, the first observation of related viral genes in a eucaryotic DNA virus, the demonstration of splicing in the Polydnaviridae, and the cloning and mapping of one of the largest SH DNA segments of the CsV genome.
Base Sequence↗
Segment W of Campoletis sonorensis virus: expression, gene products, and organization.
Campoletis sonorensis virus (CsV, Polydnaviridae) is a segmented double-stranded DNA virus which has an apparently symbiotic relationship with the parasitic wasp, Campoletis sonorensis. CsV replicates in the oviducts of the parasitic wasp and is injected into the wasp's host, Heliothis virescens (Lepidoptera; Noctuiidae), during oviposition. In the parasitized lepidopteran host, the virus has a dramatic effect on host physiology and viral gene products are believed to play an essential role in the survival of the parasitic wasp's egg and larva. In the current study, we used Northern blot analyses to examine expression from segment W in the parasitized host and in the parasitic wasp. Segment W hybridized primarily to two relatively abundant mRNAs (1.6 and 1.0 kb) from the parasitized host. These 1.6- and 1.0-kb mRNAs, which were previously shown to be transcribed from two closely related genes (WHv1 and WHv2) on segment W (G. W. Blissard, O. P. Smith, and M. D. Summers, 1987, Virology 160, 120-134) increased in relative abundance between 2 and 24 hr postparasitization (pp) and were detected throughout parasitization (8 days). To study the proteins encoded by these closely related genes, the open reading frame from each of the related genes was cloned into a baculovirus expression vector. By pulse labeling in the presence and absence of tunicamycin, we examined secretion and glycosylation of these CsV proteins in infected lepidopteran cells (Spodoptera frugiperda). Expression of segment W in the oviducts of the female wasp was also examined. Segment W hybridized to at least five CsV mRNAs on Northern blots of poly(A) mRNA from C. sonorensis oviducts. To identify specific CsV mRNAs and map putative viral genes expressed in wasp oviduct tissues, segment W was used to screen a cDNA library of C. sonorensis oviduct mRNAs. Three cDNAs were used to identify CsV mRNAs by Northern blot analyses and to map the locations of three putative CsV genes on segment W. Cross-hybridization within the CsV genome was examined with cloned segment W and with the three cloned cDNAs.
Amino Acid Sequence↗
Identification and comparison of Campoletis sonorensis virus transcripts expressed from four genomic segments in the insect hosts Campoletis sonorensis and Heliothis virescens.
The Campoletis sonorensis virus (CsV; Polydnaviridae) genome consists of at least 28 closed circular superhelical (SH) DNAs. In this study we used complete clones of four SH DNAs to analyze viral transcription both in the adult parasitic wasp host Campoletis sonorensis (Ichneumonidae) and in the lepidopteran host, Heliothis virescens (Noctuidae). CsV genes are expressed in parasitized H. virescens, but no viral transcripts had been characterized from C. sonorensis until this study. The clones of the SH DNAs B, H, M, and O1 were used to probe Northern blots of poly(A)+ RNA isolated from C. sonorensis reproductive tissue and from parasitized H. virescens larvae. All four SH DNAs hybridized to viral transcripts. SH-H,-M, and -O1 hybridized to messages expressed in both hosts. SH-B and -M hybridized to transcripts that were detected only in either C. sonorensis reproductive tissue or parasitized H. virescens larvae. These results suggest that some CsV genes are expressed in a host-specific manner. In a previous study we identified a family of imperfectly conserved tandemly repeated 540-bp repeat elements on SH-B,-H and -O1 (D. A. Theilmann and M. D. Summers, 1987 J. Virol. 61; 2589-2598). Hybridization of the 540-bp repeat regions to Northern blots showed that they were all homologous to viral transcripts. A cDNA clone of a mRNA that is transcribed from the 540-bp repeat region of SH-B was isolated from a lambda gt 10 library and completely sequenced. The sequence data revealed that the 540-bp repeat element was contained within the open reading frame of this gene. These results indicate that transcribed sequences homologous to the 540-bp repeat elements represent a second gene family to be identified within the CsV genome.
Amino Acid Sequence↗
Expression and hemocyte-targeting of a Campoletis sonorensis polydnavirus cysteine-rich gene in Heliothis virescens larvae.
The polydnavirus associated with the parasitic wasp Campoletis sonorensis is injected into the lepidopteran insect, Heliothis virescens, during parasitization, after which viral gene products suppress the cellular immune system of the hosts. Four related cysteine-rich polydnavirus gene have been identified in parasitized H. virescens larvae and grouped into a family. In this study, we investigated the expression and hemocyte targeting of the cysteine-rich VHv1.4 protein. Full-length and truncated VHv1.4 proteins were produced in a bacterial expression system, and the purified proteins were used to raise polyclonal antisera. In immunoblots the VHv1.4 protein was detected in parasitized insects as early as 6 h and throughout the entire course of parasitism. The VHv1.4 protein appeared predominantly in the plasma fraction of hemolymph from parasitized larvae, suggesting that this protein is secreted. The VHv1.4 protein expressed from a recombinant baculovirus was secreted in two lepidopteran cell lines and in larvae injected with the recombinant virus. Digestion with endoglycosidases suggests that the VHv1.4 protein is glycosylated at multiple N-glycosylation sites. Immunofluorescence assays showed that the VHv1.4 protein binds to the hemocytes, most notably the granulocytes, in H. virescens larvae. After binding, the VHv1.4 protein was internalized, probably by endocytosis. Specific binding of the VHv1.4 to granulocytes implies an important function in the suppression of host cellular encapsulation response.
Animals↗
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.
Amino Acid Sequence↗
Plasmatocyte spreading peptide does not induce Microplitis demolitor polydnavirus-infected plasmatocytes to spread on foreign surfaces.
Capsule formation by the moth Pseudopulsia includens requires that plasmatocytes change from being nonadhesive cells in circulation to strongly adhesive cells capable of attaching to the foreign target and one another. This change in adhesive state is induced by Plasmatocyte Spreading Peptide (PSP1); a 23 amino acid peptide isolated from P. includens plasma. Plasmatocytes from hosts parasitized by Microplitis demolitor remain in a nonadhesive state after infection by Microplitis demolitor polydnavirus (MdPDV). This alteration in plasmatocyte function prevents P. includens from encapsulating the developing parasitoid. In the current study, we examined whether MdPDV infection eliminates PSP1-responsive plasmatocytes from circulation or disrupts the ability of PSP1 to induce adhesion and spreading of plasmatocytes to foreign surfaces. In vivo experiments revealed that infection of P. includens by MdPDV induced an increase in the total number of hemocytes in circulation but reduced the proportion of hemocytes in circulation that were plasmatocytes. However, plasmatocytes normally capable of responding to PSP1 were not eliminated from circulation. Both in vivo and in vitro experiments indicated that plasmatocytes inoculated with MdPDV lost the capacity to respond to PSP1 4-6 h post-infection. Infection of P. includens with MdPDV reduced expression levels of prepro-PSP1 mRNA in hemocytes but did not appear to alter expression levels in fat body.
Animals↗
Isomeric and quaternary properties of homogenous 33 kDa protein from the venom of Chelonus near curvimaculatus.
The 33,000 Dalton venom protein of Chelonus near curvimaculatus was characterized for structural properties of charge, quaternary associations, and relationship to polydnavirus encoded proteins. Homogenous isoforms of the protein were isolated from the venom by sequential steps of 1) microdissection, 2) separation based on charge (Mono-Q column HPLC or narrow-range electrofocusing), and 3) centrifugal filtration based on molecular weight using Centricon microconcentrators. The purified protein dimerized under native conditions, and this quaternary association became denaturation resistant under certain conditions. Chemical modification of lysine epsilon amino groups did not disrupt such dimerization. The cDNA for the protein did not possess high similarity to any sequence encoded in the polydnavirus, as indicated by results of Southern blotting, but does possess similarity in its repeats to the repeats of the immunologically protective surface glycoprotein of Leishmania amazonensis.
Amino Acid Sequence↗
Transformation of gypsy moth (Lymantria dispar) cell lines by infection with Glyptapanteles indiensis polydnavirus.
Glyptapanteles indiensis, a species of braconid parasitic wasp, infects its host Lymantria dispar (gypsy moth) with a polydnavirus (GiPDV) to suppress the host immune system during parasitization. Here it is shown that GiPDV can infect L. dispar cell lines and that a portion of the GiPDV genome is stably maintained in infected cells. Results of Southern hybridization analyses suggested that this portion of the GiPDV genome is integrated into the L. dispar cellular genome. This is the first report of an insect viral DNA molecule that can apparently integrate into lepidopteran insect cells.
Animals↗
Functional significance of parasitism-induced suppression of juvenile hormone esterase activity in developmentally delayed Choristoneura fumiferana larvae.
The parasitic wasp Tranosema rostrale transmits a polydnavirus (PDV) to its host, Choristoneura fumiferana, during oviposition. Last-instar C. fumiferana larvae parasitized by T. rostrale early in the stadium fail to undergo metamorphosis, and injection of the wasp's calyx fluid (CxF; contains PDV) into healthy caterpillars induces a dose-dependent delay in initiation of metamorphosis (D. Doucet and M. Cusson, 1996, Entomol. Exp. Appl. 81, 21-30). In the present work, parasitization and injection of CxF (0.5 female equivalent) on the first day of the last stadium both prevented the rise in hemolymph 20-hydroxyecdysone (20HE) titer observed between day 4 and day 7 in control and saline-injected larvae. Similarly, juvenile hormone esterase (JHE) activity was depressed following parasitization or CxF injection, whereas control larvae displayed a peak on day 4. However, neither parasitism nor injection of CxF on day 1 prevented the JH-producing glands from turning off during the first half of the last stadium. Likewise, low but clearly detectable JH titers were observed in the first hours following the molt but very low titers, at or near the detection limit of our radioimmunoassay, were seen in both control and parasitized larvae on day 4. Prothoracic glands showed no apparent sign of degeneration 4 days after injection of CxF but had significantly smaller cells than saline-injected larvae 7 days postinjection. It is not clear whether this was a direct effect of T. rostrale PDV. Thus, disruption of spruce budworm metamorphosis by T. rostrale CxF involves depression of 20HE titers but is not associated with a measurable increase in the level of JH, as shown for some other host-parasitoid systems. In view of the latter observation, we put forward three hypotheses regarding the functional significance of the observed suppression of JHE activity in developmentally arrested C. fumiferana larvae.
Animals↗
A polydnavirus from the spruce budworm parasitoid, Tranosema rostrale (Ichneumonidae).
The calyx epithelium of the campoplegine wasp, Tranosema rostrale, contains typical ichneumonid polydnaviruses (PVs) that display an apparently uncommon association with the egg chorion. The latter structure features fine hair-like projections, longest around the egg's apices. In the lumen of the ovary, T. rostrale virus becomes lodged between these projections and forms a particulate coat around the egg. In the host, Choristoneura fumiferana, projections and associated virions are observed in close contact with basement membranes of fat body and muscle tissues, to which the eggs rapidly become attached following introduction into the host hemocoel. We discuss the implications of this unusual virus-chorion association in terms of immune protection, delivery of virus to specific host tissues, and the evolution of PVs.
Animals↗
Effects of Glyptapanteles liparidis (Hym.: Braconidae) parasitism, polydnavirus, and venom on development of microsporidia-infected and uninfected Lymantria dispar (Lep.: Lymantriidae) larvae.
Effects of parasitism, polydnavirus, and venom of the endoparasitoid Glyptapanteles liparidis on Lymantria dispar larvae infected with the microsporidium Vairimorpha sp. and uninfected hosts were studied. We tested the impact on growth and development of hosts, as well as on microsporidian infection. Both parasitism and polydnavirus/venom treatment alone caused a slight increase in growth rate and relative growth rate in uninfected fourth instar hosts. This effect was more pronounced with the addition of Vairimorpha infection. With no parasitism, however, infection reduced host growth markedly. Microsporidiosis delayed larval molts of L. dispar, and additional polydnavirus/venom treatment or parasitization induced significantly earlier molting. Polydnavirus/venom treatment of uninfected L. dispar resulted in prolonged larval development due to supernumerary molts and in higher pupal mortality. Infected larvae treated with polydnavirus/venom died earlier than infected larvae that were not treated and produced more Vairimorpha spores per unit fresh mass of the host.
Animals↗
An abundantly expressed hemolymph glycoprotein isolated from newly parasitized Manduca sexta larvae is a polydnavirus gene product.
Cotesia congregata polydnavirus (CcPDV) is essential for successful parasitism of Manduca sexta larvae by the braconid wasp C. congregata. CcPDV virions are present in large numbers in the oviducts of C. congregata and injected with eggs into the hemocoel of M. sexta larvae during parasitization. Injection of sucrose density purified virions into nonparasitized larvae causes several of the parasitism-induced alterations in the physiology of host larvae that occur as a result of natural parasitism, including the synthesis of novel hemolymph proteins and abrogation of the host's immune response against the developing parasites. One of these proteins, early-expressed protein 1 (EP1), is a 190-kDa molecule which constitutes up to 5% of the total hemolymph protein by 24 hr following oviposition by the wasp. Using N-terminal sequence data for EP1 to construct primers for use in the polymerase chain reaction, we amplified and cloned a cDNA corresponding to the gene encoding EP1. This cDNA hybridized to DNA of the CcPDV genome, but not to DNA isolated from M. sexta larvae, suggesting that EP1 is a CcPDV gene product. A cDNA clone was isolated from an expression library generated from RNA extracted from newly parasitized M. sexta larvae. Sequence analysis of the cDNA clone revealed the presence of an open reading frame of 819 bp encoding a protein of 30.7 kDa. In vitro transcription/translation of the cDNA clone produced a protein of approximately 31 kDa, which was immunoprecipitated by EP1-specific polyclonal antiserum generated against purified deglycosylated EP1. EP1-like sequences also were amplified from male wasp genomic DNA, suggestive of integration of EP1-like sequences in the genome. This report constitutes the first evidence that a specific protein isolated from a parasitized host insect is a wasp polydnavirus gene product.
Amino Acid Sequence↗
Related RNAs in lepidopteran cells after in vitro infection with Hyposoter didymator virus define a new polydnavirus gene family.
In the present study, we describe the isolation and the characterization of three different Hyposoter didymator virus (HdV) lepidopteran host-expressed genes, the products of which might interfere with the host physiology during parasitism. In this report, we study the expression of HdV genes in Sf9 cells infected with HdV since results indicate that the Sf9 model mimics to some extent the in vivo model and may be utilized to study expression of HdV genes in lepidopteran host cells. This system allowed us to isolate three HdV-specific cDNAs, termed M24, M27, and M40. cDNA nucleotide sequence analysis demonstrated significant regions of homology. The three cDNAs displayed repeated sequences arranged in tandem array that might have evolved through domain duplication. Similar to other previously described polydnavirus host-expressed genes, two intron positions have been found in the M24 leader region. The cDNAs corresponded to RNAs of 1.5, 1.6, and 2.3 kb that are also detected in parasitized Spodoptera littoralis larvae. They are encoded by different genes likely located on different HdV DNA molecules. Corresponding RNAs are detected early postinfection and remain detectable for at least 10 days postinfection. They encode secreted glycine- and proline-rich proteins. An antiserum raised against a baculovirus recombinant M24-encoded protein detected similar proteins in the culture medium of infected lepidopteran cells and in parasitized host hemolymph. We propose that the three cloned genes belong to an HdV gene family specifically expressed in parasitized lepidopteran hosts.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
Co-replication of a reovirus and a polydnavirus in the ichneumonid parasitoid Hyposoter exiguae.
A recently established colony of the ichneumonid parasitoid, Hyposoter exiguae, was found to carry both a reovirus (HeRV) and a polydnavirus (HePDV). Morphogenesis of these viruses was observed in all cells comprising the ovarian calyx epithelium, apparently without detrimental effect to the parasitoid. While polydnavirus replication in H. exiguae was restricted to the calyx region, HeRV was detected in ovarioles, oviducts, midguts, malpighian tubules, and accessory glands associated with the male reproductive system. In addition, HeRV was able to infect the fat body of parasitized host larvae and to establish a persistent infection in vitro. Electron microscopy revealed that both viruses were released into the calyx fluid compartment exclusively by budding, a phenomenon rarely observed among the Reoviridae; HeRV envelopes thus obtained, however, appeared to be subsequently shed within the oviducts. HeRV particles were concentrated to near homogeneity by differential centrifugation; mature virions consisted of seven to eight structural polypeptides and 10 dsRNA genome segments. Prominent spikes were observed at the vertices of icosahedral core particles. Most, but not all, individuals comprising the H. exiguae colony appeared to be infected with HeRV, suggesting a commensal relationship between wasp and virus; however, while this association is of obvious benefit to the virus, it seems unlikely that any advantage accrues to the parasitoid which carries it.
Animals↗
Promoter studies of a polydnavirus gene from Cotesia rubecula (Hym: Braconidae).
The Cotesia rubecula polydnavirus gene, CrV1, is expressed in a highly transient fashion. Within four hours after egg deposition and virus infection, tissues of the host caterpillar, Pieris rapae, express high levels of the transcript. Twelve hours after infection no transcripts are visible. We have previously shown that the CrV1 secreted protein is mainly produced in host haemocytes. In haemocytes, immune functions such as phagocytosis and cell spreading are abolished by destabilization of the cell cytoskeleton. To test whether the observed down-regulation of CrV1 transcripts is mediated by transcriptional control or by other factors, such as the disruption of cytoskeleton in CrV1-inactivated cells, we cloned the promoter and the 3' untranslated region of the CrV1 gene to study CrV1 expression. The promoter region of the CrV1 gene was cloned into baculovirus expression systems along with the CAT reporter gene. Molecular analyses showed that the CAT gene under the control of CrV1 promoter is expressed as early as 2 h post infection and continues until late phase of infection suggesting that down-regulation of CrV1 expression in host haemocytes is perhaps mediated by post-transcriptional mechanisms.
Animals↗
Transformation of lepidopteran and coleopteran insect cell lines by Glyptapanteles indiensis polydnavirus DNA.
Recently investigators showed that polydnavirus DNA from the parasitic wasp Glyptapanteles indiensis could transform gypsy moth L. dispar cell lines in vitro (McKelvey et al., 1996). Here we show GiPDV DNA is capable of transforming in vitro to varying degrees lepidopteran (IPLB-TN-R2, IPLB-SF-21, IAL-PID2, IPLB-HvT1) and coleopteran (IPLB-DU182E) insect cell lines derived from various somatic tissue types. An insect cell line derived from dipteran Aedes albopictus (C7/10) could not be transformed with G. indiensis polydnavirus.
Aedes↗