Cryo-EM structure reveals cylindrical nucleocapsids from two polydnaviruses.

Bracovirus is one of the two polydnavirus genera. Here, we used a cryo-EM analysis to reveal the near-native morphology of two nucleocapsid-containing model bracoviruses: Microplitis bicoloratus bracovirus (MbBV) and Microplitis mediator bracovirus (MmBV). MbBV and MmBV nucleocapsids have discernable cap structures in two distal regions with relatively high electron density. Adjacent to the end-cap structures are two electron-lucent rings. Some nucleocapsids were uniformly electron-dense and had a distinctive "helix-tail-like structure". Cryo-EM revealed inconsistent nucleocapsid diameters of 34-69.9 nm in MbBV and 46-69.9 nm in MmBV, and the largest observed cylindrical area length was expanded to 126 nm.

Ultrastructural and genomic characterization of a second banchine polydnavirus confirms the existence of shared features within this ichnovirus lineage.

Polydnaviruses (PDVs) are symbiotic viruses carried by endoparasitic wasps and transmitted to caterpillar hosts during parasitization. Although they share several features, including a segmented dsDNA genome, a unique life cycle where replication is restricted to the wasp host, and immunodepressive/developmental effects on the caterpillar host, PDVs carried by ichneumonid and braconid wasps (referred to as ichnoviruses and bracoviruses, respectively) have different evolutionary origins. In addition, ichnoviruses (IVs) form two distinct lineages, with viral entities found in wasps belonging to the subfamilies Campopleginae and Banchinae displaying strikingly different virion morphologies and genomic features. However, the current description for banchine IVs is based on the characterization of a single species, namely that of the Glypta fumiferanae IV (GfIV). Here we provide an ultrastructural and genomic analysis of a second banchine IV isolated from the wasp Apophua simplicipes, and we show that this virus shares many features with GfIV, including a multi-nucleocapsid virion, an aggregate genome size of ~300 kb, genome segments <5 kb, an impressively high degree of genome segmentation and a very similar gene content (same gene families in both viruses). Altogether, the data presented here confirm the existence of shared characteristics within this banchine IV lineage.

Polydnavirus hidden face: the genes producing virus particles of parasitic wasps.

Very few obligatory relationships involve viruses to the remarkable exception of polydnaviruses (PDVs) associated with tens of thousands species of parasitic wasps that develop within the body of lepidopteran larvae. PDV particles, injected along with parasite eggs into the host body, act by manipulating host immune defences, development and physiology, thereby enabling wasp larvae to survive in a potentially harmful environment. Particle production does not occur in infected tissues of parasitized caterpillars, but is restricted to specialized cells of the wasp ovaries. Moreover, the genome enclosed in the particles encodes almost no viral structural protein, but mostly factors used to manipulate the physiology of the parasitized host. We recently unravelled the viral nature of PDVs associated with braconid wasps by characterizing a large set of nudivirus genes residing permanently in the wasp chromosome(s). Many of these genes encode structural components of the bracovirus particles and their expression pattern correlates with particle production. They constitute a viral machinery comprising a large number of core genes shared by nudiviruses and baculoviruses. Thus bracoviruses do not appear to be nudiviruses remnants, but instead complex nudiviral devices carrying DNA for the delivery of virulence genes into lepidopteran hosts. This highlights the fact that viruses should no longer be exclusively considered obligatory parasites, and that in certain cases they are obligatory symbionts.

Microplitis demolitor bracovirus genome segments vary in abundance and are individually packaged in virions.

Polydnaviruses (PDVs) are distinguished by their unique association with parasitoid wasps and their segmented, double-stranded (ds) DNA genomes that are non-equimolar in abundance. Relatively little is actually known, however, about genome packaging or segment abundance of these viruses. Here, we conducted electron microscopy (EM) and real-time polymerase chain reaction (PCR) studies to characterize packaging and segment abundance of Microplitis demolitor bracovirus (MdBV). Like other PDVs, MdBV replicates in the ovaries of females where virions accumulate to form a suspension called calyx fluid. Wasps then inject a quantity of calyx fluid when ovipositing into hosts. The MdBV genome consists of 15 segments that range from 3.6 (segment A) to 34.3 kb (segment O). EM analysis indicated that MdBV virions contain a single nucleocapsid that encapsidates one circular DNA of variable size. We developed a semi-quantitative real-time PCR assay using SYBR Green I. This assay indicated that five (J, O, H, N and B) segments of the MdBV genome accounted for more than 60% of the viral DNAs in calyx fluid. Estimates of relative segment abundance using our real-time PCR assay were also very similar to DNA size distributions determined from micrographs. Analysis of parasitized Pseudoplusia includens larvae indicated that copy number of MdBV segments C, B and J varied between hosts but their relative abundance within a host was virtually identical to their abundance in calyx fluid. Among-tissue assays indicated that each viral segment was most abundant in hemocytes and least abundant in salivary glands. However, the relative abundance of each segment to one another was similar in all tissues. We also found no clear relationship between MdBV segment and transcript abundance in hemocytes and fat body.

Disruption effect of Microplitis bicoloratus polydnavirus EGF-like protein, MbCRP, on actin cytoskeleton in lepidopteran insect hemocytes.

Microplitis bicoloratus is a braconid endoparasitic wasp associated with the polydnavirus named Microplitis bicoloratus bracovirus (MbBV). Parasitism of Spodoptera litura larvae leads to an impaired cellular immune response and to the disappearance of the 42 kDa actin in host hemocytes. In this work, we investigated if the absence of actin in blood cells was related to MbBV infection. An MbBV gene similar to egf-like genes identified in another bracovirus was partially cloned and named Mbcrp1. The full-length gene, named Mbcrp, is transcribed throughout the course of parasitism in host hemocytes and the 30 kDa MbCRP protein was detected in hemocytes 6-7 d post-parasitization. The Mbcrp1 gene contains the cysteine-rich trypsin inhibitor-like (TIL) domain coding sequence and the expression of recombinant MbCRP1 inhibited the expression of the 42 kDa actin in Hi5 cells. The 34.1 kDa MbCRP1-green fluorescent protein fusion protein locate specifically in the cytoplasm. These results suggest that expression of MbCRP in lepidopteran insect cells is related to the disruption of the actin cytoskeleton.

Morphological and genomic characterization of the polydnavirus associated with the parasitoid wasp Glyptapanteles indiensis (Hymenoptera: Braconidae).

Glyptapanteles indiensis polydnavirus (GiPDV) is essential for successful parasitization of the larval stage of the lepidopteran Lymantria dispar (gypsy moth) by the endoparasitic wasp Glyptapanteles indiensis. This virus has not been characterized previously. Ultrastructural studies of GiPDV showed that virions had a rod-like or rectangular form and each contained as many as ten nucleocapsids enclosed by a single unit membrane envelope. Field inversion gel electrophoresis (FIGE) analysis of the virus genomic DNA revealed that GiPDV had a segmented genome composed of 13 dsDNA segments, ranging in size from approximately 11 kb to more than 30 kb. Four genomic segments were present in higher molar concentration than the others. Further characterization of the GiPDV genome yielded several cDNA clones which derived from GiPDV-specific mRNAs, and Northern blot analysis confirmed expression of isolated cDNA clones in the parasitized host. Each was present on more than one GiPDV genomic DNA segment, suggesting the existence of related sequences among DNA segments. It has been proposed previously that in polydnavirus systems, genome segmentation, hypermolar ratio segments and segment nesting may function to increase the copy number of essential genes and to increase the levels of gene expression in the absence of virus replication. The present data support this notion and suggest that GiPDV morphology and genomic organization may be intrinsically linked to the function and evolutionary strategies of the virus.

Origin and evolution of polydnaviruses by symbiogenesis of insect DNA viruses in endoparasitic wasps.

During oviposition, many endoparasitic wasps inject virus-like particles into their insect hosts that enable these parasitoids to evade or directly suppress their hosts' immune system, especially encapsulation by hemocytes. These particles are defined as virions that belong to viruses of the two genera that comprise the family Polydnaviridae, bracoviruses (genus Bracovirus) transmitted by braconid wasps, and ichnoviruses (genus Ichnovirus) transmitted by ichneumonid wasps. Structurally, bracovirus virions resemble nudivirus and baculovirus virions (family Baculoviridae), and ichnovirus virions resemble those of ascoviruses (family Ascoviridae). Whereas nudiviruses, baculoviruses and ascoviruses replicate their DNA and produce progeny virions, polydnavirus DNA is integrated into and replicated from the wasp genome, which also directs virion synthesis. The structural similarity of polydnavirus virions to those of viruses that attack the wasps' lepidopteran hosts, along with polydnavirus transmission and replication biology, suggest that these viruses evolved from insect DNA viruses by symbiogenesis, the same process by which mitochondia and chloroplasts evolved from bacteria. Molecular evidence supporting this hypothesis comes from similarities among structural proteins of ascoviruses and the Campoletis sonorensis ichnovirus. Implications of this hypothesis are that polydnaviruses evolved from viruses, but are no longer viruses, and that DNA packaged into polydnavirus virions is not viral genomic DNA per se, but rather wasp genomic DNA consisting primarily of wasp genes and non-coding DNA. Thus, we suggest that a better understanding of polydnaviruses would result by viewing these not as viruses, but rather as a wasp organelle system that evolved to shuttle wasp genes and proteins into hosts to evade and suppress their immune response.

Polydnavirus replication: the EP1 segment of the parasitoid wasp Cotesia congregata is amplified within a larger precursor molecule.

Polydnaviruses are unique viruses: they are essential for successful parasitism by tens of thousands of species of parasitoid wasps. These viruses are obligatorily associated with the wasps and are injected into the host during oviposition. Molecular analyses have shown that each virus sequence in the segmented polydnavirus genome is present in the wasp DNA in two forms: a circular form found in the virus particles and an integrated form found in the wasp chromosomes. Recent studies performed on polydnaviruses from braconid wasps suggested that the circular forms were excised from the chromosome. The different forms of the EP1 circle of Cotesia congregata polydnavirus during the pupal-adult development of the parasitoid wasp were analysed. Unexpectedly, an off-size fragment formerly used to diagnose the integration of the EP1 sequence into wasp genomic DNA was found to be amplified in female wasps undergoing virus replication. The EP1 sequence is amplified within a larger molecule comprising at least two virus segments. The amplified molecule is different from the EP1 chromosomally integrated form and is not encapsidated into virus particles. These findings shed light on a new step towards EP1 circle production: the amplification of virus sequences preceding individual circle excision.

Detailed characterization of polydnavirus immunoevasive proteins in an endoparasitoid wasp.

Polydnaviruses are a unique group of insect viruses in terms of their obligate and symbiotic associations with some parasitic wasps. The Cotesia kariyai polydnavirus (CkPDV) replicates only in ovarian calyx cells of C. kariyai female wasps and is injected into the wasp's host, the armyworm Pseudaletia separata, along with the eggs. A previous study indicated the possibility that one of the CkPDV surface proteins mediates immunoevasion by the wasp from the encapsulation reaction of the host insect's hemocytes. This protein was named immunoevasive protein (IEP). The present studies substantially confirmed the previous observation by showing that an anti-IEP IgG neutralizes immunoevasive activity on the wasp eggs. Further, we isolated the IEP homologue (IEP-2) cDNA and IEP (IEP-1) cDNA, sequenced them and found that both are cysteine-rich proteins, each containing epidermal growth factor (EGF)-like repeats. IEP genes were not found to reside in the CkPDV genome, but in the wasp chromosomal DNA. IEPs are synthesized in the female reproductive tract and their expression was detected from 4 days after pupation, 1 day later than expression of the virus capsid proteins. In situ hybridization and immunocytochemistry indicated that the lateral oviduct cells of the reproductive tracts produce IEP-1/IEP-2 mRNAs and secrete the proteins into the oviduct. These data suggest that the expression pattern and localization of IEPs are different from other components of CkPDV virions.

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.

Ichnovirus infection of an established gypsy moth cell line.

In the present study, a lepidopteran cell line (Ld-652Y, from the gypsy moth, Lymantria dispar) exposed to Hyposoter fugitivus polydnavirus (HfPV) was found to display a variety of cytopathic effects. These included a transient inhibition of cell proliferation, rounding up, aggregation and apoptosis. In addition, unusual paracrystalline structures appeared within the lumen of the rough endoplasmic reticulum; similar structures were observed in the spherulocytes of parasitized Malacosoma disstria. Following Coomassie Blue staining, two new cell-associated polypeptides were detected; one of these, an 8 kDa polypeptide, could also be observed following exposure of LD-652Y cells to media taken from infected cultures or to cell-free haemolymph from parasitized M. disstria. After a period of 2-4 weeks, the L. dispar cell cultures were observed to largely recover from the effects of exposure to virus, and resumed proliferation; "transformed' cell populations tended to form aggregates, and adhered less tightly to the substrate. Viral DNA was stably maintained in all recovered cell lines, possibly in chromosomally integrated form.

Polydnavirus of the parasitic wasp Chelonus inanitus (Braconidae): characterization, genome organization and time point of replication.

Ultrastructural analysis of the polydnavirus of the braconid wasp Chelonus inanitus revealed that virions consist of one cylindrical nucleocapsid enveloped by a single unit membrane. Nucleocapsids have a constant diameter of 33.7 +/- 1.4 nm and a variable length of between 8 and 46 nm. Spreading of viral DNA showed that the genome consists of circular dsDNA molecules of variable sizes and measurement of the contour lengths indicated sizes of between 7 and 31 kbp. When virions were exposed to osmotic shock conditions to release the DNA, only one circular molecule was released per particle suggesting that the various DNA molecules are singly encapsidated in this bracovirus. The viral genome was seen to consist of at least 10 different segments and the aggregate genome size is in the order of 200 kbp. By partial digestion of viral DNA with HindIII or EcoRI in the presence of ethidium bromide and subsequent ligation with HindIII-cut pSP65 or EcoRI-cut pSP64 and transfection into Escherichia coli, libraries of 103 HindIII and 23 EcoRI clones were obtained. Southern blots revealed that complete and unrearranged segments were cloned with this approach, and restriction maps for five segments were obtained. Part of a 16.8 kbp segment was sequenced, found to be AT-rich (73%) and to contain six copies of a 17 bp repeated sequence. The development of the female reproductive tract in the course of pupal-adult development of the wasp was investigated and seen to be strictly correlated with the pigmentation pattern. By the use of a semiquantitative PCR, replication of viral DNA was observed to initiate at a specific stage of pupal-adult development.

Microplitis demolitor polydnavirus infects and expresses in specific morphotypes of Pseudoplusia includens haemocytes.

Microplitis demolitor is a polydnavirus-carrying wasp that parasitizes the larval stage of Pseudoplusia includens. M. demolitor eggs are never encapsulated by host haemocytes when coinfected with its associated polyndnavirus (MdPDV) whereas eggs are encapsulated within 36 h when injected into hosts without virus. In this study, infection of specific classes of P. includens haemocytes by MdPDV was examined. Electron microscopic studies indicated that MdPDV entered all haemocyte morphotypes. Northern blot analysis revealed that similar size classes of viral mRNAs were produced in granular cells, plasmatocytes and spherule cells. Expression of a 1.6 kb MdPDV mRNA in haemocytes from parasitized hosts was detectable by in situ hybridization at 2 h post-parasitism (p.p.) and continued through until day 6 p.p. By 12 h p.p., viral expression was detected in greater than 80% of the haemocytes in circulation but thereafter the percentage of haemocytes exhibiting a hybridization signal declined. Similar patterns were observed in haemocytes from larvae injected with calyx fluid or MdPDV plus venom. Granular cells and plasmatocytes from unparasitized larvae were purified on Percoll cushions and maintained in vitro. Both morphotypes were successfully infected with MdPDV and exhibited changes in morphology and adhesiveness very similar to cells from parasitized hosts. Cell-free plasma from parasitized larvae had a variable effect on haemocyte adhesion. Haemocytes cultured in plasma from 1 or 4 day p.p. larvae rapidly spread whereas cells cultured in 7 day p.p. plasma did not. Reciprocally, adhesion of haemocytes from parasitized larvae could not be rescued by cell-free plasma from unparasitized larvae. Together, these data suggest that disruption of the host encapsulation response is medicated primarily by direct infection of granular cells and plasmatocytes by MdPDV.