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Polydnaviruses of Braconid Wasps Derive from an Ancestral Nudivirus

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Science  13 Feb 2009:
Vol. 323, Issue 5916, pp. 926-930
DOI: 10.1126/science.1166788

Abstract

Many species of parasitoid wasps inject polydnavirus particles in order to manipulate host defenses and development. Because the DNA packaged in these particles encodes almost no viral structural proteins, their relation to viruses has been debated. Characterization of complementary DNAs derived from braconid wasp ovaries identified genes encoding subunits of a viral RNA polymerase and structural components of polydnavirus particles related most closely to those of nudiviruses—a sister group of baculoviruses. The conservation of this viral machinery in different braconid wasp lineages sharing polydnaviruses suggests that parasitoid wasps incorporated a nudivirus-related genome into their own genetic material. We found that the nudiviral genes themselves are no longer packaged but are actively transcribed and produce particles used to deliver genes essential for successful parasitism in lepidopteran hosts.

Comparative genomic studies have highlighted the role of symbiotic associations in evolution (1). Polydnaviruses (PDVs) are virus-like particles associated with wasp species that parasitize lepidopteran larvae. PDV particles are injected along with the eggs of the wasp into the lepidopteran larvae (or eggs) and express proteins that interfere with host immune defenses, development, and physiology; this interference enables wasp larvae to survive and develop within the host (2). Viral particle production occurs exclusively in a specialized region of the wasp ovaries (the calyx), and the vertically transmitted virus does not initiate particle production in the infected host tissues (3). The viral genome packaged in the particles is composed of multiple double-stranded DNA (dsDNA) circles, and it is surprising that it encodes almost no viral structural proteins, although it harbors immunosuppressive genes that are expressed in the host and are essential for successful parasitism (4, 5) (see PDV description at www.ictvonline.org). Because of this lack of genes coding for structural proteins, it has been debated whether PDVs are of viral origin or a “genetic secretion” of the wasp (6, 7).

PDVs are classified as either bracoviruses or ichnoviruses, when associated with braconid or ichneumonid wasps, respectively. Detailed phylogenetic studies have shown that the bracovirus-associated wasps form a monophyletic group known as the microgastroid complex (8), and it has been hypothesized that there has been a single integration event of a viral genome, as a provirus, in the microgastroid lineage. This predicts that vertically transmitted viral DNA may have been maintained because of its contribution to successful parasitism and that PDVs have contributed to the diversification of the microgastroid complex of at least 17,500 species (8).

The sequence of the DNA packaged in Cotesia congregata bracovirus (CcBV) comprises 560 kb, organized in 30 circles of dsDNA (4), and encodes several products functionally resembling virulence factors used by parasites or bacterial pathogens of vertebrates (911). However, the origin of the protein components of PDV particles remains unknown. The few CcBV sequences with significant similarity to viral genes correspond mostly to remnants of mobile elements inserted randomly into the chromosomal form of CcBV and are thus not informative about the origin of bracoviruses (4). A viral genome without genes involved in particle production seems paradoxical. However, it may be that the genes involved in particle morphogenesis have lost the ability to be incorporated into the particles injected into the host, because the particles are exclusively produced in wasp ovaries and PDVs are only transmitted in their integrated chromosomal form. In accordance with this theory, a gene encoding an ichnovirus structural protein was identified in the wasp genome (12).

Regardless of where the genes are located, structural proteins of the particles are expressed in virus-producing cells. We therefore searched for virus-related genes expressed in wasp pupal ovaries at times when virus particle production is highest. We studied the braconid wasps Chelonus inanitus (Cheloninae) and Cotesia congregata (Microgastrinae) belonging to different bracovirus-associated subfamilies (13). We also sequenced cDNAs from the wasp Hyposoter didymator, associated with an ichnovirus but belonging to the same superfamily (Ichneumonoidea). We sequenced 5000 expressed sequence tags (ESTs) from the ovaries of each species and identified a set of nudivirus-related genes expressed by the braconid wasp ovaries (Table 1, A and B). The predicted products of these genes are related to 22 nudivirus genes (e values from 1e–65 to 1.1, see Table 1), 13 of which are conserved between nudiviruses and baculoviruses. In ichneumonid wasp ovaries, we detected mRNAs coding for characterized ichnovirus proteins but no genes related to known viruses. This suggests that the viral machinery of ichnovirus production may differ from that of bracoviruses. The recent discovery of a possible new lineage of insect viruses, represented by the Glossina pallidipes salivary gland hypertrophy virus (14), illustrates that the diversity of insect viruses is not completely known. This could explain our inability to identify viral genes in ichneumonid sequences. Few nudivirus genomes have been sequenced, and little is known about the function of their proteins, except by inference from studies on baculoviruses. Nudiviruses share half of the core set of essential genes conserved among baculoviruses (15, 16). The nudivirus-related gene products we identified in the braconid ovaries have been associated with different functions in baculoviruses (1720)(Table 1) including subunits of the viral RNA polymerase (LEF-4, LEF-8, and p47), proteins involved in particle assembly and packaging [38K (Ac98), VLF-1, and VP91], and envelope proteins of particles released after the death of infected insects (p74, PIF-1, PIF-2, and ODV-E56). In addition, we characterized several genes coding for variants of ODV-E66, a conserved envelope protein encoded by lepidopteran baculoviruses and a nudivirus (Table 1). We found no viral genes involved in DNA replication, which suggests that these mRNAs are rare or that host genes are involved.

Table 1.

Nudivirus-related genes expressed in braconid wasp ovaries and the presence of their products in bracovirus particles. Genes characterized in both Cotesia congregata (A) and Chelonus inanitus (B) are shaded in gray. Gene products identified as components of purified CcBV and CiBV particles by proteomic analyses are shown in red. HzNV-1, Heliothis zea nudivirus-1; GbNV, Gryllus bimaculatus nudivirus; OrNV, Oryctes rhinoceros nudivirus; PmV, Penaeus monodon virus (shrimp nudivirus); AdorNPV, Adoxophyes orana nucleopolyhedrovirus. Dash indicates a transcript not found among the cDNAs sequenced. “No hit” indicates a protein having no blast hit but similar to a nudivirus-related protein of the other species. See table S1 for more details.

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The nudivirus Heliothis zea nudivirus-1 (HzNV-1) was originally identified as a persistent viral infection in a cell line from adult ovarian tissue of the moth Heliothis zea (21) and is closely related to HzNV-2, a sexually transmitted virus replicating in insect reproductive tissues (22). The genomes of HzNV-1; of Gryllus bimaculatus nudivirus (GbNV), a virus infecting the fat body of crickets; and of Oryctes rhinoceros nudivirus (OrNV), a biological control agent used to protect palm trees against the rhinoceros beetle have been sequenced (15, 23, 24). Partial data are also available for a shrimp nudivirus, designated here as PmV (GenBank accession number: 160432003). In braconid wasp ovaries, we identified eight ESTs similar to nudivirus-specific genes; two are shared between several nudiviruses (HzNVorf64-like and HzNVorf144-like); five have been found only in HzNV-1 (HzNVorf9-like, HzNVorf89-like, HzNVorf106-like, HzNVorf128-like, and HzNVorf140-like); and one is specific to the shrimp nudivirus (PmV-like). All braconid viral products have less than 60% similarity with nudiviral proteins (Table 1, A and B, e values 6e–20 to 1.1), which is not surprising, considering that the ancestral nudivirus is hypothesized to have integrated into the wasp genome 100 million years ago (25).

The prevalence of nudiviruses in hymenoptera is unknown, but the fact that nudivirus transcripts were not detected in the ichneumonid species indicates that parasitic wasps are not universally infected. The isolation of the nudivirus-related p74 genes from eight Cotesia species using wasps collected in different parts of the world (fig. S1) indicated that this gene is stably associated with these wasps and does not belong to a pathogenic virus present in our laboratory strain. We could also show by sequencing Cotesia congregata large genomic DNA regions that at least 10 nudivirus-related genes are chromosomally integrated. Five genes were found to be clustered (38K, HzNVorf9-like1, HzNVorf89-like, pif-3, and PmV-like); others were dispersed (HzNVorf128-like, lef-8, odv-e56-1, p74, and HzNVorf140-like) and flanked by wasp genomic DNA (Fig. 1). Quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) showed that the expression of nudivirus-related genes increases in female wasp pupae (Fig. 2), coinciding with the onset of bracovirus replication (26). Moreover, expression in the ovaries is confined to the calyx region where particles are produced (Fig. 2).

Fig. 1.

Organization of nudivirus-related genes in the wasp genome (Cotesia congregata). Five genes (38K, HzNVorf9-like1, HzNVorf89-like, pif-3, and PmV-like) are organized as a cluster that is likely to constitute a remnant of the virus integrated into the ancestral wasp genome. Two genes (HzNVorf128-like and lef-8) are located within the same 60-kb chromosomal region; other genes (odv-e56-1, p74, and HzNVorf140-like) are dispersed and located in regions containing Cotesia congregata putative homologs of insect genes (in blue) and/or remnants of mobile elements (in gray). For nudivirus-related genes, the presence of sequences found in promoters of baculovirus genes transcribed by the cellular RNA polymerase (E) or baculovirus RNA polymerase (L) is indicated (19). E indicates the presence of a TATAA sequence with a CA(T/G)T or a CGTGC transcription start site 20 to 40 nucleotides downstream. L indicates the presence of a (A/T/G)TAAG motif within 300 nucleotides upstream of the translation start codon. CDS, coding sequence; ORF, open reading frame. Acyrthosiphon pisum (pea aphid), Drosophila ananassae (fruit fly), and Nasonia vitripennis (parasitoid wasp).

Fig. 2.

Nudivirus-related gene expression correlates temporally with bracovirus particle production and occurs in the same tissue. (A) Real-time RT-PCR indicates that three nudivirus-related genes(HzNVorf89-like coding for a structural component of CcBV particles, p74, and odv-e56-1) are induced in female pupae from day 4, coincident with the initiation of particle production as detected by transmission electron microscopy (TEM) and PCR (26). The induction of p47, coding for one of the viral RNA polymerase subunits, occurs earlier, which would be expected if the viral RNA polymerase controls the expression of some nudivirus-related genes. Although baculovirus regulatory sequences are present in some promoters (see Fig. 1), they appear to be too short and, therefore, would lack the specificity necessary to selectively express chromosomally integrated genes. The four nudivirus-related genes are specifically expressed in the calyx region of the ovaries where bracovirus particles are produced, but not in the oviducts (B) nor in males (A). Asterisk indicates expression too low to be indicated. On wasp ovaries: o, oviduct; c, calyx.

To confirm that the nudivirus-related genes produce bracoviruses, we analyzed proteins from purified particles by tandem mass spectrometry (MS/MS). We identified the products of six genes as components of the particles in both CcBV and CiBV (Chelonus inanitus bracovirus); the protein ODV-E66 in CcBV particles; and the proteins ODV-E56, p74, PIF-1, and PIF-2 in CiBV (indicated in red in Table 1, see table S2 for detailed results). Altogether, 20 different nudivirus-related gene products were identified as components of bracovirus particles, which demonstrated a functional link between the nudiviral machinery and bracoviruses.

Chelonus inanitus and Cotesia congregata belong to the most distantly related subfamilies of bracovirus-associated wasps (25), which suggests that the nudiviral machinery is present in all these species. Accordingly, we amplified the most conserved nudivirus-related genes (HzNVorf9-like1 and HzNVorf128-like) from most wasp subfamilies of the microgastroid complex (Fig. 3). We conclude that these genes were present before the radiation of the complex and originated from a virus integrated in a chromosome of the ancestral wasp. The cluster of genes present in Cotesia congregata could constitute the remnants of the genome of this ancestral virus. The level of similarity between HzNVorf9-like1 and HzNVorf128-like products from Chelonus inanitus and Cotesia congregata reaches 80% and thus indicates a strong conservation of their functions (Table 1). Some proteins show a lower conservation (for example for p74, 46% similarity), which suggests these sequences may be involved in more specific interactions with the host and have had to evolve more rapidly due to selective pressures associated with infection.

Fig. 3.

Conservation of the machinery producing bracovirus particles among microgastroid wasps. The tree and dates of radiations are taken from (25). (A) Nudivirus-related genes amplified with DNA from species belonging to different subfamilies: C. congregata (Microgastrinae), Toxoneuron nigriceps (Cardiochilinae), Mirax sp. (Miracinae), Epsilogaster sp. (Mendesellinae), Sania sp. (Khoikhoiinae), and C. inanitus (Cheloninae). (B) Alignment of HzNVORF9-like1 and HzNVORF128-like proteins deduced from the amplified sequences. (C) Bracovirus particles visualized by TEM from C. congregata, Mirax sp., and C. inanitus. n, Nucleocapsid. The particles contain one (CiBV) or several nucleocapsids (CcBV and MspBV), dispersed (CcBV) or organized (MspBV).

The overall conservation of the nudiviral machinery encoded by the wasps contrasts sharply with the lack of similarity found between the DNA enclosed in CiBV and CcBV particles. No common genes were found between CiBV and other bracovirus genomes (27); only sequences involved in the production of the circular dsDNAs of the particles (excision sequences) were conserved (28, 29). Furthermore, packaged bracovirus genomes do not contain any nudivirus-related genes. We hypothesize that shortly after initial integration of the nudivirus ancestor, viral DNA might have been replaced by wasp DNA in the particles (possibly by translocation of sequences allowing excision and encapsidation) and that most genes promoting parasitism were acquired later and independently in bracovirus-associated wasps.

It is well documented that genes of viral origin are used by eukaryotes to ensure physiological functions, such as the syncytins involved in trophoblast differentiation, which originated from retroviral envelope proteins independently acquired by primates and mice (30). However, the bracovirus-wasp associations represent the only example, so far, of the incorporation of genes encoding a complex viral machinery that allows its eukaryotic host to transfer and express heterologous genes in target organisms. In this regard, unraveling bracovirus particle assembly could contribute to the design of new vectors for gene therapy.

Supporting Online Material

www.sciencemag.org/cgi/content/full/323/5916/926/DC1

Material and Methods

Fig. S1

Tables S1 to S5

References

References and Notes

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