Bruce A. Webb

Polydnavirus Biology, Genome Structure, and Evolution

Polydnaviruses are an unusual group of insect viruses that have an obligate mutualistic association with certain parasitic wasps. Polydnaviruses are unique both in terms of their association with parasitic Hymenoptera and because of their profusely segmented DNA genomes. These viruses are named on the basis of their unique polydisperse DNA genomes (i.e., poly -DNA-viruses), but their life cycles are equally distinctive (Stoltz et al., 1995). In this chapter, a general overview of polydnaviruses is provided but is biased by the preponderance of molecular studies performed in a single species, the Campoletis sonorensis polydnavirus (CsPDV). CsPDV is the type species member of the ichnoviruses, one of the two major polydnavirus groups. Bracoviruses, which comprise the other major group, have distinctive morphologies and appear to be evolutionarily unrelated to the ichnoviruses (Whitfield, 1997), making it likely that significant differences will continue to be found in the organization and function of the two polydnavirus genera. While attempting to provide comprehensive coverage of the field, I have deliberately focused on more recent work. For more comprehensive consideration of earlier developments, the reader should consult reviews by Stoltz and Vinson (1979a), Fleming (1992), Stoltz and Whitfield (1992), Beckage (1993), Fleming and Krell (1993), Strand and Pech (1995a), and Lavine and Beckage (1996).

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.

Perspectives on polydnavirus origins and evolution.

Publisher Summary This chapter discusses the origin and evolution of Polydnavirus (PDV) that exists in obligate mutualisms with some parasitic Hymenoptera. A given PDV species replicates only in a single wasp species, and all members of that wasp species carry the associated virus. Two genera of PDVs are recognized––the bracoviruses (BVs) and the ichnoviruses (IVs)–– associated with braconid and ichneumonid wasps, respectively. The PDV genera have similar genome organization and replication/transmission pathways but have no known genetic similarity. Viral gene expression in insects parasitized by PDV-carrying wasps causes physiological alterations that are essential for parasitoid survival and development. PDV life cycles have been described as having “two arms.” PDVs replicate from proviral DNA in specialized cells of the wasp oviduct. Virus replication is first detected in the late pupal stage with virus released from calyx cells by budding or cell lysis and accumulating to high concentrations in the oviduct lumen. PDVs provide an interesting system for viral evolutionary studies. The unique life cycle involves coevolution with two hosts––one mutualistic and one pathogenic––thus, imposing both reductive and diversifying selection pressures on viral genes.

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 hosts 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.

Factors mediating short- and long-term immune suppression in a parasitized insect

Abstract Suppression of the host immune response after parasitization is essential for the survival of many endoparasitic hymenoptera. In Heliothis virescens larvae parasitized by Campoletis sonorensis , factors have been isolated that rapidly and persistently suppress the immune response to parasite eggs. When C. sonorensis eggs are washed to remove protective factors from the female reproductive tract, H. virescens larvae encapsulate many of the eggs within 3 h. Ovarian proteins are introduced with the parasite egg and rapidly but transiently alter hemocyte morphology and disable the immune response. Campoletis sonorensis polydnavirus (CsPDV) is also introduced during parasitization and disrupts hemocyte morphology at 24 h and encapsulation at later times (Edson et al. , 1981). However, CsPDV-encoded proteins are not detected before 5–6 h after parasitization and hemocyte morphology is not altered at 3 h after virus injection. Therefore, it is unlikely that CsPDV protects parasite eggs from encapsulation in the early stages after parasitization. We hypothesize that in parasitized insects, the rapid short-term immunosuppressive activity of ovarian proteins complements the slower, long-term immune suppression that is provided by CsPDV following infection of host cells and expression of functionally active viral genes.

Genomic and Morphological Features of a Banchine Polydnavirus: Comparison with Bracoviruses and Ichnoviruses

ABSTRACT Many ichneumonid and braconid endoparasitoids inject a polydnavirus (PDV) into their caterpillar hosts during oviposition. The viral entities carried by wasps of these families are referred to as “ichnoviruses” (IVs) and “bracoviruses” (BVs), respectively. All IV genomes characterized to date are found in wasps of the subfamily Campopleginae; consequently, little is known about PDVs found in wasps of the subfamily Banchinae, the only other ichneumonid taxon thus far shown to carry these viruses. Here we report on the genome sequence and virion morphology of a PDV carried by the banchine parasitoid Glypta fumiferanae. With an aggregate genome size of ∼290 kb and 105 genome segments, this virus displays a degree of genome segmentation far greater than that reported for BVs or IVs. The size range of its genome segments is also lower than those in the latter two groups. As reported for other PDVs, the predicted open reading frames of this virus cluster into gene families, including the protein tyrosine phosphatase (PTP) and viral ankyrin (ank) families, but phylogenetic analysis indicates that ank genes of the G. fumiferanae virus are not embedded within the IV lineage, while its PTPs and those of BVs form distinct clusters. The banchine PDV genome also encodes a novel family of NTPase-like proteins displaying a pox-D5 domain. The unique genomic features of the first banchine virus examined, along with the morphological singularities of its virions (IV-like nucleocapsids, but enveloped in groups like some of the BVs), suggest that they could have an origin distinct from those of IVs and BVs.

INTERACTION OF A WASP OVARIAN PROTEIN AND POLYDNAVIRUS IN HOST IMMUNE SUPPRESSION

During parasitization of Heliothis virescens, Campoletis sonorensis deposits an egg along with venom, polydnavirus particles (CsPDV), and ovarian proteins (OPs). Oviposited eggs are not encapsulated, while washed eggs are encapsulated rapidly by H. virescens. Early protection from encapsulation is afforded by a group of 29-36 kD OP glycoforms. These glycoforms are endocytosed by host hemocytes within 30 min post-parasitization (pp) and disrupt hemocyte spreading behavior and egg encapsulation through at least 24 h p.i. Purified CsPDV does not protect eggs from encapsulation early, but disrupts hemocyte spreading and egg encapsulation from 24 h through at least 5 days p.i. Functional activity of CsPDV appears to be correlated with time-dependent accumulation of virus-specific proteins in parasitized insects. We propose that the fast-acting 29-36 kD OP protects Campoletis eggs from encapsulation until accumulation of CsPDV proteins which sustain immunosuppression.

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.

Polydnavirus infection inhibits synthesis of an insect plasma protein, arylphorin

The wasp Campoletis sonorensis injects a segmented, double-stranded DNA polydnavirus (CsPDV) along with its egg during parasitization of Heliothis virescens larvae. After parasitization, CsPDV protects the wasp egg and larva by selectively disabling the hosts cellular immune response. Other host physiological systems including growth and development are affected to the apparent benefit of the parasite. To begin the characterization of the biochemical effects and mode of action of CsPDV on host growth, the titre of a developmentally regulated insect storage protein, arylphorin, was studied. Parasitized or virus-infected insects had substantially less circulating arylphorin than control insects. Fat bodies from parasitized larvae also synthesized less arylphorin in vitro. However, Northern blots of total RNA from parasitized and non-parasitized, control insects showed that the arylphorin transcript level was unaffected by parasitization suggesting a biochemical block at the translational level. In vitro translation followed by immunoprecipitation of arylphorin indicated that the mRNA was present and translatable at equal levels in both parasitized and control insects. Injection of purified virus elicited the response observed in naturally parasitized larvae, demonstrating that the effect on arylphorin synthesis is mediated, either directly or indirectly, by polydnavirus gene product(s).

Iκβ-Related vankyrin Genes in the Campoletis sonorensis Ichnovirus: Temporal and Tissue-Specific Patterns of Expression in Parasitized Heliothis virescens Lepidopteran Hosts

ABSTRACT Polydnaviruses (PDVs) are unusual insect viruses that occur in obligate symbiotic associations with parasitic ichneumonid (ichnoviruses, or IVs) and braconid (bracoviruses, or BVs) wasps. PDVs are injected with eggs, ovarian proteins, and venom during parasitization. Following infection of cells in host tissues, viral genes are expressed and their products function to alter lepidopteran host physiology, enabling endoparasitoid development. Here we describe the Campoletis sonorensis IV viral ankyrin (vankyrin) gene family and its transcription. The seven members of this gene family possess ankyrin repeat domains that resemble the inhibitory domains of the Drosophila melanogaster NF-κβ transcription factor inhibitor (Iκβ) cactus. vankyrin gene expression is detected within 2 to 4 h postparasitization (p.p.) in Heliothis virescens hosts and reaches peak levels by 3 days p.p. Our data indicate that vankyrin genes from the C. sonorensis IV genome are differentially expressed in the tissues of parasitized hosts and can be divided into two subclasses: those that target the host fat body and those that target host hemocytes. Polyclonal antibodies raised against a fat-body targeting vankyrin detected a 19-kDa protein in crude extracts prepared from the 3 days p.p. fat body. Vankyrin-specific Abs localized to 3-day p.p. fat-body and hemocyte nuclei, suggesting a role for vankyrin proteins in the nuclei of C. sonorensis IV-infected cells. These data are evidence for divergent tissue specificities and targeting of multigene families in IVs. We hypothesize that PDV vankyrin genes may suppress NF-κβ activity during immune responses and developmental cascades in parasitized lepidopteran hosts of C. sonorensis.