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.

Identification of bracovirus particle proteins and analysis of their transcript levels at the stage of virion formation.

Polydnaviruses (PDVs) are unique symbiotic viruses associated with parasitic wasps; they replicate only in the calyx cells of a wasp's ovaries and are transferred at oviposition along with the parasitoid egg into the lepidopteran host. The DNA packaged in the viral particles encodes factors that manipulate the host's immune defences and development to benefit the parasitoid. PDVs are found in two subfamilies of ichneumonids (ichnoviruses) and in braconids of the microgastroid complex (bracoviruses). We recently showed that the latter derive from an ancestral nudivirus, as 24 nudivirus-related genes were identified in ovaries of two distantly related braconids at the stage of virion formation. Here, we present a comprehensive analysis of the viral particle proteins of the Chelonus inanitus bracovirus (CiBV). Proteins of purified CiBV particles were analysed by mass spectrometry and amino acid sequences matched to the existing ovarian-cDNA database. In addition, transcript quantities of identified genes were measured by quantitative real-time PCR in female pupae at the onset and peak of virion formation and at corresponding stages in male pupae. This combined approach allowed the identification of 44 CiBV particle proteins: 16 were nudivirus-related, three had similarity to ovarian proteins of another braconid, 11 had similarity to cellular proteins and 14 had no similarity to known proteins. The transcripts of all of them increased in female, but not male, pupae. These data confirm the important contribution of nudivirus genes but also indicate the presence of many lineage- or species-specific proteins possibly involved in the parasitoid-host interaction.

Viral cystatin evolution and three-dimensional structure modelling: a case of directional selection acting on a viral protein involved in a host-parasitoid interaction.

BACKGROUND: In pathogens, certain genes encoding proteins that directly interact with host defences coevolve with their host and are subject to positive selection. In the lepidopteran host-wasp parasitoid system, one of the most original strategies developed by the wasps to defeat host defences is the injection of a symbiotic polydnavirus at the same time as the wasp eggs. The virus is essential for wasp parasitism success since viral gene expression alters the immune system and development of the host. As a wasp mutualist symbiont, the virus is expected to exhibit a reduction in genome complexity and evolve under wasp phyletic constraints. However, as a lepidopteran host pathogenic symbiont, the virus is likely undergoing strong selective pressures for the acquisition of new functions by gene acquisition or duplication. To understand the constraints imposed by this particular system on virus evolution, we studied a polydnavirus gene family encoding cyteine protease inhibitors of the cystatin superfamily. RESULTS: We show that cystatins are the first bracovirus genes proven to be subject to strong positive selection within a host-parasitoid system. A generated three-dimensional model of Cotesia congregata bracovirus cystatin 1 provides a powerful framework to position positively selected residues and reveal that they are concentrated in the vicinity of actives sites which interact with cysteine proteases directly. In addition, phylogenetic analyses reveal two different cystatin forms which evolved under different selective constraints and are characterized by independent adaptive duplication events. CONCLUSION: Positive selection acts to maintain cystatin gene duplications and induces directional divergence presumably to ensure the presence of efficient and adapted cystatin forms. Directional selection has acted on key cystatin active sites, suggesting that cystatins coevolve with their host target. We can strongly suggest that cystatins constitute major virulence factors, as was already proposed in previous functional studies.

Characterization of a protein tyrosine phosphatase gene CvBV202 from Cotesia vestalis polydnavirus (CvBV).

Cotesia vestalis (Haliday) (Hymenoptera: Braconidae) is an endoparasitoid of the larval stage of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae) and injects a polydnavirus (CvBV) into its host during oviposition. In this paper we characterize CvBV202 and its product. CvBV202 is located on segment S2 of CvBV genome; it has a size of 984 bp and encodes a putative protein of 328 amino acids, including protein phosphatase domain and tyrosine-specific protein phosphatase domain. Gene transcripts were detected in extracts of the host as early as 2 h post-parasitization (p.p.) and continued to be detected for 6 days. Tissue-specific patterns of this gene expression showed that CvBV202 had a close relationship with the host's physiological alternations including immunosuppression, modulation of hormone titer, and nutrition metabolism. The protein was detected in the parasitized hosts at 12 h p.p. using western blot assay. The product of CvBV202 was found to be around 59 kDa, much larger than the predicted molecular weight of 37.8 kDa, suggesting that post-translational modification of CvBV202 occurs in host cells, corresponding with the existence of many post-translational modification sites. Immunofluorescence staining and confocal laser scanning microscopy revealed that CvBV202 and the fused protein eGFP-CvBV202 were observed both in the nuclear region and cytoplasm of the hemocytes of the naturally parasitized host larvae and rBac-eGFP-CvBV202-infected Tn-5B1-4 cells, respectively.

Characterization of a novel gene encoding ankyrin repeat domain from Cotesia vestalis polydnavirus (CvBV).

Cotesia vestalis (Haliday) is an endoparasitoid of Plutella xylostella (L.) larvae and injects a polydnavirus (CvBV) into its host during oviposition. In this report we describe the characterization of a gene (CvBV805) and its products. CvBV805 is located on the segment S8 of CvBV genome; it has a size of 909 bp and encodes a predicted protein of 125 amino acids. This protein contains an ankyrin repeat domain with a high degree of similarity with IkappaB-like genes. Gene transcripts were detected in extracts of the host as early as 2 h post-parasitization (p.p.) and continued to be detected through 24 h. Tissue-specific expression patterns showed that CvBV805 might be involved in early host immunosuppression. CvBV805 was detected in parasitized hosts at 12 h p.p. and in rBac-eGFP-CvBV805-infected Tn-5B1-4 cells at 72 h.p.i. by using western blots analysis. The size of the protein expressed in the host hemocytes and infected Tn-5B1-4 cells was 17 kDa and 56 kDa (including eGFP), respectively, which nearly corresponded with the predicted molecular weight (14.31 kDa) of CvBV805, suggesting that the protein did not undergo extensive post-translational modification. The protein was confirmed to be present within the nuclear region in hemocytes of the parasitized P. xylostella larvae at 48 h p.p. using confocal laser scanning microscopy.

A viral histone H4 encoded by Cotesia plutellae bracovirus inhibits haemocyte-spreading behaviour of the diamondback moth, Plutella xylostella.

Histone H4 is highly conserved and forms a central-core nucleosome with H3 in eukaryotic chromatin. Its covalent modification at the protruding N-terminal region from the nucleosomal core can change the chromatin conformation in order to regulate gene expression. A viral H4 was found in the genome of Cotesia plutellae bracovirus (CpBV). The obligate host of the virus is an endoparasitoid wasp, C. plutellae, which parasitizes the diamondback moth, Plutella xylostella, and interrupts host development and immune reactions. CpBV has been regarded as a major source for interrupting the physiological processes during parasitization. CpBV H4 shows high sequence identity with the amino acid sequence of P. xylostella H4 except for an extended N-terminal region (38 aa). This extended N-terminal CpBV H4 contains nine lysine residues. CpBV H4 was expressed in P. xylostella parasitized by C. plutellae. Western blot analysis using a wide-spectrum H4 antibody showed two H4s in parasitized P. xylostella. In parasitized haemocytes, CpBV H4 was detected predominantly in the nucleus and was highly acetylated. The effect of CpBV H4 on haemocytes was analysed by transient expression using a eukaryotic expression vector, which was injected into non-parasitized P. xylostella. Expression of CpBV H4 was confirmed in the transfected P. xylostella by RT-PCR and immunofluorescence assays. Haemocytes of the transfected larvae lost their spreading ability on an extracellular matrix. Inhibition of the cellular immune response by transient expression was reversed by RNA interference using dsRNA of CpBV H4. These results suggest that CpBV H4 plays a critical role in suppressing host immune responses during parasitization.

Genome organization of the Chelonus inanitus polydnavirus: excision sites, spacers and abundance of proviral and excised segments.

Polydnaviruses are only found in symbiotic association with parasitic wasps within the families Ichneumonidae and Braconidae (ichnoviruses and bracoviruses). They have a segmented genome consisting of circular double-stranded DNA. In the proviral linear form they are integrated in the wasp's genome; in two bracoviruses, segments were found to be clustered. Proviral segments have direct terminal repeats. Segment excision has been proposed to occur through juxtaposition of these repeats by formation of a loop and recombination; one copy of the repeat then ends up in the circular segment and one in the rejoined DNA. Here we analysed the excision/circularization site of four segments of the Chelonus inanitus bracovirus (CiV) and found that they are similar to the two already known sites; on the basis of the combined data an extended excision site motif was found. Analyses of segment flanking sequences led to the first identification of one complete and several partial spacers between proviral segments in a polydnavirus. The spacer between the proviral segments CiV14 and CiV22.5 has a length of 2065 bp; the terminal repeats of CiV14 and CiV22.5 were seen to have an opposite orientation and from this a model on the spacial organization of the loops of the proviral cluster is proposed. Through various approaches it was shown that spacers are not excised or injected into the host. Measurement of relative abundances of various segments in proviral and excised form indicates for the first time that abundant segments are present in multiple copies in the proviral form.

Identification of host translation inhibitory factor of Campoletis sonorensis ichnovirus on the tobacco budworm, Heliothis virescens.

Parasitization of a wasp, Campoletis sonorensis, against the larvae of Heliothis virescens depresses synthesis of specific host proteins related to growth and immunity. It has been suggested that the inhibition of host gene expression is targeted at a posttranscriptional level. This study aimed to verify the identity of host translation inhibitory factor (HTIF) derived from wasp parasitization. To identify HTIF, the proteins in the parasitized host were fractionated using different protein purification methods, and each fraction's HTIF activity was assessed. In the course of the protein purification steps, HTIF activity was highly correlated with the fractions containing VHv 1.4 protein, which has a conserved cysteine-motif and is encoded in C. sonorensis ichnovirus (CsIV). Purified VHv 1.4 protein using an immunoaffinity column exhibited a significant HTIF effect, while the heat-inactivated VHv 1.4 did not. Both recombinant VHv 1.4 and VHv 1.1 (another cys-motif protein encoded in CsIV) proteins were synthesized in Sf 9 cells through a baculovirus expression system. The purified recombinant VHv 1.4 and VHv 1.1 exhibited significant HTIF activities in a nanomolar range. However, VHv1.4 protein showed about four times higher HTIF activity than did VHv 1.1 protein. Both HTIFs acted directly on translation machinery because they inhibited a cell-free in vitro translation system using rabbit reticulocyte lysate. Both HTIFs are likely to discriminate specific target mRNAs because they inhibited translation of RNA extracts from the Tn 368 cell line, but not from Sf 9 cells. In addition, they inhibited translation of RNAs from fat body, hemocytes, and testis, but not from epidermis, gut, labial gland, and nerve tissues of H. virescens. These results indicate that both cys-motif proteins of VHv 1.4 and VHv 1.1 play a role as HTIF in C. sonorensis parasitization.

Toxoneuron nigriceps polydnavirus encodes a putative aspartyl protease highly expressed in parasitized host larvae.

Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae) is an endophagous parasitoid of larval stages of the tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae). This parasitoid is associated with a polydnavirus (TnBV), injected at oviposition along with the egg, and involved in the disruption of host immune reaction and endocrine balance. This paper reports the molecular characterization of TnBV2, one of the most abundant genes in the TnBV genome. TnBV2 expression produces a mature 0.6 kb transcript in fat body, prothoracic glands and haemocytes, as early as 6 h after parasitoid oviposition. Only in haemocytes a specific longer transcript of 2.5 kb is found 24 h after parasitization. The putative translation product of TnBV2 contains a retroviral type aspartyl protease domain. The possible origin and functional role of this TnBV gene are discussed.

Solution structure of the carboxyl-terminal cysteine-rich domain of the VHv1.1 polydnaviral gene product: comparison with other cystine knot structural folds.

Polydnaviruses are an unusual group of insect viruses that have an obligate symbiotic association with certain parasitic wasps. These viruses are transmitted with the wasp egg during oviposition into lepidopteran insects, enabling the survival and development of the egg inside the host larvae. We report the three-dimensional structure of a novel polydnaviral cysteine-rich motif (cys-motif), identified as the carboxyl-terminal domain of a two cys-motif containing polydnaviral VHv1.1 gene product, abbreviated "C-term VHv1.1". This 65-residue domain was identified experimentally by limited proteolysis of the full-length protein and was subsequently cloned in a bacterial expression system for NMR studies. The C-term VHv1.1 3D structure was determined in solution by two-dimensional (1)H NMR spectroscopy. Calculation of the structure was based on a total of 300 upper distance restraints and 20 dihedral angle constraints, and resulted in an ensemble of 25 representative conformers with an average rmsd of 0.47 A from the mean structure for core backbone atoms. The protein core is made of a four beta-strand scaffold held together in a compact structure by three disulfide bonds, which form a cystine knot. The four beta-strands are arranged in an unusual configuration to form a triple-stranded beta-sheet and double-stranded beta-sheet. Comparison with other classes of cystine knots provides indication that C-term VHv1.1 represents a new and distinct cystine knot motif. This analysis provides a structural basis for interpretation of the genetic and amino acid sequence data classifying polydnavirus gene products as members of cysteine-rich protein families.

Characterization of two novel Microplitis demolitor polydnavirus mRNAs expressed in Pseudoplusia includens haemocytes.

The braconid wasp Microplitis demolitor carries M. demolitor polydnavirus (MdPDV) and parasitizes the larval stage of the moth Pseudoplusia includens. M. demolitor injects MdPDV into P. includens larvae when it lays an egg and the virus infects various cells including haemocytes. Two new MdPDV transcripts expressed in host haemocytes were characterized in this study. Screening of an MdPDV-infected haemocyte cDNA library identified a 0.4 kb cDNA encoding a predicted protein of 103 amino acids which was named Egf0. 4. This protein contained a cysteine-rich epidermal growth factor (EGF)-like motif at its N terminus that was similar to the EGF-like domains in the previously identified MdPDV genes egf1.5 and egf1.0. Sequencing of the genomic clone pMd-10 indicated that it contained the egf0.4 gene, which consisted of two introns and three exons. This gene was located on MdPDV segment O and appeared to exist in multiple copies. A nucleic acid and expression screen identified a 1. 8 kb cDNA encoding a predicted protein of 515 amino acids designated Glc1.8. This protein consisted of a heavily glycosylated central core of six tandemly arranged repeats flanked by hydrophobic N- and C-terminal domains. Northern blotting and in situ hybridization studies indicated that both egf0.4 and glc1.8 were expressed in MdPDV-infected host haemocytes. Immunocytochemical studies also indicated that Glc1.8 localized to the cell surface.

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 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.