Functional characterization of Microplitis demolitor bracovirus genes that encode nucleocapsid components.

IMPORTANCE: Understanding how bracoviruses (BVs) function in wasps is of broad interest in the study of virus evolution. This study characterizes most of the Microplitis demolitor bracovirus (MdBV) genes whose products are nucleocapsid components. Results indicate several genes unknown outside of nudiviruses and BVs are essential for normal capsid assembly. Results also indicate most MdBV tyrosine recombinase family members and the DNA binding protein p6.9-1 are required for DNA processing and packaging into nucleocapsids.

Microplitis bicoloratus parasitism promotes cyclophilin D-p53 interaction to induce apoptosis of hemocytes in Spodoptera litura.

Microplitis bicoloratus parasitism can induce apoptosis of hemocytes in the M. bicolortus host, Spodoptera litura. However, it is unclear how M. bicolortus parasitism regulates host signaling pathways to induce apoptosis. Expression of cyclophilin D (CypD) and p53 was significantly upregulated in S. litura hemocytes at 6 days postparasitization. In the parasitized hemocytes, there was mitochondrial membrane potential (△Ψm ) loss, cytochrome c (Cyt C) release from mitochondria, and caspase-3 activation. These occurred while hemocytes were undergoing upregulation of CypD and p53. Parasitism also promoted the interaction between CypD and p53. CypD silencing could rescue the apoptotic phenotypes induced by parasitism, but had no effect on apoptosis in unparasitized S. litura. These findings suggest that the CypD-p53 pathway may be an important component of the parasitism-induced immunosuppressive response and establish a basis for further studies of parasitoid/host interactions.

Cky811 protein expressed by polydnavirus and venom gland of Cotesia kariyai regulates the host Mythimna separata larvae immune response function of C-type lectin responsible for foreign substance recognition which suppresses its melanization and encapsulation.

Cotesia kariyai (Ck) larvae implanted into the body cavity of the Mythimna separata (armyworm) larvae get melanized and encapsulated after adhesion by hemocytes called hyperspread cells (HSCs). The present study showed that HSCs could not adhere to the implanted Ck larvae in armyworm larvae after injection of Ck polydnavirus (CkPDV) + venom (V), thus melanization and encapsulation could not occur. A C-type lectin called Mys-IML of the host armyworm larvae was considered to be involved in the recognition of foreign substances which always expressed in hemocytes. The CkPDV DNA encodes a C-type lectin called Cky811 that has high amino acid homology to Mys-IML. HSCs did not adhere when CkPDV + V was mixed with the hemolymph of armyworm larvae on glass slides and incubated in vitro, but the addition of anti-Cky811 antibody enabled HSCs to adhere. The messenger RNA (mRNA) expression of Mys-IML in armyworm larvae injected with CkPDV + V became undetectable by 6 h. On the contrary, Cky811 mRNA was well expressed in the hemocytes of armyworm larvae injected with CkPDV + V from 0.5 to 6 h. Cky811 protein was also detected in the crude extracts from Ck venom gland + Ck venom reservoir, suggesting that these proteins regulate foreign substance recognition by the armyworm within 0.5 h. These results suggest that CkPDV + V suppresses mRNA expression of Mys-IML, and that Cky811 protein expressed in hemocytes regulates foreign substance recognition of Mys-IML, resulting in inhibition of the downstream reaction steps: HSCs adhesion, melanization, and encapsulation.

Suppressive activity of a viral histone H4 against two host chromatin remodelling factors: lysine demethylase and SWI/SNF.

Histone H4, a nucleosome subunit in eukaryotes, plays crucial roles in DNA package and regulation of gene expression through covalent modification. A viral histone H4 encoded in Cotesia plutellae bracovirus (CpBV), a polydnavirus, is called CpBV-H4. It is highly homologous to other histone H4 proteins excepting 38 extra amino acid residues in the N terminus. CpBV-H4 can form octamer with other histone subunits and alter host gene expression. In this study, CpBV-H4 was transiently expressed in a natural host (Plutella xylostella) and its suppressive activity on host gene expression was evaluated by the suppressive subtractive hybridization (SSH) technique. The SSH targets down-regulated by CpBV-H4 were read with the 454 pyrosequencing platform and annotated using the genome of P. xylostella. The down-regulated genes (610 contigs) were annotated in most functional categories based on gene ontology. Among these SSH targets, 115 genes were functionally distinct, including two chromatin remodelling factors: a lysine-specific demethylase (Px-KDM) and a chromatin remodelling complex [Px-SWI/SNF (SWItch/Sucrose Non-Fermentable)]. Px-KDM was highly expressed in all tested tissues during the entire larval period. Suppression of Px-KDM expression by specific RNA interference (RNAi) significantly (P<0.05) reduced haemocyte nodule formation in response to immune challenge and impaired both larval and pupal development. Px-SWI/SNF was expressed in all developmental stages. Suppression of Px-SWI/SNF expression by RNAi reduced cellular immune response and interfered with adult metamorphosis. These results suggest that CpBV-H4 can alter host gene expression by interfering with chromatin modification and remodelling factors in addition to its direct epigenetic control activity.

Polydnaviral ankyrin proteins aid parasitic wasp survival by coordinate and selective inhibition of hematopoietic and immune NF-kappa B signaling in insect hosts.

Polydnaviruses are mutualists of their parasitoid wasps and express genes in immune cells of their Lepidopteran hosts. Polydnaviral genomes carry multiple copies of viral ankyrins or vankyrins. Vankyrin proteins are homologous to IκB proteins, but lack sequences for regulated degradation. We tested if Ichnoviral Vankyrins differentially impede Toll-NF-κB-dependent hematopoietic and immune signaling in a heterologous in vivo Drosophila, system. We first show that hematopoiesis and the cellular encapsulation response against parasitoid wasps are tightly-linked via NF-κB signaling. The niche, which neighbors the larval hematopoietic progenitors, responds to parasite infection. Drosophila NF-κB proteins are expressed in the niche, and non cell-autonomously influence fate choice in basal and parasite-activated hematopoiesis. These effects are blocked by the Vankyrin I²-vank-3, but not by P-vank-1, as is the expression of a NF-κB target transgene. I²-vank-3 and P-vank-1 differentially obstruct cellular and humoral inflammation. Additionally, their maternal expression weakens ventral embryonic patterning. We propose that selective perturbation of NF-κB-IκB interactions in natural hosts of parasitic wasps negatively impacts the outcome of hematopoietic and immune signaling and this immune deficit contributes to parasite survival and species success in nature.

Polydnavirus Ank proteins bind NF-κB homodimers and inhibit processing of Relish.

Recent studies have greatly increased understanding of how the immune system of insects responds to infection, whereas much less is known about how pathogens subvert immune defenses. Key regulators of the insect immune system are Rel proteins that form Nuclear Factor-κB (NF-κB) transcription factors, and inhibitor κB (IκB) proteins that complex with and regulate NF-κBs. Major mortality agents of insects are parasitoid wasps that carry immunosuppressive polydnaviruses (PDVs). Most PDVs encode ank genes that share features with IκBs, while our own prior studies suggested that two ank family members from Microplitis demolitor bracovirus (MdBV) (Ank-H4 and Ank-N5) behave as IκB mimics. However, the binding affinities of these viral mimics for Rel proteins relative to endogenous IκBs remained unclear. Surface plasmon resonance (SPR) and co-immunoprecipitation assays showed that the IκB Cactus from Drosophila bound Dif and Dorsal homodimers more strongly than Relish homodimers. Ank-H4 and -N5 bound Dif, Dorsal and Relish homodimers with higher affinity than the IκB domain of Relish (Rel-49), and also bound Relish homodimers more strongly than Cactus. Ank-H4 and -N5 inhibited processing of compound Relish and reduced the expression of several antimicrobial peptide genes regulated by the Imd signaling pathway in Drosophila mbn2 cells. Studies conducted in the natural host Pseudoplusia includens suggested that parasitism by M. demolitor also activates NF-κB signaling and that MdBV inhibits this response. Overall, our data provide the first quantitative measures of insect and viral IκB binding affinities, while also showing that viral mimics disable Relish processing.

Analysis of gene transcription and relative abundance of the cys-motif gene family from Campoletis sonorensis ichnovirus (CsIV) and further characterization of the most abundant cys-motif protein, WHv1.6.

The cys-motif gene family associated with Campoletis sonorensis ichnovirus contains 10 members, WHv1.6, WHv1.0, VHv1.1, VHv1.4, AHv1.0, A'Hv0.8, FHv1.4, LHv2.8, UHv0.8, and UHv0.8a. The results of this study indicated that, within the encapsidated virion, WHv1.6 is the most abundant cys-motif gene, while the combined AHv genes are the least abundant. During parasitization of Heliothis virescens by Campoletis sonorenis, WHv1.6 transcripts were the mostly highly expressed, while the combined UHv genes had the lowest expression. Further proteomic analysis of WHv1.6 showed that it accumulates at high levels in parasitized plasma by 6 h, and is detectable in the haemocytes, fat body, malpighian tubules, nerve cord and epidermis by 2 days after parasitization. Localization experiments led us to conclude that WHv1.6 interacts with the cell membrane along with other organelles within a virus-infected cell and prevents immunocytes from spreading or adhering to a foreign surface. Similarly to VHv1.4 and VHv1.1, WHv1.6 is able to inhibit the translation of haemocyte and Malpighian tubule RNAs. Our results showed that the expression of cys-motif genes during parasitization is related to the gene copy number of each gene within the encapsidated virion and may also be dependent upon cis-regulatory element activity in different target tissues. In addition, WHv1.6 plays a major role in inhibiting the cellular encapsulation response by H. virescens.

Identification of an in vitro interaction between an insect immune suppressor protein (CrV2) and G alpha proteins.

The protein CrV2 is encoded by a polydnavirus integrated into the genome of the endoparasitoid Cotesia rubecula (Hymenoptera:Braconidae:Microgastrinae) and is expressed in host larvae with other gene products of the polydnavirus to allow successful development of the parasitoid. CrV2 expression has previously been associated with immune suppression, although the molecular basis for this was not known. Here, we have used time-resolved Förster resonance energy transfer (TR-FRET) to demonstrate high affinity binding of CrV2 to Gα subunits (but not the Gßγ dimer) of heterotrimeric G-proteins. Signals up to 5-fold above background were generated, and an apparent dissociation constant of 6.2 nm was calculated. Protease treatment abolished the TR-FRET signal, and the presence of unlabeled CrV2 or Gα proteins also reduced the TR-FRET signal. The activation state of the Gα subunit was altered with aluminum fluoride, and this decreased the affinity of the interaction with CrV2. It was also demonstrated that CrV2 preferentially bound to Drosophila Gα(o) compared with rat Gα(i1). In addition, three CrV2 homologs were detected in sequences derived from polydnaviruses from Cotesia plutellae and Cotesia congregata (including the immune-related early expressed transcript, EP2). These data suggest a potential mode-of-action of immune suppressors not previously reported, which in addition to furthering our understanding of insect immunity may have practical benefits such as facilitating development of novel controls for pest insect species.

Evolutionary mechanisms driving the evolution of a large polydnavirus gene family coding for protein tyrosine phosphatases.

BACKGROUND: Gene duplications have been proposed to be the main mechanism involved in genome evolution and in acquisition of new functions. Polydnaviruses (PDVs), symbiotic viruses associated with parasitoid wasps, are ideal model systems to study mechanisms of gene duplications given that PDV genomes consist of virulence genes organized into multigene families. In these systems the viral genome is integrated in a wasp chromosome as a provirus and virus particles containing circular double-stranded DNA are injected into the parasitoids' hosts and are essential for parasitism success. The viral virulence factors, organized in gene families, are required collectively to induce host immune suppression and developmental arrest. The gene family which encodes protein tyrosine phosphatases (PTPs) has undergone spectacular expansion in several PDV genomes with up to 42 genes. RESULTS: Here, we present strong indications that PTP gene family expansion occurred via classical mechanisms: by duplication of large segments of the chromosomally integrated form of the virus sequences (segmental duplication), by tandem duplications within this form and by dispersed duplications. We also propose a novel duplication mechanism specific to PDVs that involves viral circle reintegration into the wasp genome. The PTP copies produced were shown to undergo conservative evolution along with episodes of adaptive evolution. In particular recently produced copies have undergone positive selection in sites most likely involved in defining substrate selectivity. CONCLUSION: The results provide evidence about the dynamic nature of polydnavirus proviral genomes. Classical and PDV-specific duplication mechanisms have been involved in the production of new gene copies. Selection pressures associated with antagonistic interactions with parasitized hosts have shaped these genes used to manipulate lepidopteran physiology with evidence for positive selection involved in adaptation to host targets.

A Polydnavirus Protein Tyrosine Phosphatase Negatively Regulates the Host Phenoloxidase Pathway.

Polydnavirus (PDV) is a parasitic factor of endoparasitic wasps and contributes greatly to overcoming the immune response of parasitized hosts. Protein tyrosine phosphatases (PTPs) regulate a wide variety of biological processes at the post-transcriptional level in mammals, but knowledge of PDV PTP action during a parasitoid−host interaction is limited. In this study, we characterized a PTP gene, CvBV_12-6, derived from Cotesia vestalis bracovirus (CvBV), and explored its possible regulatory role in the immune response of the host Plutella xylostella. Our results from qPCR show that CvBV_12-6 was highly expressed in hemocytes at an early stage of parasitization. To explore CvBV_12-6 function, we specifically expressed CvBV_12-6 in Drosophila melanogaster hemocytes. The results show that Hml-Gal4 > CvBV_12-6 suppressed the phenoloxidase activity of hemolymph in D. melanogaster, but exerted no effect on the total count or the viability of the hemocytes. In addition, the Hml-Gal4 > CvBV_12-6 flies exhibited decreased antibacterial abilities against Staphylococcus aureus. Similarly, we found that CvBV_12-6 significantly suppressed the melanization of the host P. xylostella 24 h post parasitization and reduced the viability, but not the number, of hemocytes. In conclusion, CvBV_12-6 negatively regulated both cellular and humoral immunity in P. xylostella, and the related molecular mechanism may be universal to insects.

The impact on microtubule network of a bracovirus IkappaB-like protein.

Polydnavirus-encoded IkappaB-like proteins are similar to insect and mammalian IkappaB, and an immunosuppressive function in the host cells has been inferred to these proteins. Here we show that the expression of one of these IkappaB-like viral genes, the TnBVank1, in the Drosophila germline affects the localization of gurken, bicoid, and oskar mRNAs whose gene products are relevant for proper embryonic patterning. The altered localization of these mRNAs is suggestive of general defects in the intracellular, microtubule-based, trafficking routes. Analysis of microtubule motor proteins components such as the dynein heavy chain and the kinesin heavy chain revealed defects in the polarized microtubule network. Interestingly, the TnBVANK1 viral protein is uniformly distributed over the entire oocyte cortex, and appears to be anchored to the microtubule ends. Our data open up a very interesting issue on novel function(s) played by the ank gene family by interfering with cytoskeleton organization.

MicroRNAs from Snellenius manilae bracovirus regulate innate and cellular immune responses of its host Spodoptera litura.

To avoid inducing immune and physiological responses in insect hosts, parasitoid wasps have developed several mechanisms to inhibit them during parasitism, including the production of venom, specialized wasp cells, and symbioses with polydnaviruses (PDVs). These mechanisms alter the host physiology to give the wasp offspring a greater chance of survival. However, the molecular mechanisms for most of these alterations remain unclear. In the present study, we applied next-generation sequencing analysis and identified several miRNAs that were encoded in the genome of Snellenius manilae bracovirus (SmBV), and expressed in the host larvae, Spodoptera litura, during parasitism. Among these miRNAs, SmBV-miR-199b-5p and SmBV-miR-2989 were found to target domeless and toll-7 in the host, which are involved in the host innate immune responses. Microinjecting the inhibitors of these two miRNAs into parasitized S. litura larvae not only severely decreased the pupation rate of Snellenius manilae, but also restored the phagocytosis and encapsulation activity of the hemocytes. The results demonstrate that these two SmBV-encoded miRNAs play an important role in suppressing the immune responses of parasitized hosts. Overall, our study uncovers the functions of two SmBV-encoded miRNAs in regulating the host innate immune responses upon wasp parasitism.

Polydnavirus Innexins Disrupt Host Cellular Encapsulation and Larval Maturation.

Polydnaviruses are dsDNA viruses associated with endoparasitoid wasps. Delivery of the virus during parasitization of a caterpillar and subsequent virus gene expression is required for production of an amenable environment for parasitoid offspring development. Consequently, understanding of Polydnavirus gene function provides insight into mechanisms of host susceptibility and parasitoid wasp host range. Polydnavirus genes predominantly are arranged in multimember gene families, one of which is the vinnexins, which are virus homologues of insect gap junction genes, the innexins. Previous studies of Campoletis sonorensis Ichnovirus Vinnexins using various heterologous systems have suggested the four encoded members may provide different functionality in the infected caterpillar host. Here, we expressed two of the members, vnxG and vnxQ2, using recombinant baculoviruses in susceptible host, the caterpillar Heliothis virescens. Following intrahemocoelic injections, we observed that >90% of hemocytes (blood cells) were infected, producing recombinant protein. Larvae infected with a vinnexin-recombinant baculovirus exhibited significantly reduced molting rates relative to larvae infected with a control recombinant baculovirus and mock-infected larvae. Similarly, larvae infected with vinnexin-recombinant baculoviruses were less likely to survive relative to controls and showed reduced ability to encapsulate chromatography beads in an immune assay. In most assays, the VnxG protein was associated with more severe pathology than VnxQ2. Our findings support a role for Vinnexins in CsIV and more broadly Ichnovirus pathology in infected lepidopteran hosts, particularly in disrupting multicellular developmental and immune physiology.

Interactions of Vank proteins from Microplitis bicoloratus bracovirus with host Dip3 suppress eIF4E expression.

Microplitis bicoloratus bracovirus (MbBV) inhibits the immune response of the host Spodoptera litura by disrupting nuclear factor (NF)-κB signaling and downstream gene expression. However, the underlying molecular mechanisms are not well understood. Herein, we report that viral ankyrin (Vank) proteins interacted with host dorsal-interacting protein 3 (Dip3) to selectively inhibit the transcription of eukaryotic translation initiation factor 4 E (eIF4E). Dip3 and Vank proteins were co-expressed and colocalized in the nucleus. Furthermore, ectopic expression of Dip3 rescued the transcription of some NF-κB-dependent genes suppressed by Vank proteins, including eIF4E. Co-immunoprecipitation and pull-down assays confirmed that Vank proteins interacted with and bound to full-length Dip3, which including MADF, DNA-binding protein, BESS, and protein-protein interaction motifs as well as non-motif sequences. In vivo, RNAi-mediated dip3 silencing decreased eIF4E levels and was accompanied by an immunosuppressive phenotype in S. litura. Our results provided novel insights into the regulation of host transcription during immune suppression by viral proteins that modulate nuclear NF-κB signaling.

A viral histone H4 suppresses expression of a transferrin that plays a role in the immune response of the diamondback moth, Plutella xylostella.

A transferrin (Tf) gene has been predicted from an expressed sequence tag of the diamondback moth, Plutella xylostella. It encodes 681 amino acid residues that share 80-90% sequence homologies with other lepidopteran Tfs. The gene was constitutively expressed in all developmental stages of P. xylostella. Double-stranded RNA (dsRNA) specific to the Tf gene was prepared and microinjected into the larvae. We hypothesize that the dsRNA treatment suppressed the Tf gene expression level and it significantly inhibited haemocyte nodule formation in response to bacterial challenge. The larvae treated with dsRNA also showed a significantly enhanced susceptibility to an entomopathogenic bacterium, Bacillus thuringiensis. An endoparasitoid wasp, Cotesia plutellae, parasitized the larvae of P. xylostella, which showed significant reduction of Tf expression. The suppression of Tf expression was mimicked by transient expression of a viral gene CpBV-H4, encoded in the symbiotic virus of C. plutellae. A truncated form of CpBV-H4 prepared by deleting an extended N-terminal 38 amino acid residue lost its inhibitory activity against the Tf gene expression. These results suggest that Tf of P. xylostella plays an immunological role in P. xylostella and that the suppression of its expression in the parasitized larvae is caused by a viral histone H4 in an epigenetic mode.

Transient transcription of a putative RNase containing BEN domain encoded in Cotesia plutellae bracovirus induces an immunosuppression of the diamondback moth, Plutella xylostella.

A polydnavirus, Cotesia plutellae bracovirus (CpBV), possesses segmented genome located on chromosome(s) of an endoparasitoid wasp, C. plutellae. An episomal viral segment (CpBV-S3) consists of 11,017 bp and encodes two putative open reading frames (ORFs). ORF301 shows amino acid sequence homologies (28-50%) with RNase T2s of various organisms. It also contains BEN domain in C-terminal region. ORF302 is a hypothetical gene, which is also found in other bracoviruses. Both genes were expressed in larvae of Plutella xylostella parasitized by C. plutellae. Their expressions were detected in all tested tissues including hemocyte, fat body, gut, and epidermis. To analyze effects of these genes on the parasitism, the segment of CpBV-S3 was injected to nonparasitized larvae of P. xylostella, in which the two genes were expressed at least for 4 days post-injection. The larvae injected with CpBV-S3 exhibited significant immunosuppression, such as reduction in total hemocyte population and impairment in nodule formation behavior of hemocytes in response to bacterial challenge. Each gene expression in the treated larvae was inhibited by co-injecting respective double strand RNA (dsRNA) specific to each ORF. Injection of dsRNA of ORF301 could rescue the immunosuppression of the viral segment-treated larvae, while dsRNA specific to ORF302 did not. These results suggest that a putative RNase fused with a BEN domain encoded in CpBV-S3 plays a parasitic role in inducing host immunosuppression in the parasitism.

Snellenius manilae bracovirus suppresses the host immune system by regulating extracellular adenosine levels in Spodoptera litura.

Sufficient energy supply to the host immune system is important for resisting pathogens. Therefore, during pathogen infection, the host metabolism is reassigned from storage, growth, and development to the immune system. Previous studies in Drosophila melanogaster have demonstrated that systemic metabolic switching upon an immune challenge is activated by extracellular adenosine signaling, modulating carbohydrate mobilization and redistributing energy to the hemocytes. In the present study, we discovered that symbiotic virus (SmBV) of the parasitoid wasp Snellenius manilae is able to down-regulate the extracellular adenosine of its host, Spodoptera litura, to inhibit metabolism switching. The decreased carbohydrate mobilization, glycogenolysis, and ATP synthesis upon infection results in the host being unable to supply energy to its immune system, thus benefitting the development of wasp larvae. When we added adenosine to the infected S. litura larvae, we observed enhanced host immune responses that decreased the pupation rate of S. manilae. Previous studies showed that after pathogen infection, the host activates its adenosine pathway to trigger immune responses. However, our results suggest a different model: we found that in S. manilae, SmBV modulates the host adenosine pathway such that wasp eggs and larvae can evade the host immune response.

N-terminal tail of a viral histone H4 encoded in Cotesia plutellae bracovirus is essential to suppress gene expression of host histone H4.

An endoparasitoid wasp, Cotesia plutellae, possesses a symbiotic bracovirus (CpBV), which facilitates parasitism of a specific host, such as larvae of the diamondback moth, Plutella xylostella. A viral histone H4 (CpBV-H4) has been found in the CpBV genome and its gene product plays a role in impairing the host insect cellular immune response. Based on its high similarity to histone H4 of P. xylostella apart from its extended N-terminal tail, it has been suspected to alter host gene expression. Histone subunits were purified from parasitized P. xylostella larvae and found to contain both host and viral H4s, confirming a previous report of a possible epigenetic mode of action. Moreover, this study showed that the host H4 levels in the parasitized larvae clearly decreased during the parasitization period, whereas CpBV-H4 levels maintained a significant level without significant changes. To understand the decrease of host H4 levels, transcription levels of host H4 were monitored by quantitative reverse-transcriptase PCR (RT-PCR) and showed a significant decrease in parasitized P. xylostella larvae, whereas no significant change of the mRNA level was detected in nonparasitized larvae. This transcriptional control of host H4 expression was also observed by inducing transient expression of CpBV-H4 in nonparasitized P. xylostella. Moreover, co-injection of CpBV-H4 and its specific double-stranded RNA recovered the host H4 expression level. To identify a functional domain of CpBV-H4 involved in the transcriptional control, the extended N-terminal tail of CpBV-H4 was removed by preparing a truncated viral H4 construct in an expression vector by deleting the N-terminal tail of 38 amino acid residues and inducing its expression in nonparasitized P. xylostella larvae. The truncated CpBV-H4 clearly lost its inhibitory effects on host H4 transcription. Moreover, the presence of CpBV-H4 affects the spreading of host haemocytes by an epigenetic effect, which is at least partly restored in larvae expressing the truncated version of CpBV-H4. This study suggests that the viral H4 encoded in CpBV can alter host gene expression with its extended N-terminal tail.

Characterization of an IkappaB-like gene in Cotesia vestalis polydnavirus.

Cotesia vestalis (Braconidae, Hymenoptera) depends mainly on 3 regulatory factors to manipulate its host's development and immune response, including polydnavirus, venom, and teratocytes, among which polydnavirus plays a key role in suppressing the host immune system. In the present work, we cloned the full sequence of gene CvBV-ank2, encoding an IkappaB-like protein in C. vestalis polydnavirus (CvBV). The full sequence of CvBV-ank2 is 955 bp, encoding 162 amino acids with a calculated molecular mass of 18,355 Da. CvBV-ank2 shares high similarity with the exon I and exon II of CvBV-ank1, which is on the same segment with CvBV-ank2. This result suggests that gene duplication might occur in CvBV-ank1 and CvBV-ank2. Phylogenetic analysis indicated that CvBV-ank2 and CvBV-ank1, both on segment CvBV-S2, are, respectively, closely related with CcBV-26.3 and CcBV-26.2, both on segment Circle26 of C. congregata polydnavirus (CcBV). BLAST search using the sequence of segment CvBV-S2 as a query revealed that segment CvBV-S2 shares 90% max identity with segment Circle26 of CcBV over 67% query coverage. These results demonstrate that there is not only gene similarity, but also segment similarity between CvBV and CcBV. Transcripts of CvBV-ank2 were detected as early as 0.5 h post-parasitization and continued to be detected for 6 days, indicating that CvBV-ank2 might be involved in the early protection of the parasitoid egg.

A viral lectin encoded in Cotesia plutellae bracovirus and its immunosuppressive effect on host hemocytes.

An endoparasitoid wasp, Cotesia plutellae, induces immunosuppression of the host diamondback moth, Plutella xylostella. To identify an immunosuppressive factor, the parasitized hemolymph of P. xylostella was separated into plasma and hemocyte fractions. When nonparasitized hemocytes were overlaid with parasitized plasma, they showed significant reduction in bacterial binding efficacy. Here, we considered a viral lectin previously known in other Cotesia species as a humoral immunosuppressive candidate in C. plutellae parasitization. Based on consensus regions of the viral lectins, the corresponding lectin gene was cloned from P. xylostella parasitized by C. plutellae. Its cDNA is 674 bp long and encodes 157 amino acid residues containing a signal peptide (15 residues) and one carbohydrate recognition domain. Open reading frame is divided by one intron (156 bp) in its genomic DNA. Amino acid sequence shares 80% homology with that of C. ruficrus bracovirus lectin and is classified into C-type lectin. Southern hybridization analysis indicated that the cloned lectin gene was located at C. plutellae bracovirus (CpBV) genome. Both real-time quantitative RT-PCR and immunoblotting assays indicated that CpBV-lectin showed early expression during the parasitization. A recombinant CpBV-lectin was expressed in a bacterial system and the purified protein significantly inhibited the association between bacteria and hemocytes of nonparasitized P. xylostella. In the parasitized P. xylostella, CpBV-lectin was detected on the surface of parasitoid eggs after 24 h parasitization by its specific immunostaining. The 24 h old eggs were not encapsulated in vitro by hemocytes of P. xylostella, compared to newly laid parasitoid eggs showing no CpBV-lectin detectable and easily encapsulated. These results support an existence of a polydnaviral lectin family among Cotesia-associated bracovirus and propose its immunosuppressive function.