Antiviral activity of the inducible humoral immunity and its suppression by eleven BEN family members encoded in Cotesia plutellae bracovirus

Comparative Biochemistry and Physiology, Part A 179 (2015) 44–53

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Antiviral activity of the inducible humoral immunity and its suppression by eleven BEN family members encoded in Cotesia plutellae bracovirus Md. Ramjan Ali, Yonggyun Kim ⁎ Department of Bioresource Sciences, Andong National University, Andong 760-749, Republic of Korea

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Article history: Received 1 August 2014 Received in revised form 29 August 2014 Accepted 4 September 2014 Available online 16 September 2014 Keywords: Antimicrobial peptide Antiviral BEN Immunosuppression Prophenoloxidase RNA interference

a b s t r a c t Upon parasitization by some endoparasitoids, polydnaviruses (PDVs) play a crucial role in inducing host immunosuppression. This study reports a novel immunosuppressive activity against humoral immune responses by BEN family genes encoded in Cotesia plutellae bracovirus (CpBV). A total of 11 BEN family members are encoded in 10 different CpBV DNA segments. When the CpBV segments were individually injected, specific BEN genes were expressed and suppressed the expression of antimicrobial peptide (AMP) and prophenoloxidase genes following bacterial challenge. The suppressive activities of the BEN genes were reversed by injection of the double-stranded RNA (dsRNA) specific to each BEN gene. The suppression of the AMP gene expressions by the BEN genes was also confirmed using an inhibition zone assay against Gram-positive and Gram-negative bacterial growth. The significance of the suppressive activity of BEN genes against humoral immune responses was analyzed in terms of suppression of antiviral activity by the host humoral immunity. When CpBV was incubated with the plasma obtained from the larvae challenged with bacteria, the immunized plasma severely impaired the expression activity of the viral genes. However, an expression of BEN gene significantly rescued the viral gene expression by suppressing humoral immune response. These results suggest that BEN family genes of CpBV play a crucial role in defending the antiviral response of the parasitized Plutella xylostella by inhibiting humoral immune responses. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Polydnaviruses (PDVs) are a group of insect DNA viruses that are mutually associated with some endoparasitoid wasps (Webb et al., 2000). PDVs are classified into two genera, ichnovirus (IV) and bracovirus (BV), depending on the viral morphology and host wasp family (Webb and Strand, 2005). Both IV and BV have been regarded to be independently originated due to their difference in genome composition (Webb et al., 2000). An analysis of the ovarian transcripts of braconid wasps containing BVs indicates that BVs are originated from an ancestral nudivirus (Bézier et al., 2009a). However, a similar approach in ichneumonid wasp containing IV did not find any nudiviral rudiments supporting an independent origin of BV and IV (Volkoff et al., 2010). The entire PDV genome is segmented and located on host wasp chromosome(s) in a proviral form (Stoltz, 1990; Bézier et al., 2009b). During viral replication at late pupal development, the ovarian calyx cells produce PDV particles and release them to the lateral oviduct lumen (Wyler and Lanzrein, 2003). The viral genome is divided into “encapsidated” and “non-encapsidated” parts during replication ⁎ Corresponding author at: Department of BioSciences, Andong National University, Andong 760-749, Korea. Tel.: +82 54 820 5638. E-mail address: [email protected] (Y. Kim).

http://dx.doi.org/10.1016/j.cbpa.2014.09.004 1095-6433/© 2014 Elsevier Inc. All rights reserved.

(Bézier et al., 2009b; Burke and Strand, 2012). The “non-encapsidated” proviral genes contribute to produce the viral particles by expressing capsid and assembly factors (Burke et al., 2013). In the meantime, the “encapsidated” proviral genes are mostly associated with the regulation of the parasitized host physiology (Beck et al., 2011; Strand et al., 2013). An endoparasitoid wasp, Cotesia plutellae, parasitizes young larvae of the diamondback moth, Plutella xylostella (Bae and Kim, 2004). The parasitized larvae undergo an immunosuppressive state and exhibit a prolonged larval period by approximately two days at 25 °C (Ibrahim and Kim, 2006; Kwon et al., 2010). A specific PDV called C. plutellae bracovirus (CpBV) has been identified from C. plutellae and plays a crucial role in the parasitism (Kim et al., 2007). The encapsidated CpBV genome was sequenced and annotated to encode 157 genes (Chen et al., 2011). Almost half of the genes are grouped into different PDV canonical gene families proposed by Kroemer and Webb (2004), but the others remain hypothetical genes. Most canonical PDV gene families encoded in the encapsidated CpBV genome have been experimentally assessed in their functions with respect to host immunosuppression (Gad and Kim, 2008; Ibrahim and Kim, 2008; Kwon and Kim, 2008; Nalini et al., 2008; Bae and Kim, 2009; Park and Kim, 2012). In addition, some non-canonical (hypothetical) genes are also under strong positive selection process probably to meet the functional differentiation to defend various and specific host immune responses (Jancek et al., 2013).

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A novel PDV gene family, BEN (BANP, E5R and NAC1), has been proposed in the CpBV genome, which contains eleven members encoded in 10 CpBV segments (Chen et al., 2011; Ali and Kim, 2012). The transient expression of BEN family genes significantly suppressed hemocyte nodule formation in response to bacterial challenge and played a crucial role in inducing a significant suppression in cellular immunity (Ali and Kim, 2012). Furthermore, a BEN family member (CpBV-ORF301) suppresses both cellular and humoral immune responses of host insects by inhibiting specific mRNA expression probably with nuclease activity of its RNase T2 domain (Park and Kim, 2010, 2012). The inhibition of cellular immune response helps to protect the wasp egg and larva from a fatal encapsulation behavior of host hemocytes. However, the inhibition of humoral immune response by CpBV has been poorly understood in the biological significance. In the meantime, some inducible humoral immune responses have been suggested to be associated with antiviral responses of insects (Imler and Eleftherianos, 2009). These led us to impose a hypothesis that BEN family members of CpBV inhibit host humoral immune response to suppress the host antiviral activity. In this study, we analyzed the suppressive effect of BEN family members on expression of antimicrobial peptide (AMP) and prophenoloxidase (PPO) genes of P. xylostella. To test the hypothesis, changes of gene expressions of four AMPs and PPO in response to bacterial challenge were monitored after transient expression of BEN genes. Furthermore, their specific immunosuppressive activities were tested by individual RNA interference (RNAi) of BEN family members. Finally, we analyzed the antiviral responses of the induced humoral immunity on the CpBV infection to understand the physiological significance of the suppressive activity of BEN family genes. 2. Materials and methods

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amplification. No DNA contamination was confirmed by PCRs using the RNA templates. 2.4. RT-PCR Eleven CpBV-BEN genes, four AMP genes, PPO gene, and other CpBV genes were amplified using gene-specific primers (Table S1) under the following conditions: a pre-denaturation step (94 °C, 3 min), followed by 35 amplification cycles (denaturation at 94 °C for 1 min, annealing for 1 min at temperatures as described in Table S1, chain extension at 72 °C for 1 min) and final extension at 72 °C for 10 min. To confirm the cDNA preparation, β-actin expression was analyzed with primers (5′-ATGTACCCTGGTATTGCTCA-3′ and 5′-GGACGATAGAGGGGCCAG AC-3′) by RT-PCR. 2.5. Preparation of individual viral segments containing different CpBV-BEN genes and microinjection BEN domain-containing DNA segments of CpBV were cloned using a plasmid capture system (Choi et al., 2005). For transient expression, each viral DNA segment was mixed with Metafectene PRO transfection reagent (Biontex, Planegg, Germany) according to manufacturer's instruction. Briefly, DNA segment (≈ 200 ng/μL) was mixed with the transfection reagent at 1:1 (v/v) ratio and incubated for 20 min at room temperature to allow DNA-lipid complexes to be formed before injection. Glass capillary injection needles were prepared using a Micropipette puller (PN-30, Narishige, Tokyo, Japan). The DNA-lipid complex was injected into larval hemocoel of P. xylostella through dorsal intersegmental membrane with 0.5 μL volume at a rate of 50 nL/s using a Ultra Micropump (Four) with SYS-microcontroller (World Precision Instruments, Sarasota, FL, USA). Microinjection was performed under a

2.1. Insect rearing and parasitization Larvae of P. xylostella were reared on cabbage leaves at 25 ± 1 °C and 16:8 (L:D) h photoperiod. Adults were fed 10% sucrose. Young larvae (2 days after hatch) of P. xylostella were parasitized with C. plutellae adults at 2:1 (host:wasp) ratio. The parasitized larvae were then allowed to feed cabbage leaves at the same conditions until the end of parasitoid larval development. Adults emerged from the cocoons (11 days after parasitization at 25 ± 1 °C) were collected and allowed to mate for 24 h. The mated adults were used for the parasitization. 2.2. Bacterial culture Bacillus subtilis ATCC6633 and Escherichia coli Top10 (Invitrogen, Carlsbad, CA, USA) bacteria were cultured on Luria-Bertani (LB) agar plates and LB liquid broth at 37 °C. After overnight culture with LB broth, the bacterial cells were collected by centrifugation at 5000 ×g for 10 min and were resuspended with 100 mM phosphate-buffered saline (PBS, pH 7.4) for injection. Bacteria to be injected were counted using a hemocytometer (Superior, Marienfeld, Germany) with the help of a tally counter. 2.3. cDNA preparation of P. xylostella transcripts Total RNAs of P. xylostella larvae were extracted using Trizol (Invitrogen) according to manufacturer's instruction and digested with DNase (Promega, Madison, WI, USA). After the inactivation of DNase at 65 °C for 15 min, first strand cDNA was synthesized by reverse-transcription of RNAs (1 μg) using RT-Premix oligo-dT (5′CCAGTGAGCAGAGTGACGA GGACTCGAGCTCAAGCTTTTTTTTTTTTTTTT3′) kit (Intron Biotechnology, Daejeon, Korea) according to the manufacturer's instruction and subsequently treated with RNase H (Promega). The synthesized cDNA was used as a template for PCR

Fig. 1. Influence of the parasitism by Cotesia plutellae on expression of humoral factors of Plutella xylostella larvae. (A) Diagram of experimental trials. Nonparasitized (NP) individuals spent 8 days (‘D8’) for larval period, while parasitized (P) individuals spent 10 days (‘D10’) and died. Parasitization occurred at the second day (‘D2’). All bacterial challenges began at the third instar at the fifth day (‘D5’). Each bacterial challenge used 5 × 104 cells of Escherichia coli. (B) Expression of five humoral factors at 8 h after the bacterial challenge. The immune genes analyzed in this RT-PCR are cecropin (‘PxCec’), gloverin (‘PxGlv’), hemolin (‘PxHem’), lysozyme (‘PxLys’), and prophenoloxidase (‘PxPPO’). The expressions of different periods in the parasitized larvae are individually compared with those of the corresponding periods in the nonparasitized larvae. All cDNA templates were confirmed by expression of β-actin.

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stereomicroscope (Olympus S730, Tokyo, Japan). After injection, the larvae were fed cabbage and cultured at 25 °C. The gene expression was analyzed by RT-PCR using gene-specific primers described in Table S1. Larvae injected with a mixture of ‘PBS + transfection’ reagent served as a control. 2.6. Suppression of AMP gene expression by transient expression of CpBV-BEN members A transient expression of CpBV-BEN was induced as described above. After 36 h or 48 h, expressions of AMP and PPO genes were induced by injecting 5 × 104 cells of E. coli in a volume of 0.5 μL to larval hemocoel. After 8 h incubation, the total RNAs were extracted from the treated larvae with Trizol (Invitrogen). Inhibitory effects of CpBV-BEN members on AMP and PPO gene expressions were analyzed by RT-PCR using gene-specific primers described in Table S1. 2.7. RNA interference (RNAi) For RNAi of CpBV-BEN genes, specific double-stranded RNAs (dsRNAs) were prepared with Megascript RNAi kit (Ambion, Austin,

TX, USA) according to method as described by Ali and Kim (2012). For dsRNA treatment, a mixture of dsRNA and Metafectene PRO transfection reagent at 1:1 (v/v) ratio was microinjected as described above with a volume of 200 nL into the hemocoel of each P. xylostella larva. The test larvae were pre-injected with the recombinant CpBV-BEN DNA segments at 48 h before the RNAi treatment. After 12 h incubation at 25 °C, the RNAi effect on the targeting BEN gene expression was assessed by RT-PCR using the gene-specific primers described in Table S1. Under RNAi conditions, the immune challenge with bacteria described above was performed and incubated for 8 h at 25 °C to induce expressions of AMP and PPO genes. 2.8. Antibacterial activity by inhibition zone assay Antimicrobial activity of the plasma of P. xylostella was analyzed by an inhibition zone assay described by Haine et al. (2008). Early third instar larvae of P. xylostella were injected with CpBV-BEN DNA segments. After 36 h, BEN gene-specific dsRNAs were injected and incubated for 12 h at 25 °C. To induce humoral immunity, the larvae were further injected with E. coli (5 × 104 cells/larva) using a microinjector as described above. Plasma was collected at 8 h post-bacterial injection

Fig. 2. Suppressive activities of 10 CpBV segments (‘S3–S51’) encoding 11 BEN family members (‘301-5101’) on the expression of three humoral factors in response to bacterial challenge. (A) Transient expression of 11 BEN family genes by microinjection of their corresponding viral segments (50 ng DNA per larva) to nonparasitized third instar larvae of Plutella xylostella. Expression analysis was performed by RT-PCR at different time after injection (‘TAI’). ‘Con’ represents control by injecting the test larvae with PBS + transfection reagent mixture. Expression of β-actin confirms the integrity of cDNA preparation. (B–D) Effect of the viral segment (‘VS’) injection on expression of three humoral factors of cecropin (‘PxCec’), gloverin (‘PxGlv’), and prophenoloxidase (‘PxPPO’). The analyses of humoral factors were conducted at 8 h after the bacterial challenge. The bacterial challenge was conducted with Escherichia coli (5 × 104 cells per larva) at 36 h or 48 h after the VS injection.

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Fig. 2 (continued).

and combined with anticoagulant buffer (ACB). ACB was freshly prepared by dissolving 4 mg L-cysteine hydrochloride (Sigma, USA) in 5 mL Tris-buffered saline (50 mM Tris/HCl, pH 7.5, 100 mM glucose, 5 mM KCl, 2.5 mM MgCl2, and 50 mM NaCl). The collected plasma was centrifuged at 2000 rpm for 3 min to remove fat body or debris. Plasma (10 μL) was treated on paper disc and incubated 8 h at 4 °C for diffusion. Before the placement of the treated disc, the culture plate was plated with 100 μL target bacteria (5 × 105 cfu/mL). Then, the treated disc was put on the plate and cultured for 18 h at 37 °C. Each experiment was independently replicated three times. Diameter of the resulting inhibition zone was measured and compared with control. Kanamycin (30 μg/disc) was used as a positive inhibition reference. 2.9. Isolation, UV inactivation, and plasma treatment of CpBV CpBV was isolated from the ovary of adult C. plutellae females according to the method of Ali et al. (2013). Briefly, 10 female wasps were dissected under a stereomicroscope and the isolated ovaries were washed with PBS and broken at the ovarian calyx area using a sharp tip needle. CpBV suspension in PBS was collected with pipette

and centrifuged at 800 g for 3 min to exclude large cellular debris. The supernatant was then passed through 0.45 μm filter (Pall Corporation, Ann Arbor, MI, USA) using a 3 mL syringe. Viral particles were collected by centrifugation at 12,500 g for 30 min and resuspended in PBS. To inactivate CpBV with UV irradiation, the suspension of active CpBV with PBS was frozen using dry ice-ethanol and irradiation was subsequently performed under germicidal Halogen lamp (Sankyo Denki, Hiratsuka, Japan) for 4 h under CN-TFX (115 V, 60 Hz) UV radiation tube (Vil Ber Lourate, Marne La Vallee, France). For plasma treatment, the suspension of active CpBV was mixed with plasma collected from larvae of P. xylostella in 2:1 volume ratio (0.2 female equivalent virus dose per μL) and incubated for 1 h at 25 °C. All treated and control CpBV samples were micro-injected in a volume of 0.5 μL into hemocoel of the early 4th instar larvae of P. xylostella. The viral activity of CpBV after plasma treatment was assessed in CpBV gene expression as well as its induction of host immunosuppression by measuring increase of susceptibility against the injection of non-pathogenic bacterium. To investigate the viral inactivation by irradiation or plasma treatment, three CpBV genes (CpBV-ORF301, CpBV-H4,

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and CpBV-ELP1 in Table S1) were analyzed by RT-PCR from the treated larvae. To assess the susceptibility change to E. coli, the larvae were pretreated with the virus and subsequently were injected with the bacteria (E. coli, 5 × 106 cfu per larva) at 3 h post-pretreatment. The larvae were fed cabbage and reared at 25 °C to monitor pathogenicity until 24 h after the bacterial injection. Each bacterial treatment used 10 larvae and was replicated three times. 2.10. Statistical analysis All assays were performed with independent replications and plotted by mean ± standard deviation using Sigma plot (version 10.0, Systat Software, Richmond, CA, USA). Statistical differences of means were analyzed by Duncan's multiple range (post hock) test after one way analysis of variance using PROC GLM of SAS Enterprise Guide 4.3 program (SAS Institute Inc., Cary, NC, USA). 3. Results 3.1. Suppression of humoral immunity in the parasitized host In response to parasitization by C. plutellae, the parasitized P. xylostella larvae were analyzed in induction of AMP and PPO gene expressions (Fig. 1). Young larvae were parasitized by C. plutellae (Fig. 1A). Since then, bacterial challenges were performed at different time periods until late larval stages of P. xylostella. Four AMPs and PPO were analyzed in their expressions at 8 h after the bacterial challenge (Fig. 1B). In nonparasitized larvae, all these humoral factors were expressed following the bacterial challenge. However, in parasitized larvae, the expressions of cecropin, gloverin, and PPO were highly

suppressed, while hemolin and lysozyme were less affected. Subsequent analyses of BEN family genes used transcriptions of cecropin, gloverin, and PPO. 3.2. All CpBV segments containing BEN family genes suppress the expression of the humoral factors To analyze the effects of BEN family genes on expression of the three immune genes, CpBV segments containing BEN family genes were individually injected to nonparasitized larvae to express their own BEN genes (Fig. 2). Each segment contains a BEN gene except segment number 33 (‘S33’) containing two BEN genes. When the CpBV segments were injected, the BEN genes were expressed at least for 24 h postinjection (Fig. 2A). Then we analyzed the suppressive activities of different BEN family genes on the three immune genes at 36 h and 48 h post-injection, respectively (Fig. 2B–D). Three immune genes were inducible in their expressions after the bacterial challenge. However, most larvae pre-injected with CpBV segments encoding BEN family genes did not effectively express the three immune genes as early as 36 h post-injection. The suppressive effect on the expression of the immune genes was remarkable in all the injected segments 48 h post-injection. 3.3. RNAi of BEN gene expression rescued the inducible expressions of humoral immune factors To determine the individual functions of BEN family members, the dsRNA specific to each BEN family gene was injected to the larvae treated with the CpBV DNA segments (Fig. 3). Each dsRNA specifically suppressed the corresponding target mRNA among all mRNAs of the

Fig. 3. RNA interference of 11 BEN family members (‘301-5101’) with specific double-stranded RNAs (dsRNAs) in Plutella xylostella larvae injected with each of 10 BEN-containing CpBV segments (‘S3-S51’). Each viral segment was injected to third instar larvae (50 ng DNA per larva). After 48 h, each specific dsRNA was injected in 100 ng per larva. After another 12 h, the total RNA was extracted for RT-PCR analysis. To confirm the specific inhibition of target BEN gene by the corresponding dsRNA, all the predicted genes in the viral segments were analyzed in their expression after the dsRNA injection.

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encoding genes in each segment. For example, the injection of S3 segment (‘CpBV-S3’) induced both ORF301 and ORF302 genes encoded in the segment. However, the co-injection of dsRNA specific to ORF301 along with S3 (‘CpBV-S3 + dsRNA301’) specifically suppressed the expression of ORF301 BEN family gene. In S5 segment (‘CpBV-S5’) injection, the ORF503 was not expressed in control. Similarly, ORF5105 and ORF5107 were not expressed in control. Under these RNAi conditions of different BEN family genes, all three immune genes suppressed by the expression of BEN family genes were assessed in recovery of their expressions (Fig. 4). Any expression of 11 BEN family genes (‘301-5101’) by injection of viral segment (‘VS’) inhibited the inducible expression of cecropin (‘PxCec’) (Fig. 4A). However, the co-injection of the dsRNAs specific to the BEN family genes rescued the gene expression in response to bacterial challenge. Similarly, the expressions of gloverin (‘PxGlv’, Fig. 4B) and prophenoloxidase (‘PxPPO’, Fig. 4C) were rescued by the specific RNAi against BEN family genes. These RNAi experiment supported the suppressive function of BEN family genes in transcription of the humoral immune genes. The inhibitory effect of different BEN family genes on expression of antimicrobial genes was then assessed in humoral immune responses using an antibacterial assay (Fig. 5). The humoral immune response was analyzed using an inhibition zone assay against the growth of Gram-positive (B. subtilis) and Gram-negative (E. coli) bacteria. All injections of different CpBV segments significantly inhibited the humoral immunity to prevent the bacterial growth. However, the addition of the specific dsRNA to the CpBV DNA segment rescued the humoral immune responses. These results indicated that BEN family genes suppressed the humoral immune responses. 3.4. Significance of humoral immunity in antiviral activity The suppressive activity of BEN family genes on humoral immunity was assessed in suppressing antiviral activity induced by P. xylostella

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larvae (Fig. 6). The viral activity was measured in the expression of the encoded genes and the immunosuppression. When the purified CpBV was injected to nonparasitized larvae, the selected three CpBV genes (ORF301, CpBV-H4, and CpBV-ELP1) were expressed as early as 3 h after the CpBV injection and maintained their expression at least for 24 h (Fig. 6A). Thus, the purified CpBV was active in terms of expression of its encoded genes. However, when the virus was exposed to UV, it completely lost its expression activity of the three CpBV genes tested in this assay (Fig. 6B). As it was shown that the plasma of P. xylostella exhibited humoral immunity, which was suppressed by expression of BEN family genes as seen in Fig. 5. When the virus was incubated with the plasma collected from naive larvae, it still expressed the three CpBV genes with a little depressed activity, in which the transcript abundances of CpBV-H4 and CpBV-ELP1 were significantly decreased, suggesting the presence of some antiviral activity in the naïve plasma. However, when the virus was incubated with the plasma collected from the larvae challenged with bacteria, it severely lost the expression activity of the three CpBV genes. However, the plasma collected from the larvae injected with CpBV-S3 along with the bacterial challenge remarkably rescued the expression activity of the viral genes. The antiviral activity of the humoral immune response was further supported by the immunosuppressive activity due to CpBV injection (Fig. 6C). Infact CpBV significantly suppressed the host immune responses and allowed the nonpathogenic bacterial growth to kill the host larvae with more than 90% mortality. However, UV-treated CpBV failed to inhibit host immune responses and did not kill the host larvae following bacterial challenge. When CpBV was incubated with the plasma collected from the larvae challenged with bacteria, it significantly lost the inhibitory activity on host immune responses and was much less effective to suppress host immune response because more than 50% individuals treated survived after the bacterial challenge. Again, the plasma collected from the larvae injected with CpBV-S3 followed by bacterial challenge enhanced the mortality by suppressing humoral immune responses.

Fig. 4. Recovery in expressions of three humoral factors by RNA interference of BEN family gene expressions in response to bacterial challenge in Plutella xylostella injected with BENcontaining CpBV viral segments (‘VS’). The third instar larvae were injected with different VSs (50 ng DNA per larva) containing different BEN family genes (‘301-5101’). After 36 h, a dsRNA (100 ng per larva) specific to a partial open reading frame (‘ORF’) sequence of the injected VS-corresponding BEN family gene was injected and incubated for 12 h. Thereafter, 5 × 104 cells of Escherichia coli were injected to each larva. After 8 h, the total RNA was extracted for RT-PCR analysis of three humoral factors: cecropin (‘PxCec’), gloverin (‘PxGlv’), and prophenoloxidase (‘PxPPO’).

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4. Discussion The suppression of humoral immunity following BEN gene expressions enhanced the expressions of CpBV genes. On the other hand, RNAi of BEN genes adversely affected the CpBV gene expressions. Furthermore, the negative factor(s) against CpBV gene expressions was involved in the plasma and was potentiated by bacterial challenge. These results suggest that the humoral factors including AMPs and PPO act as antiviral agents against CpBV. In general, the antiviral responses in insects have been known as RNAi against RNA viruses, apoptosis mostly against DNA viruses, and inducible humoral responses (Imler and Eleftherianos, 2009). In response to PDV including CpBV, which is a dsDNA virus, apoptosis and humoral immunity may be induced to suppress the viral activity. Apoptosis can be explained by two different ways in PDV infection: virulence induced by PDV against host insect and antiviral response induced by host insect against PDV. The virulence

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aspect is well demonstrated in several PDVs. The infection of Microplitis demolitor BV induced apoptosis of granulocytes of the larvae to suppress cellular immune responses (Strand and Pech, 1995). Similarly, Campoletis sonorensis IV (CsIV) also appears to induce the apoptosis of plasmatocytes (Davies and Vinson, 1988; Webb and Luckhart, 1996). The hemocyte apoptosis was also observed in Spodoptera litura parasitized by Microplitis bicoloratus due to the symbiotic PDV (Luo and Pang, 2006). Interestingly, co-infection with baculovirus containing an inhibitor of apoptosis suppressed the hemocyte apoptosis (Luo and Pang, 2006). Alternatively, the direct contribution of PDVs to defend host antiviral apoptosis was demonstrated by the anti-apoptotic action of a viral IκB encoded in CsIV, in which the viral factor inhibited nuclear and internucleosomal degradation and caspase activity in the cells induced to undergo apoptosis (Fath-Goodin et al., 2009). Indeed, a viral IκB encoded in CpBV was inserted to a baculovirus and orally fed to larvae of P. xylostella (Bae and Kim, 2009). The recombinant

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Fig. 5. Suppression of antimicrobial activity of Plutella xylostella by BEN family members. In the first control group, the third instar larvae were challenged with Escherichia coli (5 × 104 cells per larva) to induce antimicrobial peptide (‘AMP’) gene expression. In the second group, larvae were injected with each CpBV segment (‘S3-S51’) containing different BEN family genes and followed by bacterial challenge at 48 h post-injection. In the third group, larvae were injected with the CpBV segments. After 36 h, dsRNA (100 ng per larva) specific to the injected VScorresponding BEN family gene was injected and incubated for 12 h. Thereafter, 5 × 104 cells of E. coli were injected to each larva. At 8 h after the bacterial challenge, hemolymph was collected and analyzed for the antibacterial growth analysis. The plasma was used for inhibition zone assay against Gram-positive bacterium, B. subtilis and Gram-negative bacterium, E. coli. Each treatment was independently replicated three times. Different letters above standard deviation bars indicate significant difference among means at Type I error = 0.05 (LSD test).

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Fig. 5 (continued).

baculovirus significantly reduced the midgut melanotic response, which was highly induced at the treatment of wild-type baculovirus (Bae and Kim, 2009). Thus, the apoptosis can be regarded as a main antiviral response against PDV infection in some cases. This current study showed that inducible humoral responses play a crucial role in the antiviral responses, which is significantly inhibited by suppressive activity of BEN family genes against humoral gene expression. A main inducible humoral defense against viral infection is expressed by genes under control of Janus kinase-signal transduction and activators of transcription (JAK-STAT) signaling pathway (Lin et al., 2004; Dostert et al., 2005). Any mutation of signal components in the JAK-STAT pathway enhanced the viral infectivity in Drosophila melanogaster (Hedges and Johnson, 2008). However, little is known in any functional study of JAK-STAT signal on the antiviral response of the host infected with PDVs. Interestingly, the endoparasitoids induce the expression of host AMPs at parasitization. Indeed, the parasitism by Diadegma semiclausum up-regulated the AMP expression of P. xylostella (Etebari et al., 2011). CpBV inhibits the AMP expression of P. xylostella parasitized by C. plutellae (Shrestha et al., 2009; Barandoc et al., 2010). Parasitism by M. demolitor also activates NF-κB signaling, which is subsequently suppressed by its symbiotic PDV using the viral IκBs (Bitra et al., 2012). In Drosophila, there is no clear evidence to link Toll/Imd pathways to antiviral immunity, though Drosophila X virus induces AMP genes using a unique pathway of Toll signaling (Zambon et al., 2005). Thus, the suppression of AMP gene expressions induced by BEN family genes can be considered as the viral defense against host antiviral activity. Results of our current study supported the hypothesis of the antiviral activity of the BEN family genes. Plasma extracted from the larvae challenged by bacteria possessed antibacterial activity along with the induction of AMP and PPO expressions and significantly inactivated CpBV with respect to the viral expression in treated larvae of P. xylostella. Indeed, cecropin exhibits an antiviral activity by inhibiting viral multiplication of the arenavirus Junin virus, presumably through interfering with virus-cell fusion (Albiol Matanic and Castilla, 2004; Lee et al., 2004). Gloverin also possesses an antiviral

activity against baculovirus presumably by disruption of membrane integrity of budded viral form (Moreno-Habel et al., 2012). PO activity is also associated with antiviral immunity against the budded form of baculovirus in Heliothis virescens presumably by production of POdependent H2O2 (Shelby and Popham, 2006). Thus, PDVs inhibit PO activity to protect the host parasitoid eggs as well as the virus from the fatal melanization mediated by the enzyme (Bae and Kim, 2004; Beck and Strand, 2007). These explain the significance of the inhibitory activity of BEN family genes against AMP and PO expressions with respect to protecting CpBV from the antiviral activities. BEN gene family was originally identified from the analysis of transcription regulation and chromatin remodeling, in which the unique α-helical motif is found in diverse animal and viral proteins, such as cnidarian NEMVEDRAFT protein, human NAC1 protein, and viral E5R protein (Abhiman et al., 2008). Especially, BEN proteins of PDVs share sequence homologies with NAC1 proteins that possess POZ domain interacting with transcriptional repressor, such as histone deacetylase, or directly binding to target DNA, suggesting that CpBV-BEN family members can directly or indirectly control host gene expression. Furthermore, some BEN family members also encode RNase T2 domain (Ali and Kim, 2012), suggesting the putative role of BEN family members to degrade host specific mRNAs. Indeed, Park and Kim (2012) showed that the viral RNase suppresses the expression of AMP genes in P. xylostella. Thus the down-regulation of host humoral immune response may be explained by the protein–protein or protein–chromatin interactions of CpBV-BEN family members. The identification of host molecular target(s) interacting with CpBV-BEN family members isnecessary to understand the viral activity. In P. xylostella parasitized by C. plutellae, various immunosuppressive factors have been isolated from CpBV, teratocytes, and ovarian proteins, in which almost half of CpBV genes (a half of putative 156 genes) have been considered to be immunosuppressants (Kim et al., 2007). Teratocyte also seems to secrete various immunosuppressants (Basio and Kim, 2005; Ali et al., 2013). Ovarian proteins of C. plutellae also possess immunosuppressive activity against hemocyte behaviors

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some AMP genes (lysozyme and hemolin) in the larvae parasitized by C. plutellae were not inhibited in their inducible expression in response to bacterial challenge, though other humoral factors were severely inhibited in their expressions. Alternatively, the additional immune factors should be originated from C. plutellae under a complete immunosuppression of host immunity. A recent study on C. plutellae showed that the parasitized larvae express different defensin types of AMPs presumably to protect against any pathogenic microbes (Wang et al., 2013). In summary, this study reports a novel function of BEN family members in terms of host immunosuppression in humoral responses. In addition, this immunosuppressive activity of BEN family members can be considered to protect the CpBV viral activity from the host antiviral response associated with inducible humoral immune responses.

(A)

(B)

Acknowledgments This study was supported by a grant to YK from National Research Foundation (NRF-2013R1A1A2A10058197) of Korea. It was also partially supported by Biogreen project of Rural Development Administration (PJ009020), Suweon, Korea.

(C) a

100

a

Mortality (%)

80

Appendix A. Supplementary data

b Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cbpa.2014.09.004.

60

c 40

References

d

20 0

d PBS

d

d

d

PL E. coli PDV

V PD

UV

PD

V

PL

V PD

PD

PL

V

(B

AC

) PL

PD

(S

3+

BA

C)

V

+ E. coli Fig. 6. Suppression of antiviral activity of humoral immunity of Plutella xylostella by a BEN family gene. (A) Time course of expression of three different CpBV genes, such as ORF301 (‘CpBV-BENORF301’), a viral histone H4 (‘CpBV-H4’), and EP1-like protein 1 (‘CpBV-ELP1’), after injection of CpBV (‘PDV’, 0.1 female equivalent (FE) CpBV per larva). Expression of β-actin confirms the cDNA integrity. (B) Effect of humoral immunity on expression of the three CpBV genes. PDV was treated as described in Materials and methods with UV (‘PDVUV’), immunized plasma (‘PDVPL(BAC)’) or CpBV-S3 + immunized plasma (‘PDVPL(S3 + BAC)’). For PDVPL, CpBV was incubated with non-immunized PL. For PDVPL(BAC), the third instar larvae of P. xylostella were treated by injection of 5 × 104 cells of Escherichia coli. After 8 h at 25 °C, plasma (‘PL’) was collected and incubated with CpBV for 1 h at 25 °C. PDVPL(S3 + BAC) represents an incubation of CpBV in plasma collected from larvae that were treated with CpBV-S3 (50 ng per larva) and the bacterial injection after 48 h of the segment injection. PDVUV represents an incubation of CpBV under UV light for 4 h. PDV represents a control CpBV without any treatment. All treated PDV samples were injected to the third instar larvae at 0.1 FE CpBV dose. After 3 h postinjection, the expressions of the three CpBV genes were analyzed by RT-PCR. (C) Susceptibility of P. xylostella larvae treated with different PDV samples to nonpathogenic bacterial infection. Different PDV samples described above were injected to third instar larvae in 0.1 FE. ‘Con’ represents PBS injection. To assess the susceptibility to bacterial infection, the larvae pretreated with the virus were injected with the bacteria (E. coli, 5 × 106 cfu per larva) at 3 h post-pretreatment. The larvae were fed cabbage and reared at 25 °C to monitor pathogenicity until 24 h after the bacterial injection. Each treatment was replicated three times with 10 larvae per replication. Different letters above standard deviation bars indicate significant difference among means at Type I error = 0.05 (LSD test).

P. xylostella (Basio and Kim, 2006). Altogether, the larvae of P. xylostella parasitized by C. plutellae should undergo a severe immunosuppressive state in both cellular and humoral immune responses. Then how do the host and parasitized larvae defend any infection of other pathogenic microbes? This may require an ideal optimization in the suppressive intensity of the parasitoid wasp against host immunity. In this study,

Abhiman, S., Iyer, L.M., Aravind, L., 2008. BEN: a novel domain in chromatin factors and DNA viral proteins. Bioinformatics 24, 458–461. Albiol Matanic, V.C., Castilla, V., 2004. Antiviral activity of antimicrobial cationic peptides against Junin virus and herpes simplex virus. Int. J. Antimicrob. Agents 23, 382–389. Ali, M.R., Kim, Y., 2012. A novel polydnaviral gene family, BEN, and its immunosuppressive function in larvae of Plutella xylostella parasitized by Cotesia plutellae. J. Invertebr. Pathol. 110, 389–397. Ali, M.R., Seo, J., Lee, D., Kim, Y., 2013. Teratocyte-secreting proteins of an endoparasitoid wasp, Cotesia plutellae, prevent host metamorphosis by altering endocrine signals. Comp. Biochem. Physiol. A 166, 251–262. Bae, S., Kim, Y., 2004. Host physiological changes due to parasitism of a braconid wasp Cotesia plutellae on diamondback moth Plutella xylostella. Comp. Biochem. Physiol. 138A, 39–44. Bae, S., Kim, Y., 2009. IkB genes encoded in Cotesia plutellae bracovirus suppress an antiviral response and enhance baculovirus pathogenicity against the diamondback moth, Plutella xylostella. J. Invertebr. Pathol. 102, 79–87. Barandoc, K.P., Kim, J., Kim, Y., 2010. Cotesia plutellae bracovirus suppresses expression of an antimicrobial peptide, cecropin, in the diamondback moth, Plutella xylostella, challenged by bacteria. J. Microbiol. 48, 117–123. Basio, N.A.M., Kim, Y., 2005. A short review of teratocyte and their characters in Cotesia plutellae (Braconidae:Hymenoptera). J. Asia Pac. Entomol. 8, 211–217. Basio, N.A.M., Kim, Y., 2006. Additive effect of teratocyte and calyx fluid from Cotesia plutellae on immunosuppression of Plutella xylostella. Physiol. Entomol. 31, 341–347. Beck, M.H., Strand, M.R., 2007. A novel polydnavirus protein inhibits the insect prophenoloxidase activation pathway. Proc. Natl. Acad. Sci. U. S. A. 104, 19267–19272. Beck, M.H., Zhang, S., Bitra, K., Burke, G.R., Strand, M.R., 2011. The encapsidated genome of Microplitis demolitor bracovirus integrates into the host Pseudoplusia includens. J. Virol. 85, 11685–11696. Bézier, A., Annaheim, M., Herbiniere, J., Wetterwald, C., Gyapay, G., Bernard-Samain, S., Wincker, P., Roditi, I., Heller, M., Belghazi, M., Pfister-Wilhem, R., Periquet, G., Dupuy, C., Huguet, E., Volkoff, A.N., Lanzrein, B., Drezen, J.M., 2009a. Polydnaviruses of braconid wasps derive from an ancestral nudivirus. Science 323, 926–930. Bézier, A., Herbiniere, J., Lanzrein, B., Drezen, J.M., 2009b. Polydnavirus hidden face: the genes producing virus particles of parasitic wasps. J. Invertebr. Pathol. 101, 194–203. Bitra, K., Suderman, R.J., Strand, M.J., 2012. Polydnavirus Ank proteins bind NF-κB homodimers and inhibit processing of relish. PLoS Pathog. 8, e1002722. Burke, G.R., Strand, M.R., 2012. Deep sequencing identifies viral and wasp genes with potential roles in replication of Microplitis demolitor bracovirus. J. Virol. 86, 3293–3306. Burke, G.R., Thomas, S.A., Eum, J.H., Strand, M.R., 2013. Mutualistic polydnaviruses share essential replication gene functions with pathogenic ancestors. PLoS Pathog. 9, e1003348. Chen, Y., Gao, F., Ye, X., Wei, S., Shi, M., Zheng, H., Chen, X., 2011. Deep sequencing of Cotesia vestalis bracovirus reveals the complexity of a polydnavirus genome. Virology 414, 42–50. Choi, J.Y., Roh, J.Y., Kang, J.N., Shim, H.J., Woo, S.D., Jin, B.R., Li, M.S., Je, Y.H., 2005. Genomic segments cloning and analysis of Cotesia plutellae polydnavirus using plasmid capture system. Biochem. Biophys. Res. Commun. 332, 487–493.

M.R. Ali, Y. Kim / Comparative Biochemistry and Physiology, Part A 179 (2015) 44–53 Davies, D.H., Vinson, S.B., 1988. Interference with function of plasmatocytes of Heliothis virescens in vivo by calyx fluid of the parasitoid Campoletis sonorensis. Cell Tissue Res. 251, 467–475. Dostert, C., Jouanguy, E., Irving, P., Troxler, L., Galiana-Arnoux, D., Hetru, C., Hoffmann, J.A., Imler, J.L., 2005. The JAK-STAT signalling pathway is required but not sufficient for the antiviral response of Drosophila. Nat. Immunol. 6, 946–953. Etebari, K., Palfreyman, R.W., Schlipalius, D., Nielsen, L.K., Glatz, R.V., Asgari, S., 2011. Deep sequencing-based transcriptome analysis of Plutella xylostella larvae parasitized by Diadegma semiclausum. BMC Genomics 12, 446–463. Fath-Goodin, A., Kroemer, J.A., Webb, B.A., 2009. Campoletis sonorensis ichnovirus vankyrin protein P-vank-I inhibits apoptosis in insect Sf9 cells. Insect Mol. Biol. 18, 497–506. Gad, W., Kim, Y., 2008. A viral histone H4 encoded by Cotesia plutellae bracovirus inhibits haemocyte-spreading behavior of the diamondback moth, Plutella xylostella. J. Gen. Virol. 89, 931–938. Haine, E.R., Pollitt, L., Moret, Y., Siva Jothy, M.T., Rolff, J., 2008. Temporal patterns in immune responses to a range of microbial insults (Tenebrio molitor). J. Insect Physiol. 54, 1090–1097. Hedges, L.M., Johnson, K.N., 2008. Induction of host defense responses by Drosophila C virus. J. Gen. Virol. 89, 1497–1501. Ibrahim, A.M., Kim, Y., 2006. Parasitism by Cotesia plutellae alters the hemocyte population and immunological function of the diamondback moth, Plutella xylostella. J. Insect Physiol. 52, 943–950. Ibrahim, A.M., Kim, Y., 2008. Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses. Naturwissenschaften 95, 25–32. Imler, J.L., Eleftherianos, I., 2009. Drosophila as a model for studying antiviral defense. In: Rolft, J., Reynolds, S.E. (Eds.), Insect Infection and Immunity. Oxford University Press, Oxford, UK, pp. 49–68. Jancek, S., Bézier, A., Gayral, P., Paillusson, C., Kaiser, L., Dupas, S., Le Ru, B.P., Barbe, V., Periquet, G., Drezen, J.M., Herniou, E.A., 2013. Adaptive selection on bracovirus genomes drives the specialization of Cotesia parasitoid wasps. PLoS One 8, e64432. Kim, Y., Choi, Y., Je, Y.H., 2007. Cotesia plutellae bracovirus genome and its function in altering insect physiology. J. Asia Pac. Entomol. 10, 181–191. Kroemer, J.A., Webb, B.A., 2004. Polydnavirus gene and genomes: emerging gene families and new insights into polydnavirus replications. Annu. Rev. Entomol. 49, 431–456. Kwon, B., Kim, Y., 2008. Transient expression of an EP1-like gene encoded in Cotesia plutellae bracovirus suppresses the hemocyte population in the diamondback moth, Plutella xylostella. Dev. Comp. Immunol. 32, 932–942. Kwon, B., Song, S., Choi, J.Y., Je, Y.H., Kim, Y., 2010. Transient expression of specific Cotesia plutellae bracoviral segments induces prolonged larval development of the diamondback moth, Plutella xylostella. J. Insect Physiol. 56, 650–658. Lee, D.G., Park, Y., Jin, I., Hahm, K.S., Lee, H.H., Moon, Y.H., Woo, E.R., 2004. Structureantiviral activity relationships of cecropin A-magainin 2 hybrid peptide and its analogues. J. Pept. Sci. 10, 298–303. Lin, C.C., Chou, C.M., Hsu, Y.L., Lien, J.C., Wang, Y.M., Chen, S.T., Tsai, S.C., Hsiao, P.W., Huang, C.J., 2004. Characterization of two mosquito STATs AaSTAT and CtSTAT. Differential regulation of tyrosine phosphorylation and DNA binding acting by lipopolysaccharide treatment and by Japanese encephalitis virus infection. J. Biol. Chem. 279, 3308–3317.

53

Luo, K.J., Pang, Y., 2006. Spodoptera litura multicapsid nucleopolyhedrovirus inhibits Microplitis bicoloratus polydnavirus-induced host granulocytes apoptosis. J. Insect Physiol. 52, 795–806. Moreno-Habel, D.A., Biglang-awa, I.M., Dulce, A., Luu, D.D., Garcia, P., Weers, P.M., Haas-Stapleton, E.J., 2012. Inactivation of the budded virus of Autographa californica M nucleopolyhedrovirus by gloverin. J. Invertebr. Pathol. 110, 92–101. Nalini, M., Choi, J.Y., Je, Y.H., Hwang, I., Kim, Y., 2008. Immunoevasive property of a polydnaviral product, CpBV-lectin, protects the parasitoid egg from hemocytic encapsulation of Plutella xylostella (Lepidoptera: Yponomeutidae). J. Insect Physiol. 54, 1125–1131. Park, B., Kim, Y., 2010. Transient transcription of a putative RNase containing BEN domain encoded in Cotesia plutellae bracovirus induces an immunosuppression of the diamondback moth, Plutella xylostella. J. Invertebr. Pathol. 105, 156–163. Park, B., Kim, Y., 2012. Immunosuppression induced by expression of viral RNase enhances susceptibility of Plutella xylostella to microbial pesticides. Insect Sci. 19, 47–54. Shelby, K.S., Popham, H.J., 2006. Plasma phenoloxidase of the larval tobacco budworm, Heliothis virescens, is virucidal. J. Insect Sci. 6, 1–12. Shrestha, S., Kim, H.H., Kim, Y., 2009. An inhibitor of NF-κB encoded in Cotesia plutellae bracovirus inhibits expression of antimicrobial peptides and enhances pathogenicity of Bacillus thuringiensis. J. Asia Pac. Entomol. 12, 277–283. Stoltz, D.B., 1990. Evidence for chromosomal transmission of polydnavirus DNA. J. Gen. Virol. 71, 1051–1056. Strand, M.R., Pech, L.L., 1995. Microplitis demolitor polydnavirus induces apoptosis of a specific haemocyte morphotype in Pseudoplusia includens. J. Gen. Virol. 76, 283–291. Strand, M.R., Pennacchio, F., Denlinger, D.L., 2013. Immune interactions between insects and their natural antagonists: a workshop honoring Professor Stuart E. Reynolds. J. Insect Physiol. 59, 121–240. Volkoff, A.N., Jouan, V., Urbach, S., Samain, S., Bergoin, M., Wincker, P., Demettre, E., Cousserans, F., Provost, B., Coulibly, F., Legeai, F., Béliveau, C., Cusson, M., Gyapay, G., Drezen, J.M., 2010. Analysis of virion structural components reveals vestiges of the ancestral ichnovirus genome. PLoS Pathog. 6, e1000923. Wang, Z.Z., Shi, M., Zhao, W., Bian, Q.L., Ye, G.Y., Chen, X.X., 2013. Identification and characterization of defensin genes from the endoparasitoid wasp Cotesia vestalis (Hymenoptera: Braconidae). J. Insect Physiol. 59, 1095–1103. Webb, B.A., Luckhart, S., 1996. Factors mediating short- and long-term immune suppression in a parasitized insect. J. Insect Physiol. 42, 33–40. Webb, B., Strand, M.R., 2005. The biology and genomics of polydnaviruses. In: Iatrou, K., Gilbert, L., Gill, S. (Eds.), Comprehensive Molecular Insect Science vol. 6. Elsevier, Amsterdam, pp. 323–360. Webb, B.A., Beckage, N.E., Hayakawa, Y., Krell, P.J., Lanzrein, B., Stoltz, D.B., Strand, M.R., Summers, M.D., 2000. Polydnaviridae. In: van Regenmortel, M.H.V., Faquet, C.M., Bishop, D.H.L., Carstens, E.B., Estes, M.K., Lennon, S.M., Maniloff, J., Mayo, M.A., McGeoch, D.J., Pringle, C.R., Wickner, R.B. (Eds.), Virus Taxonomy. Academic Press, New York, pp. 253–260. Wyler, T., Lanzrein, B., 2003. Ovary development and polydnavirus morphogenesis in the parasitic wasp Chelonus inanitus. II. Ultrastructural analysis of calyx cell development, virion formation and release. J. Gen. Virol. 84, 1151–1163. Zambon, R.A., Nandakumar, M., Vakharia, V.N., Wu, L.P., 2005. The Toll pathway is important for an antiviral response in Drosophila. Proc. Natl. Acad. Sci. U. S. A. 102, 7257–7262.