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. 2025 May 20;33(4):1541–1553. doi: 10.1111/1744-7917.70073

BmADARa cooperatively inhibits BmNPV proliferation through the interaction of its dsRBD2 with BmDcr‐2‐DEXHc in silkworm, Bombyx mori

Song Jiang 1,#, Chong‐Jun Ye 2,#, Yu‐Chen Wu 1,##, Ruo‐Yun Shi 1, Yu‐Long Yu 1, Syeda Saneela 1, Dan Liang 3, Yan‐Jiao Huang 1,4,5, Xia‐Ming Shi 1,4,5, Yan Meng 1,4,5,✉
PMCID: PMC13440982  PMID: 40394905

Abstract

Adenosine deaminases that act on RNA (ADARs) are RNA editing enzymes capable of converting adenosine into inosine at specific sites within double‐stranded RNA (dsRNA), widely distributed across various animal species. Dicer (Dcr), a member of the RNase III family and a crucial component of the RNA‐induced silencing complex (RISC), allows ADAR to participate in innate immunity through Dcr‐2 in Drosophila. Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the viruses that can cause substantial economic losses to the sericulture industry upon infecting silkworm. Knocking down the expression of BmDcr‐2 in silkworm enhances the proliferation of BmNPV. Our previous research revealed the existence of a predominantly expressed subtype, ADARa, in silkworm (BmADARa), which shares homology with Drosophila ADAR. It remains unclear whether BmADARa can also participate in innate immunity through BmDcr‐2. Initially, through bacterial challenge experiments, we found that BmADARa exhibited the highest responsiveness to BmNPV stimulation. Further studies demonstrated that BmADARa, in conjunction with BmDcr‐2‐DEXHc (DEAD‐box helicase domain), collectively inhibits the proliferation of BmNPV. BmADARa interacts with the DEXHc domain of BmDcr‐2 through its dsRNA binding domain 2 (dsRBD2), thereby enhancing its ability to inhibit BmNPV proliferation. These results lay a foundation for the study of the function and molecular mechanism of BmADARa in innate immunity, and provide a new experimental ideas for antiviral research in B. mori.

Keywords: BmADARa, BmDcr‐2, BmNPV proliferation, RNAi pathway, silkworm

In silkworms, when BmNPV infects cells, the expression level of BmADARa increases with the infection time. The elevated concentration of BmADARa facilitates the binding of dsRBD2 to BmDcr‐2‐DEXHc, enhancing their relative binding affinity (solid arrow). The interaction between BmADARa and BmDcr‐2 improves the ability of BmDcr‐2 to recognize viral dsRNA, increasing the rate of dsRNA cleavage into siRNA. Additionally, during the formation of RISC, the BmADARa‐BmDcr‐2 heterodimer may also maintain the stability and function of RISC, promoting the cleavage of viral RNA by RISC and thereby inhibiting viral replication (dashed arrow).

graphic file with name INS-33-1541-g002.jpg

Introduction

Adenosine deaminases that act on RNA (ADARs) catalyze the deamination of adenosine residues to inosine in double‐stranded RNA (dsRNA), a process known as A‐to‐I RNA editing. ADARs can encode a limited number of neurotransmitter and ion channel genes, thus increasing their functional diversity (Gerber & Keller, 2001; Hood & Emeson, 2012); however, this editing predominantly occurs in noncoding regions (Ramaswami et al., 2012). ADARs share a similar structure at the N‐terminal with 2 or 3 dsRNA binding domains (dsRBD) while highly conserving adenosine deaminase domains at the C‐terminal. There are 3 types of ADARs in mammals (Nishikura, 2010): ADAR1, which regulates immune responses (Liddicoat et al., 2015); ADAR2, which primarily regulates functions related to the nervous system (Hartner et al., 2004; Liddicoat et al., 2015); and ADAR3, which is exclusively expressed in the nervous system, with only functional dsRBD, thus lacking editing activity (Kurup et al., 2022). Both the p150 and p110 isoforms of ADAR1 and ADAR2 can induce deamination of viral dsRNA.

Dicer being a member of the RNase III gene family, processes the precursor microRNAs (pre‐miRNAs) into mature microRNAs (miRNAs) (Krol et al., 2010) with a key role of stabilizing the structure and function of intermediate RNA‐induced silencing complexes (RISCs) during RISC assembly. In Drosophila, there are 2 Dicer proteins: Dicer‐1 (Dcr‐1) and Dicer‐2 (Dcr‐2) (Kim et al., 2006). Dcr‐1 cleaves pre‐miRNAs to produce mature miRNAs, thereby regulating the expression of endogenous genes. Dcr‐2 is essential for antiviral defense, and flies lacking functional Dcr‐2 are more susceptible to viral infections such as Flock House virus (FHV) (Han et al., 2011). Studies have reported that Drosophila ADAR (dADAR) participates in innate immunity via Dcr‐2 (Deng et al., 2020). It has been found that A‐to‐I RNA editing of some primary miRNAs (pri‐miRNAs) can inhibit miRNA processing (Yang et al., 2006; Kawahara et al., 2008) or prevent RISC assembly (Iizasa et al., 2010). Interestingly, ADAR1 can interact with Dicer to form a heterodimer, increasing Dicer's cleavage rate and promoting miRNA assembly into RISC (Ota et al., 2013). Similarly, the silkworm genome contains 2 Dicer genes, BmDcr‐1, and BmDcr‐2. BmDcr‐2 includes DEAD‐box helicase domain (DEXHc), ERCC4‐related helicase domain (MPH1), Dicer dimerization domain (dimer), ribonuclease III domain (RIBOc), and PAZ domain (PAZ) (Li et al., 2020). Its functions are analogous to those of Dicer‐2 in mammals and Drosophila.

During the growth and development of silkworm, infections by various pathogenic microorganisms can cause significant losses to sericulture (Chen & Lu, 2018). The Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most severe pathogenic viruses in the sericulture industry, leading to substantial economic losses worldwide (Kleino & Silverman, 2014). During the BmNPV infection cycle, 2 distinct viral particle phenotypes are produced: occlusion‐derived virus (ODV) and budded virus (BV) (Jiang et al., 2012; L. Jiang & Xia, 2014). ODV initiates primary infection targeting midgut epithelial cells of silkworm, facilitating its spread through populations, while BV specifically infects various endogenous tissues and cells of the host (Slack & Arif, 2007). Following BmNPV infection, the expression level of the Dcr‐2 gene is significantly increased in immune organs such as the midgut and hemocytes of silkworm (Li et al., 2020).

In earlier research, our laboratory was the first to clone the full‐length sequence of BmADARa. We found that BmADARa is similar to dADAR and both are homologous of mammalian ADAR2, sharing some functions with mammalian ADAR1. Tissue expression profiling and western blot analysis revealed that BmADARa is highly expressed in the fat body (Ye et al., 2020). Based on these findings, we hypothesize that the high expression of BmADARa in the fat body is somewhat related to the innate immunity of silkworm. ADAR‐mediated innate immune response has been extensively studied in mammals, but the innate immunity regulated by BmADARa in silkworm has rarely been reported.

RNA interference (RNAi), IMD, TOLL, and Janus kinase/signal transduction and transcriptional activator (JAK/STAT) pathways are the main innate immune pathways in silkworm (Liu et al., 2015; Hua et al., 2018; Zhao et al., 2018; Wang et al., 2019; Swevers et al., 2020). But how BmADARa is involved in innate immunity remains to be explored. The results of this study found that BmADARa and BmDcr‐2 co‐inhibited the proliferation of BmNPV, and confirmed that BmADARa participated in the RNAi pathway through interaction with BmDcr‐2. It laid a foundation for the study of the function and molecular mechanism of BmADARa in innate immunity, and provided a new experimental idea for the study of antivirals of silkworm.

Materials and methods

Silkworm and cell culture, transfection

B. mori of strain p50 were reared on fresh mulberry leaves at 25 ± 1 °C, 75% ± 5% relative humidity under a 12 h light : 12 h dark photoperiod. The larval head, epidermis, fat body, midgut, silk gland, Malpighian tubule, trachea, and gonads were collected on ice. Samples were stored at −80 °C. The BmN was routinely maintained in our laboratory and cultured in TC‐100 insect medium (Ximeijie, China) with 10% fetal bovine serum (ExCell, China) and 1% penicillin–streptomycin (Hyclone, USA) at 27 °C (Zhang et al., 2023). Cells were transfected using Neofect DNA transfection reagent (NEOFECT, Beijing, China) according to the manufacturer's instructions.

Microbial pathogens

Bacillus thuringiensis (B. thuringiensis, Gram‐positive), Escherichia coli (E. coli, Gram‐negative), and Beauveria bassiana (B. bassiana, Fungi) were preserved in our laboratory. Purified BmNPV (BV) was kindly provided by Prof. Jia‐Ping Xu, Anhui Agricultural University, Hefei, China.

Challenge with microbial pathogens

A total of 180 larvae (1st day of the 5th instar) were divided into 6 groups and injected with 6 µL of B. thuringiensis (109 colony‐forming units [cfu]/mL), E. coli (109 cfu/mL), B. bassiana (109 cfu/mL), and BmNPV (1 × 106 Occlusion Bodies(OBs)/mL); sterile phosphate‐buffered saline (PBS) and Tc‐100 were used as a control. The hemocytes, midgut, and fat body were collected at 6, 9, 12, 24, 36, 48, and 72 h post‐infection (hpi). RNA of the hemocytes, midgut, and fat body was isolated according to the method by Zhang et al. (2023).

RNA isolation, complementary DNA (cDNA) synthesis, DNA extraction, and polymerase chain reaction (PCR)

Total RNA was extracted from various treated tissues from the 5th instar larvae stage and BmN cells, using TRIzol reagent (Sangon, China) according to the manufacturer's instruction. Subsequently, the RNA was treated with DNase I (TaKaRa, China) to remove genomic DNA. One microgram of total RNA was used to generate first‐stand cDNA with the PrimeScript RT reagent kit (TaKaRa, China) according to the manufacturer's instruction for cDNA synthesis.

DNA was extracted from treated BmN cells by using genomic DNA (gDNA) extract buffer (1 mol/L Tris‐HCl pH 8.0, 0.5 mol/L ethylenediaminetetraacetic acid [EDTA] pH 8.0, 10% sodium dodecyl sulfate [SDS]) according to the method reported by Brevnov et al. (2009). DNA concentrations were measured by a NanoDrop instrument. BmNPV proliferation in BmN cells was determined by estimating viral DNA using specific primers of the VP39 gene and GAPDH was chosen as the reference gene by quantitative real‐time (qRT)‐PCR using SYBR green PCR master mix (TransGen, China).

Thereafter, the expression profiles of BmADARa and BmDCR‐2 in different treated tissues and BmN cells were analyzed by qRT‐PCR. The PCR conditions were set up as recommended by the suppliers, and rp49 was chosen as the reference gene. Open reading frames or fragments of BmADARa and BmDcr‐2 were obtained by RT‐PCR using rTaq enzyme (TaKaRa, China) according to the manufacturer's instructions. The primers used in the experiments are listed in Table S1.

Construction of interference and overexpression vectors

The short hairpin RNAs (shRNAs) against candidate genes were designed on a website (http://rnaidesigner.lifetechnologies.com), and the primers were synthesized by Sangon Company (Shanghai, China). The designed shRNAs were connected with B. mori U6 promoter by PCR. The PCR products were electrophoresed on 1% agarose gel and isolated using the DNA Purification Kit (Promega, USA). Subsequently, the products were inserted into the PXL‐BacII‐IE1‐DsRed2 Real vector by KpnI and ApaI restriction enzymes.

On the other hand, stable clones were established, in which target gene expression was increased through T4 DNA Ligase. The PCR products were ligated into the pMD‐18T vector, subsequently ligated into pIZ‐V5/His and psl1180‐mCherry after digestion with BamHI and XbaI restriction enzymes. The primers used in the experiments are listed in Table S1.

BmADARa knockdown in silkworm larvae and BmNPV infection

dsRNA against BmADARa was generated using the L4440 Express dsRNA System according to the method reported by Ahn et al. (2019). A total of 30 larvae (1st d of the 5th instar) were injected with 5 mg of dsBmADARa. dsEGFP was used as a control. The hemolymph, fat body, and midgut tissues were collected at 24 hpi to assess RNAi efficiency. Subsequently, larvae were subjected to RNAi, and then all larvae were injected with 5 µL of BmNPV suspended in sterile water (1 × 106 OBs/mL) at 24 hpi of dsRNA as previously described (Slack & Arif, 2007). The fat body was collected at 24 hpi. Genomic DNA and RNA of the fat body were isolated.

For BmNPV infection: 10 µL of culture medium containing BV (1.0 × 108 pfu/mL) was added to the above‐mentioned BmN cells that had been successfully transfected. At 6, 12, 24, 36, and 48 hpi, total DNA of infected cells was isolated to detect the proliferation of BV. VP39 expression was determined to analyze the abundance of viral DNA.

Co‐immunoprecipitation (co‐IP), protein extraction, and western blot analysis

For co‐IP, cells were resuspended in lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris‐HCl pH 7.5, 0.5% TritonX‐100, and 10% glycerol), supplemented with complete protease and phosphatase inhibitor cocktail (Beyotime, Shanghai, China). After the removal of insoluble material, the supernatants were incubated with anti‐FLAG (1: 100, Solarbio, Beijing, China) followed by the addition of Protein A+G Agarose (Beyotime, Shanghai, China).

Total protein from different tissues of silkworm or BmN cells was extracted using RIPA lysis buffer (Beyotime, Shanghai, China), and the concentration was estimated by bicinchoninic assay.

Separation, transfer, and immunoblot analysis of proteins was performed as described in previous reports (Liu et al., 2015). The blots were probed with the following primary antibodies overnight at 4 °C: rabbit polyclonal anti‐VP39 (1: 500, HUABIO, Hangzhou, China), rabbit polyclonal anti‐Myc (1: 2 000, HUABIO, Hangzhou, China), mouse monoclonal anti‐β‐Tubulin (1: 5 000, TransGen, Beijing, China), mouse monoclonal anti‐Flag (1: 2 000, Solarbio, Beijing, China), mouse monoclonal anti‐V5 (1: 2 000, Sangon, Shanghai, China), and the s2nd antibody of goat anti‐rabbit/mouse immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP: Sangon, Shanghai, China) was diluted 1: 5 000 in the same blocking buffer. The final detection was performed by using Enhanced HRP‐DAB Chromogenic Substrate Kit (TIANGEN, Beijing, China) and band intensities were quantified using the software Image J.

Subcellular localization

BmN cells were inoculated into 6‐well cell culture plates (2 × 105 cells/well) with glass slides. Transient transfection was performed when the cells were 60% clustered. Seventy‐two h after transfection, the cells were washed with PBS, fixed in 4% paraformaldehyde, permeabilized with 0.05% Triton X‐100, and stained with 4ʹ,6‐diamidino‐2‐phenylindole (DAPI) according to Ye et al. (2020), and then photographed using a Nikon Eclipse TE 2000‐E confocal microscope (Nikon, Japan). Each image shown is a representative example of n ≥ 3. Transfection of psl1180‐mCherry was used as a control.

Statistical analysis

All data were calculated as the mean ± standard deviation. Differences between samples were analyzed by one‐way analysis of variance. Means were compared by least significant difference tests. All statistical calculations were run using Data Procession System software (version 8.01), and differences with P < 0.05 or P < 0.01 were considered statistically significant.

Results

Response of BmADARa to BmNPV infection

To confirm the role BmADARa plays in the innate immunity of silkworm, 4 types of pathogenic microorganisms were introduced to silkworm: Gram‐positive B. thuringiensis, Gram‐negative E. coli, the fungus B. bassiana, and BmNPV. qRT‐PCR examination reveals the expression patterns of BmADARa in the fat body, hemolymph, and midgut tissues after immune stimulation. Our results showed a reduced rate of expression of BmADARa, at least by 27% in the fat body after stimulation with B. thuringiensis, E. coli, and B. bassiana (Fig. 1A, B, and C). In contrast, BmADARa expression levels significantly increased after BmNPV infection, with at least a 5 fold increase (Fig. 1D). While in the hemolymph and midgut, the response of BmADARa to the 4 pathogenic microorganisms was similar to that observed in the fat body (Fig. S1 and S2). These findings evidently associate BmADARa in the innate immunity of silkworm, and significant changes in expression observed after BmNPV infection.

Fig. 1.

Fig. 1

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The expression pattern of BmADARa in the fat body of silkworm after stimulation with 4 microbial pathogens. Day 1 5th instar larvae were injected with inactivated (A) Bacillus thuringiensis, (B) Escherichia coli, (C) Beauveria bassiana, and (D) Bombyx mori nucleopolyhedrovirus (BmNPV); sterile phosphate‐buffered saline (PBS) was used as a control. The expression levels of the BmADARa gene in the fat body were determined at 6−72 h post‐infection (hpi) by real‐time polymerase chain reaction (RT‐PCR). Three independent biological replicates were performed for each experiment, and the data are presented as the mean ± standard error of the mean. Bars labeled with different letters are significantly different (P < 0.05).

To further investigate the impact of BmADARa in response to BmNPV infection, we collected BmN cells at 6, 12, 24, 36, and 48 hpi with BmNPV. Results from qRT‐PCR and western blot analysis showed a continuous and significant increase in the abundance of VP39, particularly notable after 24 hpi (Fig. 2A and B). The expression level of BmADARa also increased with BmNPV proliferation, but it only showed a significant increase of about 2 fold at 24 hpi (Fig. 2C). This indicates the function of BmADARa is predominantly in the later stages of BmNPV infection.

Fig. 2.

Fig. 2

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BmADARa expression levels after Bombyx mori nucleopolyhedrovirus (BmNPV) infection of BmN cells. (A−B) The abundance of BmNPV in infected BmN cells was assessed by analyzing relative transcription and protein levels of VP39 at 6, 12, 24, 36, and 48 h post‐infection (hpi). (C) The expression levels of BmADARa in the BmN cells infected with BmNPV. Three independent biological replicates were performed for each experiment, and the data are presented as the mean ± standard error of the mean. Bars labeled with different letters are significantly different (P < 0.05).

BmADARa inhibits BmNPV proliferation

The above results confirmed that BmADARa responds to BmNPV infection, but the specific role of BmADARa in BmNPV infection remains unclear. To investigate it, BmADARa was overexpressed in BmN cells for 48 h and then cells were collected at 12, 24, 36, and 48 hpi with BmNPV. Results showed the overexpression of BmADARa significantly reduced VP39 nucleic acid and protein levels at 36 hpi (Fig. 3A, B, and C). Conversely, when BmADARa expression was knocked down in BmN cells, the results were opposite: VP39 nucleic acid levels significantly increased at 24 hpi. Western blot analysis revealed a prominent increase in VP39 protein levels at 36 hpi (Fig. 3D, E, and F).

Fig. 3.

Fig. 3

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Effect of BmADARa overexpression and knockdown on Bombyx mori nucleopolyhedrovirus (BmNPV) proliferation in BmN cells and fat body. (A) BmADARa overexpression in BmN cells was confirmed by analyzing the transcript level of BmADARa. (B−C) The abundance of BmNPV in BmADARa‐OE (overexpression) BmN cells was assessed by analyzing relative transcription and protein levels of VP39 at 12, 24, 36, 48, and 72 j post‐infection (hpi). (D) BmADARa knockdown in BmN cells was confirmed by analyzing the transcript level of BmADARa. (E−F) The abundance of BmNPV in BmADARa‐KD (knockdown) BmN cells was assessed by analyzing relative transcription and protein levels of VP39 at 12, 24, 36, 48, and 72 hpi. (G) BmADARa knockdown in fat body was confirmed by analyzing the transcript level of BmADARa. (H−I) The abundance of BmNPV in dsBmADARa fat body was assessed by analyzing relative transcription and protein levels of VP39 at 12, 24, 36, 48, and 72 hpi. Three independent biological replicates were performed for each experiment, and the data are presented as the mean ± standard error of the mean. The asterisks represent significant differences (*P < 0.05, **P < 0.01, and ***P < 0.001, ns, not significant).

Similar to the results of cellular interference, after injecting dsRNA‐BmADARa (dsBmADARa) into silkworm for 6 h, fat body samples were collected at 12, 24, 36, 48, and 72 hpi with BmNPV. The efficiency of dsBmADARa post‐infection was first confirmed (Fig. 3G). Subsequently, it was observed that VP39 expression levels significantly increased, starting at 12 hpi (Fig. 3H). Western blot analysis showed a significant increase in VP39 at 48 hpi (Fig. 3I). These results indicate that BmADARa can inhibit BmNPV proliferation.

BmADARa and BmDcr‐2 cooperatively inhibit BmNPV proliferation

To investigate whether BmADARa inhibits BmNPV proliferation through the innate immune pathways of silkworm, we examined the expression levels of key genes in the RNAi, IMD, TOLL, and JAK/STAT pathways in the fat body of silkworm infected with BmNPV for 48 h after dsBmADARa treatment. The key genes in these 4 major innate immune pathways were all upregulated (Fig. S3), with significant changes observed in the key genes of the RNAi and JAK/STAT pathways, where the highest upregulation exceeded 2 fold (Fig. S3A and B). This indicates that BmADARa inhibits BmNPV proliferation through the innate immune pathways.

According to reports, ADAR mutant fruit flies affect innate immunity through Dcr‐2 (Deng et al., 2020), and the helicase domain of Drosophila Dcr‐2 mediates its antiviral activity (Deddouche et al., 2008). Additionally, interfering with the expression of BmDcr‐2 in silkworm promotes the proliferation of BmNPV (Li et al., 2020). To investigate whether BmADARa is involved in the process by which BmDcr‐2 suppresses BmNPV proliferation, we constructed an expression vector for the ERCC4‐related helicase domain (MPH1) of BmDcr‐2 (BmDcr‐2‐MPH1) (Fig. 4A). First, we confirmed that BmDcr‐2‐MPH1 can inhibit BmNPV proliferation with an efficiency of approximately 64%, slightly higher than the 56% inhibition efficiency of BmADARa (Fig. 4B, C, D, and E). This suggests that the helicase domain of BmDcr‐2 has antiviral activity against BmNPV. Co‐transfection of BmDcr‐2‐MPH1 and BmADARa revealed a significant inhibition in BmNPV proliferation, with an efficiency of approximately 77% (Fig. 4E), indicating remarkably increased efficiency of co‐inhibition of BmNPV proliferation by BmDcr‐2‐MPH1 and BmADARa.

Fig. 4.

Fig. 4

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Bombyx mori adenosine deaminases that act on RNA (BmADARa) cooperates with B. mori Dicer‐2 (BmDcr‐2)‐MPH1/BmDcr‐2‐DEXHc to inhibit the proliferation of B. mori nucleopolyhedrovirus (BmNPV). (A) BmDcr‐2, BmDcr‐2‐MPH1, and BmDcr‐2‐DEXHc (DEAD‐box helicase domain) patterns. (B−C) The overexpression efficiency of BmADARa and BmDcr‐2‐MPH1 co‐expressed in BmN cells at 36 h post‐infection (hpi) was confirmed by analyzing the transcript level of BmADARa and BmDcr‐2‐MPH1, respectively. (D) The abundance of BmNPV in BmADARa and BmDcr‐2‐MPH1 BmN cells were determined in protein expression levels of BmADARa, BmDcr‐2‐MPH1 and VP39 at 36 hpil β‐Tubulin was used as a control. (E) Quantitative densitometry of VP39 protein expression in nuclear fractions from (D). (F−G) The overexpression efficiency of BmADARa and BmDcr‐2‐DEXHc co‐expressed in BmN cells at 36 hpi was confirmed by analyzing the transcript level of BmADARa and BmDcr‐2‐DEXHc, respectively. (H) The abundance of BmNPV in BmADARa and BmDcr‐2‐MPH1 in BmN cells were determined in the protein expression levels of BmADARa, BmDcr‐2‐DEXHc, and VP39 at 36 hpi; β‐Tubulin was used as a control. (I) Quantitative densitometry of VP39 protein expression in nuclear fractions from (H). Three independent biological replicates were performed for each experiment, and the data are presented as the mean ± standard error of the mean. Bars labeled with different letters are significantly different (P < 0.05).

BmDcr‐2‐MPH1 contains a DEXH domain (BmDcr‐2‐DEXHc) (Fig. 4A). To confirm whether BmDcr‐2‐DEXHc is the core region responsible for the antiviral activity against BmNPV, we performed western blot analysis. We found that after overexpression, BmDcr‐2‐DEXHc exhibited a similar inhibition efficiency against BmNPV proliferation as BmADARa, at approximately 20% (Fig. 4F, G, H, and I). This demonstrates that DEXHc is crucial for the antiviral activity against BmNPV. Moreover, when BmDcr‐2‐DEXHc was co‐transfected with BmADARa, the inhibition efficiency against BmNPV proliferation reached approximately 71% (Fig. 4I), indicating that the antiviral activity against BmNPV is synergistically enhanced by BmDcr‐2‐DEXHc and BmADARa working in concert.

The relative affinity between BmADARa and BmDcr‐2‐DEXHc increases after BmNPV infection

To further investigate how BmDcr‐2‐DEXHc and BmADARa jointly enhance the antiviral activity against BmNPV, we initially conducted subcellular co‐localization studies, which confirmed that BmADARa and BmDcr‐2‐DEXHc co‐localize around the nucleus (Fig. 5A), suggesting potential interaction between them. To confirm the interaction between BmADARa and BmDcr‐2‐DEXHc, we performed co‐IP experiments, identifying their mutual interaction (Fig. 5B). Furthermore, the interaction between BmADARa and BmDcr‐2‐MPH1 has also been confirmed (Fig. S4), demonstrating that DEXHc is the key region for interaction with BmADARa. Moreover, after BmNPV infection, the relative binding affinity between BmADARa and BmDcr‐2‐DEXHc significantly increased (Fig. 5B). These results indicate that BmADARa and BmDcr‐2‐DEXHc collectively inhibit BmNPV proliferation by increasing their binding affinity.

Fig. 5.

Fig. 5

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Subcellular localization and co‐immunoprecipitation (co‐IP) of Bombyx mori adenosine deaminases that act on RNA (BmADARa) and B. mori Dicer‐2 (BmDcr‐2)‐DEXHc (DEAD‐box helicase domain) in the BmN cells. (A) BmADARa and BmDcr‐2‐DEXHc co‐localizes with 4ʹ,6‐diamidino‐2‐phenylindole (DAPI)‐stained nuclei. Psl1180‐mCherry was used as a control. Scale bar is 10 µm. (B) BmADARa and BmDcr‐2‐DEXHc were transiently expressed in BmN cells and co‐immunoprecipitated using a Flag‐Tag antibody. Western blots were probed with Myc‐Tag, or Flag‐Tag antibodies. Mouse immunoglobulin G (IgG) antibodies were used as background control in the IP. The relative binding affinity analysis of the ratio of IP group of BmADARa/BmDcr‐2‐DEXHc is shown in the bar chart. Three independent biological replicates were performed for each experiment, and the data are presented as the mean ± standard error of the mean. The asterisks represent significant differences (**P < 0.01).

Interaction between BmADARa‐dsRBD2 and BmDcr‐2‐DEXHc

BmADARa contains 2 dsRNA binding domains (dsRBD) and 1 adenosine deaminase domain (AD). To determine which domain of BmADARa interacts with BmDcr‐2‐DEXHc, we divided BmADARa into ΔAD, which contains dsRBD, and AD, which contains only AD (Fig. 6A). Co‐IP results showed that ΔAD interacts with BmDcr‐2‐DEXHc, whereas AD does not, indicating that dsRBD is the critical region for interaction with BmDcr‐2‐DEXHc (Fig. 6B). To further elucidate the mechanism of interaction between the 2 dsRBD domains of BmADARa and BmDcr‐2‐DEXHc, we individually deleted these 2 dsRBD domains, obtaining dsRBD1 deletion mutant ΔdsRBD1 and dsRBD2 deletion mutant ΔdsRBD2. The results showed that ΔdsRBD1 still interacts with BmDcr‐2‐DEXHc, whereas ΔdsRBD2 loses its ability to interact with BmDcr‐2‐DEXHc (Fig. 6C). These findings reveal that BmADARa‐dsRBD2 functions as the critical interaction domain with BmDcr‐2‐DEXHc.

Fig. 6.

Fig. 6

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The domain of Bombyx mori adenosine deaminases that act on RNA (BmADARa) interacting with B. mori Dicer‐2 (BmDcr‐2)‐DEXHc (DEAD‐box helicase domain) was determined. (A) ΔRBD1 (RNA binding domain 1), ΔRBD2, ΔAD (adenosine deaminase domain), and AD mutation patterns of BmADARa. (B) BmDcr‐2‐DEXHc, and ΔAD and AD were transiently expressed in BmN cells and co‐immunoprecipitated (co‐IP) using a Flag‐tag antibody. Western blots were probed with Myc‐Tag or FLAG‐Tag antibodies. Mouse immunoglobulin G (IgG) antibodies were used as background control in the IP. (C) BmDcr‐2‐DEXHc, and ΔRBD1 and ΔRBD2 were transiently expressed in BmN cells and Co‐IP using a Flag‐tag antibody. Western blots were probed with V5‐Tag or FLAG‐Tag antibodies. Mouse IgG antibodies were used as background control in the IP.

Discussion

ADAR, as an RNA editing enzyme, plays a crucial role in immunity. In mammals, the knockout of ADAR1 results in high embryonic mortality due to immune system dysregulation (Hartner et al., 2004), while in South American shrimp, ADAR inhibits virus replication by editing viral miRNAs (Cui et al., 2015). However, there have been few reports on the immune response regulated by ADAR in insects. In this study, we examined a supportive response of BmADARa toward different pathogens.Particularly, after silkworm were infected with BmNPV, the expression of BmADARa significantly increases in the hemolymph, midgut, and fat body (Fig. S1D, S2D, and S3D). ADAR1 has been involved directly or indirectly in the regulation of various viral infections such as human immunodeficiency virus, hepatitis C virus, and COVID‐19 (Pfaller et al., 2021). We also observed that BmADARa responds to BmNPV infection in BmN cells (Fig. 2). Interestingly, in the fat body, the expression of BmADARa significantly increases during the early and middle stages of BmNPV infection (6 and 12 hpi) (Fig. 1D). These results demonstrate the involvement of BmADARa in the process of BmNPV infection.

The antiviral immune response mechanism of silkworm mainly relies on innate immunity pathways, specifically including RNAi, IMD, TOLL, and JAK/STAT pathways (Jiang, 2021). Our findings indicate that when BmADARa expression is knocked down in BmN cells and fat body, key genes in the JAK/STAT, IMD, TOLL, and RNAi pathways are significantly upregulated (Fig. S3). This suggests that BmADARa participates in the process of BmNPV infection through these innate immune pathways.

During the process of silkworm infection with BmNPV, some responsive genes promote BmNPV proliferation (Liang et al., 2022; Mei et al., 2023) while others inhibit it (Kang et al., 2021; Lin et al., 2023). Our results demonstrate that after overexpression of BmADARa, the expression level of VP39 significantly decreases at 36 hpi (Fig. 3A, B, and C), indicating that BmADARa inhibits BmNPV proliferation. In silkworm, reducing the expression level of Dcr‐2 can moderately increase the production of viral gDNA (Li et al., 2020). We speculate that BmADARa may inhibit BmNPV proliferation through BmDcr‐2. We observed that the key gene BmDcr‐2 in the RNAi pathway was significantly upregulated in the fat body after knocking down BmADARa and infecting with BmNPV (Fig. S3A). This suggests that BmADARa may participate in the RNAi pathway through BmDcr‐2 to inhibit BmNPV proliferation.

In mammals, the antiviral function of ADAR1 occurs through Melanoma differentiation‐associated protein‐5 (MDA5) recognizing unedited viral dsRNA, which then enters the MDA5‐MAVS (mitochondrial antiviral‐signaling) pathway for antiviral defense. However, insects lack homologs of the DExD/H‐box RNA helicases found in the cytoplasmic dsRNA sensor MDA5 in mammals. The closest homolog in insects is the DEXHc of Dicer‐2, which is considered an innate immune sensor for certain specific viral RNAs (Galiana‐Arnoux et al., 2006; Deddouche et al., 2008). Additionally, it has been found that ADAR1 forms a complex through direct protein‐protein interaction with Dicer. ADAR1 also increases the rate of Dicer cleavage of precursor miRNA and promotes the loading of miRNA into RISC, thereby playing a role in miRNA processing and the RNAi mechanism (Ota et al., 2013). Our results showed that after overexpressing the BmDcr‐2 fragment and BmADARa, followed by BmNPV infection, the expression level of the viral protein VP39 was significantly reduced compared to that observed when only the BmDcr‐2 fragment or BmADARa was overexpressed and infected with BmNPV (Fig. 4). This indicates that BmADARa cooperates with BmDcr‐2‐DEXHc to inhibit BmNPV proliferation. Overexpression of BmDcr‐2‐MPH1 followed by BmNPV infection resulted in an approximately 20% increase in the efficiency of inhibiting BmNPV proliferation compared to its co‐expression with BmADARa (Fig. 4E). And overexpression of BmDcr‐2‐DEXHc followed by BmNPV infection led to an approximately 50% increase in the efficiency of inhibiting BmNPV proliferation compared to its co‐expression with BmADARa (Fig. 4I). The co‐expression of BmDcr‐2‐DEXHc and BmADARa demonstrated a higher efficiency in inhibiting BmNPV proliferation. These findings indicate that DEXHc is a crucial region for BmDcr‐2 and BmADARa to cooperatively inhibit BmNPV proliferation.

BmADARa mediates protein‐RNA and protein‐protein interactions through its dsRBD (Jiang et al., 2024). The DExDH‐box RNA helicase has the ability to bind to dsRNA, allowing BmADARa to interact with BmDcr‐2‐MPH1 and BmDcr‐2‐DEXHc. We further confirmed that it is the dsRBD2 of BmADARa that interacts with BmDcr‐2‐DEXHc (Fig. 6). Interestingly, the relative binding affinity between BmADARa and BmDcr‐2‐DEXHc significantly increases after BmNPV infection (Fig. 5B). There are many factors that affect protein interactions, and protein concentration is a key condition among them. BmNPV infection promotes an increase in the expression levels of BmADARa and BmDcr‐2 genes (Figs. 1 and S3), which may facilitate the heterodimerization of BmADARa and BmDcr‐2‐DEXHc. Therefore, we have once again demonstrated that BmADARa cooperates with the BmDcr‐2‐DEXHc‐dependent RNAi pathway to inhibit BmNPV proliferation.

RNAi is an important antiviral defense strategy, typically involving the recognition of viral dsRNA by the key gene Dicer2 enzyme, which processes it into small interfering RNA (siRNA). One of the double‐stranded siRNAs binds with Ago2 to form RISC, which then guides RISC to the viral RNA target through base pairing. Subsequently, Ago2 cleaves the viral RNA, thereby inhibiting viral replication (Kingsolver et al., 2013; Bronkhorst & Van Rij, 2014; Marques & Imler, 2016). Overexpression of Ago2 and Dicer2 can enhance the sensitivity of silkworm to dsRNA (You et al., 2020). In transgenic silkworm, the expression of dsRNA targeting viral genes such as BmNPV (Jiang et al., 2013), BmCPV (Jiang et al., 2017), and BmBDV (Sun et al., 2018) significantly reduces the viral messenger RNA (mRNA) content and the mortality rate of silkworm infected with the virus. Based on previous reports, we propose a model illustrating how BmADARa inhibits BmNPV proliferation through BmDcr‐2 (Fig. 7). As the infection time of BmNPV in cells increases, the expression level of BmADARa also rises. This enhances the relative binding affinity between BmADARa‐dsRBD2 and BmDcr‐2‐DEXHc, promoting BmDcr‐2 to recognize the viral dsRNA and cleave it into siRNA. Subsequently, the siRNA serves as a ligand for RISC, guiding RISC to the viral RNA target through base pairing. Then, RISC cleaves the viral RNA, thereby inhibiting viral replication. During the formation of RISC, the BmADARa‐BmDcr‐2 heterodimer may also maintain the stability and function of RISC.

Fig. 7.

Fig. 7

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A model to explain the function of Bombyx mori adenosine deaminases that act on RNA (BmADARa) in the B. mori nucleopolyhedrovirus (BmNPV) proliferation process. In silkworm, when BmNPV infects cells, the expression level of BmADARa increases with the infection time. The elevated concentration of BmADARa facilitates the binding of double‐stranded RNA (dsRNA) binding domain 2 (dsRBD2) to B. mori Dicer‐2 ‐ DEAD‐box helicase domain (BmDcr‐2‐DEXHc), enhancing their relative binding affinity (solid arrow). The interaction between BmADARa and BmDcr‐2 improves the ability of BmDcr‐2 to recognize viral dsRNA, increasing the rate of dsRNA cleavage into small interfering RNA (siRNA). Additionally, during the formation of RNA‐induced silencing complex (RISC), the BmADARa‐BmDcr‐2 heterodimer may also maintain the stability and function of RISC, promoting the cleavage of viral RNA by RISC and thereby inhibiting viral replication (dashed arrow).

Disclosure

The authors have declared that no competing interests exist.

Supporting information

Fig. S1 The expression pattern of BmADARa in the hemolymph of silkworm after stimulation with 4 microbial pathogens.

Fig. S2 The expression pattern of BmADARa in the midgut of silkworm after stimulation with 4 microbial pathogens.

Fig. S3 The effects of BmADARa knockdown and Bombyx mori nucleopolyhedrovirus (BmNPV) infection on the RNA interference (RNAi), IMD, TOLL, and Janus kinase/signal transduction and transcriptional activator (JAK/STAT) pathways in the fat body.

Fig. S4 Co‐immunoprecipitation of Bombyx mori adenosine deaminases that act on RNA (BmADARa) and B. mori Dicer‐2 (BmDcr‐2)‐MPH1 in the BmN cells.

INS-33-1541-s001.docx (2.3MB, docx)

Table S1 Oligonucleotides of primers in polymerase chain reaction.

INS-33-1541-s002.docx (20KB, docx)

Acknowledgments

This work was supported by National Natural Science Foundation of China (Grant No. 32372947), Anhui Provincial Natural Science Foundation (Grant No. 2308085MC100), and the International Science & Technology Cooperation Plan of Anhui Province (Grant No. 2022h11020020) to Y. Meng. We would like to thank Prof. Jia‐Ping Xu (Anhui Agricultural University, Hefei, China) for kindly providing the BmNPV virus and VP39 antibody.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1 The expression pattern of BmADARa in the hemolymph of silkworm after stimulation with 4 microbial pathogens.

Fig. S2 The expression pattern of BmADARa in the midgut of silkworm after stimulation with 4 microbial pathogens.

Fig. S3 The effects of BmADARa knockdown and Bombyx mori nucleopolyhedrovirus (BmNPV) infection on the RNA interference (RNAi), IMD, TOLL, and Janus kinase/signal transduction and transcriptional activator (JAK/STAT) pathways in the fat body.

Fig. S4 Co‐immunoprecipitation of Bombyx mori adenosine deaminases that act on RNA (BmADARa) and B. mori Dicer‐2 (BmDcr‐2)‐MPH1 in the BmN cells.

INS-33-1541-s001.docx (2.3MB, docx)

Table S1 Oligonucleotides of primers in polymerase chain reaction.

INS-33-1541-s002.docx (20KB, docx)

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