Identification of BmSP25 gene in Bombyx mori with antiviral function against BmNPV

Yonghong Zhang; Yajin Li; Zhengqin Wang; Jiafu Luo; Lingli Li; Hongrui Zhang

doi:10.1371/journal.pone.0345502

Abstract

Bombyx mori serine protease (BmSP) is constitute a gene family of proteolytic enzymes characterized by serine residues at their active sites and play critical roles in physiological processes, including digestion, growth and development, and immune responses. As a member of the BmSP family, BmSP25 exhibits differential expression following Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Our result demonstrated that BmNPV infection upregulated BmSP25 expression in both resistant (SuN) and susceptible (P50) silkworm strains, with a more pronounced response observed in SuN than in P50. To further investigate its function, siRNA-mediated knockdown of BmSP25 in BmN cells promoted the proliferation of recombinant BV-EGFP virus, whereas overexpression of BmSP25 significantly suppressed BmNPV replication. To validate its antiviral activity at the organismal level, transgenic silkworm strains overexpressing BmSP25 (BmSP25-OE) and BmSP25 knockout strains (BmSP25-KO) were generated. Following oral inoculation with BmNPV, viral proliferation was significantly inhibited in the BmSP25-OE strain, whereas viral replication was notably enhanced in the BmSP25-KO strain. This study is the first to clearly demonstrate the anti-BmNPV function of BmSP25 in silkworms, providing a foundation for further elucidation of its role in host immune defense mechanisms and identifying a potential genetic target for molecular breeding aimed at improving disease resistance in silkworms.

Citation: Zhang Y, Li Y, Wang Z, Luo J, Li L, Zhang H (2026) Identification of BmSP25 gene in Bombyx mori with antiviral function against BmNPV. PLoS One 21(3): e0345502. https://doi.org/10.1371/journal.pone.0345502

Editor: Jisheng Liu, Guangzhou University, CHINA

Received: January 5, 2026; Accepted: March 5, 2026; Published: March 27, 2026

Copyright: © 2026 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting information files.

Funding: This research was supported by Pre Research Project of Yunnan Academy of Agricultural Sciences (Grant number: 2024KYZX-01).

Competing interests: The authors have declared that no competing interests exist.

Introduction

The silkworm, Bombyx mori L. is an economically important insect and a representative Lepidopteran model organism [1,2]. During sericulture, silkworms are highly susceptible to infection by multiple viruses, among which with double-stranded DNA (dsDNA) and double-stranded RNA (dsRNA) viruses pose particularly serious threats. Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), a dsRNA virus, infects midgut epithelial cells of silkworm and subsequently induces host innate immune response, including upregulation of the BmToll-2 gene [3]. Among dsDNA viruses, BmNPV also triggers immune responses in the midgut following oral infection. Silkworms are frequently infected with BmNPV during sericulture, leading to blood-type grasserie and severe losses in silk production. As a major viral pathogen in sericulture, yet effective control measures remain lacking, posing a substantial challenge to the industry. BmNPV belongs to the family Baculoviridae, featuring and is characterized by rod-shaped enveloped virions containing a closed circular double-stranded DNA genome of approximately 130 kb [4,5]. Viral occlusion bodies (OBs) enter the midgut lumen of silkworms through ingestion. Under alkaline digestive conditions, OBs dissolve and release virions, which subsequently binding to columnar epithelial cells of the midgut and spread to other healthy tissues [6]. The silkworm midgut therefore functions as the first barrier against BmNPV infection and represents a critical site for virus host interactions. Previous studies have identified several midgut-expressed proteins exhibiting with anti-BmNPV activity [7–12]. Consequently, elucidating the roles of genes highly expressed genes in midgut during BmNPV infection is essential crucial for understanding the immune defense mechanisms of silkworms.

Different silkworm strains display distinct levels of resistance to BmNPV infection. For example, KN [13], NB [14], and A35 [15] are highly resistant strains, whereas 306 and P50 are susceptible. With advances in silkworm molecular biology, the functions of several genes and proteins associated with BmNPV resistance have been characterized elucidated. For instance, knockout of the peptidoglycan recognition protein 2 (BmPGRP2−2) gene suppresses BmNPV proliferation in both cell lines and silkworm larvae, thereby reducing host mortality [16]. In contrast, overexpression of BmAtlastin-n [17], silkworm lysozyme (BMC-LZM) [18], and silkworm alkaline trypsin A (BmTA) [19] in larvae or cells enhances resistance to BmNPV. Additionally, silkworm serine/threonine protein phosphatase 2A (BmPP2A) has been shown to possess anti-BmNPV activity [20]. Conversely, proteins such as receptor expression-enhancing protein a (BmREEPa), which interacts with GP64 [21], the E3 ubiquitin-protein ligase SINAL10 which binds to GP64 [22], and the autophagy-related protein ATG13 [23] have been demonstrated promote BmNPV proliferation in BmN cells.

Serine proteases are widely distributed among insects and play essential roles in intestinal protein digestion. The midgut of Lepidopteran larvae contains a complex repertoire of proteolytic enzymes with diverse substrate distinct specificities, including trypsin, chymotrypsin, elastase, cathepsin-B-like proteases, aminopeptidases, and carboxypeptidases, which collectively participate in protein digestion [24]. In the larval intestinal environment, serine proteases predominate, accounting for approximately 95% of total digestive enzyme activity [25]. Serine proteases are endopeptidases characterized by a serine residue at the active site that functions as a nucleophile. These enzymes exhibit diverse biological functions, being directly involved in critical physiological processes such as protein metabolism, digestion, blood coagulation, apoptosis, immune regulation, development, and fertilization [26,27], while regulating multiple synergistic processes through proteolysis. Zhao et al. [28] identified 143 serine protease-related genes in the silkworm genome database based on amino acid sequence homology with serine proteases from other species, designated BmSP1 to BmSP143, including 51 serine protease (BmSPs) and 92 serine protease homologs (BmSPHs). Members of the BmSP family exhibit functional diversity, with distinct expression patterns across different tissues and developmental stages of the silkworm, thereby fulfilling a wide range performing diverse roles [25]. Previous studies have shown that certain BmSP family members are specifically expressed in the silkworm midgut. For example, BmSP2 plays an important role in defense against BmNPV [8], whereas BmSP36 and BmSP141 are primarily involved in food digestion [29,30]. In the humoral immune response of silkworms, BmSPs play crucial roles by participating in the prophenoloxidase (proPO) activation cascade. These proteases convert inactive proPO into active phenoloxidase (PO), which catalyzes the synthesis of quinones and melanin to combat pathogenic microorganisms [31]. BmSPs typically contain a clip domain and possess highly conserved active-site motifs, including TAAHC, DIAL, and GDSGGP [32]. These structural features enable the activation of proPO through cleavage at specific amino acid residues at the carboxyl terminus [33]. Tanaka et al. [34] identified multiple BmSPs and BmSPHs involved in silkworm immune responses, some of which are specifically expressed in midgut tissues and primarily function in food digestion under normal conditions. However, upon BmNPV infection, the transcriptional levels of these genes are altered. Previous studies have demonstrated that several members of the BmSP gene family exhibit induced following expression BmNPV infection [35,36]. Given that the midgut serves as the first barrier against BmNPV invasion and that certain some midgut-specific genes have established roles in digestion, their precise functions in immune responses remain incompletely understood. Therefore, comprehensive investigation of serine protease genes if of substantial scientific importance.

Following BmNPV infection, 18 silkworm serine protease (SP) and serine protease homolog (SPH) genes exhibit varying degrees of upregulation or downregulation. This study focuses on BmSP25, a gene that is highly expressed in the midgut and significantly upregulated upon infection [28,37]. First, the differential expression of BmSP25 between the resistant strain SuN and the susceptible strain P50 was analyzed following BmNPV infection. Subsequently the effects of BmSP25 overexpression or knockdown on BmNPV proliferation were examined. In addition, the expression patterns and subcellular distribution of the BmSP25 protein were analyzed, and potential interacting proteins were screened. Through these approaches, this study systematically elucidates the molecular mechanism by which BmSP25 contributes to resistance against BmNPV.

Materials and methods

Silkworm rearing and viral particles

The BmNPV-susceptible silkworm strain P50 (LC₅₀ = 2.47 × 10⁵ OBs/mL), the BmNPV-resistant strain SuN (LC₅₀ = 1.43 × 10⁷ OBs/mL), and the BmNPV-Baoshan strain were maintained at the Sericulture and Apiculture Research Institute, Yunnan Academy of Agricultural Science, Honghe, China. Silkworms were reared to the first day of the fifth instar prior to viral infection. Each treatment was conducted in triplicate with 100 larvae per replicate. The experimental group was orally inoculated with 5 μL of BmNPV suspension (1.21 × 10⁶ PIB/mL), whereas the control group received an equal volume of ddH₂O. After inoculation, larvae were fed fresh mulberry leaves. Midgut tissues were collected at multiple time points (6 h, 12 h, 24 h, 48 h, 72 h, and 96 h), carefully freed of peritrophic membranes and contents, immediately frozen in liquid nitrogen, and stored at −80℃ until use. A recombinant virus expressing enhanced green fluorescent protein (BV-EGFP) was provided by the State Key Laboratory of Resource Insects, Southwest University, Chongqing, China. The BV-EGFP titer (OBs/mL) was determined using the endpoint dilution method.

BmN cell culture and transfection

The silkworm ovarian cell line BmN was cultured at 28°C in TC-100 medium supplemented with 10% (v/v) fetal bovine serum and 1% penicillin–streptomycin. Transfections were performed using Cellfectin™ II Reagent (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s instructions. Cells were seeded in six-well plates at 60 ～ 80% confluency. Two hours prior to transfection, the culture medium was replaced with serum- and antibiotic-free TC-100 medium following three washes. For preparation of the transfection complexes, 2 μg of plasmid DNA or 20 μM siRNA was mixed with 100 μL of serum-free dilution medium, followed by the addition of 2 μL of transfection reagent and incubation at room temperature for 30 min. The resulting mixture was added to the cell culture wells and gently mixed. Fluorescence images were captured using an Olympus inverted fluorescence microscope (IX73, Olympus, Japan).

Bioinformatics analysis

The coding sequence of the BmSP25 gene was obtained from SilkBase (https://silkbase.ab.a.u-tokyo.ac.jp/). The nucleotide and amino acid sequence of BmSP25 were analyzed using DNAMAN 10 (Lynnon Corporation, Quebec, Canada). Conserved motifs were predicted using on the SMART server (http://smart.embl-heidelberg.de/), and signal peptide prediction was performed with SignalP 5.0 (https://services.healthtech.dtu.dk/services/SignalP-5.0/).

RNA isolation and cDNA synthesis

Total RNA was extracted from silkworm midgut tissues using the Takara MiniBEST Universal RNA Extraction Kit (Takara, Osaka, Japan). RNA concentration and purity (A260/A280 ratio) were determined using a NanoPhotometer N50T (IMPLEN, Munich, Germany), and RNA integrity was assessed by 1% agarose gel electrophoresis. First-strand cDNA was synthesized using the PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, Osaka, Japan) and stored at −20°C until further analysis.

Quantitative reverse transcription PCR (RT-qPCR) analysis

RT-qPCR was performed to determine the relative expression levels of target genes using primers listed in Table 1. Reactions were prepared using the SYBR Premix Ex Taq™ II (Tli RNaseH Plus) Kit (Takara, Osaka, Japan) in a 20 μL, containing10 μL SYBR premix, 0.4 μL ROX Reference Dye, 0.4 μL each of forward and reverse primers (10 μmol/L), 1 μL of cDNA template, and 7.8 μL ddH₂O. The thermal cycling conditions were as follows: 95°C for 5 min; followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. Each sample was analyzed in triplicate. Relative gene expression level was calculated using the 2^-ΔΔCt method [38], with BmGAPDH used as the internal reference gene. Based on the standard curves constructed for the BmNPV vp39 and gp41 genes in this study (S1 Fig), absolute quantification was employed to viral copy numbers in different samples.

Download:

Table 1. Primer used in RT-qPCR.

https://doi.org/10.1371/journal.pone.0345502.t001

Prokaryotic expression and antibody preparation

Specific primers for BmSP25 were designed as follow: F: 5′-gatcGGATCCTTCACACTGCCACTGCACGAAAACC-3′ R: 5′-gcAGATCTTTACAGGTGCTGATTGAAGAAGTTC-3′ (underlined sequences indicate BamH I and Bgl II restriction sites). The target fragment was amplified using P50 midgut cDNA as the template, cloned into the pMD19-T vector, subsequently subcloned into the pET-30a vector via double digestion. The recombinant plasmid was transformed into Escherichia. coli BL21 cells. The recombinant protein was purified using Ni-NTA agarose resin (Qiagen, Hilden, Germany) and used to immunize injected into New Zealand white rabbits for the production of polyclonal antibodies.

Western blot analysis

Midgut tissues were dissected, and total protein was extracted as previously described [39]. Cells were lysed using lysis buffer (Beyotime, China), washed twice with PBS, and denatured in 5 × SDS-PAGE loading buffer (Beyotime) at 100°C. Protein sample were separated by 12% SDS-PAGE and transferred onto PVDF membranes. Membranes were incubated with primary antibodies (anti-BmSP25, 1:5000; anti-β-actin3, Beyotime, 1:5000) for 1 h at 25°C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Beyotime). Protein signals were detected using an ECL Western Blot Detection System (Bio-Rad), and grayscale values were quantified using AlphaEase FC software.

Effect of BmSP25 RNAi and overexpression on BV-EGFP proliferation in BmN cells

BmSP25 targeting siRNAs were synthesized by General Biosystems (Chuzhou, China) and reconstituted to a final concentration of 20 μM using RNase-free H₂O. The experimental groups consisted of BmN cells transfected with BmSP25 siRNA, whereas the control groups included untransfected cells, cells transfected with negative control siRNA, and cells transfected with DsRed siRNA. After 24 h of incubation with BmSP25 siRNA, total RNA was extracted and reverse-transcribed into cDNA. RT-PCR was performed to evaluate the RNA interference efficiency of BmSP25 and to identify the optimal siRNA fragment. Subsequently cells were infected with BV-EGFP and observed at 24, 48, 72, and 96 hour post-infection (hpi). Genomic DNA was extracted, and viral copy numbers were quantified by RT-qPCR targeting the vp39 gene. For overexpression analysis, the coding sequence of BmSP25 was amplified using the following primers: F: 5′-gatcGGATCCATGAAGACATTCGTTGCGACC-3′, R:5′-gcGCGGCCGCTTAAAGGTGTTGATTGAAGAAATTC-3′ (underlined sequences indicate BamH I and Not I restriction sites). The amplified fragment was first cloned into the pSL1180 intermediate vector and subsequently sub-cloned into the pIZT/V5-His-mCherry vector to generate the recombinant plasmid pIZT/V5-His-mCherry+hr3-i.e.,1p-BmSP25. BmN cells were transfected with the constructed plasmid and harvested 48 h later for RNA and DNA extraction. The overexpression efficiency of BmSP25 was assessed by RT-qPCR, and viral copy numbers were determined as described above.

Effect of BmSP25 overexpression and knockout on BmNPV proliferation in silkworms

The transgenic overexpression vector pBac[A3p-EGFP-SV40 + BmP2p-BmSP25-SV40] was constructed by inserting the BmSP25 expression cassette into the pBac[A3p-EGFP-SV40] backbone. For gene knockout, sgRNA sequences targeting BmSP25 were cloned into a U6 promoter-driven vector, and the resulting cassette was inserted into pBac[A3p-DsRed-SV40] to generate pBac[A3p-DsRed-SV40 + U6-sgRNA-SV40]. The silkworm strain 305 was used for generating transgenic silkworms. Both vectors were co-injected with the helper plasmid pHA3PIG into silkworm eggs (G0). G1 larvae expressing fluorescent markers were selected for subsequent experiment. Total RNA was extracted from the midgut tissues of BmSP25 overexpressing (BmSP25-OE, green fluorescent) and negative control individuals at the fifth instar on day 3, and the transcriptional level of the BmSP25 gene was analyzed by RT-qPCR. BmSP25-OE and wild-type silkworms were orally inoculated at the 5^th-1 d instar with 5 μL of BmNPV (1.21 × 10⁶ PIB/mL) or ddH₂O. Silkworms were maintained on fresh mulberry leaves, and daily mortality was recorded. Midgut tissues were collected at 24, 48, 72, and 96 hpi for DNA extraction and viral DNA quantification by RT-qPCR. For knockout experiments, BmSP25-sgRNA transgenic larvae (red fluorescent) were crossed with Cas9-overexpressing larvae (Cas9-OE, green fluorescent). Hybrid offspring positive for both fluorescent markers were selected, and genomic DNA was extracted. PCR amplification and clone sequencing of the target site were performed to evaluate knockout efficiency. The knockout efficiency of BmSP25 was further assessed by RT-qPCR using midgut tissues from the third day of the fifth instar. Double-fluorescent offspring were selected for viral challenge, and viral replication was evaluated as described above.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8. Differences between groups were evaluated using Student’s t-test. A P value< 0.05 was considered statistically indicated significant, and a P value < 0.01 considered highly significant (*P < 0.05, **P < 0.01, ***P < 0.001). All data were presented as the mean ± SD from at least three independent experiments.

Results

Characterization of the BmSP25 sequence

The cDNA sequence (KWMTBOMO10987) of BmSP25 contains an open reading frame (ORF) of 885 bp, encoding a protein of 294 amino acids with a predicted molecular weight of 30.86 kDa and an isoelectric point of 8.19. The first 17 amino acid residues at the N-terminus of BmSP25 constitute a signal peptide (positions 1–17), indicating that BmSP25 is a secreted protein (Fig 1A). Conserved domain analysis using SMART software revealed that BmSP25 contains a trysin-like serine protease domain (Tryp_SPc) spanning 54–290 (Fig 1B). Three highly conserved motifs—TAAHC, DVAV, and GDSGGP—were identified in the deduced amino acid (Fig 1C), confirming that BmSP25 belongs to the serine protease family.

Download:

Fig 1. Bioinformatics analysis of BmSP25.

(A) The ORF nucleotide sequence of BmSP25 and its deduced amino acid sequence. BmSP25 protein signal peptide (1–17) was represented by the rectangle. The Tryp_SPc domain is shaded gray. (B) Functional domain prediction of BmSP25 by using SMART online software. (C) Three conserved domains of TAAHC, DVAV and GDSGGPL were labeled the red underline. The GenBank number of each sequence was as follows: Bombyx mori (this study), Mamestra configurata (ADM35105.1), Heliothis virescens (AFM28248.1), Spodoptera frugiperda (AIR09775.1), Helicoverpa armigera (ADI32882.1).

https://doi.org/10.1371/journal.pone.0345502.g001

BmSP25 exhibited specific expression patterns in response to BmNPV infection

The dynamic changes in BmSP25 transcription and protein expression in midgut tissues of silkworm strains with different levels of resistance were comparatively analyzed at multiple time points following BmNPV infection. In the susceptible strain P50, the transcriptional level of BmSP25 was upregulated between 6 and 24 hpi. In contrast, in the resistant strain SuN, BmSP25 expression showed a time-dependent and significant increase throughout the course of infection. Under uninfected conditions, the basal transcriptional level of BmSP25 in the resistant strain SuN was significantly higher than that in the susceptible strain P50 at all examined time points. Furthermore, the expression difference between the two strains remained significant from 24 to 96 h after infection (Fig 2A). At the protein level, BmSP25 expression in the resistant strain SuN was significantly higher than that in the susceptible strain P50 as early as 24 hpi following BmNPV infection, and the overall expression pattern was largely consistent with the transcriptional data (Figs 2B, S2, S1 Table). These findings suggested that BmSP25 had been involved in the immune response of the silkworm midgut to BmNPV infection.

Download:

Fig 2. Expression analysis of BmSP25 in silkworm strains SuN and P50 after BmNPV infection.

(A) RT-qPCR analysis of BmSP25 mRNA transcriptional levels in SuN and P50 strains; (B) Western blot analysis of BmSP25 protein expression changes in SuN and P50 strains before and after BmNPV infection. Significant differences were indicated by different letter (P < 0.05). “+” and “-”: BmNPV-fed infection group and non-fed control group, respectively.

https://doi.org/10.1371/journal.pone.0345502.g002

BmSP25 interference affected the susceptibility of BmN cells to BmNPV

To investigate the effect of BmSP25 interference, negative control siRNA and three siRNA fragments targeting specific regions of the BmSP25 gene were transiently transfected into BmN cells to suppress BmSP25 expression. Cells were collected 24 hpi, and the expression level of BmSP25 was analyzed by RT-qPCR. The results showed that the siRNA targeting the BmSP25–268 site achieved the most significant interference efficiency, resulting in a markedly lower expression level of the target gene compared with in normal BmN cells (Fig 3A). Subsequently, BmN cells transfected with BmSP25 siRNA were infected with the recombinant virus BV-EGFP after 24 h. Control groups included cells transfected with DsRed siRNA followed by viral infection, cells infected with BV-EGFP alone (without transfection), and untreated BmN cells (without transfection or viral infection). To assess the impact of BmSP25 interference on viral infection, viral proliferation was evaluated. As shown in Fig 3B, the number of green fluorescence-positive cells in the BmSP25 interference group was significantly higher than that in the control groups. Consistently, the expression level of the viral gene vp39 in the BmSP25 interference group was significantly upregulated compared with that in normal BmN cells (Fig 3C). These results indicate that suppression of BmSP25 expression in BmN cells facilitates BmNPV infection.

Download:

Fig 3. Effect of BmNPV transduction in BmSP25-RNAi BmN cells.

(A) Verification of BmSP25 gene interference effect; (B) Fluorescence observation of BmN cells infected with BV-EGFP at 72 hpi, scale bar = 200 μm; (C) Analysis of vp39 expression levels in BmN cells infected with BV-EGFP at different time points, the qPCR employed an absolute quantification method. Significant differences were indicated by different letter (P < 0.05). * Indicates significant differences at P < 0.05, ** indicates significant differences at P < 0.01 with respect to the control.

https://doi.org/10.1371/journal.pone.0345502.g003

BmSP25 overexpression enhanced the susceptibility of BmN cells to BmNPV invasion

Successful overexpression of BmSP25 in BmN cells was confirmed by RT-qPCR analysis (Fig 4A), BmN cells overexpressing BmSP25 were subsequently infected with the recombinant virus BV-EGFP at 48 hpi. Normal BmN cells served as the uninfected control. Two experimental groups were established: BmN cells infected with BV-EGFP alone and BmN cells transfected with either pIZT-hr3-i.e.,1p-BmSP25/V5-His-mCherry or pIZT/V5-His-mCherry vector followed by BV-EGFP infection. At 48 hpi, fluorescence microscopy and RT-qPCR were performed to evaluate the expression levels of EGFP and vp39. Red fluorescence was observed in both BmSP25-overexpressing cells (BmN^{pIZT-hr3-i.e.,1p-BmSP25/V5-His-mCherry}) and vector control cells (BmN^{pIZT/V5-His-mCherry}) (Fig 4B). Green fluorescence signals were detected at multiple time points following viral infection. At 48 and 72 hpi, the green fluorescence intensity in BmSP25-overexpressing cells was notably weaker than that in control cells. RT-qPCR analysis demonstrated that EGFP expression at 48 hpi was significantly lower in BmSP25-overexpressing cells than in control cells (Fig 4C). Similarly, vp39 expression was significantly reduced in BmSP25-overexpressing BmN cells compared to controls (Fig 4D). In addition, the number of viral particles in the culture supernatant of BmSP25-overexpressing BmN cells was significantly lower than that in the control group. Collectively, these results demonstrate that BmSP25 overexpression suppresses BmNPV proliferation in BmN cells.

Download:

Fig 4. Effect of BmNPV transduction in BmSP25 overexpression BmN cells.

(A) The transcriptional level of BmSP25 in BmN cells transfected with pIZT-hr3-i.e.,1p-BmSP25/V5-His-mCherry at 48 h after transfection; (B) Green fluorescence in cells at 48 hpi with BmNPV, Scale bar = 200 μm; (C) Analysis of EGFP expression in BmN cells 24, 36, 48 and 72 hpi with BmNPV, C1 represented normal BmN cells infected with BV-EGFP, while C2 represented BmN cells transfected with the pIZT/V5-His-mCherry vector that did not contain the BmSP25 gene; (D) Analysis of vp39 expression in BmN cells 24, 48 and 72 hpi after infection with BmNPV, the qPCR employed a relative quantification method; C: BmN + pIZT/V5-His-mCherry vector. Significant differences are indicated by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001), ns: no significant.

https://doi.org/10.1371/journal.pone.0345502.g004

BmSP25 overexpression inhibited the invasion of BmNPV into silkworm individuals

A BmSP25-OE transgenic silkworm strain was generated using a piggyBac transposon vector. Under fluorescence microscopy, ubiquitous green fluorescence was observed in positive transgenic individuals (S3 Fig). RT-qPCR analysis revealed that the expression level of BmSP25 in the midgut tissues of positive transgenic individuals was significantly higher than that in negative individuals (Fig 5A). To further evaluate the effect of BmSP25 overexpression on BmNPV infection, the first day of the fifth-instar larvae were starved for 24 h, after which positive transgenic positive individuals and negative controls were orally inoculated with occlusion-derived virus (ODV). Mortality was continuously monitored and recorded. The cumulative mortality rates in the non-inoculated negative and positive control groups were 2.5% and 2.1%, respectively, with no significant difference observed. Following after viral inoculation, however, the cumulative mortality rate reached 54.58% in the negative experimental group and 37.92% in the positive experimental group, indicating a significantly enhanced resistance to BmNPV infection in BmSP25-overexpressing silkworms (Fig 5B). The transcriptional levels of the BmNPV genes vp39 and gp41 in midgut tissues were measured at 24, 48, 72, and 96 hpi. Viral copy numbers were calculated based on the expression levels of vp39 and gp41. RT-qPCR analysis showed that at 72 and 96 hpi, the transcriptional levels of both vp39 and gp41 were significantly lower in the BmSP25 transgenic strain were than in negative controls (Fig 5C, 5D). These results indicated that BmSP25 overexpression in vivo effectively inhibited BmNPV proliferation and replication.

Download:

Fig 5. The effect of transgenic silkworms overexpressing BmSP25 on the proliferation of BmNPV.

(A) Analysis of transcription levels of BmSP25 between BmSP25-OE strain and negative individuals; (B) Western Blot Analysis of BmSP25 protein expression between transgenic silkworm and negative individuals; (C) Statistical mortality rate of silkworm larvae after oral administration of BmNPV; (D) The copies of BmNPV vp39 gene; (E) The copies of BmNPV gp41 gene; “+” and “-”: BmNPV-fed infection group and non-fed control group, respectively. Significant differences are indicated by asterisks (*P < 0.05; ***P < 0.001).

https://doi.org/10.1371/journal.pone.0345502.g005

BmSP25 knockdown enhanced the invasion of BmNPV into silkworm individuals

The BmSP25 knockout hybrid (BmSP25-sgRNA × Cas9) exhibited both red and green fluorescence (S4 Fig). Sequencing analysis confirmed that the two target sites within the BmSP25 gene were edited by Cas9, resulting in various base deletions and mutations (S5 Fig), thereby verifying the successful generation of targeted knockout line. In BmSP25 knockout silkworms (BmSP25-KO), the expression level of the target gene in midgut tissues of positive individuals was significantly lower than that in negative individuals (Fig 6A). To further assess the effect of BmSP25 knockout on silkworm resistance to BmNPV, negative control individuals and BmSP25-KO silkworms were reared under normal conditions until the onset of the fifth instar and then orally inoculated with BmNPV. Using standard curves established for the BmNPV vp39 and gp41 genes, the transcriptional levels of these viral genes in midgut tissues were measured at 24, 48, 72, and 96 hpi, and viral copy numbers were calculated accordingly. RT-qPCR results showed that the transcriptional levels of both vp39 and gp41 in the BmSP25-KO strain were significantly higher than those in negative controls (Fig 6B, 6C). In addition, the cumulative mortality rate of the BmSP25-KO strain was higher than that of the BmSP25-sgRNA strain (Fig 6D). These findings demonstrate that knockdown of BmSP25 in vivo enhances BmNPV proliferation and replication, further confirming the critical role of BmSP25 in the host response to BmNPV infection.

Download:

Fig 6. The impact of the BmSP25-KO knockout strain of silkworm on the proliferation of BmNPV.

(A) Analysis of transcription levels of BmSP25 in BmSP25-KO strain and negative individuals; (B) The copies of BmNPV vp39 gene; (C) The copies of BmNPV gp41 gene; (D) Statistical mortality rate of silkworm larvae after oral administration of BmNPV; “+” and “-”: BmNPV-fed infection group and non-fed control group, respectively. Significant differences are indicated by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001).

https://doi.org/10.1371/journal.pone.0345502.g006

Discussion

Serine proteases are a class of endopeptidases characterized by the presence of a serine residue at their active site and perform diverse functions primarily related to development, digestion, and immunity [28,33,40]. In insects, serine proteases and their homologs (SPs/SPHs) are mainly involved in developmental processes and immune responses, playing central roles in immune defense networks—including the melanization cascade, prophenoloxidase activation, and the production of cytokine-like signaling molecules. BmSP25 contains a Tryp_SPc domain, suggesting its potential involvement in food digestion in silkworms [37]. In the present study, we first observed that BmNPV infection induced the upregulation of BmSP25, with both basal and induced expression levels being higher in the resistant SuN strain than in the susceptible P50 strain. This expression pattern strongly suggests that BmSP25 may act as an important response factor in silkworm defense against BmNPV. To verify its function, gain- and loss-of-function experiments were conducted in BmN cells. The results demonstrated that knockdown of BmSP25 promoted viral proliferation, whereas its overexpression significantly suppressed viral replication. This bidirectional evidence clearly establishes the antiviral function of BmSP25 at the cellular level.

Previous studies have shown that certain silkworm digestive enzymes participate in defense responses against BmNPV and that overexpression of endogenous antiviral genes can enhance antiviral capacity. In this study, BmSP25 was specifically overexpressed in the silkworm midgut using a piggyBac transgenic vector driven by the BmP2 promoter. Overexpression of BmSP25 resulted in a reduction in viral DNA copies in BmN cells, indicating that, similar to other antiviral proteins, BmSP25 can inhibit BmNPV replication. The outer structure of BmNPV mainly consists of the capsid and envelope proteins [41]. After incubation with recombinant BmTA and BmSP142 proteins [19,42], viral DNA copy numbers in silkworm larvae and BmN cells were significantly reduced. It has been hypothesized that these proteins may induce proteolytic cleavage of viral structural proteins, thereby disrupt viral integrity and reducing infectivity. Although the present study did not examine the effects of co-incubating recombinant BmSP25 with BmNPV virions either in vivo or in vitro, the high similarity in functional domains and active sites among BmSP25, BmSP142, and BmTA suggests that BmSP25 may mediate antiviral effects through a comparable proteolytic mechanism.

This study revealed an immune-related phenotype of the BmSP25 gene in defense against BmNPV infection in silkworms. Although BmSP25 has primarily been annotated as a digestion-related gene, its altered expression following viral infection and its regulatory effects on viral proliferation suggest that BmSP25 may belong to a class of “dual-function” genes in insects that are involved in both metabolism and immune regulation. Similar phenomena have been reported in Drosophila, in which certain digestive enzyme genes have evolved to acquire pathogen-recognition or immunomodulatory functions through structural adaptations [43]. In Drosophila, clip-domain serine proteases, such as Persephone, Grass, Spirit, and SPE (Spätzle-processing enzyme), participate in activation of the Toll immune signaling pathway, ultimately inducing the expression of antimicrobial peptides. These proteases contain trypsin-like serine protease domains [44], which not only degrade ingested proteins but also play key roles in immune signal transduction. The precursor Spätzle protein is cleaved by serine proteases in the hemolymph to generate active Spätzle, which subsequently binds to the membrane receptor Toll, thereby activating the Toll immune signaling pathway [45–47]. Therefore, BmSP25 may represent an evolutionary adaptation in insects, reflecting a “digestion–immunity coupling” defense strategy that is particularly suited for protection against orally acquired pathogens.

The BmSP25 gene was knocked out in silkworms using transgenic technology. Guided by BmSP25-sgRNA, the Cas9 protein successfully cleaved the target site of the BmSP25 gene. Compared with normal silkworms, the BmSP25-KO strain exhibited significantly higher expression of the viral proliferation-related gene vp39 at 48 h after oral inoculation with BmNPV, indicating that disruption of BmSP25 promoted viral replication in the host. Furthermore, analysis of cumulative mortality throughout the entire fifth instar stage showed that the mortality rate of the BmSP25-KO strain was higher than that of normal silkworms, suggesting that loss of BmSP25 reduced the host defense capacity against BmNPV. However, the difference in mortality between the experimental and control groups did not reach statistical significance. Possible explanations include: (I) the editing efficiency or mutation type at the selected knockout target site may not have completely disrupted gene function; (II) some mutations resulted in frameshifts that were multiples of three, potentially allowing residual or truncated protein function; and (III) functional compensation by other genes following loss of BmSP25 may have masked significant changes in resistance in conventional mortality assays. Through both cellular- and individual-level overexpression and knockdown experiments, we found that overexpression of BmSP25 significantly reduced BmNPV replication efficiency, whereas interference with or knockout of the gene increased viral susceptibility. These results indicate that BmSP25 plays a critical role in antiviral immunity. This study not only identifies BmSP25 as a novel key gene involved in silkworm resistance to BmNPV but also confirms its important function through multi-level experimental evidence. Collectively, these findings provide new insights into the immune defense mechanisms of silkworms against DNA viruses and establish a theoretical basis, as well as a valuable candidate target, for breeding highly disease-resistant silkworm varieties through gene editing or molecular marker-assisted selection.

Supporting information

S1 Fig. The standard curve of vp39 and gp41 gene.

https://doi.org/10.1371/journal.pone.0345502.s001

(TIF)

S2 Fig. The densitometric intensity of the western blot was analyzed by using AlphaEase FC software.

Significant differences were indicated by different letter (P < 0.05).

https://doi.org/10.1371/journal.pone.0345502.s002

(TIF)

S3 Fig. Images of positive transgenic silkworm of adults between bright and green lights.

Positive individuals show green fluorescence in the silkworm body, scale bar = 2 mm.

https://doi.org/10.1371/journal.pone.0345502.s003

(TIF)

S4 Fig. Observation of different fluorescence in larvae of the silkworm BmSP25-KO knockout strain.

The BmSP25-KO strain exhibited both red and green fluorescence, the BmSP25-sgRNA strain showed red fluorescence, and the Cas9 strain displayed green fluorescence, the negative individual silkworms didn’t carry these two fluorescent markers, scale bar = 2 mm.

https://doi.org/10.1371/journal.pone.0345502.s004

(TIF)

S5 Fig. DNA sequencing analysis of the CRISPR/Cas9 editing target genes.

305C/WT: Negative individual; sgRNA: Individual containing BmSP25 sgRNA, site1: Knockout site 1 of BmSP25 gene exon1, site2: Knockout site 1 of BmSP25 gene exon4; Cas9: Individual expressing only Cas9 protein; KO 1# ～ 10#: Double-fluorescent individual.

https://doi.org/10.1371/journal.pone.0345502.s005

(TIF)

S1 Table. Gray value of the specimen.

https://doi.org/10.1371/journal.pone.0345502.s006

(DOCX)

S1 Data. Original data.

https://doi.org/10.1371/journal.pone.0345502.s007

(ZIP)

Acknowledgments

We would like to thank RenZe Zhang of State Key Laboratory of Resource Insects, Southwest University for microscopic injection of silkworm eggs.

References

1. Goldsmith MR, Shimada T, Abe H. The genetics and genomics of the silkworm, Bombyx mori. Annu Rev Entomol. 2005;50:71–100. pmid:15355234
- View Article
- PubMed/NCBI
- Google Scholar
2. Shao Q, Yang B, Xu Q, Li X, Lu Z, Wang C, et al. Hindgut innate immunity and regulation of fecal microbiota through melanization in insects. J Biol Chem. 2012;287(17):14270–9. pmid:22375003
- View Article
- PubMed/NCBI
- Google Scholar
3. Feng Z, Li Z, He Q, Deng B, Ma H, Chen W, et al. Transcriptional activation of BmToll9-2 to exogenous dsRNA in the larvae of Bombyx mori. J Insect Physiol. 2025;165:104860. pmid:40701287
- View Article
- PubMed/NCBI
- Google Scholar
4. Jiang L, Xia Q. The progress and future of enhancing antiviral capacity by transgenic technology in the silkworm Bombyx mori. Insect Biochem Mol Biol. 2014;48:1–7. pmid:24561307
- View Article
- PubMed/NCBI
- Google Scholar
5. Chen S, Hou C, Bi H, Wang Y, Xu J, Li M, et al. Transgenic Clustered Regularly Interspaced Short Palindromic Repeat/Cas9-Mediated Viral Gene Targeting for Antiviral Therapy of Bombyx mori Nucleopolyhedrovirus. J Virol. 2017;91(8):e02465-16. pmid:28122981
- View Article
- PubMed/NCBI
- Google Scholar
6. Wang P, Granados RR. An intestinal mucin is the target substrate for a baculovirus enhancin. Proc Natl Acad Sci U S A. 1997;94(13):6977–82. pmid:9192677
- View Article
- PubMed/NCBI
- Google Scholar
7. Ponnuvel KM, Nakazawa H, Furukawa S, Asaoka A, Ishibashi J, Tanaka H, et al. A lipase isolated from the silkworm Bombyx mori shows antiviral activity against nucleopolyhedrovirus. J Virol. 2003;77(19):10725–9. pmid:12970462
- View Article
- PubMed/NCBI
- Google Scholar
8. Nakazawa H, Tsuneishi E, Ponnuvel KM, Furukawa S, Asaoka A, Tanaka H, et al. Antiviral activity of a serine protease from the digestive juice of Bombyx mori larvae against nucleopolyhedrovirus. Virology. 2004;321(1):154–62. pmid:15033574
- View Article
- PubMed/NCBI
- Google Scholar
9. Selot R, Kumar V, Shukla S, Chandrakuntal K, Brahmaraju M, Dandin SB, et al. Identification of a soluble NADPH oxidoreductase (BmNOX) with antiviral activities in the gut juice of Bombyx mori. Biosci Biotechnol Biochem. 2007;71(1):200–5. pmid:17213661
- View Article
- PubMed/NCBI
- Google Scholar
10. Ponnuvel KM, Nithya K, Sirigineedi S, Awasthi AK, Yamakawa M. In vitro antiviral activity of an alkaline trypsin from the digestive juice of Bombyx mori larvae against nucleopolyhedrovirus. Arch Insect Biochem Physiol. 2012;81(2):90–104. pmid:22898997
- View Article
- PubMed/NCBI
- Google Scholar
11. Zhang S-Z, Zhu L-B, You L-L, Wang J, Cao H-H, Liu Y-X, et al. A Novel Digestive Proteinase Lipase Member H-A in Bombyx mori Contributes to Digestive Juice Antiviral Activity Against B. mori Nucleopolyhedrovirus. Insects. 2020;11(3):154. pmid:32121517
- View Article
- PubMed/NCBI
- Google Scholar
12. Lü P, Xia H, Gao L, Pan Y, Wang Y, Cheng X, et al. V-ATPase Is Involved in Silkworm Defense Response against Bombyx mori Nucleopolyhedrovirus. PLoS One. 2013;8(6):e64962. pmid:23823190
- View Article
- PubMed/NCBI
- Google Scholar
13. Bao Y-Y, Tang X-D, Lv Z-Y, Wang X-Y, Tian C-H, Xu YP, et al. Gene expression profiling of resistant and susceptible Bombyx mori strains reveals nucleopolyhedrovirus-associated variations in host gene transcript levels. Genomics. 2009;94(2):138–45. pmid:19389468
- View Article
- PubMed/NCBI
- Google Scholar
14. Zhou Y, Gao L, Shi H, Xia H, Gao L, Lian C, et al. Microarray analysis of gene expression profile in resistant and susceptible Bombyx mori strains reveals resistance-related genes to nucleopolyhedrovirus. Genomics. 2013;101(4):256–62. pmid:23434630
- View Article
- PubMed/NCBI
- Google Scholar
15. Cheng Y, Wang X, Du C, Gao J, Xu J. Expression analysis of several antiviral related genes to BmNPV in different resistant strains of silkworm, Bombyx mori. J Insect Sci. 2014;14:76. pmid:25373223
- View Article
- PubMed/NCBI
- Google Scholar
16. Jiang L, Liu W, Guo H, Dang Y, Cheng T, Yang W, et al. Distinct Functions of Bombyx mori Peptidoglycan Recognition Protein 2 in Immune Responses to Bacteria and Viruses. Front Immunol. 2019;10:776. pmid:31031766
- View Article
- PubMed/NCBI
- Google Scholar
17. Liu T-H, Dong X-L, Pan C-X, Du G-Y, Wu Y-F, Yang J-G, et al. A newly discovered member of the Atlastin family, BmAtlastin-n, has an antiviral effect against BmNPV in Bombyx mori. Sci Rep. 2016;6:28946. pmid:27353084
- View Article
- PubMed/NCBI
- Google Scholar
18. Chen T-T, Tan L-R, Hu N, Dong Z-Q, Hu Z-G, Jiang Y-M, et al. C-lysozyme contributes to antiviral immunity in Bombyx mori against nucleopolyhedrovirus infection. J Insect Physiol. 2018;108:54–60. pmid:29778904
- View Article
- PubMed/NCBI
- Google Scholar
19. Cao H-H, Zhang S-Z, Zhu L-B, Wang J, Liu Y-X, Wang Y-L, et al. The digestive proteinase trypsin, alkaline A contributes to anti-BmNPV activity in silkworm (Bombyx mori). Dev Comp Immunol. 2021;119:104035. pmid:33535067
- View Article
- PubMed/NCBI
- Google Scholar
20. Hu Z-G, Dong Z-Q, Dong F-F, Zhu Y, Chen P, Lu C, et al. Identification of a PP2A gene in Bombyx mori with antiviral function against B. mori nucleopolyhedrovirus. Insect Sci. 2020;27(4):687–96. pmid:31070299
- View Article
- PubMed/NCBI
- Google Scholar
21. Dong X, Liu T, Wang W, Pan C, Wu Y, Du G, et al. BmREEPa Is a Novel Gene that Facilitates BmNPV Entry into Silkworm Cells. PLoS One. 2015;10(12):e0144575. pmid:26656276
- View Article
- PubMed/NCBI
- Google Scholar
22. Feng M, Kong X, Zhang J, Xu W, Wu X. Identification of a novel host protein SINAL10 interacting with GP64 and its role in Bombyx mori nucleopolyhedrovirus infection. Virus Res. 2018;247:102–10. pmid:29447976
- View Article
- PubMed/NCBI
- Google Scholar
23. Xiao Q, Wang L, Zhou X-L, Zhu Y, Dong Z-Q, Chen P, et al. BmAtg13 promotes the replication and proliferation of Bombyx mori nucleopolyhedrovirus. Pestic Biochem Physiol. 2019;157:143–51. pmid:31153462
- View Article
- PubMed/NCBI
- Google Scholar
24. Terra WR, Ferreira C. Insect digestive enzymes: properties, compartmentalization and function. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry. 1994;109(1):1–62.
- View Article
- Google Scholar
25. Srinivasan A, Giri AP, Gupta VS. Structural and functional diversities in lepidopteran serine proteases. Cell Mol Biol Lett. 2006;11(1):132–54. pmid:16847755
- View Article
- PubMed/NCBI
- Google Scholar
26. Di Cera E. Serine proteases. IUBMB Life. 2009;61(5):510–5. pmid:19180666
- View Article
- PubMed/NCBI
- Google Scholar
27. Gohara DW, Di Cera E. Allostery in trypsin-like proteases suggests new therapeutic strategies. Trends Biotechnol. 2011;29(11):577–85. pmid:21726912
- View Article
- PubMed/NCBI
- Google Scholar
28. Zhao P, Wang G-H, Dong Z-M, Duan J, Xu P-Z, Cheng T-C, et al. Genome-wide identification and expression analysis of serine proteases and homologs in the silkworm Bombyx mori. BMC Genomics. 2010;11:405. pmid:20576138
- View Article
- PubMed/NCBI
- Google Scholar
29. Liu H-W, Li Y-S, Tang X, Guo P-C, Wang D-D, Zhou C-Y, et al. A midgut-specific serine protease, BmSP36, is involved in dietary protein digestion in the silkworm, Bombyx mori. Insect Sci. 2017;24(5):753–67. pmid:27311916
- View Article
- PubMed/NCBI
- Google Scholar
30. Liu HW, Li YS, Tang X, Zhang XL, Zhao P. Characterization and expression analysis of serine protease BmSP141 from the silkworm (Bombyx mori) in response to starvation. Scientia Agric Sinica. 2016;49(6):1207–18.
- View Article
- Google Scholar
31. Cerenius L, Lee BL, Söderhäll K. The proPO-system: pros and cons for its role in invertebrate immunity. Trends Immunol. 2008;29(6):263–71. pmid:18457993
- View Article
- PubMed/NCBI
- Google Scholar
32. Perona JJ, Craik CS. Structural basis of substrate specificity in the serine proteases. Protein Sci. 1995;4(3):337–60. pmid:7795518
- View Article
- PubMed/NCBI
- Google Scholar
33. Zou Z, Lopez DL, Kanost MR, Evans JD, Jiang H. Comparative analysis of serine protease-related genes in the honey bee genome: possible involvement in embryonic development and innate immunity. Insect Mol Biol. 2006;15(5):603–14. pmid:17069636
- View Article
- PubMed/NCBI
- Google Scholar
34. Tanaka H, Ishibashi J, Fujita K, Nakajima Y, Sagisaka A, Tomimoto K, et al. A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori. Insect Biochem Mol Biol. 2008;38(12):1087–110. pmid:18835443
- View Article
- PubMed/NCBI
- Google Scholar
35. Jiang L, Goldsmith MR, Xia Q. Advances in the Arms Race Between Silkworm and Baculovirus. Front Immunol. 2021;12:628151. pmid:33633750
- View Article
- PubMed/NCBI
- Google Scholar
36. Xia Q, Cheng D, Duan J, Wang G, Cheng T, Zha X, et al. Microarray-based gene expression profiles in multiple tissues of the domesticated silkworm, Bombyx mori. Genome Biol. 2007;8(8):R162. pmid:17683582
- View Article
- PubMed/NCBI
- Google Scholar
37. Zhang YH, Zhu F, Tang FF, Shao YL, Bai XR. Transcription analysis of serine protease gene BmSP25 in Bombyx mori and its immune reponse. J Southern Agric. 2017;48(6):1093–8.
- View Article
- Google Scholar
38. Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods. 2001;25(4):402–8.
- View Article
- Google Scholar
39. Zhang S-Z, Wang J, Zhu L-B, Toufeeq S, Xu X, You L-L, et al. Quantitative label-free proteomic analysis reveals differentially expressed proteins in the digestive juice of resistant versus susceptible silkworm strains and their predicted impacts on BmNPV infection. J Proteomics. 2020;210:103527. pmid:31610263
- View Article
- PubMed/NCBI
- Google Scholar
40. Rawlings ND, Tolle DP, Barrett AJ. Evolutionary families of peptidase inhibitors. Biochem J. 2004;378(Pt 3):705–16. pmid:14705960
- View Article
- PubMed/NCBI
- Google Scholar
41. Tang Q, Li G, Yao Q, Chen L, Lv P, Lian C, et al. Bm91 is an envelope component of ODV but is dispensable for the propagation of Bombyx mori nucleopolyhedrovirus. J Invertebr Pathol. 2013;113(1):70–7. pmid:23391406
- View Article
- PubMed/NCBI
- Google Scholar
42. Li G, Zhou Q, Qiu L, Yao Q, Chen K, Tang Q, et al. Serine protease Bm-SP142 was differentially expressed in resistant and susceptible Bombyx mori strains, involving in the defence response to viral infection. PLoS One. 2017;12(4):e0175518. pmid:28414724
- View Article
- PubMed/NCBI
- Google Scholar
43. Lemaitre B, Hoffmann J. The host defense of Drosophila melanogaster. Annu Rev Immunol. 2007;25:697–743. pmid:17201680
- View Article
- PubMed/NCBI
- Google Scholar
44. Piao S, Song Y-L, Kim JH, Park SY, Park JW, Lee BL, et al. Crystal structure of a clip-domain serine protease and functional roles of the clip domains. EMBO J. 2005;24(24):4404–14. pmid:16362048
- View Article
- PubMed/NCBI
- Google Scholar
45. El Chamy L, Leclerc V, Caldelari I, Reichhart J-M. Sensing of “danger signals” and pathogen-associated molecular patterns defines binary signaling pathways “upstream” of Toll. Nat Immunol. 2008;9(10):1165–70. pmid:18724373
- View Article
- PubMed/NCBI
- Google Scholar
46. Kambris Z, Brun S, Jang I-H, Nam H-J, Romeo Y, Takahashi K, et al. Drosophila immunity: a large-scale in vivo RNAi screen identifies five serine proteases required for Toll activation. Curr Biol. 2006;16(8):808–13. pmid:16631589
- View Article
- PubMed/NCBI
- Google Scholar
47. Ligoxygakis P, Pelte N, Hoffmann JA, Reichhart J-M. Activation of Drosophila Toll during fungal infection by a blood serine protease. Science. 2002;297(5578):114–6. pmid:12098703
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Goldsmith MR, Shimada T, Abe H. The genetics and genomics of the silkworm, Bombyx mori. Annu Rev Entomol. 2005;50:71–100. pmid:15355234
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Shao Q, Yang B, Xu Q, Li X, Lu Z, Wang C, et al. Hindgut innate immunity and regulation of fecal microbiota through melanization in insects. J Biol Chem. 2012;287(17):14270–9. pmid:22375003
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Feng Z, Li Z, He Q, Deng B, Ma H, Chen W, et al. Transcriptional activation of BmToll9-2 to exogenous dsRNA in the larvae of Bombyx mori. J Insect Physiol. 2025;165:104860. pmid:40701287
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Jiang L, Xia Q. The progress and future of enhancing antiviral capacity by transgenic technology in the silkworm Bombyx mori. Insect Biochem Mol Biol. 2014;48:1–7. pmid:24561307
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Chen S, Hou C, Bi H, Wang Y, Xu J, Li M, et al. Transgenic Clustered Regularly Interspaced Short Palindromic Repeat/Cas9-Mediated Viral Gene Targeting for Antiviral Therapy of Bombyx mori Nucleopolyhedrovirus. J Virol. 2017;91(8):e02465-16. pmid:28122981
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Wang P, Granados RR. An intestinal mucin is the target substrate for a baculovirus enhancin. Proc Natl Acad Sci U S A. 1997;94(13):6977–82. pmid:9192677
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Ponnuvel KM, Nakazawa H, Furukawa S, Asaoka A, Ishibashi J, Tanaka H, et al. A lipase isolated from the silkworm Bombyx mori shows antiviral activity against nucleopolyhedrovirus. J Virol. 2003;77(19):10725–9. pmid:12970462
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Nakazawa H, Tsuneishi E, Ponnuvel KM, Furukawa S, Asaoka A, Tanaka H, et al. Antiviral activity of a serine protease from the digestive juice of Bombyx mori larvae against nucleopolyhedrovirus. Virology. 2004;321(1):154–62. pmid:15033574
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. Selot R, Kumar V, Shukla S, Chandrakuntal K, Brahmaraju M, Dandin SB, et al. Identification of a soluble NADPH oxidoreductase (BmNOX) with antiviral activities in the gut juice of Bombyx mori. Biosci Biotechnol Biochem. 2007;71(1):200–5. pmid:17213661
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. Ponnuvel KM, Nithya K, Sirigineedi S, Awasthi AK, Yamakawa M. In vitro antiviral activity of an alkaline trypsin from the digestive juice of Bombyx mori larvae against nucleopolyhedrovirus. Arch Insect Biochem Physiol. 2012;81(2):90–104. pmid:22898997
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Zhang S-Z, Zhu L-B, You L-L, Wang J, Cao H-H, Liu Y-X, et al. A Novel Digestive Proteinase Lipase Member H-A in Bombyx mori Contributes to Digestive Juice Antiviral Activity Against B. mori Nucleopolyhedrovirus. Insects. 2020;11(3):154. pmid:32121517
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref12] 12. Lü P, Xia H, Gao L, Pan Y, Wang Y, Cheng X, et al. V-ATPase Is Involved in Silkworm Defense Response against Bombyx mori Nucleopolyhedrovirus. PLoS One. 2013;8(6):e64962. pmid:23823190
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref13] 13. Bao Y-Y, Tang X-D, Lv Z-Y, Wang X-Y, Tian C-H, Xu YP, et al. Gene expression profiling of resistant and susceptible Bombyx mori strains reveals nucleopolyhedrovirus-associated variations in host gene transcript levels. Genomics. 2009;94(2):138–45. pmid:19389468
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref14] 14. Zhou Y, Gao L, Shi H, Xia H, Gao L, Lian C, et al. Microarray analysis of gene expression profile in resistant and susceptible Bombyx mori strains reveals resistance-related genes to nucleopolyhedrovirus. Genomics. 2013;101(4):256–62. pmid:23434630
View Article
PubMed/NCBI
Google Scholar

[54] View Article

[55] PubMed/NCBI

[56] Google Scholar

[ref15] 15. Cheng Y, Wang X, Du C, Gao J, Xu J. Expression analysis of several antiviral related genes to BmNPV in different resistant strains of silkworm, Bombyx mori. J Insect Sci. 2014;14:76. pmid:25373223
View Article
PubMed/NCBI
Google Scholar

[58] View Article

[59] PubMed/NCBI

[60] Google Scholar

[ref16] 16. Jiang L, Liu W, Guo H, Dang Y, Cheng T, Yang W, et al. Distinct Functions of Bombyx mori Peptidoglycan Recognition Protein 2 in Immune Responses to Bacteria and Viruses. Front Immunol. 2019;10:776. pmid:31031766
View Article
PubMed/NCBI
Google Scholar

[62] View Article

[63] PubMed/NCBI

[64] Google Scholar

[ref17] 17. Liu T-H, Dong X-L, Pan C-X, Du G-Y, Wu Y-F, Yang J-G, et al. A newly discovered member of the Atlastin family, BmAtlastin-n, has an antiviral effect against BmNPV in Bombyx mori. Sci Rep. 2016;6:28946. pmid:27353084
View Article
PubMed/NCBI
Google Scholar

[66] View Article

[67] PubMed/NCBI

[68] Google Scholar

[ref18] 18. Chen T-T, Tan L-R, Hu N, Dong Z-Q, Hu Z-G, Jiang Y-M, et al. C-lysozyme contributes to antiviral immunity in Bombyx mori against nucleopolyhedrovirus infection. J Insect Physiol. 2018;108:54–60. pmid:29778904
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref19] 19. Cao H-H, Zhang S-Z, Zhu L-B, Wang J, Liu Y-X, Wang Y-L, et al. The digestive proteinase trypsin, alkaline A contributes to anti-BmNPV activity in silkworm (Bombyx mori). Dev Comp Immunol. 2021;119:104035. pmid:33535067
View Article
PubMed/NCBI
Google Scholar

[74] View Article

[75] PubMed/NCBI

[76] Google Scholar

[ref20] 20. Hu Z-G, Dong Z-Q, Dong F-F, Zhu Y, Chen P, Lu C, et al. Identification of a PP2A gene in Bombyx mori with antiviral function against B. mori nucleopolyhedrovirus. Insect Sci. 2020;27(4):687–96. pmid:31070299
View Article
PubMed/NCBI
Google Scholar

[78] View Article

[79] PubMed/NCBI

[80] Google Scholar

[ref21] 21. Dong X, Liu T, Wang W, Pan C, Wu Y, Du G, et al. BmREEPa Is a Novel Gene that Facilitates BmNPV Entry into Silkworm Cells. PLoS One. 2015;10(12):e0144575. pmid:26656276
View Article
PubMed/NCBI
Google Scholar

[82] View Article

[83] PubMed/NCBI

[84] Google Scholar

[ref22] 22. Feng M, Kong X, Zhang J, Xu W, Wu X. Identification of a novel host protein SINAL10 interacting with GP64 and its role in Bombyx mori nucleopolyhedrovirus infection. Virus Res. 2018;247:102–10. pmid:29447976
View Article
PubMed/NCBI
Google Scholar

[86] View Article

[87] PubMed/NCBI

[88] Google Scholar

[ref23] 23. Xiao Q, Wang L, Zhou X-L, Zhu Y, Dong Z-Q, Chen P, et al. BmAtg13 promotes the replication and proliferation of Bombyx mori nucleopolyhedrovirus. Pestic Biochem Physiol. 2019;157:143–51. pmid:31153462
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref24] 24. Terra WR, Ferreira C. Insect digestive enzymes: properties, compartmentalization and function. Comparative Biochemistry and Physiology Part B: Comparative Biochemistry. 1994;109(1):1–62.
View Article
Google Scholar

[94] View Article

[95] Google Scholar

[ref25] 25. Srinivasan A, Giri AP, Gupta VS. Structural and functional diversities in lepidopteran serine proteases. Cell Mol Biol Lett. 2006;11(1):132–54. pmid:16847755
View Article
PubMed/NCBI
Google Scholar

[97] View Article

[98] PubMed/NCBI

[99] Google Scholar

[ref26] 26. Di Cera E. Serine proteases. IUBMB Life. 2009;61(5):510–5. pmid:19180666
View Article
PubMed/NCBI
Google Scholar

[101] View Article

[102] PubMed/NCBI

[103] Google Scholar

[ref27] 27. Gohara DW, Di Cera E. Allostery in trypsin-like proteases suggests new therapeutic strategies. Trends Biotechnol. 2011;29(11):577–85. pmid:21726912
View Article
PubMed/NCBI
Google Scholar

[105] View Article

[106] PubMed/NCBI

[107] Google Scholar

[ref28] 28. Zhao P, Wang G-H, Dong Z-M, Duan J, Xu P-Z, Cheng T-C, et al. Genome-wide identification and expression analysis of serine proteases and homologs in the silkworm Bombyx mori. BMC Genomics. 2010;11:405. pmid:20576138
View Article
PubMed/NCBI
Google Scholar

[109] View Article

[110] PubMed/NCBI

[111] Google Scholar

[ref29] 29. Liu H-W, Li Y-S, Tang X, Guo P-C, Wang D-D, Zhou C-Y, et al. A midgut-specific serine protease, BmSP36, is involved in dietary protein digestion in the silkworm, Bombyx mori. Insect Sci. 2017;24(5):753–67. pmid:27311916
View Article
PubMed/NCBI
Google Scholar

[113] View Article

[114] PubMed/NCBI

[115] Google Scholar

[ref30] 30. Liu HW, Li YS, Tang X, Zhang XL, Zhao P. Characterization and expression analysis of serine protease BmSP141 from the silkworm (Bombyx mori) in response to starvation. Scientia Agric Sinica. 2016;49(6):1207–18.
View Article
Google Scholar

[117] View Article

[118] Google Scholar

[ref31] 31. Cerenius L, Lee BL, Söderhäll K. The proPO-system: pros and cons for its role in invertebrate immunity. Trends Immunol. 2008;29(6):263–71. pmid:18457993
View Article
PubMed/NCBI
Google Scholar

[120] View Article

[121] PubMed/NCBI

[122] Google Scholar

[ref32] 32. Perona JJ, Craik CS. Structural basis of substrate specificity in the serine proteases. Protein Sci. 1995;4(3):337–60. pmid:7795518
View Article
PubMed/NCBI
Google Scholar

[124] View Article

[125] PubMed/NCBI

[126] Google Scholar

[ref33] 33. Zou Z, Lopez DL, Kanost MR, Evans JD, Jiang H. Comparative analysis of serine protease-related genes in the honey bee genome: possible involvement in embryonic development and innate immunity. Insect Mol Biol. 2006;15(5):603–14. pmid:17069636
View Article
PubMed/NCBI
Google Scholar

[128] View Article

[129] PubMed/NCBI

[130] Google Scholar

[ref34] 34. Tanaka H, Ishibashi J, Fujita K, Nakajima Y, Sagisaka A, Tomimoto K, et al. A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori. Insect Biochem Mol Biol. 2008;38(12):1087–110. pmid:18835443
View Article
PubMed/NCBI
Google Scholar

[132] View Article

[133] PubMed/NCBI

[134] Google Scholar

[ref35] 35. Jiang L, Goldsmith MR, Xia Q. Advances in the Arms Race Between Silkworm and Baculovirus. Front Immunol. 2021;12:628151. pmid:33633750
View Article
PubMed/NCBI
Google Scholar

[136] View Article

[137] PubMed/NCBI

[138] Google Scholar

[ref36] 36. Xia Q, Cheng D, Duan J, Wang G, Cheng T, Zha X, et al. Microarray-based gene expression profiles in multiple tissues of the domesticated silkworm, Bombyx mori. Genome Biol. 2007;8(8):R162. pmid:17683582
View Article
PubMed/NCBI
Google Scholar

[140] View Article

[141] PubMed/NCBI

[142] Google Scholar

[ref37] 37. Zhang YH, Zhu F, Tang FF, Shao YL, Bai XR. Transcription analysis of serine protease gene BmSP25 in Bombyx mori and its immune reponse. J Southern Agric. 2017;48(6):1093–8.
View Article
Google Scholar

[144] View Article

[145] Google Scholar

[ref38] 38. Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods. 2001;25(4):402–8.
View Article
Google Scholar

[147] View Article

[148] Google Scholar

[ref39] 39. Zhang S-Z, Wang J, Zhu L-B, Toufeeq S, Xu X, You L-L, et al. Quantitative label-free proteomic analysis reveals differentially expressed proteins in the digestive juice of resistant versus susceptible silkworm strains and their predicted impacts on BmNPV infection. J Proteomics. 2020;210:103527. pmid:31610263
View Article
PubMed/NCBI
Google Scholar

[150] View Article

[151] PubMed/NCBI

[152] Google Scholar

[ref40] 40. Rawlings ND, Tolle DP, Barrett AJ. Evolutionary families of peptidase inhibitors. Biochem J. 2004;378(Pt 3):705–16. pmid:14705960
View Article
PubMed/NCBI
Google Scholar

[154] View Article

[155] PubMed/NCBI

[156] Google Scholar

[ref41] 41. Tang Q, Li G, Yao Q, Chen L, Lv P, Lian C, et al. Bm91 is an envelope component of ODV but is dispensable for the propagation of Bombyx mori nucleopolyhedrovirus. J Invertebr Pathol. 2013;113(1):70–7. pmid:23391406
View Article
PubMed/NCBI
Google Scholar

[158] View Article

[159] PubMed/NCBI

[160] Google Scholar

[ref42] 42. Li G, Zhou Q, Qiu L, Yao Q, Chen K, Tang Q, et al. Serine protease Bm-SP142 was differentially expressed in resistant and susceptible Bombyx mori strains, involving in the defence response to viral infection. PLoS One. 2017;12(4):e0175518. pmid:28414724
View Article
PubMed/NCBI
Google Scholar

[162] View Article

[163] PubMed/NCBI

[164] Google Scholar

[ref43] 43. Lemaitre B, Hoffmann J. The host defense of Drosophila melanogaster. Annu Rev Immunol. 2007;25:697–743. pmid:17201680
View Article
PubMed/NCBI
Google Scholar

[166] View Article

[167] PubMed/NCBI

[168] Google Scholar

[ref44] 44. Piao S, Song Y-L, Kim JH, Park SY, Park JW, Lee BL, et al. Crystal structure of a clip-domain serine protease and functional roles of the clip domains. EMBO J. 2005;24(24):4404–14. pmid:16362048
View Article
PubMed/NCBI
Google Scholar

[170] View Article

[171] PubMed/NCBI

[172] Google Scholar

[ref45] 45. El Chamy L, Leclerc V, Caldelari I, Reichhart J-M. Sensing of “danger signals” and pathogen-associated molecular patterns defines binary signaling pathways “upstream” of Toll. Nat Immunol. 2008;9(10):1165–70. pmid:18724373
View Article
PubMed/NCBI
Google Scholar

[174] View Article

[175] PubMed/NCBI

[176] Google Scholar

[ref46] 46. Kambris Z, Brun S, Jang I-H, Nam H-J, Romeo Y, Takahashi K, et al. Drosophila immunity: a large-scale in vivo RNAi screen identifies five serine proteases required for Toll activation. Curr Biol. 2006;16(8):808–13. pmid:16631589
View Article
PubMed/NCBI
Google Scholar

[178] View Article

[179] PubMed/NCBI

[180] Google Scholar

[ref47] 47. Ligoxygakis P, Pelte N, Hoffmann JA, Reichhart J-M. Activation of Drosophila Toll during fungal infection by a blood serine protease. Science. 2002;297(5578):114–6. pmid:12098703
View Article
PubMed/NCBI
Google Scholar

[182] View Article

[183] PubMed/NCBI

[184] Google Scholar

Figures

Abstract

Introduction

Materials and methods

Silkworm rearing and viral particles

BmN cell culture and transfection

Bioinformatics analysis

RNA isolation and cDNA synthesis

Quantitative reverse transcription PCR (RT-qPCR) analysis

Prokaryotic expression and antibody preparation

Western blot analysis

Effect of BmSP25 RNAi and overexpression on BV-EGFP proliferation in BmN cells

Effect of BmSP25 overexpression and knockout on BmNPV proliferation in silkworms

Statistical analysis

Results

Characterization of the BmSP25 sequence

BmSP25 exhibited specific expression patterns in response to BmNPV infection

BmSP25 interference affected the susceptibility of BmN cells to BmNPV

BmSP25 overexpression enhanced the susceptibility of BmN cells to BmNPV invasion

BmSP25 overexpression inhibited the invasion of BmNPV into silkworm individuals

BmSP25 knockdown enhanced the invasion of BmNPV into silkworm individuals

Discussion

Supporting information

S1 Fig. The standard curve of vp39 and gp41 gene.

S2 Fig. The densitometric intensity of the western blot was analyzed by using AlphaEase FC software.

S3 Fig. Images of positive transgenic silkworm of adults between bright and green lights.

S4 Fig. Observation of different fluorescence in larvae of the silkworm BmSP25-KO knockout strain.

S5 Fig. DNA sequencing analysis of the CRISPR/Cas9 editing target genes.

S1 Table. Gray value of the specimen.

S1 Data. Original data.

Acknowledgments

References