Introduction
The silkworm (Bombyx mori), a silk-spitting cocooning economic insect that feeds on mulberry leaves, is a fully domesticated Lepidoptera insect that plays an important role in the economic development of many developing countries. It also has important scientific research value and is used as a model organism for basic research and applied biotechnology, and is an important model for genetic and immunological studies (Goldsmith et al., Reference Goldsmith, Shimada and Abe2005; Jiang and Xia, Reference Jiang and Xia2014). The silkworm (Bombyx mori) is susceptible to pathogenic microorganisms such as bacteria, fungi, microsporidia, and viruses during sericulture production, which can seriously damage the sericulture economy (Jiang et al., Reference Jiang, Liu, Guo, Dang, Cheng, Yang, Sun, Wang, Wang, Xie and Xia2019). Among them, Bombyx mori nuclear polyhedrosis virus (BmNPV) poses the greatest threat to the silkworm (Jiang et al., Reference Jiang, Zhao, Cheng, Sun, Peng, Dang, Wu, Wang, Jin, Lin and Xia2013). Viruses rely on host cellular mechanisms for their own reproduction by promoting cellular anabolism to produce the biomolecules required for viral particle replication and assembly. Thus, viral infection can reprogram host cell metabolism to create an optimal environment for virus production (Polcicova et al., Reference Polcicova, Badurova and Tomaskova2020; Thai et al., Reference Thai, Graham, Braas, Nehil, Komisopoulou, Kurdistani, McCormick, Graeber and Christofk2014; Thaker et al., Reference Thaker, Ch’ng and Christofk2019). Currently, there are no effective treatments to cope with virus disease in domestic silkworms (Lü et al., Reference Lü, Pan, Yang, Zhu, Li, Guo, Yao and Chen2018). In recent years, anti-BmNPV strategies have been mainly through overexpression of host antiviral proteins and knockdown of key viral genes associated with replication and proliferation of BmNPV (Dong et al., Reference Dong, Dong, Yu, Huang, Jiang, Hu, Chen, Lu and Pan2018; Chen et al., Reference Chen, Hou, Bi, Wang, Xu, Li, James, Huang and Tan2017; Isobe et al., Reference Isobe, Kojima, Matsuyama, Quan, Kanda, Tamura, Sahara, Asano and Bando2004; Jiang et al., Reference Jiang, Cheng, Zhao, Yang, Wang, Jin, Lin, Xiao and Xia2012; Liu et al., Reference Liu, Dong, Pan, Du, Wu, Yang, Chen, Lu and Pan2016; Zhang et al., Reference Zhang, Wang, Lu, Hu, Xue, Cao and Gong2014). In addition, many endogenous housekeeping proteins have been found to have antiviral activity, including BmAtlastin-n (Liu et al., Reference Liu, Dong, Pan, Du, Wu, Yang, Chen, Lu and Pan2016), BmRas3 (Xia et al., Reference Xia, Jiang, Yu, Jia, Wang, Shen, Zhao and Lu2024), BmTPT (Jia et al., Reference Jia, Wang, Jiang, Zhao, Shen, Zhang, Qiu, Wang, Lu and Xia2024), BmFoxO (Kang et al., Reference Kang, Wang, Liang, Tang, Zhang, Wang, Zhao and Lu2021), BmPEPCK-2 (Xiong et al., Reference Xiong, Lei, Zhao and Guan2011), BmPP2A (Hu et al., Reference Hu, Dong, Dong, Zhu, Chen, Lu and Pan2020) and BmIML-2 (Mei et al., Reference Mei, Li, Peng, Wang, He, Qiu, Xia, Zhao and Shen2022).
Aminoacyl-tRNA synthetases (aaRSs) are important housekeeping enzymes involved in the translation of mRNAs (Xu et al., Reference Xu, Love, Ma, Blum, Bronskill, Bernstein, Grey, Hofmann, Camerman and Wong1989). The catalytic activity of aaRSs plays an important role in maintaining cell viability; however, they are also multifunctional proteins regulated by a variety of control mechanisms (Fuchs et al., Reference Fuchs, Schene, Kok, Jansen, Nikkels, van Gassen, Terheggen-Lagro, van der Crabben, Hoeks, Niers, Wolf, de Vries, Koolen, Houwen, Mulder and van Hasselt2019; Kim et al., Reference Kim, You and Hwang2011; Lee et al., Reference Lee, Cho, Park and Kim2004; Yao and Fox, Reference Yao and Fox2013). Most aaRSs are transferred from the cytoplasm to the nucleus or extracellular space in response to specific stimuli and are involved in a variety of biological processes. The expansion of aaRSs’ functions has been linked to a number of biological activities, including transcription, translation, cell death and survival, immunity to microorganisms, chemotaxis and inflammation, angiogenesis, IFN-γ and p53 signaling, mTOR signaling, and tumorigenesis (Guo and Schimmel, Reference Guo and Schimmel2013; Kise et al., Reference Kise, Lee, Park, Fukai, Sengoku, Ishii, Yokoyama, Kim and Nureki2004; Kisselev et al., Reference Kisselev, Frolova and Haenni1993; Sajish et al., Reference Sajish, Zhou, Kishi, Valdez, Kapoor, Guo, Lee, Kim, Yang and Schimmel2012; Wakasugi, Reference Wakasugi2007).
Tryptophanyl-tRNA synthetase (WARS) belongs to the family of class Ic aaRSs (Rubio Gomez and Ibba, Reference Rubio Gomez and Ibba2020), which are important enzymes that catalyze the ligation of tryptophan (Trp) to its cognate tRNA during translation. The specific recognition by WARS of its substrates, Trp and tRNA, is essential for maintaining the fidelity of protein synthesis (Shen et al., Reference Shen, Zhou, Yang, Yu, Dong and Ding2008; Yang et al., Reference Yang, Guo, Kapoor, Ewalt, Otero, Skene, McRee and Schimmel2007; Zhou et al., Reference Zhou, Dong, Shen, Zhong and Ding2010). Partha Seshaiah and Deborah J. Andrew identified highly expressed Tryptophanyl-tRNA Synthetase in the salivary glands of Drosophila, which is the first time that WARS has been cloned in insects (Seshaiah and Andrew, Reference Seshaiah and Andrew1999). Recently, WARS was reported to play an important role in innate immunity against bacterial infections (Ahn et al., Reference Ahn, Park, Choi, Park, Rhee, Kang, Ahn, Lee, Lee, Inn, Cho, Park, Park, Park, Lee, Park, Kwon, Shim, Han, Kim, Lee, Jeon, Huh, Jin and Kim2016). However, its specific role in the response to viral infections has not been elucidated in detail. Boasso et al (Reference Boasso, Herbeuval, Hardy, Winkler and Shearer2005) first reported that the CTLA-4-B7 system regulates IDO and WARS expression in CD4+, CD8+ T cells, linking the two tryptophan metabolizing enzymes to T-cell immunity. According to Ahn et al (Reference Ahn, Park, Choi, Park, Rhee, Kang, Ahn, Lee, Lee, Inn, Cho, Park, Park, Park, Lee, Park, Kwon, Shim, Han, Kim, Lee, Jeon, Huh, Jin and Kim2016), secreted WARS is a major defense against invading pathogens. WARS-stimulated immune cells exhibit secretion of antiviral cytokines increased and facilitated activation of key signaling pathways of the antiviral immune response, ultimately leading to reduced viral replication in vitro and in vivo (Lee et al., Reference Lee, Lee, Uddin, Kim, Kim, Chathuranga, Chathuranga, Jin, Kim, Kim and Lee2019). However, WARS has been rarely studied in silkworms and even insects, and even less so in antiviral studies.
The Phosphatidylinositol 3 kinase/Serine-threonine kinase (PI3K-Akt) pathway is an intracellular signaling pathway that promotes metabolism, proliferation, cell survival, growth, and angiogenesis by responding to extracellular signals (Asati et al., Reference Asati, Mahapatra and Bharti2016). PI3K-Akt signaling has been required for efficient replication of baculoviruses (Xiao et al., Reference Xiao, Yang, Weng, Lin, Yuan, Yang and Pang2009). Hepatitis B virus HBx protein (Chung et al., Reference Chung, Lee, Ko and Kim2003, p. 53) and hepatitis C virus NS5A protein (Cheng et al., Reference Cheng, Zhang, Yang, Zhao, Peng, Tian, Xiao, Chung and Gong2015) have inhibited PTEN expression, eliminated its inhibitory effect on the PI3K/Akt signaling pathway, activated Akt phosphorylation, and promoted cell survival. The PI3K-Akt pathway has been involved in host manipulation by Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) (Li et al., Reference Li, Liu, Yu, Rensing and Wang2022). PI3K, a kinase capable of phosphorylating the hydroxyl group at the 3-position of the inositol ring of phosphatidylinositol (Porta et al., Reference Porta, Paglino and Mosca2014), was first found to be associated with the transforming ability of viral oncoproteins in 1987 (Kaplan et al., Reference Kaplan, Whitman, Schaffhausen, Pallas, White, Cantley and Roberts1987; Whitman et al., Reference Whitman, Kaplan, Schaffhausen, Cantley and Roberts1985). Several studies have clarified that PI3K signaling plays a key role in metabolism, inflammation, cell survival and carcinogenesis (Vanhaesebroeck et al., Reference Vanhaesebroeck, Guillermet-Guibert, Graupera and Bilanges2010). In the PI3K-Akt pathway, the 3-site phosphate group of PIP3 binds to PDK1 and Akt proteins and recruits Akt proteins at the cell membrane, allowing PDK1 to enter the ‘activation loop’ and phosphorylate T308, leading to partial activation of PKB/Akt (Andjelković et al., Reference Andjelković, Alessi, Meier, Fernandez, Lamb, Frech, Cron, Cohen, Lucocq and Hemmings1997; Scheid et al., Reference Scheid, Parsons and Woodgett2005). Activated Akt regulates the function of a variety of substrates that are involved in the regulation of cell survival, cell cycle progression and cell growth (Fresno Vara et al., Reference Fresno Vara, Casado, de Castro, Cejas, Belda-Iniesta and González-Barón2004). First, Akt enhances cell survival by blocking the function and apoptotic process of the pro-apoptotic protein Caspcase9 (Obenauer et al., Reference Obenauer, Cantley and Yaffe2003), and inhibits the expression of the transcription factor FoxO to regulate metabolism, apoptosis and cell proliferation (Laplante and Sabatini, Reference Laplante and Sabatini2012; Xiong et al., Reference Xiong, Tao, DePinho and Dong2013); and cell growth is promoted by inhibiting TSC2 and indirectly activating serine/threonine-protein kinase (mTORC1). mTORC1 is a key regulator of translation initiation and key regulator of ribosome biogenesis and plays an evolutionarily conserved role in cell growth control (Wang et al., Reference Wang, Harris and Lawrence2008). It activates S6K and eukaryotic initiation factor 4E (elF4E) binding protein 1 (4E-BP1), and S6K activates ribosomal S6 to promote protein synthesis and cell growth (Liu et al., Reference Liu, Xie, Wan and Deng2020; Manning and Toker, Reference Manning and Toker2017)
At present, the function of BmWARS in the Bombyx mori has not been reported, and the effect of BmWARS on BmNPV replication and proliferation has not been addressed. In order to explore the effect of BmWARS on BmNPV infection and replication, firstly, BmWARS was cloned and identified from the midgut of Bombyx mori, and the similarity between BmWARS and other species was analyzed by bioinformatics, as well as by tissue expression profiling and induced expression profiling. Further, the effect of BmWARS on BmNPV infection was analyzed by means of overexpression (OE) and siRNA BmWARS in BmN cells; and the subcellular localization of BmWARS was analyzed by immunofluorescence; the effect of BmWARS on the PI3K-Akt signaling pathway was also investigated. The results of this paper suggest that BmWARS can inhibit BmNPV infection and replication by suppressing the PI3K-Akt pathway and thus inhibit BmNPV infection and replication.
Materials and methods
Silkworm species, cell culture and viruses
The domestic silkworm species p50 was kept in our laboratory and reared in mulberry leaves at a temperature of 26 ± 1°C and relative humidity of 70-85%. A 1.0 × 107 OB/mL (occlusion body) wild-type baculovirus was applied to mulberry leaves to infect silkworm larvae orally. BmN cells of Bombyx mori were kept in our laboratory and cultured in TC-100 (Livning Biotechnology Co., Ltd.) medium containing 10 % (v/v) fetal bovine serum (Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, Grand Island, NY, USA). BmNPV-egfp baculoviruses expressing the egfp marker gene were kept in our laboratory for evaluation of viral infection and propagation. BmN cells were infected with BmNPV-egfp at multiplicity of infection (MOI) 5.
Bioinformatics analysis of BmWARS
Protein sequence, structure and 2-D electrophoresis data were analyzed using EXPASY (https://web.expasy.org/translate/). SignalP-6.0 server (https://services.healthtech.dtu.dk/services/SignalP − 6.0/) was used to predict signal peptide shear sites and their location in the amino acid sequence. Prediction of conserved structural domains and motifs was performed on the TMHMM-2.0 website (https://services.healthtech.dtu.dk/services/TMHMM-2.0/). A neighbor-joining method MEGA 6.0 was used to generate the phylogenetic tree of BmWARS (bootstrap = 1000 replications, Poisson model, uniform rates).
RNA extraction and cDNA synthesis
Ice dissections were performed to obtain hemolymph, midgut, head, tracheal clusters, fat body, malpighian tubule, ventral nerve cord, silk gland, testis, ovary, and epidermis tissues from 1 to 5 d after feeding at age five. At the same time, the first feeding of mulberry leaves at five years of age added 107 OB/mL of virus. Total RNA from the above tissues was extracted with TRIzol reagent (TIANGEN, Biotech Co., Ltd., Beijing, China), respectively, and then the first strand cDNA was synthesized according to the instructions of FastKing RT Kit (TIANGEN, China).
Vector construction and siRNA synthesis
The synthetic primer pairs 5ʹ-CCAAGCTTATGAACTTGA TTAAAAGAAGTAT-3ʹ, 5ʹ-CGGATATCTTGAGTGGCGGGCT CTGGGTT-3ʹ were designed according to the sequence of BmWARS published in the GenBank database (accession no. XM_004927121) (the underline is the Hind III, EcoR V restriction endonuclease site; Table 1), the ORF region of BmWARS was obtained by cloning the cDNA of p50 midgut tissue of the silkworm as template, and the amplified product was ligated with pMD18-T vector and sequenced for verification. The obtained T clone containing the target gene and pIZT/V5-His-mCherry were double digested with Hind III and EcoR V, respectively, and the target fragments were recovered, respectively, ligated with T4 DNA ligase and transformed into DH5α receptor cells, and the positive recombinant plasmid, pIZT/V5-His-mCherry-BmWARS, was obtained and then sequenced for verification.
The primers for gene cloning, RT-PCR and siRNA used in this study
The siRNA interfering with the expression of BmWARS was designed and synthesized by Suzhou Gemma Genetics Co Ltd (Suzhou, China), and its oligonucleotide sequences were Bm WARS-270-S (5ʹ-GCAGAACCCUGAAGUAUUATT-3ʹ), BmW ARS-270-AS (5ʹ-UAAUACUUCAGGGUUCUGCTT-3ʹ). The negative control sequences were NC-S (5ʹ-UUCUCCGAAC GUGUCACGUTT-3ʹ), NC-AS (5ʹ-ACGUGACACGUUCGGA GAATT-3ʹ). And the sequences are shown in Table 1.
Cell transfection and viral infection
The BmN cells were evenly spread over the cell culture plate, and when the cells were stably attached to the wall and the density was about 60%, the recombinant plasmid and siRNA were transfected into the cells according to the methods of DNA transfection reagent EntransterTM-H4000 (Engreen Biosystem, Beijing, China) and RNA transfection reagent (synthesized by Suzhou Gemma Genetics Co., Ltd., China), respectively. After 4 h of transfection, the cell culture medium was changed to continue the culture. After 24 h of cell transfection treatment, BmNPV-egfp of MOI 5 was added, and the amounts of EGFP-positive cells in each group of cells at 24, 48, and 72 h post-infection was observed and analyzed under a fluorescence inverted microscope, and the cells were collected and kept for reserve.
Cellular genomic DNA extraction
The medium was aspirated from the cell culture plates and cells were collected by adding an appropriate volume of DNA extraction buffer (10 mM Tris-HCl, 100 mM EDTA, 100 mM NaCl, 0.5% SDS; pH = 8.0). After centrifugation, the supernatant was collected and an equal volume of phenol (pH 7.7-pH 8.1) was added and shaken and mixed, after centrifugation again, the supernatant was collected and a 24:1 mixture of chloroform and isopropanol was added and mixed, after centrifugation again, the supernatant was collected and 2 times the volume of anhydrous ethanol containing 3 M sodium acetate (NaAc) was added for precipitation and purification, and finally, the precipitate was lysed in RNase water.
Quantitative real-time PCR (qRT-PCR)
qRT-PCR was performed according to the instruction method. qRT-PCR was carried out in a 10 μL reaction volume, with 2 × SYBR premix Ex TaqTM reagent (Nanjing Novozymes Bio-technology Co., Ltd., Nanjing, China) implemented through a LightCycle 96 real-time fluorescence quantitative PCR instrument (Roche, Switzerland). Three experimental replicates were performed for each set of samples. The reaction conditions were 95°C reaction for 10 min, followed by 40 cycles of 95°C reaction for 5 s, 60°C reaction for 10 s and 72°C reaction for 10 s. The reaction conditions were as follows. Three biological replicates or three technical replicates were done for each experiment, and the expression or copy number of their genes was calculated by 2-∆∆Ct. BmWARS-specific primers (forward 5ʹ-TAAGTTCAGCCAGTAATGTCG-3ʹ, reverse 5ʹ-GCTCCCCAAACAGTACGC-3ʹ) were used for qPCR using the viral VP39 gene (forward 5ʹ-AAGAAGGAGGACGGCAGCAT-3ʹ, reverse 5ʹ-ATCTCGCCAGAAATCCAATAAAAC-3ʹ) was used to quantify the viral DNA load (Xia et al., Reference Xia, Jiang, Yu, Jia, Wang, Shen, Zhao and Lu2024). The Bombyx mori glyceraldehyde-3-phosphate dehydrogenase (BmGAPDH) gene (forward 5ʹ-TTCATGCCACAACTGCTACA-3ʹ, reverse 5ʹ-AGTCAGCTTGCCATTAAGAG-3ʹ) was used as an internal reference (Table 1).
Immunofluorescence localization of BmWARS
BmN cells were fixed on a crawler sheet, and after transfecting the cells with the pIZT/V5-His-mCherry-BmWARS recombinant vector for 48 h, the cells were washed three times with PBS for 3 min each time, fixed with 4% paraformaldehyde for 20 min, the cells were washed three times with PBS, and the cells were permeabilized with 0.5% Triton X-100 (PBS preparation) for 20 min at room temperature, and washed with PBS as described above. The permeabilized cells were closed with 5% BSA prepared in PBST for 1 h. The cells were conjugated to His tag on the recombinant plasmid with mouse anti-6xHis primary antibody (1:800 in 5% BSA) (Sangon Bioengineering (Shanghai) Co., Ltd., Shanghai, China), incubated overnight at 4°C, and washed 3 times with PBS. Cells were incubated with green fluorescence-labeled goat anti-mouse secondary antibody (Shanghai Bioscience Biological Co., Ltd., Shanghai, China) (1:400 in 5% BSA) for 2 h at room temperature, then washed 3 times with PBS and nuclei were stained with 4ʹ,6-diamidino-2-phenylindole (DAPI) (Sangon Bioengineering (Shanghai) Co., Ltd., Shanghai, China) for 15 min (Avoiding light), then washed with PBS for 3 times, blotted on absorbent paper, added a drop of anti-fluorescence attenuating sealer, covered the cell side with the sealer, sealed the edges with fingernail polish, and observed the cells under a fluorescent inverted micro-oil microscope and photographed.
Western blot
The cell samples were treated at a ratio of hydration solution per 1 mg/10 ul, and after resting for 5 min, 1/2 volume of protein Loading buffer was added, and boiled for 3 min for hybridization protein validation. The proteins were migrated onto the PVDF membrane, which was closed with 5% skimmed milk powder solution for 1 h, and washed three times with TBST, in which anti-VP39 (rabbit polyclonal antibody, 1:3000, Shanghai Youke Biotechnology Co., Ltd., Shanghai, China), the phosphorylation of Akt (p-Akt) and Akt (rabbit polyclonal antibody, 1:2000, Cell Signaling Technology, MA, USA) were used as primary antibodies overnight at 4°C, and microtubule protein Tubulin (1:2000) (Beyotime Biotechnology, Shanghai, China) was used as internal reference protein, washed three times again by TBST, incubated with goat anti-rabbit IgG/alkaline phosphatase-labeled secondary antibody (1:2000, Beyotime Biotechnology, Shanghai, China) at room temperature for 2 h. The antibody was washed three times again by TBST and incubated with IgG/alkaline Phosphatase Colorimetric Kit (Beyotime Biotechnology, Shanghai, China) to avoid light to develop the color, and then photographed and stored.
Annexin V-FITC/PI apoptosis assay
According to the above method, pIZT/V5-His-mCherry-BmWARS (BmWARS-OE) and pIZT/V5-His-mCherry (control) were transfected into BmN cells, and then infected with BV at a MOI of 5, and normal cultured cells were used as negative controls. After 72 h of BV infection, the cells were harvested with PBS and stained with Annexin V-FITC and PI as described in the instructions for use of the Annexin V-FITC apoptosis detection kit (Beyotime Biotechnology, Shanghai). Finally, apoptotic cells were analyzed by flow cytometry (Becton, Dickinson and Company, USA) and processed using FlowJo software.
MTT assay for cell proliferation
According to the above method, pIZT/V5-His-mCherry-BmWARS, pIZT/V5-His-mCherry, siRNA-BmWARS-270 (siRNA-270), and siRNA-NC (NC) were transfected into BmN cells, respectively, and cell proliferation was determined by MTT reduction assay. 48 h after transfection, cells were made into cell suspension and introduced into 96-well cell culture plates, and 10 μL MTT reagent was added to each well (MTT kit from Beyotime Biotechnology). The plates were returned to the cell culture incubator to continue incubation for 4 h, and then 100 μL Formazan dissolution solution was added to each well. When all Formazan was dissolved, the plate cover was removed and the absorbance at 570 nm of each well was measured in a microtitre plate reader.
Cell cycle assays
Cells were collected 48 h after transfection with pIZT/V5-His-mCherry-BmWARS and pIZT/V5-His-mCherry and washed twice with pre-cooled PBS. The cell pellet was resuspended in 300 μL of pre-cooled PBS to completely suspend the cells to a single-cell state. The resuspended cells were added to a pre-cooled solution of 700 μL of 99.7% anhydrous ethanol (final ethanol concentration of 70%), mixed thoroughly to avoid cell clumping, fixed at 4°C for 12 h, and centrifuged to collect the cells. The centrifuged cells were washed with 1 mL of PBS and 400 μL of PI staining solution was added. After staining for 30 min, the cell cycle was analyzed by flow cytometry.
Data analysis
The qRT-PCR data were statistically analyzed by GraphPad Prism 5 (GraphPad, San Diego, CA, USA). The significance of the experimental results was also analyzed using the t-test method that comes with the software. Significant differences were indicated when p < 0.05, p < 0.01 for significant differences, and p < 0.001 for highly significant differences. ‘*’ is used to indicate ‘p < 0.05,’ ‘**’ is used to indicate ‘p < 0.01’ and ‘***’ is used to indicate ‘p < 0.001’ in the graphs. All results are expressed as three replicated experiments and results are expressed as mean ± standard deviation (S.D.).
Results and analysis
Sequence characterization and bioinformatics analysis of BmWARS
BmWARS was cloned from the p50 midgut of Bombyx mori and characterized, and the coding sequence of BmWARS was 1149 bp in length, encoding 382 amino acid residues (fig. 1A). The homology of BmWARS protein was analyzed by multiple comparisons with homologous proteins in other species, and the results showed that BmWARS was relatively conserved, and had the highest similarity with the Bombyx mandarina, Manduca sexta, and Danaus plexippus (fig. 1B).
Bioinformatics analysis, the expression profiles analysis of BmWARS and subcellular localization of BmWARS in BmN cells. (A) The asterisk below the sequences represents the consistent amino acid residues. The GenBank accession numbers are as follows: BmWARS, XP_004927178.1; BmaWARS, XP_028030485.1; MsWARS, XP_037294780.1; VtWARS, XP_026499919.1; VaWARS, XP_047534634.1. (B) The phylogenetic relationships of B. mori WARS with other homologous sequences. (C) The mRNA levels of BmWARS in various tissues (3rd day of 5th instar); (D-E) Inducible expression profiles of BmWARS in midgut and silk gland upon BmNPV infection. Margin of error bars reflect the mean ± S.D. of three independent values. Asterisks show significant differences against controls by unpaired t-test. (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001). (F) Subcellular localization of BmWARS in BmN cells. The cells were transfected with pIZT/V5-His-mCherry-BmWARS recombinant vector. After 48 hours of culture, the cells were fixed, permeable, blocked, bound with antibody and stained, and the nuclei were labeled with DAPI. From left to right: antibody-labeled BmWARS; DAPI-stained nuclei; fusion map. The scale bar represents 20 µm.
The evolutionary tree of BmWARS was constructed using the neighbor-joining method, and the evolutionary relationships of BmWARS were analyzed. As shown in fig. 1B, BmWARS showed the highest similarity with Bombyx mandarina, with 98.95 %, followed by higher similarity with Danaus plexippus and Manduca sexta, with 73.78 % and 70.09 %, respectively.
Tissue and induced expression profiling of BmWARS
Eleven tissues of hemolymph, midgut, head, tracheal clusters, fat body, malpighian tubule, ventral nerve cord, silk gland, testis, ovary, and epidermis of Bombyx mori at age 5 days 1-5 were obtained by dissection, and the total RNA of each tissue was extracted and reverse-transcribed into cDNA. qRT-PCR was performed to detect the level of expression of BmWARS in all tissues of Bombyx mori. The results showed that BmWARS was expressed in all tissues of Bombyx mori, and there was obvious differential expression in different tissues (fig. 1C). After transoral infection of p50 silkworms with suspended polyhedrosis virus (107 OB/mL) solution evenly coated with mulberry leaves, the transcript level of BmWARS in the silk gland and midgut of the immunized tissue in silkworms showed a significant increase compared with that of the control group not infected with BmNPV (fig. 1D, E).
Immunofluorescence localization analysis of BmWARS
Localization of BmWARS in cells was detected by immunofluorescence and fluorescence inverted micro-oil microscopy. Cells were transfected with pIZT/V5-His-mCherry-BmWARS recombinant plasmid with a His tag, and the His tag on the recombinant plasmid was conjugated with a murine 6xHis primary antibody, and then the overexpressed BmWARS was de-labeled with goat anti-mouse secondary antibody with a green fluorescent marker, while the nuclei of the cells were rendered 4ʹ,6-Diamidino-2-phenylindole (DAPI) blue color. The results showed that BmWARS was mainly distributed in the cytoplasm (fig. 1F).
Overexpression of BmWARS inhibited the proliferation of BmNPV
Plasmid pIZT/V5-His-mCherry and recombinant plasmid pIZT/V5-His-mCherry-BmWARS overexpressing BmWARS were transfected into BmN cells separately, and qRT-PCR assay revealed that the transcript levels of BmWARS in cells with BmWARS-OE were extremely significantly higher than that in the control group (fig. 2A). At 24 h after plasmid transfection, 2 groups of cells were infected with equal amounts of BmNPV-egfp with MOI 5, and the infection status of BmN cells at 24, 48, and 72 h after viral infection was analyzed. The results showed that at 24, 48, and 72 h after viral infection, EGFP-positive cells in the cells of BmWARS-OE were significantly reduced compared with those of the control group (fig. 2B). Viral replication was detected by qRT-PCR using the BmNPV nuclear coat protein gene VP39 as a template. The results showed that the number of BmNPV in the BmWARS-OE group was extremely significantly lower than that in the control group at 24,48, and 72 h after virus infection (fig. 2C). We further detected the protein expression of viral late-expressed VP39 (39 K protein) by Western blot. The result was that the expression of VP39 was significantly inhibited in BmWARS-OE cells 48 h after infection with the virus (fig. 2D). In conclusion, overexpression of BmWARS significantly inhibited BmNPV infection and replication.
Overexpression of BmWARS inhibited BmNPV infection and replication. (A) Transcriptional level of BmWARS in BmN transfected with piZT/V5-His-mCherry-BmWARS (BmWARS-OE) or pIZT/V5-His-mCherry (control). (B) Fluorescence image of virus positive cells (egfp) of BmN cells in BmWARS-OE or control at 24, 48 and 72 hours after infection with the same amount of BmNPV (scale bar = 100 μm). (C) The DNA abundance of BmNPV in BmWARS-OE or control BmN cells at 24, 48, and 72 hours. (D) The expression level of VP39 protein in BmWARS-OE and control cells. Margin of error bars reflect the mean ± S.D. of three independent values. Asterisks show significant differences against controls by unpaired t-test. (*p < 0.05; **p < 0.01; ***p < 0.001).
Interference of BmWARS promotes BmNPV proliferation
To further validate the above experimental results, siRNA was utilized to down-regulate the expression of BmWARS, and the effects on the replication and proliferation of BmNPV were detected. The synthesized siRNA-BmWARS-270, which can inhibit the expression of BmWARS, and the control siRNA-NC were transfected into BmN cells, respectively, and the change of BmWARS expression was detected by qRT-PCR. The result was that the expression of BmWARS in cells with siRNA-270 was extremely significantly lower than that of NC (fig. 3A). After 24 h of siRNA treatment, two groups of cells were infected with equal amounts of BmNPV-egfp with MOI 5, and the infection status of BmN cells at 24, 48, and 72 h after viral infection was analyzed. The results were a significant increase in fluorescence intensity in cells with siRNA-270 at 48 h and 72 h after viral infection of BmN cells (fig. 3B), a significant increase in the copy number of VP39 at 24, 48 and 72 h after infection (fig. 3C) and the VP39 protein expression at 48 h was also significantly increased (fig. 3D). In conclusion, interfering with the expression of BmWARS in cells significantly promoted the replication and proliferation of BmNPV.
Interference of BmWARS facilitated BmNPV multiplication. (A) Transcriptional level of BmWARS in BmN cells transfected with siRNA-BmWARS-270 (siRNA-270) or siRNA-EGFP-NC (NC). (B) Fluorescence image of the number of the virus positive cells (egfp) of BmN cells in siRNA-270 or NC (scale bar = 100 μm). (C) The DNA abundance of BmNPV in the cells siRNA-270 and NC. (D) The expression level of VP39 protein in BmWARS knockdown and control cells. Margin of error bars reflect the mean ± S.D. of three independent values. Asterisks show significant differences against controls by unpaired t-test. (**p < 0.01; ***p < 0.001).
BmNPV induces the expression of BmWARS and the key genes of PI3K-Akt pathway
BmN cells not infected with BmNPV were used as the control group, and BmNPV-infected BmN cells were used as the experimental group. Cells were collected at 24, 48, and 72 h after virus infection, and the expression levels of the BmWARS and the key genes of PI3K-Akt pathway, PI3K, Akt, and PDK1, were detected by qRT-PCR. The results showed that the expression levels of the BmWARS, PI3K, Akt and PDK1 were significantly up-regulated in the Bombyx mori cells after virus infection (fig. 4A–D). It indicated that BmNPV could induce the overexpression of BmWARS, and BmWARS made an immune response during virus infection of silkworms. BmNPV realized its own efficient replication and proliferation by activating the PI3K-Akt pathway.
BmNPV induces the expression of BmWARS and the key genes of PI3K-Akt pathway. (A–D) The expression levels of the BmWARS, BmPI3K, BmAkt and BmPDK1 in the Bombyx mori cells after virus infection. Margin of error bars reflect the mean ± S.D. of three independent values. Asterisks show significant differences against controls by unpaired t-test. (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001).
BmWARS regulates the expression of key genes of the PI3K-Akt pathway
BmN cells were transfected with pIZT/V5-His-mCherry, pIZT/V5-His-mCherry-BmWARS, respectively, and the cells were collected at 24, 48, and 72 h after viral infection. qRT-PCR was performed to detect the key genes of PI3K-Akt pathway, BmPI3K, BmAkt, BmPDK1, BmFoxO, BmPTEN, BmCaspase9, BmeIF4E, and BmS6. The results showed that the expression of BmPI3K, BmAkt, BmPDK1, BmeIF4E, and BmS6 was significantly lower in BmWARS-OE cells than in the control group (fig. 5A-E), and the expression of BmPTEN, BmFoxO, BmCaspase9 expression was significantly higher than that of the control group (fig. 5F–H). We further detected the protein expression of Akt and the phosphorylation of Akt (p-Akt) by Western blot. The result was that the expression of p-Akt was significantly inhibited in BmWARS-OE cells 48 h after infection with the virus (fig. 5I). To verify the above experimental results, siRNA was utilized to down-regulate the expression of BmWARS, which resulted in significantly higher expression of BmPI3K, BmAkt, BmPDK1, BmeIF4E, BmS6 in siRNA-270 cells than in the control group (fig. 6A–E), and significantly lower expression of BmPTEN, BmFoxO, BmCaspase9 than the control group (fig. 6F–H). And the p-Akt protein expression at 48 h was also significantly increased (fig. 6I). The above results suggested that BmWARS responds to BmNPV infection through the PI3K-Akt pathway.
Overexpression of BmWARS regulate expression of key genes and p-Akt protein in PI3K-Akt pathway. (A–E) The expression levels of BmPI3K, BmAkt, BmPDK1, BmeIF4E and BmS6 after overexpression of BmWARS; (F–H) The expression levels of BmPTEN, BmFoxO and BmCaspase9 after overexpression of BmWARS. (I) The expression levels of p-Akt and Akt after overexpression of BmWARS. Margin of error bars reflect the mean ± S.D. of three independent values. Asterisks show significant differences against controls by unpaired t-test. (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001).
Interference of BmWARS regulate expression of key genes and p-Akt protein in PI3K-Akt pathway. (A–E) The expression levels of BmPI3K, BmAkt, BmPDK1, BmeIF4E and BmS6 after knockdown of BmWARS; (F–H) The expression of BmPTEN, BmFoxO and BmCaspase9 after knockdown of BmWARS. (I) The expression levels of p-Akt and Akt after knockdown of BmWARS. Margin of error bars reflect the mean ± S.D. of three independent values. Asterisks show significant differences against controls by unpaired t-test. (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001).
Overexpression of BmWARS promotes apoptosis
pIZT/V5-His-mCherry-BmWARS and pIZT/V5-His-mCherry were transfected into BmN cells and apoptosis was detected by flow cytometry. The results showed that overexpression of BmWARS significantly promoted apoptosis (fig. 7A, B). Thus, the replication and proliferation of BmNPV were inhibited..
BmWARS promotes apoptosis and inhibits cell proliferation. (A) Typical representative dot plots of the distribution of normal (Annexin V−/PI−), early apoptotic (Annexin V+/PI−), late apoptotic (Annexin V+/PI+) and necrotic (Annexin V−/PI+) cells. (B) Statistical analysis of the proportion of BmN cells with late apoptosis and early apoptosis. (C, D) Effects of overexpression and knockdown of BmWARS on cell proliferation. (E) Cell cycle detection by flow cytometry after overexpression of BmWARS. (F) Statistical analysis of the proportion of cell cycle G1 phase. Margin of error bars reflect the mean ± S.D. of three independent values. Asterisks show significant differences against controls by unpaired t-test. (*p < 0.05; **p < 0.01; ***p < 0.001).
BmWARS inhibits cell proliferation
pIZT/V5-His-mCherry-BmWARS, pIZT/V5-His-mCherry, siRNA-BmWARS-270 and siRNA-NC were transfected into BmN cells, respectively, and MTT reduction assay was performed to determine cell proliferation. The results showed that overexpression of BmWARS significantly inhibited the proliferation of BmN cells (fig. 7C), while the proliferation of BmN cells after knockdown of BmWARS was significantly higher than that of the control group (fig. 7D). The results of cell cycle detection by flow cytometry showed that overexpression of BmWARS blocked the cell cycle at the G1 phase (fig. 7E, F), thus inhibiting cell proliferation.
Conclusion
The mechanism of interaction between BmNPV and the silkworm has been one of the hot spots in entomoviral research. BmNPV specifically parasitizes the silkworm and poses a great danger to the sericulture industry. In this study, we cloned a novel aminoacyl tRNA synthetase family gene from Bombyx mori, namely BmWARS, and investigated its potential function in BmNPV infection of Bombyx mori. The results showed that BmWARS could significantly inhibit the replication and proliferation of the virus in Bombyx mori. This is the first report that BmWARS is involved in the antiviral response of Bombyx mori.
Immunofluorescence localization analysis of BmWARS showed that it was mainly expressed in the cytoplasm and cell membrane of BmN cells (fig. 1F), which is consistent with its function of catalyzing the linkage of tryptophan (Trp) to its homologous tRNA in the process of protein translation, and that the ribosome is an important synthetic organelle involved in protein synthesis and transcription. The results of tissue expression profiling showed that BmWARS was expressed in all tissues of the silkworm at the 3rd of age 5 (fig. 1C), with higher expression in the testis, ovary, silk gland, and malpighian tubule, which was consistent with the physiological characteristics of the silkworm at the 3rd of age 5. During this period, the silkworm needs to synthesize a large amount of spermatozoa, eggs and silk proteins, and protein synthesis is exuberant, so the expression of BmWARS is higher in testis, ovary and silk gland. The main functions of the malpighian tubule are to remove CO and nitrogen wastes produced by metabolism and to regulate the balance of inorganic salts and water in body fluids. The high metabolism of silkworms on the 3rd of the 5th year of age may be the reason for the high expression of BmWARS in the canal. The expression of BmWARS in the midgut and silk gland of BmNPV-infected silkworms was significantly up-regulated at 2-5 d post-infection (fig. 1D, E). The midgut is an innate immune organ of Bombyx mori, which is not only a digestive organ, but also a key immune organ against various pathogenic microorganisms accompanying food intake, and is the first barrier against viral invasion via the mouth (Qian et al., Reference Qian, Guo, Zhang, Liu, Zhao, Xu and Li2022). These results implied that BmWARS may be involved as an immune protein in resistance to BmNPV in the silkworm.
In this study, we clarified the function of BmWARS against BmNPV invasion by overexpressing and interfering with the expression of BmWARS in BmN cells. The results showed that BmWARS significantly inhibited BmNPV infection and replication (fig. 2), and could significantly inhibit the expression of BmPI3K, BmAkt, BmPDK1, BmeIF4E, BmS6 and the p-Akt protein in the PI3K-Akt pathway (fig. 5A–E, I), as well as significantly increase the expression of BmPTEN, BmFoxO, and BmCaspase9 (fig. 5F–H). However, the knockdown results of BmWARS were completely opposite to the overexpression results (Figures 3, 6).
The PI3K-Akt pathway regulates cell proliferation, differentiation and apoptosis (Brunet et al., Reference Brunet, Datta and Greenberg2001; Fresno Vara et al., Reference Fresno Vara, Casado, de Castro, Cejas, Belda-Iniesta and González-Barón2004) and is involved in the regulation of protein synthesis (Stitt et al., Reference Stitt, Drujan, Clarke, Panaro, Timofeyva, Kline, Gonzalez, Yancopoulos and Glass2004; Ueki et al., Reference Ueki, Yamamoto-Honda, Kaburagi, Yamauchi, Tobe, Burgering, Coffer, Komuro, Akanuma, Yazaki and Kadowaki1998). Blocking the virus-induced PI3K/Akt signaling pathway inhibits influenza virus replication (Wu et al., Reference Wu, Yen, Chang, Peng, Hsieh, Chen, Lin and Horng2011). Cheng Zhang et al (Zhang et al., Reference Zhang, Lin, Zhao, Wang, Jiang, Ji, Li, Gao, Li and Shen2020) showed that YARS promotes gastric cancer development by activating the PI3K-Akt pathway. Both WARS and YARS belong to the family of class Ic aaRSs with high homology (Rubio Gomez and Ibba, Reference Rubio Gomez and Ibba2020). More viruses can proliferate efficiently by activating the host PI3K-Akt signaling pathway (Cooray, Reference Cooray2004; Diehl and Schaal, Reference Diehl and Schaal2013; Xiao et al., Reference Xiao, Yang, Weng, Lin, Yuan, Yang and Pang2009). Overexpression of BmFoxO promotes the expression of BmPEPCK-2 and inhibits BmNPV proliferation(Kang et al., Reference Kang, Wang, Liang, Tang, Zhang, Wang, Zhao and Lu2021). BmPGRP2-2 inhibits apoptosis and promotes viral proliferation by inhibiting the expression of BmPTEN and phosphorylating Akt (Jiang et al., Reference Jiang, Liu, Guo, Dang, Cheng, Yang, Sun, Wang, Wang, Xie and Xia2019). The PI3K/Akt signaling pathway and PTEN gene play an essential role in ‘tree-top disease’ induced by LdMNPV (Li et al., Reference Li, Liu, Yu, Rensing and Wang2022). HN1L regulates the cell cycle and induces an anti-apoptotic response to BmNPV infection (Lei et al., Reference Lei, Hu, Xue, Mao, Obeng, Quan and Yu2019). Bombyx mori cell division cycle protein 37 promotes BmNPV proliferation (Zhang et al., Reference Zhang, Wu, Chen, Liu, Dong, Lu and Pan2021). Overexpression of BmWARS was found to promote apoptosis by Annexin V-FITC/PI apoptosis assay (fig. 7A, B). MTT reduction assay for cell proliferation showed that overexpression of BmWARS significantly inhibited the proliferation of BmN cells (fig. 7C), whereas the knockdown of BmWARS significantly increased the proliferation of cells (fig. 7D). The results of cell cycle detection by flow cytometry showed that overexpression of BmWARS blocked the cell cycle at G1 phase (fig. 7E, F), thus inhibiting cell proliferation. BmWARS could inhibit the expression of BmeIF4E and BmS6, thus inhibiting protein synthesis and cell growth.
In summary, we cloned a novel aminoacyl tRNA synthetase gene, BmWARS, in the silkworm, Bombyx mori, and demonstrated that BmWARS was able to inhibit the infection and replication of BmNPV. Further studies revealed that BmWARS promotes apoptosis and inhibits cell proliferation by regulating the PI3K-Akt signaling pathway, thereby enhancing cellular immunity (fig. 8). This study lays the foundation for the study of the function of BmWARS and its mechanism against BmNPV infection. It also provides a target for creating transgenic silkworm lines resistant to viral infection.
Schematic representation of the hypothesized mechanism by which BmWARS inhibits BmNPV infection through the PI3K-Akt pathway. BmWARS inhibits BmNPV replication and proliferation by promoting apoptosis and inhibiting cell proliferation through the PI3K-Akt pathway. Black arrows indicate promotion, and red lines plus horizontal bars indicate inhibition.
Funding
This work was supported by the National Natural Science Foundation of China (Nos. 31372377, 31972616) and the China Agriculture Research System (CARS-18-ZJ0101).
Declaration of competing interest
The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper.
Data availability
Data will be made available on request.
