Bombyx mori β-1,3-Glucan Recognition Protein 4 (BmβGRP4) Could Inhibit the Proliferation of B. mori Nucleopolyhedrovirus through Promoting Apoptosis

Simple Summary In this study, a differentially expressed β-1,3-glucan recognition protein, BmβGRP4, was identified from a transcriptome database. Significant downregulation of BmβGRP4 expression was detected after BmNPV infection in the P50 larvae midgut. Subsequently, the overexpression of BmβGRP4 suppressed BmNPV proliferation in BmN cells; however, the siRNA-mediated knockdown of BmβGRP4 facilitated BmNPV proliferation in B. mori larvae. Furthermore, we demonstrated that BmβGRP4 overexpression promoted BmNPV induced cellular apoptosis. Then, we found that BmβGRP4 positively regulated BmPTEN and negatively regulated BmIAP. Consequently, we speculated that BmNPV inhibited BmβGRP4 to suppress BmPTEN and facilitate BmIAP to inhibit cell apoptosis to evade host antiviral defense. These findings will lay a foundation for further study of the functions of BmβGRP4 in response to BmNPV. Abstract β-1,3-glucan recognition proteins (βGRPs) as pattern recognition receptors (PRRs) play an important role in recognizing various pathogens and trigger complicated signaling pathways in insects. In this study, we identified a Bombyx mori β-1,3-glucan recognition protein gene named BmβGRP4, which showed differential expression, from a previous transcriptome database. The full-length cDNA sequence was 1244 bp, containing an open reading frame (ORF) of 1128 bp encoding 375 amino acids. BmβGRP4 was strongly expressed in the larval stages and highly expressed in the midgut of B. mori larvae in particular. After BmNPV infection, the expression of BmβGRP4 was reduced significantly in the midgut. Furthermore, a significant increase in the copy number of BmNPV was observed after the knockdown of BmβGRP4 in 5th instar larvae, while the overexpression of BmβGRP4 suppressed the proliferation of BmNPV in BmN cells. Subsequently, the expression analysis of several apoptosis-related genes and observation of the apoptosis morphology demonstrated that overexpression of BmβGRP4 facilitated apoptosis induced by BmNPV in BmN cells. Moreover, BmβGRP4 positively regulated the phosphatase and tensin homolog gene (BmPTEN), while expression of the inhibitor of apoptosis gene (BmIAP) was negatively regulated by BmβGRP4. Hence, we hypothesize that BmNPV infection might suppress BmPTEN and facilitate BmIAP to inhibit cell apoptosis by downregulating the expression of BmβGRP4 to escape host antiviral defense. Taken together, these results show that BmβGRP4 may play a role in B. mori response to BmNPV infection and lay a foundation for studying its functions.


Introduction
The silkworm Bombyx mori is a holometabolism lepidopteran insect, which has been domesticated for about 5700 years from the wild progenitor B. mandarina [1]. It is used as a model organism in insect genetics and immunology fundamental research [2,3]. B. mori nucleopolyhedrovirus (BmNPV) is a serious viral pathogen that specifically infects the domestic silkworm, causing severe economic loss in sericulture around the world [4]. In recent years, many genes and proteins involved in BmNPV infection have been identified by different approaches, such as serine protease-2, lipase-1, alkaline trypsin protein and NADPH oxidoreductase (BmNox) [5]. Furthermore, these genes are expressed differentially in the resistant strains compared to the susceptible strain, and the results confirm the correlation of these genes to BmNPV resistance in B. mori [6]. Previous studies attempted to explore this resistance mechanism mainly using transcriptomic or proteomic analysis [7,8]. Xue et al. [9] found that numerous differentially expressed genes were mainly involved in the cytoskeleton, transcription, translation and energy metabolism in Bm5 cells after BmNPV infection based on transcriptome analysis. Dong et al. [10] identified a receptor expression-enhancing protein (REEP) using iTRAQ-based quantitative protein expression profiling, indicating that BmREEPa was required for BmNPV infection. However, the interaction of β-1,3-glucan recognition proteins (βGRPs) with BmNPV has not yet been reported. The innate immune system is an evolutionally conserved mechanism that can protect the host from invading pathogens including viruses [11]. In invertebrates, this system is activated in the presence of cell wall components from microbes like lipopolysaccharide (LPS), β-1,3 glucan (βG) and peptidoglycan (PG), together known as pathogen-associated molecular patterns (PAMPs) [12]. PAMPs are recognized by pattern recognition receptors (PRRs) in cells of the innate immune system, which can be divided into membrane-bound PRRs, receptor kinases and the mannose receptor [13]. PRRs have been identified in many insect species, including Drosophila melanogaster and Manduca sexta [14,15]. The most important PRRs purified from invertebrates include β-1,3 glucan binding protein (β-GBP), lipopolysaccharide and β-1,3 glucan binding protein (LGBP), Gram negative bacteria binding protein (G-NBP), peptidoglycan recognition protein (PGRP) and C-type lectin [16]. PGRPs and βGRPs are pattern recognition molecules that can recognize peptidoglycans in the bacterial cell wall and β-1,3-glucan from fungi, respectively, which play an important role in innate immunity activation and regulation [17,18]. The recognition of invading pathogen cells will trigger complex signaling pathways that eventually lead to the synthesis of effector molecules such as cytokines and antimicrobial peptides. Extracellular serine proteinase systems have evolved in invertebrates to mediate these processes [19]. One of these proteinase cascades causes proteolytic activation of prophenoloxidase (proPO), and active phenoloxidase (PO) generates quinones that are intermediates for melanization [20].
To date, β-1,3-glucan recognition proteins have been identified from many invertebrate groups. Ochiai et al. [21] were the first to purify these proteins from B. mori. Huang et al. [22] cloned a β-1,3-glucan recognition protein from P. xylostella, and suggested that it played a vital role in response to the expression of antimicrobial peptide (AMP) genes. Additionally, a 52 kDa β-1,3-glucan recognition protein isolated from M. sexta was shown to aggregate bacteria and fungi to stimulate PPO activation [17]. All of these results indicate that βGRPs could be involved in insect innate immune response against pathogens. The innate immune response to a virus is critical for mobilizing protective immunity. Host cells of the innate immune system can utilize PRRs to identify viral pathogens by engaging PAMPs [23]. The antiviral innate immune response is induced depending on the recognition of viral components by host PRRs [24]. Heiberg et al. [25] revealed a toll-like receptor (TLR)mediated inflammatory response in children with chronic hepatitis B (CHB) virus infection. Tsai et al. [26] found that the expression of PGRP-SB1 and PGRP-SD was upregulated after Sigma virus infection. Gao et al. [27] also revealed that BmPGRP-S3 played an important role in the immune response of silkworms to B. mori cytoplasmic polyhedrosis virus Insects 2021, 12, 743 3 of 16 (BmCPV). However, the function of βGRPs in virus infection is still poorly understood, especially for BmNPV.
In this study, we identified a pattern recognition receptor BmβGRP4 from a B. mori transcriptome database and investigated its role in BmNPV infection. The spatiotemporal expression pattern of BmβGRP4 was analyzed and a significant downregulation was detected after BmNPV infection in the midgut. Subsequently, overexpression and siRNAmediated knockdown of BmβGRP4 were used to explore its influence on BmNPV in BmN cells and B. mori larvae. Furthermore, we demonstrated that BmβGRP4 promoted BmNPVinduced cellular apoptosis. Then, we found that BmβGRP4 positively regulated BmPTEN and negatively regulated BmIAP. Consequently, we speculated that BmNPV inhibited BmβGRP4 to suppress BmPTEN and facilitated BmIAP to inhibit cell apoptosis to evade host antiviral defense. These results would lay the foundation for further study of the functions of BmβGRP4 in response to BmNPV.

Silkworm Larvae, Cell Line and Virus Preparation
The silkworm P50 strain was maintained in the Key Laboratory of Sericulture, Anhui Agricultural University, Hefei, China. The larvae were fed using fresh mulberry leaves. The first three instar larvae were reared at 26 ± 1 • C, 75 ± 5% relative humidity with 12 h day/night cycles, and the last two instar larvae were reared at 24 ± 1 • C, with the same relative humidity and photoperiod as above. The BmN cell line was cultured in TC-100 medium (United States Biological, Swampscott, MA, USA) supplemented with 10% (volume ratio, v/v) fetal bovine serum, 100 µg mL −1 penicillin and 30 µg mL −1 streptomycin (Gibco, Rockville, MD, USA) at 27 • C.
The BmNPV T3 strain was kept in our laboratory. On the 1st day of the 5th instar, all larvae were starved for 24 h, then administered 5 µL BmNPV suspended in water (1.0 × 10 6 OBs mL −1 ) per os, and the control was treated with 5 µL sterile water. Larvae midguts were collected at different time points post infection.
Budded virus of BmNPV containing an EGFP tag (BV-EGFP) was generously donated by Doctor Xue-Yang Wang from Jiangsu University of Science & Technology and was kept in our laboratory. The culture containing BV-EGFP (1.0 × 10 8 pfu mL −1 ) was used to infect BmN cells in this study.

Identification and Phylogenetic Analysis of BmGRP4
In the previous study, a comparative transcriptome analysis was employed to identify differential expressed genes (DEGs) in response to BmNPV infection [7]. In this study, BmβGRP4 was identified from the dataset through the National Center for Biotechnology Information non-redundant (NCBI-nr) database using a basic local alignment search tool (BLAST). The presence and location of the signal peptide was predicted by the SignalP-5.0 Server (http://www.cbs.dtu.dk/services/SignalP) accessed on 5 April 2021. The molecular weight (MW) and isoelectric point (pI) of the BmβGRP4 protein were calculated using ExPASy software (http://web.expasy.org/compute_pi/). Conserved domains were determined using the NCBI conserved domain database (http://www.ncbi.nlm.nih.gov/ Structure/cdd/wrpsb.cgi) and the SMART online software (http://smart.embl-heidelberg. de/). The phylogenetic tree was constructed with MEGA X software using the neighborjoining method with 1000-fold bootstrap resampling [28].

RNA Isolation, cDNA Synthesis and RT-qPCR Analysis
Total RNA was extracted from different samples using TRIzol reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions. The first-strand cDNA was synthesized using a PrimeScript TM RT kit with gDNA Eraser (TaKaRa, Dalian, China) according to the manufacturer's instructions. Quantitative reverse transcription PCR (RT-qPCR) analysis was performed using the CFX96 real-time system (Bio-Rad, Singapore) with a SYBR Premix ExTaq TM Kit (TaKaRa), as described previously [29]. The primers used in the RT-qPCR are shown in Table S2. The 2 −∆∆Ct method was adopted to calculate the relative expression levels. In this study, B. mori glyceraldehyde-3-phosphate dehydrogenase (BmGAPDH) was used as the reference gene. The statistical significance between treatments was analyzed using SPSS Statistics software (V 26.0. IBM, NY, USA). Three biological replicates were used.

Prokaryotic Expression and Protein Purification
In order to analyze the function of BmβGRP4 at the protein level, we expressed recombinant BmβGRP4 protein using a prokaryotic expression system. The primers are listed in Table S2 (the underlined portions indicate the restriction enzyme sites). The DNA fragment contained mature BmβGRP4 and was cloned and ligated into the expression vector pET-28a (Novagen, Madison, WI, USA) with restriction enzyme sites EcoR I and Xho I. The recombinant pET-28a-BmβGRP4 plasmid was confirmed by DNA sequencing and transformed into Escherichia coli BL21 competent cells (TransGen, Beijing, China). Recombinant BmβGRP4 protein expression was induced using 1 mM isopropyl-β-thiogalactopyranoside (IPTG) at 16 • C overnight. After centrifugation at 7500× g for 5 min at room temperature, the E. coli cells were suspended in phosphate-buffered saline (PBS, pH 7.4) and disturbed by sonication on ice. After centrifugation at 12,000× g for 20 min at 4 • C, the recombinant protein insoluble pellet was purified using High Affinity Ni-NTA Resin (GenScript, Nanjing, China), according to the manufacturer's instructions. The quality of purified protein was analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with Coomassie Brilliant Blue R250 and Western blot using anti-His primary antibody (1:5000, TransGen) and horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (1:10,000, TransGen).

RNAi by Injecting Small Interfering RNA in B. mori Larvae and BmNPV Infection
The small interfering RNA (siRNA) against BmβGRP4 and siNC (negative control) were designed and synthesized by the GenePharma Biotech Company (GenePharma, Shanghai, China); the siRNA sequences are shown in Table S3. Thirty larvae on the 3rd day of the 5th instar were used for RNA interference experiments. Each larva was injected with 2 µg siRNA, and midgut tissues were obtained at 24 h and 48 h post injection. The RNAi efficiency was assessed using RT-qPCR and Western blot.
Sixty larvae were injected with two kinds of siRNAs and divided into two groups (30 for siNC and 30 for siBmβGRP4). Then, all larvae were administered with 5 µL of BmNPV suspended in water (1 × 10 6 OBs mL −1 ) per os at 24 h post injection of siRNA. The 10 larval midgut tissues in each group were obtained and pooled at 24, 48 and 72 h p.i. (hours post infection). Genomic DNA of the midgut was isolated using a Genomic DNA extraction kit (TaKaRa). The relative expression level of BmNPV VP39 was measured by RT-qPCR to indicate the abundance of virus in infected larvae. The siNC injected larvae were used as controls in these experiments. Three biologically independent samples were used.

Overexpression of BmβGRP4 in BmN Cells and BmNPV Infection
The full-length sequence of BmβGRP4 ORF was amplified from the cDNA of the B. mori midgut with OEBmβGRP4 primers (Table S2). The purified DNA fragment was ligated into a transient overexpression vector, pIZT-mCherry vector to construct an overexpression vector for pIZT-mCherry-BmβGRP4. The recombinant overexpression vector was confirmed by DNA sequencing, and transfected into BmN cells (1.0 × 10 6 cells well −1 ) on Costar 6-well cell culture clusters using Neofect TM DNA transfection reagent (NEOFECT, Beijing, China) according to the manufacturer's instructions and cultured at 27 • C. The fluorescence signal was captured using an inverted microscope DMi8 camera (Leica, Solms, Germany), followed by culturing for 48 h after transfection. BmN cells were harvested for total RNA and protein extraction to assay the overexpression efficiency of BmβGRP4 by RT-qPCR and Western blot. The pIZT-mCherry plasmid transfected BmN cells were used as a control.
The BmβGRP4 overexpressed BmN cells (approximately 5 × 10 5 cells well −1 ) were infected with 10 µL of culture medium containing BV-EGFP at a multiplicity of infection (MOI) of 2 in each well, and the pIZT-mCherry transfected cells were used as a control. The infected BmN cells were collected at 24, 48 and 72 h p.i. The change in BmNPV proliferation was determined by analyzing BmNPV VP39 mRNA and protein levels through RT-qPCR and Western blot, as well as observing the green fluorescence using an inverted microscope DMi8 camera. Western blot analysis was performed as mentioned above. The primary antibodies, rabbit anti-VP39 antiserum (1:500, prepared by our laboratory), mouse anti-β-Tubulin (1:2000, TransGen) and the HRP-conjugated goat anti-rabbit secondary antibody or goat anti-mouse secondary antibody (1:5000, TransGen) were used in this study. Three biologically independent samples were used.

Detection of Apoptosis-Related Gene Expression and Observation of Apoptosis Morphology
The expression levels of apoptosis-related genes after BmNPV-induced apoptosis in BmβGRP4 overexpressed BmN cells were detected by RT-qPCR analysis. The BmβGRP4 overexpressed BmN cells were infected with BV-EGFP at an MOI of 2 and the cells were collected at 36 h p.i. to analyze the relative expression levels of BmApaf1, BmDredd, BmBuffy, BmCaspaseNC, BmICE and BmCaspase1 through RT-qPCR; all of the primers are shown in Table S2. The pIZT-mCherry vector transfected cells were used as a control. Three biologically independent samples were used.
The apoptosis morphology of BmN cells was determined by fluorescence microscopy. First, BV-EGFP at an MOI of 2 was added to the BmβGRP4 overexpressed BmN cells. Then, at 36 h p.i., all culture medium was removed, and the cells were fixed in 4% paraformaldehyde (PFA; Sangon) for 10 min at room temperature; after the removal of all 4% PFA, the cells were washed with PBS (pH 7.4) three times for 5 min each. After washing, the cell nuclei were stained with DAPI (Beyotime, Shanghai, China) in the dark for 10 min at room temperature. Finally, the cell morphology was examined using an inverted microscope DMi8 camera. The pIZT-mCherry vector transfected BmN cells were used as a control in this experiment. Three biologically independent samples were used.

Expression Level Analysis of BmPTEN and BmIAP
According to the demonstration of Jiang et al. [30], another important pattern recognition receptor BmPGRP2-2 was induced by BmNPV, through PTEN-phosphoinositide 3-kinase (PI3K)/Akt signaling to regulate apoptosis. Hence, we speculated that the pattern recognition receptor BmβGRP4 might also regulate apoptosis in response to BmNPV infection. In addition, Chen et al. [31] found that the B. mori inhibitor of apoptosis (BmIAP) gene had an inhibitory effect on apoptosis in silkworm cells. The expression levels of BmPTEN and BmIAP after BmβGRP4 knockdown and overexpression were evaluated through RT-qPCR analysis using the specific primers of BmPTEN and BmIAP (Table S2).

Identification of BmβGRP4 and Bioinformatics Analysis
Based on the previous transcriptome database, a noteworthy DEG was filtered; we obtained the cDNA sequence of beta-1,3-glucan recognition protein 4 (βGRP4) precursor (Table S1). Based on NCBI BLAST results, the BmβGRP4 (GenBank Accession No: NM_001166142.1) was identified. The cDNA sequence of BmβGRP4 contains an ORF of 1128 bp, which encodes 375 amino acids with a predicted MW of 42.0 kDa and an isoelectric point of 6.45. In addition, the first 17 amino acid residues (MWLLTLGVVALISASKA) at the N-terminus may act as a signal peptide for secretion ( Figure 1A). Conserved domain prediction using SMART software suggested that the BmβGRP4 protein contained a Glyco_hydro_16 domain (129-283) ( Figure 1B). In order to investigate the evolutionary relationships between BmβGRP4 and those of other insects, a phylogenetic tree was constructed by the neighbor-joining method ( Figure 1C). The results show that BmβGRP4 had a high homology with βGRP from P. xylostella.

Identification of BmβGRP4 and Bioinformatics Analysis
Based on the previous transcriptome database, a noteworthy DEG was filtered; we obtained the cDNA sequence of beta-1,3-glucan recognition protein 4 (βGRP4) precursor (Table S1). Based on NCBI BLAST results, the BmβGRP4 (GenBank Accession No: NM_001166142.1) was identified. The cDNA sequence of BmβGRP4 contains an ORF of 1128 bp, which encodes 375 amino acids with a predicted MW of 42.0 kDa and an isoelectric point of 6.45. In addition, the first 17 amino acid residues (MWLLTLGVVALISASKA) at the N-terminus may act as a signal peptide for secretion ( Figure 1A). Conserved domain prediction using SMART software suggested that the BmβGRP4 protein contained a Glyco_hydro_16 domain (129-283) ( Figure 1B). In order to investigate the evolutionary relationships between BmβGRP4 and those of other insects, a phylogenetic tree was constructed by the neighbor-joining method ( Figure 1C). The results show that BmβGRP4 had a high homology with βGRP from P. xylostella.

Tissue Distribution and Developmental Stage Expression of BmβGRP4
To explore the expression profile of BmβGRP4 in different tissues, including the head, integument, midgut, hemocyte, fat body, malpighian tube, testis and ovary, the expression level of BmβGRP4 was analyzed by RT-qPCR. The results indicated that BmβGRP4 had high expression in the head, integument, hemocyte, fat body and midgut (Figure 2A). An analysis of the developmental stages revealed that BmβGRP4 was expressed higher in the larval stage compared to the adult, and the expression increased gradually at each instar ( Figure 2B).

Tissue Distribution and Developmental Stage Expression of BmβGRP4
To explore the expression profile of BmβGRP4 in different tissues, including the head, integument, midgut, hemocyte, fat body, malpighian tube, testis and ovary, the expression level of BmβGRP4 was analyzed by RT-qPCR. The results indicated that BmβGRP4 had high expression in the head, integument, hemocyte, fat body and midgut (Figure 2A). An analysis of the developmental stages revealed that BmβGRP4 was expressed higher in the larval stage compared to the adult, and the expression increased gradually at each instar ( Figure 2B).

Recombinant BmβGRP4 Expression, Purification and Antiserum Preparation
To further analyze the function of BmβGRP4, recombinant His-tagged BmβGRP4 was expressed in E. coli. The recombinant BmβGRP4 with an MW of approximately 40 kDa was detected by 12% SDS-PAGE ( Figure 3A). The recombinant protein was purified (Figure 3B) and used for antiserum preparation. The correct expression was confirmed by Western blot using the anti-His primary antibody and goat anti-mouse secondary antibody ( Figure 3C). The antiserum was confirmed to detect the protein level of BmβGRP4 by Western blot ( Figure 3D).

Expression Pattern Analysis of BmβGRP4 following BmNPV Infection
Tissue expression patterns showed that BmβGRP4 had the highest expression in the midgut. In order to investigate the possible role of BmβGRP4 in response to BmNPV infection, RT-qPCR and Western blot were performed to determine BmβGRP4 expression patterns in the midgut at different time points post infection. The results suggest that the expression level of BmβGRP4 was significantly downregulated from 12 to 72 h p.i. in the midgut compared with the control group ( Figure 4A). Additionally, the results of Western

Recombinant BmβGRP4 Expression, Purification and Antiserum Preparation
To further analyze the function of BmβGRP4, recombinant His-tagged BmβGRP4 was expressed in E. coli. The recombinant BmβGRP4 with an MW of approximately 40 kDa was detected by 12% SDS-PAGE ( Figure 3A). The recombinant protein was purified ( Figure 3B) and used for antiserum preparation. The correct expression was confirmed by Western blot using the anti-His primary antibody and goat anti-mouse secondary antibody ( Figure 3C). The antiserum was confirmed to detect the protein level of BmβGRP4 by Western blot ( Figure 3D).

Recombinant BmβGRP4 Expression, Purification and Antiserum Preparation
To further analyze the function of BmβGRP4, recombinant His-tagged BmβGRP4 was expressed in E. coli. The recombinant BmβGRP4 with an MW of approximately 40 kDa was detected by 12% SDS-PAGE ( Figure 3A). The recombinant protein was purified (Figure 3B) and used for antiserum preparation. The correct expression was confirmed by Western blot using the anti-His primary antibody and goat anti-mouse secondary antibody ( Figure 3C). The antiserum was confirmed to detect the protein level of BmβGRP4 by Western blot ( Figure 3D).

Expression Pattern Analysis of BmβGRP4 following BmNPV Infection
Tissue expression patterns showed that BmβGRP4 had the highest expression in the midgut. In order to investigate the possible role of BmβGRP4 in response to BmNPV infection, RT-qPCR and Western blot were performed to determine BmβGRP4 expression patterns in the midgut at different time points post infection. The results suggest that the expression level of BmβGRP4 was significantly downregulated from 12 to 72 h p.i. in the midgut compared with the control group ( Figure 4A). Additionally, the results of Western

Expression Pattern Analysis of BmβGRP4 following BmNPV Infection
Tissue expression patterns showed that BmβGRP4 had the highest expression in the midgut. In order to investigate the possible role of BmβGRP4 in response to BmNPV infection, RT-qPCR and Western blot were performed to determine BmβGRP4 expression patterns in the midgut at different time points post infection. The results suggest that the expression level of BmβGRP4 was significantly downregulated from 12 to 72 h p.i. in the midgut compared with the control group ( Figure 4A). Additionally, the results of Western blot analysis show that the protein levels of BmβGRP4 were also downregulated in the midgut from 24 to 72 h p.i. (Figure 4B). Based on the above results, we speculated that BmβGRP4 could respond to BmNPV infection in the B. mori midgut. blot analysis show that the protein levels of BmβGRP4 were also downregulated in the midgut from 24 to 72 h p.i. (Figure 4B). Based on the above results, we speculated that BmβGRP4 could respond to BmNPV infection in the B. mori midgut.

Knockdown of BmβGRP4 by RNAi Facilitated Virus Proliferation in B. mori Larvae
To further determine whether BmβGRP4 was related to BmNPV resistance in vivo, we examined the proliferation of BmNPV in B. mori larvae in which BmβGRP4 was depleted. The knockdown of BmβGRP4 was confirmed through analyzing transcriptional levels by RT-qPCR and translational levels by Western blot; effective for interfering with the expression of BmβGRP4 in the midgut were observed at both 24 and 48 h post siRNA injection ( Figure 5A,B). B. mori larvae were injected with siBmβGRP4 or siNC for 24 h prior to infection with BmNPV per os; compared with the control, viral loads represented by the relative expression level of VP39 were remarkably increased from 24 to 72 h p.i. ( Figure 5C). Hence, we concluded that knocking down BmβGRP4 expression by RNAi in B. mori larvae could promote BmNPV proliferation in the midgut.

Overexpression of BmβGRP4 Suppressed Virus Proliferation in BmN Cells
To further study the role of BmβGRP4 in BmNPV infection in vitro, a recombinant plasmid, pIZT-mCherry-BmβGRP4, was constructed to overexpress BmβGRP4 in BmN cells. Subsequently, the recombinant plasmid was transfected into BmN cells. At 48 h after transfection, the red fluorescence signal was observed by fluorescence microscopy ( Figure  6C); in addition, compared to the control group, the very significant upregulation of BmβGRP4 was acquired in RT-qPCR analysis and Western blot analysis ( Figure 6A,B). After infection with BV-EGFP, viral proliferation that was indicated by the green fluorescence signal was reduced significantly in BmβGRP4 overexpressed cells at 24, 48, and 72 h p.i. compared to that in the control group ( Figure 7C). Furthermore, compared with the method. Data were normalized using BmGAPDH and are represented as means ± SEM from three independent experiments. Statistical analysis was conducted using SPSS software with Student's t-test. Significant differences are indicated by ** (p < 0.01). In the Western blot analysis, BmβGRP4 was detected using a rabbit anti-BmβGRP4 antiserum and β-Tubulin was used as an internal reference. "P50-" indicates non-infection and "P50+" indicates BmNPV-infection. h p.i., hours post infection.

Knockdown of BmβGRP4 by RNAi Facilitated Virus Proliferation in B. mori Larvae
To further determine whether BmβGRP4 was related to BmNPV resistance in vivo, we examined the proliferation of BmNPV in B. mori larvae in which BmβGRP4 was depleted. The knockdown of BmβGRP4 was confirmed through analyzing transcriptional levels by RT-qPCR and translational levels by Western blot; effective for interfering with the expression of BmβGRP4 in the midgut were observed at both 24 and 48 h post siRNA injection ( Figure 5A,B). B. mori larvae were injected with siBmβGRP4 or siNC for 24 h prior to infection with BmNPV per os; compared with the control, viral loads represented by the relative expression level of VP39 were remarkably increased from 24 to 72 h p.i. ( Figure 5C). Hence, we concluded that knocking down BmβGRP4 expression by RNAi in B. mori larvae could promote BmNPV proliferation in the midgut.

Overexpression of BmβGRP4 Suppressed Virus Proliferation in BmN Cells
To further study the role of BmβGRP4 in BmNPV infection in vitro, a recombinant plasmid, pIZT-mCherry-BmβGRP4, was constructed to overexpress BmβGRP4 in BmN cells. Subsequently, the recombinant plasmid was transfected into BmN cells. At 48 h after transfection, the red fluorescence signal was observed by fluorescence microscopy ( Figure 6C); in addition, compared to the control group, the very significant upregulation of BmβGRP4 was acquired in RT-qPCR analysis and Western blot analysis ( Figure 6A,B). After infection with BV-EGFP, viral proliferation that was indicated by the green fluorescence signal was reduced significantly in BmβGRP4 overexpressed cells at 24, 48, and 72 h p.i. compared to that in the control group ( Figure 7C). Furthermore, compared with the control group, viral loads represented by the expressions of VP39 analyzed by RT-qPCR and Western blot were also remarkably decreased from 24 to 72 h p.i. (Figure 7A,B). Due to the very low expression of BmβGRP4 in BmN cells, the knockdown of BmβGRP4 could not be investigated. These results show that the overexpression of BmβGRP4 could suppress BmNPV proliferation in BmN cells.
Insects 2021, 12, x FOR PEER REVIEW 9 of 16 control group, viral loads represented by the expressions of VP39 analyzed by RT-qPCR and Western blot were also remarkably decreased from 24 to 72 h p.i. (Figure 7A,B). Due to the very low expression of BmβGRP4 in BmN cells, the knockdown of BmβGRP4 could not be investigated. These results show that the overexpression of BmβGRP4 could suppress BmNPV proliferation in BmN cells.   (C) BmNPV load change upon BmNPV infected in knockdown of BmβGRP4 larvae was determined by estimating the relative expression level of BmNPV VP39 by RT-qPCR. Data were normalized using BmGAPDH and are presented as means ± SEM from three independent experiments. Statistical analysis was conducted using SPSS software. Significant differences are indicated by ** (p < 0.01).
Insects 2021, 12, x FOR PEER REVIEW 9 of 16 control group, viral loads represented by the expressions of VP39 analyzed by RT-qPCR and Western blot were also remarkably decreased from 24 to 72 h p.i. (Figure 7A,B). Due to the very low expression of BmβGRP4 in BmN cells, the knockdown of BmβGRP4 could not be investigated. These results show that the overexpression of BmβGRP4 could suppress BmNPV proliferation in BmN cells.

BmβGRP4 Promoted BmNPV Induced Apoptosis in BmN Cells
Apoptosis plays a key role in the innate immunity of insects, serving as an important antiviral defense mechanism in insects [32,33]. To verify whether BmβGRP4 had the regulatory function in BmNPV induced apoptosis in BmN cells, we overexpressed BmβGRP4 by transfecting the recombinant pIZT-mCherry-BmβGRP4 vector in BmN cells, after which BV-EGFP was used to induce apoptosis. As shown in Figure 8, compared to the control, the relative expression levels of BmApaf1, BmDredd, BmCaspaseNC, BmICE and BmCaspase1 were upregulated, and the relative expression level of BmBuffy was downregulated at 36 h p.i. In addition, cell apoptosis level was determined by DAPI staining. Numerous apoptotic bodies were observed in the infected cells at 36 h p.i. Over 20% of the BmβGRP4 overexpressed cells had apoptotic bodies; however, only about 5% of the control cells had apoptotic bodies (Figure 9). Together, our results demonstrate that BmβGRP4 promoted BmNPV induced apoptosis in BmN cells. . White Light, optical transmission. EGFP, green fluorescence. mCherry, red fluorescence. Data were normalized using BmGAPDH and are presented as means ± SEM from three independent experiments. Statistical analysis was conducted using SPSS software. Significant differences are indicated by ** (p < 0.01).

BmβGRP4 Promoted BmNPV Induced Apoptosis in BmN Cells
Apoptosis plays a key role in the innate immunity of insects, serving as an important antiviral defense mechanism in insects [32,33]. To verify whether BmβGRP4 had the regulatory function in BmNPV induced apoptosis in BmN cells, we overexpressed BmβGRP4 by transfecting the recombinant pIZT-mCherry-BmβGRP4 vector in BmN cells, after which BV-EGFP was used to induce apoptosis. As shown in Figure 8, compared to the control, the relative expression levels of BmApaf1, BmDredd, BmCaspaseNC, BmICE and BmCaspase1 were upregulated, and the relative expression level of BmBuffy was downregulated at 36 h p.i. In addition, cell apoptosis level was determined by DAPI staining. Numerous apoptotic bodies were observed in the infected cells at 36 h p.i. Over 20% of the BmβGRP4 overexpressed cells had apoptotic bodies; however, only about 5% of the control cells had apoptotic bodies (Figure 9). Together, our results demonstrate that BmβGRP4 promoted BmNPV induced apoptosis in BmN cells.

BmβGRP4 Positively Regulated BmPTEN and Negatively Regulated BmIAP
According to the study of Jiang et al. [30], another important pattern recognition receptor BmPGRP2-2 could be induced in response to BmNPV infection, through PTENphosphoinositide 3-kinase (PI3K)/Akt signaling to regulate apoptosis. In addition, Chen et al. [31] have found that B. mori inhibitor of apoptosis (BmIAP) could inhibit apoptosis and increase BmNPV proliferation during BmNPV infection in silkworm cells. To further determine the possible way in which BmβGRP4 regulates the response to BmNPV infection, we explored the effect of BmβGRP4 on the expression of BmPTEN and BmIAP. The expression levels of BmPTEN and BmIAP were analyzed in the BmβGRP4 knockdown midgut and BmβGRP4 overexpressed BmN cells using RT-qPCR. As shown in Figure 10, the expression level of BmPTEN was significantly decreased when BmβGRP4 was knocked down in the midgut, while BmβGRP4 overexpression in BmN cells significantly increased the expression of BmPTEN. Conversely, the expression level of BmIAP was significantly increased when BmβGRP4 was knocked down, and significantly decreased when BmβGRP4 was overexpressed. These results indicate that BmPTEN is positively regulated by BmβGRP4, whereas BmIAP is negatively regulated by BmβGRP4.

BmβGRP4 Positively Regulated BmPTEN and Negatively Regulated BmIAP
According to the study of Jiang et al. [30], another important pattern recognition receptor BmPGRP2-2 could be induced in response to BmNPV infection, through PTENphosphoinositide 3-kinase (PI3K)/Akt signaling to regulate apoptosis. In addition, Chen et al. [31] have found that B. mori inhibitor of apoptosis (BmIAP) could inhibit apoptosis and increase BmNPV proliferation during BmNPV infection in silkworm cells. To further determine the possible way in which BmβGRP4 regulates the response to BmNPV infection, we explored the effect of BmβGRP4 on the expression of BmPTEN and BmIAP. The expression levels of BmPTEN and BmIAP were analyzed in the BmβGRP4 knockdown midgut and BmβGRP4 overexpressed BmN cells using RT-qPCR. As shown in Figure 10, the expression level of BmPTEN was significantly decreased when BmβGRP4 was knocked down in the midgut, while BmβGRP4 overexpression in BmN cells significantly increased the expression of BmPTEN. Conversely, the expression level of BmIAP was significantly increased when BmβGRP4 was knocked down, and significantly decreased when BmβGRP4 was overexpressed. These results indicate that BmPTEN is positively regulated by BmβGRP4, whereas BmIAP is negatively regulated by BmβGRP4. Data were normalized using BmGAPDH and are presented as means ± SEM from three independent experiments. Statistical analysis was conducted using SPSS software with Student's t-test. Significant differences are indicated by * (p < 0.05) or ** (p < 0.01).

Discussion
BmNPV is a major pathogen that specifically infects silkworms and causes serious loss in sericulture every year. The silkworm midgut is an important barrier against

Discussion
BmNPV is a major pathogen that specifically infects silkworms and causes serious loss in sericulture every year. The silkworm midgut is an important barrier against BmNPV infection [8]. Recently, many proteins related to BmNPV infection have been reported. Previously, a B. mori β-1,3-glucan recognition protein named BmβGRP4 was identified from a transcriptome database [7]. β-1,3-glucan recognition proteins (βGRPs) are known to be pattern recognition molecules that play an important role in innate immunity activation and regulation [17]. In this study, we demonstrated that BmβGRP4 showed a very signif-icant downregulation in susceptible strain P50 after BmNPV infection (Table S1); hence, we speculated that BmβGRP4 might be related to BmNPV infection. The bioinformatic analysis results show that the BmβGRP4 amino acid sequence contained the typical βGRP domains (Glycoside hydrolase family 16) and seven predicted glycosylation sites (Figure 1). Glycoside hydrolase is a widespread group of enzymes that hydrolyze the glycosidic bond between two or more carbohydrates. In other insects, M. sexta βGRP2 and Ostrinia furnacalis βGRP3 also have a conserved glycoside hydrolase family 16 domain [17]. A phylogenetic tree was generated from the analysis of amino acid sequences of the βGRPs from other insects to verify the evolutionary relationship of βGRPs; it indicated that BmβGRP4 shares a close relationship with P. xylostella βGRP. The RT-qPCR analysis showed that BmβGRP4 was highly expressed in the 1st to 5th instar larval stages and the expression level increased with instar stage. Additionally, it was expressed in almost all of the selected tissues, and was highly expressed in the head, body fat and hemocyte, especially in the midgut (Figure 2A). During the larval stage, silkworms had to eat a large number of mulberry leaves, which also meant that they would face a large number of microbial infections. For this reason, silkworms had to improve the expression of some immune-related genes to adapt to the living environment. In addition, larvae of lepidopteran insects commonly become increasingly resistant to virus infections as they age [34]. βGRPs are known as pattern recognition molecules that play an important role in innate immune response. BmβGRP4 might also be related to the innate immune response; hence, we speculated that the increased expression of BmβGRP4 with instar stage would be beneficial to the enhancement of immunity in silkworm larvae. Previous studies found that GRPs in M. sexta [35], P. interpunctella [36] and O. furnacalis [37] were mainly transcribed in the fat body and hemocyte. This indicated that BmβGRP4 might possess different functions compared to those reported in other insects.
Due to the significant decrease in BmβGRP4 transcription found from the data of the transcriptome, we consider BmβGRP4 to play an important role in BmNPV infection. The RT-qPCR analysis showed that the expression of BmβGRP4 was inhibited in the P50 strain midgut after BmNPV infection ( Figure 4A), and the Western blot analysis also showed a similar result ( Figure 4B). We speculated that BmβGRP4 might be involved in resistance to BmNPV replication in the midgut. To verify the speculation, the expression of BmNPV VP39 was detected in viral-infected knockdown of BmβGRP4 in larvae and BmβGRP4 overexpressed in BmN cells. A significant increase in replication was shown after the knockdown of BmβGRP4 in the 5th instar larvae midgut ( Figure 5), while the reduced proliferation of BmNPV was observed after the overexpression of BmβGRP4 in BmN cells ( Figure 6). Therefore, these results further confirm that BmβGRP4 was related to BmNPV infection. Previously, several proteins identified from the digestive juice of B. mori larvae showed that the overexpression of these proteins would significantly reduce the proliferation of BmNPV in BmN cells [29,38,39]. We consider BmβGRP4 to have an antiviral function.
Apoptosis plays a key role in the innate immunity of insects, serving as an important antiviral defense mechanism in insects [32,33,40]. To verify whether BmβGRP4 had a regulatory function in BmNPV induced apoptosis, we overexpressed BmβGRP4 in BmN cells and then BV-EGFP was used to induce apoptosis. As shown in Figure 8, compared to the control, the relative expression levels of several apoptosis-related genes BmApaf1, BmDredd, BmCaspaseNC, BmICE and BmCaspase1 were upregulated, while the relative expression level of BmBuffy was downregulated at 36 h p.i. In addition, cell apoptosis level was determined by DAPI staining, and more apoptotic bodies were observed in the cells BmβGRP4 overexpressed at 36 h p.i. (Figure 9). These results demonstrate that BmβGRP4 promoted BmNPV-induced apoptosis in BmN cells. According to the study of Jiang et al. [30], another important pattern recognition receptor BmPGRP2-2 could be induced to regulate apoptosis in response to BmNPV infection, through PTEN-phosphoinositide 3-kinase (PI3K)/Akt signaling. In addition, Chen et al. [31] found that the B. mori inhibitor of apoptosis (BmIAP) could inhibit apoptosis and increase BmNPV proliferation during BmNPV infection in silkworm cells. We found that BmβGRP4 positively regulated BmPTEN through the PTEN-PI3K/Akt signaling to regulate cell apoptosis and BmβGRP4 negatively regulated BmIAP to regulate cell apoptosis ( Figure 10). Hence, we hypothesize that BmβGRP4 was inhibited by BmNPV infection to suppress the expression of BmPTEN and facilitate the expression of BmIAP to inhibit apoptosis to promote viral replication ( Figure 11). The mechanism underlying the positive regulation of BmPTEN and negative regulation of BmIAP by BmβGRP4 still needs further research. Furthermore, it is still unclear whether BmβGRP4 induces apoptosis by itself or requires a pathogen to be present, and how it works.
promoted BmNPV-induced apoptosis in BmN cells. According to the study of Jiang et al. [30], another important pattern recognition receptor BmPGRP2-2 could be induced to regulate apoptosis in response to BmNPV infection, through PTEN-phosphoinositide 3-kinase (PI3K)/Akt signaling. In addition, Chen et al. [31] found that the B. mori inhibitor of apoptosis (BmIAP) could inhibit apoptosis and increase BmNPV proliferation during BmNPV infection in silkworm cells. We found that BmβGRP4 positively regulated BmP-TEN through the PTEN-PI3K/Akt signaling to regulate cell apoptosis and BmβGRP4 negatively regulated BmIAP to regulate cell apoptosis ( Figure 10). Hence, we hypothesize that BmβGRP4 was inhibited by BmNPV infection to suppress the expression of BmPTEN and facilitate the expression of BmIAP to inhibit apoptosis to promote viral replication ( Figure  11). The mechanism underlying the positive regulation of BmPTEN and negative regulation of BmIAP by BmβGRP4 still needs further research. Furthermore, it is still unclear whether BmβGRP4 induces apoptosis by itself or requires a pathogen to be present, and how it works. Figure 11. The schematic diagram shows the putative mechanism of BmβGRP4 in BmNPV infection. BmβGRP4 is suppressed by BmNPV to suppress BmPTEN and induce BmIAP, thereby inhibiting cell apoptosis. The increased cell survival is beneficial for BmNPV replication.

Conclusions
In summary, a differentially expressed β-1,3-glucan recognition protein, BmβGRP4, was identified from a transcriptome database. The significant downregulation of BmβGRP4 expression was detected after BmNPV infection in the P50 larvae midgut. Subsequently, the overexpression of BmβGRP4 suppressed BmNPV proliferation in BmN cells; however, the siRNA-mediated knockdown of BmβGRP4 facilitated BmNPV proliferation in B. mori larvae. Furthermore, we demonstrated that BmβGRP4 overexpression promoted BmNPV-induced cellular apoptosis. Then, we found that BmβGRP4 positively Figure 11. The schematic diagram shows the putative mechanism of BmβGRP4 in BmNPV infection. BmβGRP4 is suppressed by BmNPV to suppress BmPTEN and induce BmIAP, thereby inhibiting cell apoptosis. The increased cell survival is beneficial for BmNPV replication.

Conclusions
In summary, a differentially expressed β-1,3-glucan recognition protein, BmβGRP4, was identified from a transcriptome database. The significant downregulation of BmβGRP4 expression was detected after BmNPV infection in the P50 larvae midgut. Subsequently, the overexpression of BmβGRP4 suppressed BmNPV proliferation in BmN cells; however, the siRNA-mediated knockdown of BmβGRP4 facilitated BmNPV proliferation in B. mori larvae. Furthermore, we demonstrated that BmβGRP4 overexpression promoted BmNPVinduced cellular apoptosis. Then, we found that BmβGRP4 positively regulated BmPTEN and negatively regulated BmIAP. Consequently, we speculated that BmNPV inhibited BmβGRP4 to suppress BmPTEN and facilitate BmIAP to inhibit cell apoptosis to evade the host antiviral defense. The findings of this study will lay a foundation for further study of the functions of BmβGRP4 in response to BmNPV.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/insects12080743/s1, Table S1: The transcriptome data statistics of DEG BmβGRP4 , Table S2: The primers used in this study, Table S3: The siRNA sequences used in this study.