BmN Cells Research Articles

C-type lectin 5, a novel pattern recognition receptor for the JAK/STAT signaling pathway in Bombyx mori

The Janus kinase/signal transducer and activator of transcription cascade transduction (JAK/STAT) signaling pathway is highly conserved in mammals, but the pattern recognition receptors (PRRs) and their functions are unclear. We found that the expression pattern of Bombyx mori C-type lectin 5 (BmCTL 5) had a synergy relevance with the JAK/STAT signaling pathway against Beauveria bassiana. An RNAi assay, subcellular localization analysis, yeast two-hybrid technique, protein recruitment experiment and pathogen infection tests were used to explore the roles of BmCTL 5 in the JAK/STAT signaling pathway. Knock-down of the BmCTL 5 suppressed the JAK/STAT signaling pathway and the PO cascade of nodule melanization. BmCTL 5 is located in the cytomembrane and interacted with BmHOP both in yeast and B. mori ovary cells N (BmN cells). BmCTL 5 and the JAK/STAT signaling pathway was activated by B. bassiana but only slightly activated by B. mori cytoplasmic polyhedrosis virus (BmCPV), Nosema bombycis and bacteria LPS. These findings suggest that BmCTL 5 might be an important PRR for the JAK/STAT signaling pathway and may mediate the nodule melanization for fungi infection. These data provide insights into the immune mechanism of the JAK/STAT signaling pathway in insects and aid understanding of the mechanism of the JAK/STAT signaling pathway and adaptive immune systems in mammals.

Expression profile analysis of circular RNAs in BmN cells (Bombyx mori) upon BmNPV infection

The disease caused by Bombyx mori nucleopolyhedrovirus (BmNPV) has always been difficult to control, resulting in tremendous economic losses in the sericulture industry. Although much has been learned about the impact of noncoding RNAs on pathogenesis, the role of circular RNA (circRNA) in insect immunity remains unclear. To explore circRNA regulation involved in BmNPV infection, we used transcriptome analysis of BmN cells with or without BmNPV infection to generate circRNA data set. A total of 444 novel circRNAs were identified in BmN cells, with 198 pervasively distributed both in the control group and BmNPV-infection group. The host genes were enriched inMAPK signaling pathway, dorso-ventral axis formation, and ECM-receptor interaction, which were required for the normal larval growth. A total of 75 circRNAs were differentially expressed (DE) on BmNPV infection. Six downregulated circRNAs were validated by Sanger sequencing and qRT-PCR. DEcircRNA-miRNA-DEmRNA network was constructed based on the six validated circRNAs. Pathway analysis indicated that the predicted target genes were mainly enriched in the metabolic pathway and immune-related signaling pathway. Our results may provide a basis for further studies on circRNA function in BmN cells challenged by BmNPV infection and offer an insight into the molecular mechanism on silkworm-virus interaction.

Characterization of the lipidomic profile of BmN cells in response to Bombyx mori cytoplasmic polyhedrosis virus infection

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV)that belongs to the genus Cypovirus in the family of Reoviridae is one of the problematic pathogens in sericulture. In our previous study, we have found that lipid-related constituents in the host cellular membrane are associated with the BmCPV life cycle. It is important to note that the lipids not only affect the cellular biological processes, they also impact the virus life cycle. However, the intracellular lipid homeostasis in BmN cells after BmCPV infection remains unclear. Here, the lipid metabolism in BmCPV-infected BmN cells was studied by lipidomics analysis. Our results revealed that the intracellular lipid homeostasis was disturbed in BmN cells upon BmCPV infection. Major lipids constituents in cellular membrane were found to be significantly induced upon BmCPV infection, which included triglycerides, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phospholipids, glucoside ceramide, monoetherphosphatidylcholin, ceramide, ceramide phosphoethanolamine and cardiolipin. Further analysis of the pathways related to these altered lipids (such as PE and PC) showed that glycerophospholipid metabolism was one of the most enriched pathways. These results suggested that BmCPV may manipulate the lipid metabolism of cells for their own interest. The findings may facilitate a better understanding of the roles of lipid metabolic changes during virus infection in future studies.

Expressional Localization and Functionally Identifying an RNA Editing Enzyme BmADARa of the Silkworm Bombyx mori.

Simple SummaryAdenosine deaminase acting on RNA (ADAR) is a key enzyme in the editing of adenosine into inosine (A-to-I). The loss or dysfunction of ADAR enzymes in higher eukaryotes affects the editing efficiency of target genes, leading to some neurological diseases. The silkworm Bombyx mori is an oligophagous economically important insect and has been used as an important lepidoptera model insect. By far, the knowledge about A-to-I RNA editing and ADAR members in B. mori (BmADAR) is very limited. In this paper, we present a first molecular comprehensive cloning, sequence analysis of BmADAR transcripts and subcelluar localization of BmADARa. As a result, we obtained six BmADAR transcripts encoding different amino and carboxyl termini, among which BmADARa is a mainly expressed transcript with complete open reading frame. Our further investigations showed that the majority of BmADARa protein exists in the nucleus and has editing function to a specific site of the silkworm synaptotagmin I gene. Overall, by molecular cloning and functional identifing, this paper introduces the first ADAR enzyme in B. mori and contributes to further exploration of the functional domain of BmADARa and its editing substrates and target sites.The most common type of RNA editing in metazoans is the deamination of adenosine into inosine (A-to-I) catalyzed by the adenosine deaminase acting on the RNA (ADAR) family of proteins. The deletion or dysfunction of ADAR enzymes in higher eukaryotes can affect the efficiency of substrate editing and cause neurological disorders. However, the information concerning A-to-I RNA editing and ADAR members in the silkworm, Bombyx mori (BmADAR), is limited. In this study, a first molecular comprehensive cloning and sequence analysis of BmADAR transcripts was presented. A complete open reading frame (ORF) (BmADARa) was obtained using RT-PCR and RACE and its expression pattern, subcellular localization and A-to-I RNA-editing function on the silkworm synaptotagmin I (BmSyt I) were investigated. Subcellular localization analysis observed that BmADARa was mainly localized in the nucleus. To further study the A-to-I RNA-editing function of BmADARa, BmSyt I-pIZ-EGFP was constructed and co-transfected with BmADARa-pIZ-EGFP into BmN cells. The result demonstrates that BmADARa can functionally edit the specific site of BmSyt I. Taken together, this study not only provides insight into the function of the first ADAR enzyme in B. mori, but also lays foundations for further exploration of the functional domain of BmADARa and its editing substrates and target sites.

Increased expression of Suppressor of cytokine signaling 2 (BmSOCS2) is correlated with suppression of Bombyx mori nucleopolyhedrovirus replication in silkworm larval tissues and cells

The resistance of silkworm to infection by Bombyx mori nuclear polyhedrosis virus (BmNPV) is a main focus of sericultural research. Previously, a BmNPV-resistant strain, NB, was identified among a collection of Chinese silkworm strains in our lab. To better understand the molecular mechanism of NB strain resistance, the patterns of host immune response gene transcription in resistant (NB) and susceptible (306) strains were examined. Quantative real-time PCR (qRT-PCR) revealed that multiple insect innate immune signaling pathways (Toll, Imd and JAK/STAT) were strongly activated upon infection with BmNPV. Notably, Suppressor of cytokine signaling 2 (BmSOCS2) mRNA expression was significantly up-regulated in midgut tissues of the resistant NB strain, suggesting that the BmSOCS2 gene product may be involved in host immune defense against BmNPV infection. A significant inhibition of BmNPV replication was also observed in BmN cells transfected with a vector encoding BmSOCS2. The results suggest that BmSOCS2 is a key gene involved in the resistance of the NB silkworm strain to BmNPV infection.

Transcriptomic analysis reveals the regulation network of BmKrüppel homolog 1 in the oocyte development of Bombyx mori.

Krüppel homolog 1 (Kr-h1), a zinc finger transcription factor, is involved in the metamorphosis and adult reproduction of insects. However, the role of Kr-h1 in reproduction of holometabolic insects remains to be elucidated. The regulation network of Kr-h1-associated genes in the reproduction in Bombyx mori was investigated in this study. The higher expression level of BmKr-h1 in the ovaries was detected during the late pupal stage and adults. RNA interference (RNAi)-mediated depletion of BmKr-h1 in the female at day 6 of pupae resulted in abnormal oocytes at 48 h post-double-stranded RNA treatment, which showed less yolk protein deposition and partially transparent chorion. RNA-seq and subsequent differentially expressed transcripts analysis showed that knockdown of BmKr-h1 caused a decrease in the expression of 2882 genes and an increase in the expression of 2565 genes in the oocytes at day 8 of pupae. Totally, 27 genes coding for transcription factors were down-regulated, while six genes coding for other transcription factors were up-regulated. BmKr-h1 bound to the Kr-h1 binding site of the transcription factors AP-1 (activating protein-1) and FOXG1 to increase their messenger RNA transcripts in the BmN cells, respectively. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses of that positively co-expressed with AP-1 and FOXG1 transcripts showed mainly enrichment in the metabolic-related pathways, the nutrient absorption and the yolk protein absorption processes. These data suggested that BmKr-h1 might directly regulate the metabolic-related pathways, the nutrient absorption and the yolk protein absorption processes or probably through AP-1 and /or FOXG1 to regulate oocyte development.

A Novel Digestive Proteinase Lipase Member H-A in Bombyx mori Contributes to Digestive Juice Antiviral Activity Against B. mori Nucleopolyhedrovirus.

Previous studies have revealed that some proteins in Bombyx mori larvae digestive juice show antiviral activity. Here, based on the label-free proteomics data, BmLipase member H-A (BmLHA) was identified as being involved in the response to BmNPV infection in B. mori larvae digestive juice. In the present study, a gene encoding the BmLHA protein in B. mori was characterized. The protein has an open reading fragment of 999 bp, encoding a predicted 332 amino acid residue-protein with a molecular weight of approximately 35.9 kDa. The phylogenetic analysis revealed that BmLHA shares a close genetic distance with Papilio xuthus Lipase member H-A. BmLHA was highly expressed in the middle part of the B. mori gut, and the expression level increased with instar rising in larvae. There was higher expression of BmLHA in A35 than in P50 strains, and it was upregulated in both A35 and P50 strains, following BmNPV infection. The expression level of VP39 decreased significantly in appropriate recombinant-BmLHA-treated groups compared with the PBS-treated group in B. mori larvae and BmN cells. Meanwhile, overexpression of BmLHA significantly reduced the infectivity of BmNPV in BmN cells. These results indicated that BmLHA did not have digestive function but had anti-BmNPV activity. Taken together, our work provides valuable data for the clarification of the molecular characterization BmLHA and supplements research on proteins of anti-BmNPV activity in B. mori.

Identification of the nucleotide exchange factor BmGrpE and its role under high-temperature stress in silkworm, Bombyx mori.

The high-temperature stress gene GrpE plays an important role in coping with high-temperature stress. The mutation of key sites of this gene can improve the high-temperature resistance of organisms. In the present study, using complementary DNAs from the silkworm fat body as the template, the open reading frame sequence of the GrpE gene (BmGrpE) was amplified and was found to be 644 bp in length and encode a protein with a predicted molecular weight of 24.1 kDa. The presence of a binding site for the heat shock transcription factor (Hsf1) at -1440 bp upstream of its coding region indicates that BmGrpE may respond to high-temperature stress. BmGrpE was constitutively expressed throughout developmental stages, with the highest level observed in the 5th instar larvae stage. Moreover, in 5th instar larvae (the 3th day), BmGrpE was expressed in all tissues examined, with the highest levels in the fat body, silk gland, and midgut. Interestingly, under high-temperature stress, TiO2 nanoparticle treatment increased the messenger RNA levels of BmGrpE in the fat body and silk gland. After treatment with dsRNA of BmGrpE, the cell viability of BmN cells was significantly decreased under 34°C and H2 O2 stress (p < .05). Mutation of BmGrpE (H163L) enhanced the resistance of BmN cells under high-temperature stress. These results provide new clues for the study of molecular mechanisms of insect resistance to high temperatures.

Oral administration of porcine epidemic diarrhea virus spike protein expressing in silkworm pupae failed to elicit immune responses in pigs

The silkworm (Bombyx mori) and its pupae have been used for decades as nutritional additives and applied on the production of high-quality recombinant proteins via the baculovirus expression vector (BEV) system. The bio-capsule, the fat-rich body, and some body components of the silkworm pupae, which deliver antigens passing through the harsh environment of digestive tract and reaching the intestine, have been used as a vehicle for oral vaccines. In the present study, to develop a novel oral vaccine against porcine epidemic diarrhea virus (PEDV), the PEDV spike (S) protein was expressed in silkworm pupae and BmN cells using the BEV system. After three doses of oral administrations with 2-week intervals in pigs, neither PEDV S protein-specific humoral nor mucosal immune responses can be detected. The failure of eliciting the PEDV-specific immune response suggested that the BEV system using BmN cells or silkworm pupae as oral immunogen-expression vehicles was not able to overcome the immunological unresponsiveness, which was possibly due to gastrointestinal specific barriers and oral tolerance. Better strategies to enhance the delivery and immunogenicity of oral vaccines should be further investigated. Nevertheless, the PEDV S protein generated in the BmN cells and silkworm pupae herein provides an efficient tool to produce the recombinant antigen for future applications.

N 6-Methyladenosine Level in Silkworm Midgut/Ovary Cell Line Is Associated With Bombyx mori Nucleopolyhedrovirus Infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most serious pathogens in sericulture and causes huge economic loss annually. The roles of N6-methyladenosine (m6A) modification in silkworms following BmNPV infection are currently unclear. Here, methylated RNA immunoprecipitation with next-generation sequencing were applied to investigate the m6A profiles in silkworm midgut following BmNPV infection. A total of 9144 and 7384 m6A peaks were identified from the BmNPV-infected (TEST) and uninfected silkworm midguts (CON), respectively, which were distributed predominantly near stop codons. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of common m6A peaks in nuclear genes revealed that these m6A-related transcripts were associated with crucial signaling pathways. Comparative transcriptome analysis showed that 1221 differential expressed m6A peaks were identified between TEST and CON, indicating that m6A modification is regulated following BmNPV infection. GO and KEGG pathway analysis of the differentially expressed m6A peaks showed their association with signal transduction, translation, and degradation. To understand further the effect of the m6A machinery on virus infection, expression levels of m6A-related genes were altered in silencing and overexpression experiments. Expression of viral structural protein VP39 was increased in BmN cells by siRNA-mediated depletion of methyltransferase-like (METTL) enzyme genes (BmMETTL3, BmMETTL14) and cytoplasmic YTH-domain family 3 (BmYTHDF3), while the reverse results were found after overexpression of the m6A-related enzymes in BmN cells. Overall, m6A modification might be a novel epigenetic mechanism that regulation BmNPV infection and interference with this mechanism may provide a novel antiviral strategy for preventing BmNPV disease.

Analysis of lncRNA-mediated gene regulatory network of Bombyx mori in response to BmNPV infection

Bombyx mori nucleopolyhedrosis virus (BmNPV) has always been a great challenge to the development and stability of the sericulture industry. LncRNAs have been reported to play important roles in gene expression regulation, development and immune response but the roles of lncRNAs in BmNPV infection and silkworm-BmNPV interaction are not clear. We used a genome-wide transcriptome analysis to identify the lncRNAs in Bombyx mori cells (BmN cells) and analyzed the differentially expressed lncRNAs, microRNAs and protein-coding genes in silkworm cells with or without BmNPV infection. A total of 13,159 candidate lncRNAs were identified in the BmN cells, among which 4450 lncRNAs were differentially expressed (DE) with 2837 up-regulated and 1613 down-regulated. In addition, 66 differentially expressed miRNAs (DEmiRNAs) and 7448 differentially expressed mRNAs (DEmRNAs) were identified, and DElncRNA-DEmiRNA-DEmRNA regulatory network was constructed. Gene expression was variable in 4973 of predicted lncRNA cis target genes in BmNPV infected cells. KEGG pathway analysis indicated that the target genes of DElncRNAs are enriched in ubiquitin mediated proteolysis, endocytosis and lysosome pathways. qRT-PCR validated the differential expression of several lncRNAs and miRNAs. Our results suggested that DElncRNAs participate in host response to BmNPV infection via interactions with their target genes and miRNAs. Our results will help us to improve our understanding of lncRNA-mediated regulatory roles in BmNPV infection and provide new insights into silkworm-pathogen interactions.

Bombyx mori Rad23 (BmRad23) contributes to the repair of UV-damaged BmNPV

Baculoviruses have been developed as long-term and environmentally friendly biopesticides. However, solar ultraviolet radiation can reduce the activity of baculovirus. Radiation sensitive 23 (Rad23) can recognize DNA damage and is involved with nucleotide excision repair (NER). In the current study, BmRad23 was accumulated mainly within the nucleus. Host cell reactivation (HCR) assays have shown that BmRad23 significantly facilitated the expression of UV-damaged mCherry reporter gene. Reverse transcription quantitative PCR (RT-qPCR) result showed that the mRNA expression level of BmRad23 was increased in (Bombyx mori nuclear polyhedrovirus, BmNPV) BmNPV-infected BmN cells. However, the expression of BmRad23 was increased significantly when BmNPV budded viruses (BVs) or BmN cells were irradiated with UV light. Overexpression of BmRad23 promoted the mRNA levels of two UV-induced DNA damage repair genes which were from Bombyx mori and BmNPV, respectively. Meanwhile, the overexpression of BmRad23 in BmN cells was conducive to the proliferation of BmNPV and UV-damaged BmNPV. The recombinant BmNPV BVs expressing BmRad23 showed stronger resistance to UV radiation than the control virus. In conclusion, the results revealed that BmRad23 contributed to the proliferation of BmNPV and the repair of UV-damaged BmNPV, which would provide a reference for the development of efficient baculovirus pesticides against UV radiation.

Efficient Delivery of dsRNA and DNA in Cultured Silkworm Cells for Gene Function Analysis Using PAMAM Dendrimers System.

Polyamidoamine (PAMAM) dendrimers are emerging as intriguing nanovectors for nucleic acid delivery because of their unique well-defined architecture and high binding capacity, which have been broadly applied in DNA- and RNA-based therapeutics. The low-cost and high-efficiency of PAMAM dendrimers relative to traditional liposomal transfection reagents also promote their application in gene function analysis. In this study, we first investigated the potential use of a PAMAM system in the silkworm model insect. We determined the binding property of G5-PAMAM using dsRNA and DNA in vitro, and substantially achieved the delivery of dsRNA and DNA from culture medium to both silkworm BmN and BmE cells, thus leading to efficient knockdown and expression of target genes. Under treatments with different concentrations of G5-PAMAM, we evaluated its cellular cytotoxicity on silkworm cells, and the results show that G5-PAMAM had no obvious toxicity to cells. The presence of serum in the culture medium did not affect the delivery performance of DNA and dsRNA by G5-PAMAM, revealing its convenient use for various purposes. In conclusion, our data demonstrate that the PAMAM system provides a promising strategy for delivering dsRNA and DNA in cultured silkworm cells and promote its further application in individuals.

Signal transducer and activator of transcription is involved in the expression regulation of ecdysteroid-induced insulin-like growth factor-like peptide in the pupal wing disc of silkworm, Bombyx mori.

In insects, 20-hydroxyecdysone (20E) and insulin-like growth factor-like peptides (IGFLPs) regulate the development of imaginal discs. However, how IGFLPs are up-regulated to impact the development of the pupal wing disc is still unclear. In this study, we investigated the expression regulation of IGFLP in the pupal wing disc of silkworm, Bombyx mori. We confirmed that B. mori IGFLP (BmIGFLP) was mainly expressed in the pupal wing disc and the expression of BmIGFLP could be significantly induced by 20E. Bioinformatics analysis of BmIGFLP promoter sequence revealed three cis-regulation elements (CREs) of signal transducer and activator of transcription (STAT), which is a key component in the Janus-activated kinase / STAT pathway. Luciferase activity assays showed that two CREs enhanced the transcriptional activity of BmIGFLP. Electrophoretic mobility shift and chromatin immunoprecipitation assays demonstrated that BmSTAT proteins in the nuclear extracts of B. mori pupal wing discs and BmN cells could only bind to the STAT CRE3, indicating that STAT CRE3 activated by BmSTAT enhances BmIGFLP expression at pupal stages. Although 20E could not enhance the expression of BmSTAT, 20E enhanced the nucleus translocation of BmSTAT to bind with the STAT CRE3 in the BmIGFLP promoter. The increase of transcriptional activity of the STAT CRE3 by overexpression of BmSTAT and addition of 20E in BmN cells confirmed this result. Taken together, all data indicate that BmSTAT is one of the transcription factors activating 20E-induced BmIGFLP expression in the pupal wing disc.

The stability and antiapoptotic activity of Bm30K-3 can be improved by lysine acetylation in the silkworm, Bombyx mori.

Acetylation is an important, highly conserved, and reversible post-translational modification of proteins. Previously, we showed by nano-HPLC/MS/MS that many nutrient storage proteins in the silkworm are acetylated. Among these proteins, most of the known 30K proteins were shown to be acetylated, including 23 acetylated 30K proteins containing 49 acetylated sites (Kac), indicating the importance of the acetylation of 30K proteins in silkworm. In this study, Bm30K-3, a 30K protein containing three Kac sites, was further assessed in functional studies of its acetylation. Increasing the level of Bm30K-3 acetylation by adding the deacetylase inhibitor trichostatin A (TSA) increased the levels of this protein and further inhibited cellular apoptosis induced by H2 O2 . In contrast, decreasing the level of acetylation by adding the acetylase inhibitor C646 could reduce the level of Bm30K-3 and increase H2 O2 -induced apoptosis. Subsequently, BmN cells were treated with CHX and MG132, and increasing the acetylation level using TSA was shown to inhibit protein degradation and improve the stability of Bm30K-3. Furthermore, the acetylation of Bm30K-3 could compete with its ability to be ubiquitinated, suggesting that acetylation could inhibit the ubiquitin-mediated proteasome degradation pathway, improving the stability and accumulation of proteins in cells. These results further indicate that acetylation might regulate nutrition storage and utilization in Bombyx mori, which requires further study.

The Motif of 76KRKCSK in Bm65 Is an Efficient Nuclear Localization Signal Involved in Production of Infectious Virions.

orf65 (Bm65) of Bombyx mori nucleopolyhedrovirus (BmNPV) codes for a putative 104-amino-acid protein containing three cysteine residues with a putative molecular mass of 12.2 kDa. Previous studies have showed that Bm65 accumulates mainly in nucleus and involved in the repair of UV-damaged DNA. However, the mechanism of nuclear import of Bm65 remains unclear. In this study, a SDS-stable Bm65 tetramer was found in BmNPV-infected BmN cells, and alanine substitutions for the three cysteine residues did not affect the formation of Bm65 tetramer. Additionally, a basic amino acid cluster of the Bm65 protein was identified as an efficient nuclear localization signal (NLS). Firstly, transient expression of GFP-fused truncated Bm65 variants revealed that the 76KRKCSK motif functions as the NLS. This was also confirmed by alanine substitution in the 76KRKCSK motif, which caused attenuated nuclear localization of Bm65. Next, the 76KRKCSK motif-mutated bacmid was generated and the 76KRKCSK motif was also found to be important for nuclear localization of Bm65 in BmNPV-infected conditions. Lastly, analyses of flag-tagged Bm65 expressing bacmids revealed that the mutations in 76KRKCSK motif did not affect the synthesis of Bm65 tetramer, but severely impaired production levels of infectious virions. In conclusion, Bm65 exists in mainly a tetrameric form in virus-infected cells, which may be involved with production levels of infectious virions.

Efficient Expression and Processing of Ebola Virus Glycoprotein Induces Morphological Changes in BmN Cells but Cannot Rescue Deficiency of Bombyx Mori Nucleopolyhedrovirus GP64.

Ebola virus (EBOV) disease outbreaks have resulted in many fatalities, yet no licensed vaccines are available to prevent infection. Recombinant glycoprotein (GP) production may contribute to finding a cure for Ebola virus disease, which is the key candidate protein for vaccine preparation. To explore GP1,2 expression in BmN cells, EBOV-GP1,2 with its native signal peptide or the GP64 signal peptide was cloned and transferred into a normal or gp64 null Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid via transposition. The infectivity of the recombinant bacmids was investigated after transfection, expression and localization of EBOV-GP were investigated, and cell morphological changes were analyzed by TEM. The GP64 signal peptide, but not the GP1,2 native signal peptide, caused GP1,2 localization to the cell membrane, and the differentially localized GP1,2 proteins were cleaved into GP1 and GP2 fragments in BmN cells. GP1,2 expression resulted in dramatic morphological changes in BmN cells in the early stage of infection. However, GP1,2 expression did not rescue GP64 deficiency in BmNPV infection. This study provides a better understanding of GP expression and processing in BmN cells, which may lay a foundation for EBOV-GP expression using the BmNPV baculovirus expression system.

Inhibition of heat shock protein 90 suppresses Bombyx mori nucleopolyhedrovirus replication in B. mori.

Heat shock protein 90 (Hsp90) plays a very important role in facilitating the replication of many viruses. Until now, little has been known about the role of Hsp90 in Bombyx mori virus infection. In this study, we explored the role of BmHsp90 in B. mori nucleopolyhedrovirus (BmNPV) replication. We found that BmHsp90 inhibition by geldanamycin (GA) significantly reduced the BmNPV titre, the protein expression level of BmNPV nucleocapsid protein 39 (VP39) and the transcript level of BmNPV genes. Silencing the hsp90 gene in BmN cells by small interfering RNA suppressed BmNPV replication whereas overexpression of hsp90 promoted the replication of BmNPV. After inhibition of Hsp90, the expression of three key genes [signal transducing activator of transcription (stat), suppressor of cytokine signalling protein 2 (socs2), socs6] involved in the Janus kinase/STAT pathway significantly changed, with up-regulation of stat and down-regulation of socs2 and socs6. In addition, the expression of two antiapoptosis genes, BmNPV inhibitor of apoptosis protein1 (BmNPV-iap1) and Bmiap2, was greatly decreased in GA-treated cells, whereas their expression was significantly increased in hsp90-overexpressed silkworm larvae. Our results indicated that inhibition of Hsp90 can suppress BmNPV proliferation in B. mori. Our findings may provide new clues to elucidate the molecular mechanisms of silkworm-virus interactions.

Bmo-miR-2780a regulates the expression of the sericin-1 gene of Bombyx mori.

Silk production in Bombyx mori L. is largely determined by the expression of genes encoding fibroin and sericin. Here, we examined the regulatory function of a microRNA (miRNA) on silk gene expression using the sericin-1 gene (BmSer-1). First, we downloaded whole mature miRNAs of silkworm from miRBase and identified bmo-miR-2780a as a candidate miRNA for the regulation of BmSer-1 expression. We used semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) with stem-loop primers to investigate the expression profile of bmo-miR-2780a and its predicted target gene BmSer-1 in seven different tissues from 5th instar day-3 larvae, including head, fat body, anterior silk gland (ASG), middle silk gland (MSG), posterior silk gland (PSG), middle gut, and hemolymph. Our results showed that bmo-miR-2780a was specifically expressed in the MSG and that the expression level of BmSer-1 was significantly higher in the MSG than in other tissues. Recombinant plasmids carrying both pri-mir-2780a and Ser1-3'UTR were constructed and then used to cotransfect BmN cells. We further detected the effect of bmo-miR-2780a on Ser-1 in vivo. These results showed that the target gene was significantly decreased by miR-2780a compared with the control group (p < .05), thus indicating that bmo-miR-2780a might negatively regulate the expression of Ser-1.

Alternative isoforms of BmYki have different transcriptional co-activator activity in the silkworm, Bombyx mori

Yorki (Yki), a transcriptional co-activator that is a key component of the Hippo pathway, induces the transcription of a number of targets that promote cell proliferation and survival. Bombyx mori Yki3 (BmYki3), with 445 amino acid residues, facilitates cell migration and cell division, and enlarges cultured cell and wing disc size. In this study, cellular localization, transcriptional co-activator activity, cell migration, cell cycle, and cell size were characterized in alternative isoforms of BmYki. BmYki1 and BmYki3 are mainly located in the cytoplasm and nucleus, respectively, while, BmYki2 is located in both the cytoplasm and nucleus. The mutation BmYki1S97A (S97mutated to A) was transported from the cytoplasm to nucleus. Cell migration, cell cycle, and cell size could be enhanced by BmYki, however, the effect of BmYki1 and BmYki2 on cell proliferation was less compared to BmYki3. Moreover, wing discs could be enlarged by overexpressing BmYki1 or BmYki2 isoforms. Dual-luciferase reporter assay showed that BmYki3 had the highest activity to B. mori ovarian tumor gene. In BmN cells overexpressing one of the BmYki isoforms, expression levels of kibra ortholog (kibra), inhibitor of apoptosis protein (iap), four-jointed (fj), expanded (ex), crumbs (crb) and BMP and activin membrane-bound inhibitor homolog (Bmpr) genes were upregulated, while those of α-catenin (α-cat), decapentaplegic (dpp), serrate (serr) and signal transducer and activator of transcription (stat) genes were down-regulated. There was some difference in the regulation of gene expression between different isoforms. These results suggested that the activity of BmYki isoforms was different in the silkworm.