RESUMEN
Bombyx mori nuclear polyhedrosis virus (BmNPV) is one of several viruses that cause great harm to the sericulture industry, and its pathogenic mechanism is still being explored. Geldanamycin (GA), a kind of HSP90 inhibitor, has been verified to suppress BmNPV proliferation. However, the molecular mechanism by which GA inhibits BmNPV is unclear. MicroRNAs (miRNAs) have been shown to play a key role in regulating virus proliferation and host-pathogen interactions. In this study, BmN cells infected with BmNPV were treated by GA and DMSO for 72 h, respectively, then transcriptome analysis of miRNA was performed from the GA group and the control group. As a result, a total of 29 miRNAs were differentially expressed (DE), with 13 upregulated and 16 downregulated. Using bioinformatics analysis, it was found that the target genes of DEmiRNAs were involved in ubiquitin-mediated proteolysis, phagosome, proteasome, endocytosis pathways, and so on. Six DEmiRNAs were verified by quantitative reverse-transcription polymerase chain reaction. DElong noncoding RNA (DElncRNA)-DEmiRNA-messenger RNA (mRNA) regulatory networks involved in apoptosis and immune pathways were constructed in GA-treated BmN cells, which included 12 DEmiRNA, 132 DElncRNA, and 69 mRNAs. This regulatory network enriched the functional role of miRNA in the BmNPV-silkworm interactions and improved our understanding of the molecular mechanism of HSP90 inhibitors on BmNPV proliferation.
Asunto(s)
Bombyx , MicroARNs , Nucleopoliedrovirus , Animales , Benzoquinonas , Bombyx/metabolismo , Lactamas Macrocíclicas , MicroARNs/genética , MicroARNs/metabolismo , Nucleopoliedrovirus/fisiología , ARN Mensajero/metabolismo , TranscriptomaRESUMEN
Bombyx mori nucleopolyhedrosis virus (BmNPV) is one of the greatest threats to sustainable development of the sericulture industry. Circular RNA (circRNA), a type of non-coding RNA, has been shown to play important roles in gene expression regulation, immune response, and diseases. The fat body is a tissue with both metabolic and immune functions. To explore the potential immune function of circRNAs, we analyzed differentially expressed (DE)circRNAs, microRNAs(miRNAs), and mRNAs in the B. mori fat body in response to BmNPV infection using high-throughput RNA sequencing. A total of 77 DEcircRNAs, 32 DEmiRNAs, and 730 DEmRNAs that are associated with BmNPV infection were identified. We constructed a DEcircRNA/DEmiRNA/DEmRNA and DEcircRNA/DEmiRNA/BmNPV gene regulatory network and validated the differential expression of circ_0001432 and its corresponding miRNA (miR-2774c and miR-3406-5p) and mRNA (778467 and 101745232) in the network. Tissue-specific expression of circ_0001432 and its expression at different time points were also examined. KEGG pathway analysis of DEmRNAs, target genes of DEmiRNAs, and host genes of DEcircRNAs in the network showed that these genes were enriched in several metabolic pathways and signaling pathways, which could play important roles in insect immune responses. Our results suggest that circRNA could be involved in immune responses of the B. mori fat body and help in understanding the molecular mechanisms underlying silkworm-pathogen interactions.
Asunto(s)
Bombyx/genética , Bombyx/inmunología , Cuerpo Adiposo/inmunología , Redes Reguladoras de Genes/inmunología , Inmunidad Innata/genética , Nucleopoliedrovirus/fisiología , ARN Circular/genética , Animales , Bombyx/crecimiento & desarrollo , Secuenciación de Nucleótidos de Alto Rendimiento , Larva/genética , Larva/crecimiento & desarrollo , Larva/inmunología , MicroARNs/genética , MicroARNs/inmunología , ARN Circular/inmunología , ARN Circular/metabolismo , ARN Mensajero/genética , ARN Mensajero/inmunologíaRESUMEN
Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the main pathogens that seriously affect the sustainable development of sericulture industry. Inhibition of Hsp90 by Hsp90 inhibitor, geldanamycin (GA) significantly suppresses BmNPV proliferation in Bombyx mori, while the functional mechanism is not clear. LncRNA has been widely reported to play an important role in immune responses and host-virus interactions in mammalian. However, related research has been rarely reported on silkworm. In this study, firstly, we confirmed the decrease of BmNPV ORF75 protein in the BmNPV-infected BmN cells treated with GA. Next, by using a genome-wide transcriptome analysis, we compared the lncRNA and mRNA expression profiles in BmNPV infected BmN cells treated with or without GA and identified a total of 282 differentially expressed lncRNAs (DElncRNAs) and 523 DEmRNAs. KEGG pathway analysis revealed DEmRNA were mainly involved in ubiquitin mediated proteolysis, spliceosome, RNA transport and oxidative phosphorylation. Further, we selected 27 immune-related DEmRNAs, which displayed the similar changes of expression patterns on both protein level and transcript level to construct DElncRNA-DEmRNA network. In addition, based on the DElncRNA-bmo-miR-278-3p-BmHSC70 regulatory network, we explored the potential function of several lncRNAs as sponges to inhibit the regulatory effect of bmo-278-3p on Bombyx mori heat shock protein cognate 70 (BmHSC70). Our finding suggests that lncRNAs play a role in the regulation of BmNPV proliferation by Hsp90.
Asunto(s)
Benzoquinonas/farmacología , Redes Reguladoras de Genes , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Lactamas Macrocíclicas/farmacología , Nucleopoliedrovirus/fisiología , ARN Largo no Codificante/metabolismo , Animales , Secuencia de Bases , Bombyx , Línea Celular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , ARN Largo no Codificante/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
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.
Asunto(s)
Bombyx/genética , Bombyx/virología , Nucleopoliedrovirus/fisiología , ARN Circular/genética , Animales , Bombyx/inmunología , Línea Celular , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno , Transducción de SeñalRESUMEN
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.
Asunto(s)
Bombyx/genética , Redes Reguladoras de Genes , Genoma de los Insectos , MicroARNs/genética , Nucleopoliedrovirus/fisiología , ARN Largo no Codificante/genética , ARN Mensajero/genética , Animales , Bombyx/virología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , MicroARNs/metabolismo , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Bombyx mori nucleopolyhedrosis virus (BmNPV) is a major pathogen that threatens the sustainability of the sericultural industry. DNA methylation is a widespread gene regulation mode in epigenetics, which plays an important role in host immune response. Until now, little has been known about epigenetic regulation on virus diseases in insects. This study aims to explore the role of DNA methylation in BmNPV proliferation. RESULTS: Inhibiting DNA methyltransferase (DNMT) activity of silkworm can suppress BmNPV replication. The integrated analysis of transcriptomes and DNA methylomes in silkworm midguts infected with or without BmNPV showed that both the expression pattern of transcriptome and DNA methylation pattern are changed significantly upon BmNPV infection. A total of 241 differentially methylated regions (DMRs) were observed in BmNPV infected midguts, among which, 126 DMRs were hyper-methylated and 115 DMRs were hypo-methylated. Significant differences in both mRNA transcript level and DNA methylated levels were found in 26 genes. BS-PCR validated the hypermethylation of BGIBMGA014008, a structural maintenance of chromosomes protein gene in the BmNPV-infected midgut. In addition, DNMT inhibition reduced the expression of inhibitor of apoptosis family genes, iap1 from BmNPV, Bmiap2, BmSurvivin1 and BmSurvivin2. CONCLUSION: Our results indicate that DNA methylation plays positive roles in BmNPV proliferation and loss of DNMT activity could induce the apoptosis of infected cells to suppress BmNPV proliferation. Our results may provide a new idea and research direction for the molecular mechanism on insect-virus interaction.
Asunto(s)
Bombyx/virología , Metilación de ADN , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica/veterinaria , Nucleopoliedrovirus/fisiología , Animales , Bombyx/genética , Proliferación Celular/efectos de los fármacos , Células Cultivadas , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , Epigénesis Genética , Epigenómica , Regulación de la Expresión Génica , Interacciones Microbiota-Huesped , Nucleopoliedrovirus/efectos de los fármacos , Análisis de Secuencia de ARN , Replicación Viral/efectos de los fármacosRESUMEN
Heat shock protein 90, an essential chaperone responsible for the correct maturation of key proteins, has been confirmed to facilitate Bombyx mori nucleopolyhedrovirus (BmNPV) proliferation but the mechanism is not clear. In this study, we use quantitative proteomics analysis to investigate the mechanism of Hsp90 in BmNPV replication. In total, 195 differentially expressed proteins (DEPs) were identified with 136 up-regulated proteins and 59 down-regulated proteins. The protein expression level of small heat shock proteins, immune-related proteins, cellular DNA repair-related proteins and zinc finger proteins is significantly enhanced while that of protein kinases is declined. KEGG pathway analysis reveals that DEPs are involved in longevity regulating pathway, mTOR signaling pathway, FoxO signaling pathway and Toll and Imd signaling pathway. Based on the DEPs results, we speculate that inhibition of Hsp90 suppresses the BmNPV infection may because it could not only stimulate the host innate immune, induce small heat shock proteins expression to maintain the cellular proteostasis but activate host transcription factors to bind to virus DNA or protein and subsequently hinder virus replication. The results will help understand the roles of Hsp90 in BmNPV infection and shed light on new clue to illustrate the molecular mechanism of silkworm-virus interaction. SIGNIFICANCE: This is the first report on Hsp90 roles in BmNPV infection based on proteomic analysis. Our findings may provide new clue and research orientation to illustrate the molecular mechanism of silkworm-virus interaction and a set of BmHsp90 candidate clients, which may involve in BmNPV infection in BmN cells.
Asunto(s)
Bombyx/virología , Proteínas HSP90 de Choque Térmico/inmunología , Nucleopoliedrovirus , Proteómica/métodos , Virosis/etiología , Animales , Bombyx/química , Bombyx/inmunología , Regulación de la Expresión Génica , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/farmacología , Interacciones Huésped-Patógeno , Inmunidad Innata , Transducción de Señal , Virosis/inmunología , Virosis/prevención & control , Virosis/virología , Replicación ViralRESUMEN
Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen that threatens the growth and sustainability of the sericulture industry. Accumulating studies in recent years suggest that insect viruses infection can change the host microRNAs (miRNAs) expression profile and both cellular and viral miRNAs play roles in host-pathogen interactions. Until now, the functional analysis of miRNA encoded by silkworm for host-virus interaction is limited. In this study, we validate the down-regulation of bmo-miR-2819 upon BmNPV infection by qRT-PCR and confirm the BmNPV immediately early 1 gene, ie-1 is one of the targets for bmo-miR-2819 based on the results of dual luciferase report assay. Overexpression of bmo-miR-2819 can significantly decline the abundance of IE-1 protein level in BmNPV-infected silkworm larvae. Further, the expression level of polyhedrin gene and VP39 protein of BmNPV in the infected larvae after applying bmo-miR-2819 mimics was significantly decreased comparing with that of larvae with mimic control. Our results suggest that overexpression of bmo-miR-2819 could suppress BmNPV replication by down-regulating the expression of BmNPV ie-1 gene, which demonstrate that cellular miRNAs could affect virus infection by regulating the expression of virus genes.