RESUMO
Apoptosis is essential for the normal growth, development, and immunity defense of living organisms, and its function and mechanisms have been intensively studied. When viral infection occurs, apoptosis is triggered, causing programmed death of the infected cells. Meanwhile, viruses have also evolved countermeasures to inhibit apoptosis in host cells. We previously constructed a transgenic silkworm line with significantly improved resistance to Bombyx mori nucleopolyhedrovirus (BmNPV) by knocking out the BmNPV inhibitor of apoptosis 2 (iap2) gene. However, the mechanism of how IAP2 induces apoptosis still needs to be further investigated. Here, the transcriptomes of Cas9(-)/sgiap2 (-) and Cas9(+)/sgiap2(+) strains were analyzed at 48 h after BmNPV infection, and a total of 709 differential genes were obtained. A KEGG analysis revealed that the differentially expressed genes were enriched in the oxidative phosphorylation, proteasome, and ribosome pathways. In the oxidative phosphorylation pathway, 41 differentially expressed genes were downregulated, and 12 of these genes were verified by qRT-PCR. More importantly, the knockout of BmNPV iap2 led to the inhibition of the oxidative phosphorylation pathway, followed by activated oxidative stress triggered apoptosis, thereby inhibiting the replication of BmNPV in vitro and vivo. The results provide a basis for the analysis of the initiation of apoptosis that can inhibit virus proliferation, and the study presents new ideas for the subsequent creation of resistant material.
Assuntos
Bombyx , Fosforilação Oxidativa , Animais , Perfilação da Expressão Gênica , Transcriptoma , Apoptose/genética , Bombyx/metabolismoRESUMO
Ser/Thr protein phosphatase 2A (PP2A) is one of the type 2 protein phosphatases, which is required for many intracellular physiological processes and pathogen infection. However, the function of PP2A is unclear in silkworm, Bombyx mori. Here, we cloned and identified BmPP2A, a PP2A gene from B. mori, which has two HEAT domains and a high similarity to PP2A from other organisms. Our results showed that BmPP2A is localized in the cytoplasm and highly expressed in silkworm epidermis and midgut, and that Bombyx mori nucleopolyhedrovirus (BmNPV) infection induces down-regulation of BmPP2A expression. Furthermore, up-regulation of BmPP2A via overexpression significantly inhibited BmNPV multiplication. In contrast, down-regulation of BmPP2A via RNA interference and okadaic acid (a PP2A inhibitor) treatment allowed robust BmNPV replication. This is the first report of PP2A having an antiviral effect in silkworm and provides insights into the function of BmPP2A, a potential anti-BmNPV mechanism, and a possible target for the breeding of silkworm-resistant strains.
Assuntos
Bombyx/genética , Proteínas de Insetos/genética , Nucleopoliedrovírus/fisiologia , Proteína Fosfatase 2/genética , Sequência de Aminoácidos , Animais , Antivirais/metabolismo , Bombyx/crescimento & desenvolvimento , Bombyx/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Proteínas de Insetos/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Proteína Fosfatase 2/metabolismo , Regulação para CimaRESUMO
The CRISPR/Cas9 system is a powerful genetic engineering technique that has been widely used in gene therapy, as well as in the development of novel antimicrobials and transgenic insects. However, several challenges, including the lack of effective host target genes and the off-target effects, limit the application of CRISPR/Cas9 in insects. To mitigate these difficulties, we established a highly efficient virus-inducible CRISPR/Cas9 system in transgenic silkworms. This system includes the baculovirus-inducible promoter 39K, which directs transcription of the gene encoding, the Cas9 protein, and the U6 promoter which targets the sgATAD3A site of the ATPase family AAA domain-containing protein 3 (ATAD3A) gene. The double-positive transgenic line sgATAD3A×39K-Cas9 (ATAD3A-KO) was obtained by hybridization; antiviral activity in this hybrid transgenic line is induced only after Bombyx mori nucleopolyhedrovirus (BmNPV) infection. The BmNPV-inducible system significantly reduced off-target effects and did not affect the economically important characteristics of the transgenic silkworms. Most importantly, this novel system efficiently and consistently edited target genes, inhibiting BmNPV replication after the transgenic silkworms were inoculated with occlusion bodies (OBs). The suppression of BmNPV by the virus-inducible system was comparable to that of the stably expressed CRISPR/Cas9 system. Therefore, we successfully established a highly efficient BmNPV-inducible ATAD3A-KO transgenic silkworm line, with improved gene targeting specificity and antiviral efficiency. Our study thereby provides insights into the treatment of infectious diseases and into the control of insect pests.
Assuntos
Animais Geneticamente Modificados/genética , Baculoviridae/genética , Bombyx/genética , Sistemas CRISPR-Cas/genética , Animais , Bombyx/virologia , Marcação de Genes/métodos , Vetores Genéticos/genética , Nucleopoliedrovírus/genética , Controle de Pragas/métodos , Regiões Promotoras Genéticas/genética , Transcrição Gênica/genéticaRESUMO
The CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the pathogen. We recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus (BmNPV) immediate early-1 (ie-1) gene in the silkworm, respectively, and obtained four transgenic hybrid lines by G1 generation hybridization: Cas9(-)/sgRNA(-), Cas9(+)/sgRNA(-), Cas9(-)/sgRNA(+), and Cas9(+)/sgRNA(+). We demonstrated that the Cas9(+)/sgRNA(+) transgenic lines effectively edited the target site of the BmNPV genome, and large fragment deletion was observed after BmNPV infection. Further antiviral analysis of the Cas9(+)/sgRNA(+) transgenic lines shows that the median lethal dose (LD50) is 1,000-fold higher than the normal lines after inoculation with occlusion bodies. The analysis of economic characters and off-target efficiency of Cas9(+)/sgRNA(+) transgenic hybrid line showed no significant difference compared with the normal lines. Our findings indicate that CRISPR/Cas9-mediated genome engineering more effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment.
RESUMO
Baculovirus LEF-11 is a small nuclear protein that is involved in viral late gene transcription and DNA replication. However, the characteristics of its nuclear localization signal and its impact on viral DNA replication are unknown. In the present study, systemic bioinformatics analysis showed that the baculovirus LEF-11 contains monopartite and bipartite classical nuclear localization signal sequences (cNLSs), which were also detected in a few alphabaculovirus species. Localization of representative LEF-11 proteins of four baculovirus genera indicated that the nuclear localization characteristics of baculovirus LEF-11 coincided with the predicted results. Moreover, Bombyx mori nucleopolyhedrovirus (BmNPV) LEF-11 could be transported into the nucleus during viral infection in the absence of a cNLSs. Further investigations demonstrated that the NLS of BmNPV LEF-11 is important for viral DNA replication. The findings of the present study indicate that the characteristics of the baculovirus LEF-11 protein and the NLS is essential to virus DNA replication and nuclear transport mechanisms.
Assuntos
Replicação do DNA , DNA Viral/metabolismo , Sinais de Localização Nuclear , Nucleopoliedrovírus/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral , Transporte Ativo do Núcleo Celular , Animais , Bombyx , Células CultivadasRESUMO
Thermotolerance is important particularly for poikilotherms such as insects. Understanding the mechanisms by which insects respond to high temperatures can provide insights into their adaptation to the environment. Therefore, in this study, we performed a transcriptome analysis of two silkworm strains with significantly different resistance to heat as well as humidity; the thermo-resistant strain 7532 and the thermos-sensitive strain Knobbed. We identified in total 4,944 differentially expressed genes (DEGs) using RNA-Seq. Among these, 4,390 were annotated and 554 were novel. Gene Ontology (GO) analysis of 747 DEGs identified between RT_48h (Resistant strain with high-temperature Treatment for 48 hours) and ST_48h (Sensitive strain with high-temperature Treatment for 48 hours) showed significant enrichment of 12 GO terms including metabolic process, extracellular region and serine-type peptidase activity. Moreover, we discovered 12 DEGs that may contribute to the heat-humidity stress response in the silkworm. Our data clearly showed that 48h post-exposure may be a critical time point for silkworm to respond to high temperature and humidity. These results provide insights into the genes and biological processes involved in high temperature and humidity tolerance in the silkworm, and advance our understanding of thermal tolerance in insects.
Assuntos
Bombyx/genética , Bombyx/fisiologia , Perfilação da Expressão Gênica , Temperatura Alta , Umidade , Estresse Fisiológico/genética , Termotolerância/genética , Animais , Ontologia Genética , Análise de Sequência de RNA , Especificidade da EspécieRESUMO
Research on molecular mechanisms that viruses use to regulate the host apparatus is important in virus infection control and antiviral therapy exploration. Our previous research showed that the Bombyx mori nucleopolyhedrovirus (BmNPV) LEF-11 localized to dense regions of the cell nucleus and is required for viral DNA replication. Herein, we examined the mechanism of LEF-11 on BmNPV multiplication and demonstrated that baculovirus LEF-11 interacts with Bombyx mori ATAD3A and HSPD1 (HSP60) protein. Furthermore, we showed that LEF-11 has the ability to induce and up-regulate the expression of ATAD3A and HSPD1, phenomena that were both reversed upon knockdown of lef-11. Our findings showed that ATAD3A and HSPD1 were necessary and contributed to BmNPV multiplication in Bombyx mori cells. Moreover, ATAD3A was found to directly interact with HSPD1. Interestingly, ATAD3A was required for the expression of HSPD1, while the knockdown of HSPD1 had no obvious effect on the expression level of ATAD3A. Taken together, the data presented in the current study demonstrated that baculovirus LEF-11 hijacks the host ATPase family members, ATAD3A and HSPD1, efficiently promote the multiplication of the virus. This study furthers our understanding of how baculovirus modulates energy metabolism of the host and provides a new insight into the molecular mechanisms of antiviral research.
Assuntos
Adenosina Trifosfatases/metabolismo , Baculoviridae/metabolismo , Bombyx/virologia , Interações Hospedeiro-Patógeno , Proteínas de Insetos/metabolismo , Replicação Viral/fisiologia , Animais , Linhagem Celular , Imunoprecipitação , Ligação Proteica , Estabilidade Proteica , Espectrometria de Massas em TandemRESUMO
Although current antiviral strategies can inhibit baculovirus infection and decrease viral DNA replication to a certain extent, novel tools are required for specific and accurate elimination of baculovirus genomes from infected insects. Using the newly developed clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology, we disrupted a viral genome in infected insect cells in vitro as a defense against viral infection. We optimized the CRISPR/Cas9 system to edit foreign and viral genome in insect cells. Using Bombyx mori nucleopolyhedrovirus (BmNPV) as a model, we found that the CRISPR/Cas9 system was capable of cleaving the replication key factor ie-1 in BmNPV thus effectively inhibiting virus proliferation. Furthermore, we constructed a virus-inducible CRISPR/Cas9 editing system, which minimized the probability of off-target effects and was rapidly activated after viral infection. This is the first report describing the application of the CRISPR/Cas9 system in insect antiviral research. Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells provides insights to produce virus-resistant transgenic strains for future.