RESUMO
MicroRNAs (miRNAs) represent a class of short, non-coding RNAs that are widely acknowledged as crucial participants in virus-host interactions. MiR-184, a highly conserved and abundant miRNA in insects, has yet to be extensively studied for its involvement in baculovirus infection. In this study, we investigated how miR-184 affects the infection and replication of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The results indicated that after AcMNPV infection, there was an initial increase in the expression of miR-184 within 24 h, followed by a subsequent decrease. MiR-184 can inhibit AcMNPV's DNA replication and budded virus production by directly targeting four viral genes, namely ie1, ac66, p49, and lef9. Moreover, suppressing miR-184 expression enhanced the insecticidal efficacy of AcMNPV against Spodoptera exigua larvae and markedly elevated the host ATPase gene expressions. These findings showed that miR-184 had a substantial impact on the interactions between baculoviruses and insects, presenting a prospective candidate for developing highly effective miRNA-based biopesticides.
Assuntos
MicroRNAs , Nucleopoliedrovírus , Spodoptera , Replicação Viral , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/fisiologia , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Spodoptera/virologia , Spodoptera/genética , Células Sf9 , Larva/virologia , Larva/genéticaRESUMO
BACKGROUND: Normalization with respect to stable housekeeping genes is important to facilitate gene transcription regulation research and acquire more accurate quantitative polymerase chain reaction (qPCR) data. In the current study, five candidates housekeeping genes of the cotton leafworm, Spodoptera littoralis encoding for Actin (Actin), elongation factor 1-alpha (EF1α), ribosomal protein S3 (RPS3), ribosomal protein 49 (RP49), and Ubiquitin (Ubi), were evaluated as normalization housekeeping genes under Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) viral infection. METHODS AND RESULTS: The qPCR results confirmed the expression of all five housekeeping genes in S. littoralis viral infected larvae. The expression profiles of the housekeeping genes showed that the EF1α, Actin, and RP49 had the minimum average Ct values of 18.41 ± 0.66, 18.84 ± 0.90 and 19.01 ± 0.87 in all infected samples, respectively. While RPS3 and Ubi showed the maximum average Ct of 21.61 ± 0.51 and 21.11 ± 0.82, respectively. According to the results of ΔCt and geNorm analysis, EF1α was ranked as the most stable housekeeping gene during infection time-course. While by using BestKeeper, geNorm and NormFinder, the Ubi, RP49, and RPS3 showed the most genes transcription stability. The obtained results were also validated using the Cytochrome c oxidase (COX) gene transcripts in response to SpliNPV infection. CONCLUSIONS: The results revealed that EF1α and Ubi were the most stable housekeeping genes to be used for normalizing S. littoralis gene transcription regulation under SpliNPV infection. These findings, provide a significant addition for gene transcription regulation studies of S. littoralis upon infection using SpliNPV as a bio-agent.
Assuntos
Genes Essenciais , Nucleopoliedrovírus , Spodoptera , Animais , Spodoptera/genética , Spodoptera/virologia , Genes Essenciais/genética , Nucleopoliedrovírus/genética , Regulação da Expressão Gênica , Larva/genética , Larva/virologia , Transcrição Gênica/genética , Perfilação da Expressão Gênica/métodos , Proteínas de Insetos/genéticaRESUMO
An efficient gene transfer and expression tool is lacking for shrimps and shrimp cells. To solve this, this study has developed a shrimp DNA virus-mediated gene transfer and expression system, consisting of insect Sf9 cells for viral packaging, the shrimp viral vector of pUC19-IHHNV-PH-GUS and the baculoviral vector of Bacmid or Bacmid-VP28 encoding the shrimp WSSV envelope protein VP28. The pUC19-IHHNV-PH-GUS vector was constructed by assembling the genomic DNA of shrimp infectious hypodermal and hematopoietic necrosis virus (IHHNV), which has shortened inverted terminal repeats, into a pUC19 backbone, and then an expression cassette of baculoviral polyhedron (PH) promoter-driven GUS (ß-glucuronidase) reporter gene was inserted immediately downstream of IHHNV for proof-of-concept. It was found that the viral vector of pUC19-IHHNV-PH-GUS could be successfully packaged into IHHNV-like infective virions in the Sf9 cells, and the gene transfer efficiency of this system was evaluated and verified in three systems of Sf9 cells, shrimp hemolymph cells and tissues of infected shrimps, but the GUS expression could only be detected in cases where the viral vector was co-transfected or co-infected with a baculovirus of Bacmid or Bacmid-VP28 due to the Bacmid-dependence of the PH promoter. Moreover, the packaging and infection efficiencies could be significantly improved when Bacmid-VP28 was used instead of Bacmid.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos , Penaeidae , Animais , Penaeidae/virologia , Penaeidae/genética , Células Sf9 , Vetores Genéticos/genética , Baculoviridae/genética , Regiões Promotoras Genéticas , Spodoptera/virologia , Densovirinae/genética , Expressão Gênica , Vírus da Síndrome da Mancha Branca 1/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Glucuronidase/genética , Glucuronidase/metabolismoRESUMO
Virus-encoded microRNAs (miRNAs) exert diverse regulatory roles in the biological processes of both viruses and hosts. This study delves into the functions of AcMNPV-miR-2, an early miRNA encoded by Autographa californica multiple nucleopolyhedrovirus (AcMNPV). AcMNPV-miR-2 targets viral early genes ac28 (lef-6), ac37 (lef-11), ac49, and ac63. Overexpression of AcMNPV-miR-2 leads to reduced production of infectious budded virions (BVs) and diminished viral DNA replication. Delayed polyhedron formation was observed through light and transmission electron microscopy, and the larval lifespan extended in oral infection assays. Moreover, the mRNA expression levels of two Lepidoptera-specific immune-related proteins, Gloverin and Spod-11-tox, significantly decreased. These findings indicate that AcMNPV-miR-2 restrains viral load, reducing host immune sensitivity. This beneficial effect enables the virus to combat host defense mechanisms and reside within the host for an extended duration. IMPORTANCE: Virus-encoded miRNAs have been extensively studied for their pivotal roles in finetuning viral infections. Baculoviruses, highly pathogenic in insects, remain underexplored concerning their encoded miRNAs. Previous reports outlined three AcMNPV-encoded miRNAs, AcMNPV-miR-1, -miR-3, and -miR-4. This study delves into the functions of another AcMNPV-encoded miRNA, AcMNPV-miR-2 (Ac-miR-2). Through a comprehensive analysis of target gene expression, the impact on larvae, and variations in host immune-related gene expression, we elucidate a functional pathway for Ac-miR-2. This miRNA suppresses viral load and infectivity and prolongs lifespans of infected larva by downregulating specific viral early genes and host immune-related genes. These mechanisms ultimately serve the virus's primary goal of enhanced propagation. Our study significantly contributes to understanding of the intricate regulatory mechanisms of virus-encoded miRNAs in baculovirus infections.
Assuntos
Regulação Viral da Expressão Gênica , MicroRNAs , Nucleopoliedrovírus , Proteínas Virais , Replicação Viral , Nucleopoliedrovírus/genética , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Larva/virologia , Larva/genética , Células Sf9 , Carga Viral , Spodoptera/virologia , Vírion/genética , Vírion/metabolismoRESUMO
The success of using the insect cell-baculovirus expression technology (BEST) relies on the efficient construction of recombinant baculovirus with genetic stability and high productivity, ideally within a short time period. Generation of recombinant baculoviruses requires the transfection of insect cells, harvesting of recombinant baculovirus pools, isolation of plaques, and the expansion of baculovirus stocks for their use for recombinant protein production. Moreover, many options exist for selecting the genetic elements to be present in the recombinant baculovirus. This chapter describes the most commonly used homologous recombination systems for the production of recombinant baculoviruses, as well as strategies to maximize generation efficiency and recombinant protein or baculovirus production. The key steps for generating baculovirus stocks and troubleshooting strategies are described.
Assuntos
Baculoviridae , Proteínas Recombinantes , Baculoviridae/genética , Animais , Proteínas Recombinantes/genética , Vetores Genéticos/genética , Transfecção/métodos , Recombinação Homóloga , Células Sf9 , Linhagem Celular , Spodoptera/virologia , Insetos/genética , Insetos/virologiaRESUMO
The baculovirus expression vector system (BEVS) is a powerful platform for protein expression in insect cells. A prevalent application is the expression of complex protein structures consisting of multiple, interacting proteins. Coinfection with multiple baculoviruses allows for production of complex structures, facilitating structure-function studies, allowing augmentation of insect cell functionality, and production of clinically relevant products such as virus-like particles (VLPs) and adeno-associated viral vectors (AAV). Successful coinfections require the generation of robust and well-quantified recombinant baculovirus stocks. Virus production through homologous recombination, combined with rigorous quantification of viral titers, allows for synchronous coinfections producing high end-product titers. In this chapter, we describe the streamlined workflow for generation and quantification of high-quality recombinant baculovirus stocks and successful coinfection as defined by a preponderance of dually infected cells in the insect cell culture.
Assuntos
Baculoviridae , Vetores Genéticos , Proteínas Recombinantes , Baculoviridae/genética , Animais , Vetores Genéticos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Linhagem Celular , Spodoptera/virologiaRESUMO
Baculoviruses have been extensively studied for their potential in microbial pest control, but the mechanisms behind their mode of action still need to be addressed. Here we report differential expression of a cellular miRNA, Sfr-miR-184, from Sf9 cells in response to Autographa californica multicapsid Nucleopolyhedrovirus (AcMNPV) infection. Our results showed that Sfr-miR-184 is down-regulated in AcMNPV-infected cells but not with UV-inactivated virus. Prohibitin gene was determined as a target of the miRNA, which was up-regulated following AcMNPV infection. Using synthetic miRNA mimic, we found that oversupply of the miRNA resulted in decreased transcript levels of the target gene. Results suggest that Sfr-miR-184 negatively regulate prohibitin transcripts in the host cells. Antibody-mediated inhibition and silencing of the prohibitin gene revealed significant reductions in virus DNA replication suggesting a possible role for prohibitin in the virus-host interaction. These findings highlight another molecular mechanism used by baculovirus to manipulate host cells for its replication.
Assuntos
MicroRNAs , Nucleopoliedrovírus , Proibitinas , Spodoptera , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Spodoptera/virologia , Células Sf9 , Nucleopoliedrovírus/fisiologia , Replicação Viral , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Baculoviridae/genética , Baculoviridae/fisiologia , Interações Hospedeiro-PatógenoRESUMO
Purification of rAAV is a crucial unit operation of the AAV production process. It enables the capture of AAV and removal of contaminants such as host cell proteins, host cell DNA, and other cell culture-related impurities. Here we describe the purification of rAAV produced in insect cells Sf9/rBEV by immuno-affinity capture chromatography. The method is fully scale-amenable unlike other traditional purification methods based on ultracentrifugation. The method reported herein has two main steps: (1) the clarification of cell lysate by depth filtration and (2) the selective capture and single-step purification of AAV via immune-affinity chromatography. This purification method has been successfully implemented to purify the majority of wild-type AAV serotypes.
Assuntos
Cromatografia de Afinidade , Dependovirus , Dependovirus/genética , Dependovirus/isolamento & purificação , Animais , Cromatografia de Afinidade/métodos , Células Sf9 , Vetores Genéticos/genética , Humanos , Spodoptera/virologiaRESUMO
There are many methods that can be used to determine the infectious titer of your baculovirus stock. The TCID50 method is a simple end-point dilution method that determines the amount of baculovirus virus needed to produce a cytopathic effect or kill 50% of inoculated insect cells. Serial dilutions of baculovirus stock are added to Sf9 cells cultivated in 96-well plates and 3-5 days after infection, cells are monitored for cell death or cytopathic effect. The titer can then be calculated by the Reed-Muench method as described in this method.
Assuntos
Baculoviridae , Baculoviridae/genética , Animais , Células Sf9 , Efeito Citopatogênico Viral , Spodoptera/virologia , Carga Viral/métodos , Linhagem CelularRESUMO
Plaque assay method enables the quantification of infectious baculovirus when defined as plaque forming units (PFU). It allows to determine the amount of infectious virus needed to infect the cells at a specific multiplicity of infection (MOI). Serial dilutions of baculovirus stock are added to the Sf9 cells monolayer followed by addition of 5% Agarose overlay. Six days after infection clear infection halos are observed using a neutral red solution. Here we describe the quantification of recombinant baculovirus expression vector (rBEV) carrying a transgene in an rAAV expression cassette. Reproducible quantification of PFU is obtained with this method.
Assuntos
Baculoviridae , Vetores Genéticos , Ensaio de Placa Viral , Baculoviridae/genética , Células Sf9 , Ensaio de Placa Viral/métodos , Animais , Vetores Genéticos/genética , Transgenes , Vírion/genética , Dependovirus/genética , Spodoptera/virologiaRESUMO
Granuloviruses (GVs) Betabaculovirus associated with the fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), especially those of the type I, have scarcely been studied. These GVs might be an effective alternative for the biocontrol of this insect. In this study, the native GVs SfGV-CH13 and SfGV-CH28 were isolated from FAW larvae and characterized for morphology, molecular traits, and insecticidal activity. The elapsed time between symptomatic infection of larvae and stop feeding as well as the weight of larvae before death or prior to pupation were also evaluated. Both GVs had ovoid shape and a length of 0.4 µm. They had the same DNA restriction profiles and their genome sizes were about 126 kb. The symptomatic infection with the tested GVs mainly caused flaccidity of larva body and discoloration of integument. The integument lysis was only observed in 8% of infected larvae. Infected larvae gradually stopped feeding. Overall, these symptoms are characteristic of infections caused by type I GVs, which are known as monoorganotropic or slow-killing GVs. The median lethal dose (LD50) values for SfGV-CH13 and SfGV-CH28 isolates were 5.4 × 102 and 1.1 × 103 OBs/larva, respectively. The median lethal time (LT50) ranged from 17 to 24 days. LT50 values decreased as the viral dose was increased. The elapsed time from symptomatic infection until pupation and body weight of larvae (third instar) were higher with SfGV-CH28 than SfGV-CH13. Both granulovirus isolates were able to kill the FAW larvae from the 12th day.
Assuntos
Granulovirus , Larva , Spodoptera , Animais , Spodoptera/virologia , Granulovirus/genética , Larva/virologiaRESUMO
It is extremely rare that a single virus crosses host barriers across multiple kingdoms. Based on phylogenetic and paleovirological analyses, it has previously been hypothesized that single members of the family Partitiviridae could cross multiple kingdoms. Partitiviridae accommodates members characterized by their simple bisegmented double-stranded RNA genome; asymptomatic infections of host organisms; the absence of an extracellular route for entry in nature; and collectively broad host range. Herein, we show the replicability of single fungal partitiviruses in three kingdoms of host organisms: Fungi, Plantae, and Animalia. Betapartitiviruses of the phytopathogenic fungusRosellinia necatrix could replicate in protoplasts of the carrot (Daucus carota), Nicotiana benthamiana and Nicotiana tabacum, in some cases reaching a level detectable by agarose gel electrophoresis. Moreover, betapartitiviruses showed more robust replication than the tested alphapartitiviruses. One of the fungal betapartitiviruses, RnPV18, could persistently and stably infect carrot plants regenerated from virion-transfected protoplasts. Both alpha- and betapartitiviruses, although with different host preference, could replicate in two insect cell lines derived from the fall armyworm Spodoptera frugiperda and the fruit fly Drosophila melanogaster. Our results indicate the replicability of single partitiviruses in members of three kingdoms and provide insights into virus adaptation, host jumping, and evolution.
Assuntos
Daucus carota , Nicotiana , Replicação Viral , Animais , Nicotiana/virologia , Nicotiana/microbiologia , Daucus carota/virologia , Daucus carota/microbiologia , Vírus de RNA/genética , Vírus de RNA/fisiologia , Micovírus/genética , Micovírus/classificação , Micovírus/fisiologia , Filogenia , Protoplastos/virologia , Doenças das Plantas/virologia , Doenças das Plantas/microbiologia , Spodoptera/virologia , Spodoptera/microbiologiaRESUMO
Alphabaculoviruses are lethal dsDNA viruses of Lepidoptera that have high genetic diversity and are transmitted in aggregates within proteinaceous occlusion bodies. This mode of transmission has implications for their efficacy as biological insecticides. A Nicaraguan isolate of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV-NIC) comprising nine genotypic variants has been the subject of considerable study due to the influence of variant interactions on the insecticidal properties of mixed-variant occlusion bodies. As part of a systematic study on the replication and transmission of variant mixtures, a tool for the accurate quantification of a selection of genotypic variants was developed based on the quantitative PCR technique (qPCR). First, primer pairs were designed around a region of high variability in four variants named SfNic-A, SfNic-B, SfNic-C and SfNic-E to produce amplicons of 103-150 bp. Then, using cloned purified amplicons as standards, amplification was demonstrated over a dynamic range of 108-101 copies of each target. The assay was efficient (mean ± SD: 98.5 ± 0.8%), reproducible, as shown by low inter- and intra-assay coefficients of variation (<5%), and specific to the target variants (99.7-100% specificity across variants). The quantification method was validated on mixtures of genotype-specific amplicons and demonstrated accurate quantification. Finally, mixtures of the four variants were quantified based on mixtures of budded virions and mixtures of DNA extracted from occlusion-derived virions. In both cases, mixed-variant preparations compared favorably to total viral genome numbers by quantification of the polyhedrin (polh) gene that is present in all variants. This technique should prove invaluable in elucidating the influence of variant diversity on the transmission and insecticidal characteristics of this pathogen.
Assuntos
Variação Genética , Genótipo , Nucleopoliedrovírus , Reação em Cadeia da Polimerase em Tempo Real , Spodoptera , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/classificação , Nucleopoliedrovírus/isolamento & purificação , Animais , Spodoptera/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , DNA Viral/genéticaRESUMO
Invertebrate iridescent virus 6 (IIV6) is a nucleocytoplasmic insect virus and a member of the family Iridoviridae. The IIV6 genome consists of 212,482 bp of linear dsDNA with 215 non-overlapping and putative protein-encoding ORFs. The IIV6 118L ORF is conserved in all sequenced members of the Iridoviridae and encodes a 515 amino acid protein with three predicted transmembrane domains and several N-glycosylation/N-myristoylation sites. In this study, we characterized the 118L ORF by both deleting it from the viral genome and silencing its expression with dsRNA in infected insect cells. The homologous recombination method was used to replace 118L ORF with the green fluorescent protein (gfp) gene. Virus mutants in which the 118L gene sequence had been replaced with gfp were identified by fluorescence microscopy but could not be propagated separately from the wild-type virus in insect cells. Unsuccessful attempts to isolate the mutant virus with the 118L gene deletion suggested that the protein is essential for virus replication. To support this result, we used dsRNA to target the 118L gene and showed that treatment resulted in a 99% reduction in virus titer. Subsequently, we demonstrated that 118L-specific antibodies produced against the 118L protein expressed in the baculovirus vector system were able to neutralize the virus infection. All these results indicate that 118L is a viral envelope protein that is required for the initiation of virus replication.
Assuntos
Proteínas do Envelope Viral , Animais , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Fases de Leitura Aberta/genética , Replicação Viral/genética , Iridovirus/genética , Linhagem Celular , Células Sf9 , Genoma Viral/genética , Spodoptera/virologiaRESUMO
Viral infection can regulate the cell cycle, thereby promoting viral replication. Hijacking and altering the cell cycle are important for the virus to establish and maintain a latent infection. Previously, Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV)-latently infected P8-Se301-C1 cells, which grew more slowly than Se301 cells and interfered with homologous SeMNNPV superinfection, were established. However, the effects of latent and superinfection with baculoviruses on cell cycle progression remain unknown. In this study, the cell cycle profiles of P8-Se301-C1 cells and SeMNPV or Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected P8-Se301-C1 cells were characterized by flow cytometry. The results showed that replication-related genes MCM4, PCNA, and BAF were down-regulated (p < 0.05) in P8-Se301-C1 cells, and the S phase of P8-Se301-C1 cells was longer than that of Se301 cells. P8-Se301-C1 cells infected with SeMNPV did not arrest in the G2/M phase or affect the expression of Cyclin B and cyclin-dependent kinase 1 (CDK1). Furthermore, when P8-Se301-C1 cells were infected with SeMNPV after synchronized treatment with hydroxyurea and nocodazole, light microscopy and qRT-PCR analysis showed that, compared with unsynchronized cells and S and G2/M phase cells, SeMNPV-infected P8-Se301-C1 cells in G1 phase induced G2/M phase arrest, and the amount of virus adsorption and intracellular viral DNA replication were significantly increased (p < 0.05). In addition, budded virus (BV) production and occlusion body (OB)-containing cells were both increased at 120 h post-infection (p < 0.05). The expression of Cyclin B and CDK1 was significantly down-regulated at 48 h post-infection (p < 0.05). Finally, the arrest of SeMNPV-infected G1 phase cells in the G2/M phase increased BV production (p < 0.05) and the number of OB-containing cells. In conclusion, G1 phase infection and G2/M arrest are favorable to SeMNPV proliferation in P8-Se301-C1 cells, thereby alleviating the homologous superinfection exclusion. The results contribute to a better understanding of the relationship between baculoviruses and insect cell cycle progression and regulation.
Assuntos
Pontos de Checagem da Fase G2 do Ciclo Celular , Nucleopoliedrovírus , Spodoptera , Superinfecção , Replicação Viral , Animais , Nucleopoliedrovírus/fisiologia , Linhagem Celular , Spodoptera/virologia , Superinfecção/virologia , Fase G1RESUMO
Baculoviruses enter insect midgut epithelial cells via a set of occlusion-derived virion (ODV) envelope proteins called per os infectivity factors (PIFs). P74 of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), which was the first identified PIF, is cleaved by an endogenous proteinase embedded within the occlusion body during per os infection, but the target site(s) and function of the cleavage have not yet been ascertained. Here, based on bioinformatics analyses, we report that cleavage was predicted at an arginine and lysine-rich region in the middle of P74. A series of recombinant viruses with site-directed mutants in this region of P74 were generated. R325 or R334 was identified as primary cleavage site. In addition, we showed that P74 is also cleaved by brush border membrane vesicles (BBMV) of the host insect at R325 or R334, instead of R195, R196, and R199, as previously reported. Simultaneous mutations in R195, R196, and R199 lead to instability of P74 during ODV release. Bioassays showed that mutations at both R325 and R334 significantly affected oral infectivity. Taken together, our data show that both R325 and R334 of AcMNPV P74 are the primary cleavage site for both occlusion body endogenous proteinase and BBMV proteinase during ODV release and are critical for oral infection. IMPORTANCE: Cleavage of viral envelope proteins is usually an important trigger for viral entry into host cells. Baculoviruses are insect-specific viruses that infect host insects via the oral route. P74, a per os infectivity factor of baculoviruses, is cleaved during viral entry. However, the function and precise cleavage sites of P74 remain unknown. In this study, we found that R325 or R334 between the N- and C-conserved domains of P74 was the primary cleavage site by proteinase either from the occlusion body or host midgut. The biological significance of cleavage seems to be the release of the potential fusion peptide at the N-terminus of the cleaved C-terminal P74. Our results shed light on the cleavage model of P74 and imply its role in membrane fusion in baculovirus per os infection.
Assuntos
Microvilosidades , Nucleopoliedrovírus , Corpos de Oclusão Virais , Peptídeo Hidrolases , Proteínas do Envelope Viral , Animais , Microvilosidades/enzimologia , Microvilosidades/metabolismo , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/metabolismo , Nucleopoliedrovírus/fisiologia , Corpos de Oclusão Virais/enzimologia , Corpos de Oclusão Virais/metabolismo , Corpos de Oclusão Virais/virologia , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/genética , Células Sf9 , Spodoptera/citologia , Spodoptera/virologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Vírion/química , Vírion/genética , Vírion/metabolismo , Internalização do Vírus , Mutação , Boca/virologia , Especificidade por Substrato , Liberação de VírusRESUMO
The occlusion bodies of Autographa californica multiple nucleopolyhedrovirus are proteinaceous formations with significant biotechnological potential owing to their capacity to integrate foreign proteins through fusion with polyhedrin, their primary component. However, the strategy for successful heterologous protein inclusion still requires further refinement. In this study, we conducted a comparative assessment of various conditions to achieve the embedding of recombinant proteins within polyhedra. Two baculoviruses were constructed: AcPHGFP (polh+), with GFP as a fusion to wild type (wt) polyhedrin and AcΔPHGFP (polh+), with GFP fused to a fragment corresponding to amino acids 19 to 110 of polyhedrin. These baculoviruses were evaluated by infecting Sf9 cells and stably transformed Sf9, Sf9POLH, and Sf9POLHE44G cells. The stably transformed cells contributed another copy of wt or a mutant polyhedrin, respectively. Polyhedra of each type were isolated and characterized by classical methods. The fusion PHGFP showed more-efficient incorporation into polyhedra than ΔPHGFP in the three cell lines assayed. However, ΔPHGFP polyhedron yields were higher than those of PHGFP in Sf9 and Sf9POLH cells. Based on an integral analysis of the studied parameters, it can be concluded that, except for the AcΔPHGFP/Sf9POLHE44G combination, deficiencies in one factor can be offset by improved performance by another. The combinations AcPHGFP/Sf9POLHE44G and AcΔPHGFP/Sf9POLH stand out due to their high level of incorporation and the large number of recombinant polyhedra produced, respectively. Consequently, the choice between these approaches becomes dependent on the intended application.
Assuntos
Biotecnologia , Nucleopoliedrovírus , Spodoptera , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/metabolismo , Animais , Células Sf9 , Biotecnologia/métodos , Spodoptera/virologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Matriz de Corpos de Inclusão , Corpos de Oclusão Virais/metabolismo , Corpos de Oclusão Virais/genética , Linhagem Celular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Autographa californica multiple nucleopolyhedrovirus (AcMNPV) Ac93 is highly conserved in all sequenced baculovirus genomes, and it plays important roles in both the nuclear egress of nucleocapsids and the formation of intranuclear microvesicles. In this study, we characterized a cellular CRM1-dependent nuclear export signal (NES) of AcMNPV Ac93. Bioinformatic analysis revealed that AcMNPV Ac93 may contain an NES at amino acids 115-125. Green fluorescent protein (GFP) fused to the NES (GFP:NES) of AcMNPV Ac93 is localized to the cytoplasm of transfected cells. Multiple point mutation analysis demonstrated that NES is important for the nuclear export of GFP:NES. Bimolecular fluorescence complementation experiments and co-immunoprecipitation assays confirmed that Ac93 interacts with Spodoptera frugiperda CRM1 (SfCRM1). However, AcMNPV Ac34 inhibits cellular CRM1-dependent nuclear export of GFP:NES. To determine whether the NES in AcMNPV Ac93 is important for the formation of intranuclear microvesicles, an ac93-null AcMNPV bacmid was constructed; the wild-type and NES-mutated Ac93 were reinserted into the ac93-null AcMNPV bacmid. Immunofluorescence analysis showed that Ac93 and SfCRM1 were predominantly colocalized at intranuclear microvesicles in infected cells, while the construct containing point mutations at residues 123 and 125 of Ac93 resulted in a defect in budded virus production and the abolishment of intranuclear microvesicles. Together, these data demonstrate that Ac93 contains a functional NES, which is required for the production of progeny viruses and the formation of intranuclear microvesicles.IMPORTANCEAutographa californica multiple nucleopolyhedrovirus (AcMNPV) Ac93 is important for the formation of intranuclear microvesicles. However, how the baculovirus manipulates Ac93 for the formation of intranuclear microvesicles is unclear. In this study, we identified a nuclear export signal (NES) at amino acids 115-125 of AcMNPV Ac93. Our results showed that the NES is required for the interaction between Ac93 and Spodoptera frugiperda CRM1 (SfCRM1). However, AcMNPV Ac34 inhibits the nuclear export of green fluorescent protein fused to the NES. Our analysis revealed that Ac93 and SfCRM1 were predominantly colocalized at intranuclear microvesicles in AcMNPV-infected cells. Together, our results indicate that Ac93 participates in the formation of intranuclear microvesicles via the Ac93 NES-mediated CRM1 pathway.
Assuntos
Transporte Ativo do Núcleo Celular , Sinais de Exportação Nuclear , Nucleopoliedrovírus , Proteínas Virais , Animais , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Proteína Exportina 1 , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Carioferinas/metabolismo , Nucleopoliedrovírus/metabolismo , Nucleopoliedrovírus/fisiologia , Nucleopoliedrovírus/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Células Sf9 , Spodoptera/virologia , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
The lacking of stable and susceptible cell lines has hampered research on pathogenic mechanism of crustacean white spot syndrome virus (WSSV). To look for the suitable cell line which can sustain WSSV infection, we performed the studies on WSSV infection in the Spodoptera frugiperda (Sf9) insect cells. In consistent with our previous study in vitro in crayfish hematopoietic tissue cells, the WSSV envelope was detached from nucleocapsid around 2 hpi in Sf9 cells, which was accompanied with the cytoplasmic transport of nucleocapsid toward the cell nucleus within 3 hpi. Furthermore, the expression profile of both gene and protein of WSSV was determined in Sf9 cells after viral infection, in which a viral immediate early gene IE1 and an envelope protein VP28 exhibited gradually increased presence from 3 to 24 hpi. Similarly, the significant increase of WSSV genome replication was found at 3-48 hpi in Sf9 cells after infection with WSSV, indicating that Sf9 cells supported WSSV genome replication. Unfortunately, no assembled progeny virion was observed at 24 and 48 hpi in Sf9 cell nuclei as determined by transmission electron microscope, suggesting that WSSV progeny could not be assembled in Sf9 cell line as the viral structural proteins could not be transported into cell nuclei. Collectively, these findings provide a cell model for comparative analysis of WSSV infection mechanism with crustacean cells.
Assuntos
Spodoptera , Vírion , Montagem de Vírus , Replicação Viral , Vírus da Síndrome da Mancha Branca 1 , Animais , Vírus da Síndrome da Mancha Branca 1/fisiologia , Spodoptera/virologia , Células Sf9 , Vírion/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/genética , Nucleocapsídeo/metabolismo , Nucleocapsídeo/genética , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/virologia , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Genoma Viral , Linhagem CelularRESUMO
Epoxyoctadecamonoenoic acids (EpOMEs) are produced from linoleic acid by a cytochrome P450 monooxygenase (CYP) and play a crucial role in terminating excessive and unnecessary immune responses during the late infection stage in insects. This suggests that an increase in the EpOME level may enhance the virulence of insect pathogens against pests. This study tested this hypothesis using a specific inhibitor against soluble epoxide hydrolase (sEH) to degrade EpOMEs, which leads to elevated endogenous EpOME levels. A baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), was used to infect three different lepidopteran insects (Spodoptera exigua, Maruca vitrata, and Plutella xylostella) by oral feeding or hemocoelic injection treatments. Within one hour, the viral infection induced the expression of three different phospholipase A2 (PLA2) genes and, after 12 h, up-regulated the expressions of CYP and sEH genes in Spodopera exigua. As expected, AcMNPV virulence was suppressed by the addition of arachidonic acid (a catalytic product of PLA2) but was enhanced by the addition of either of the EpOME regioisomers. In addition, treatment with a specific sEH inhibitor (AUDA) increased AcMNPV virulence against three different lepidopteran insects, presumably by increasing endogenous EpOME levels. This enhanced effect of EpOMEs on virulence was further supported by specific RNA interference (RNAi), in which RNAi specific to CYP expression decreased AcMNPV virulence while a specific RNAi against sEH expression significantly enhanced virulence. In response to AcMNPV infection, TUNEL assay results showed that S. exigua larvae exhibited apoptosis in the midgut, fat body, and epidermis. Inhibition of apoptosis by a pan-caspase inhibitor, Z-VAD-FMK, significantly increased virulence. Similarly, the addition of AUDA to the viral treatment suppressed the gene expression of five inducible caspases and cytochrome C to suppress apoptosis, which led to a significant increase in the tissue viral titers. These results indicate that EpOMEs play a role in terminating excessive and unnecessary immune responses against viral infection during the late stage by down-regulating antiviral apoptosis in lepidopteran insects.