Spodoptera Frugiperda Sf9 Cells Research Articles

Autophagy induced by Vip3Aa has a pro-survival role in Spodoptera frugiperda Sf9 cells

ABSTRACT Vip3Aa is an insecticidal protein that can effectively control certain lepidopteran pests and has been used widely in biological control. However, the mechanism of action of Vip3Aa is unclear. In the present study, we showed that Vip3Aa could cause autophagy in Sf9 cells, which was confirmed by the increased numbers of GFP-Atg8 puncta, the appearance of autophagic vacuoles, and an elevated Atg8-II protein level. Moreover, we found that the AMPK-mTOR-ULK1 pathway is involved in Vip3Aa-induced autophagy, which might be associated with the destruction of ATP homeostasis in Vip3Aa-treated cells. Both the elevated p62 level and the increased numbers of GFP-RFP-Atg8 yellow fluorescent spots demonstrated that autophagy in Sf9 cells was inhibited at 24 h after Vip3Aa treatment. With the prolongation of Vip3Aa treatment time, this inhibition became more serious and led to autophagosome accumulation. Genetic knockdown of ATG5 or the use of the autophagy inhibitor 3-MA further increased the sensitivity of Sf9 cells to Vip3Aa. Overexpression of ATG5 reduced the cell mortality of Vip3Aa-treated cells. In summary, the results revealed that autophagy induced by Vip3Aa has a pro-survival role, which might be related to the development of insect resistance.

Effect of Extracts of Cupressus macrocarpa and Alpinia officinarum on Expression of Chitinase and FGF Receptor Genes in Spodoptera frugiperda Sf9 Cell Line

Effect of Extracts of Cupressus macrocarpa and Alpinia officinarum on Expression of Chitinase and FGF Receptor Genes in Spodoptera frugiperda Sf9 Cell Line

Dielectric Spectroscopy to Improve the Production of rAAV Used in Gene Therapy

The insect cell-baculovirus expression vector system is an established method for large scale recombinant adeno-associated virus (rAAV) production, largely due to its scalability and high volumetric productivities. During rAAV production it is critical to monitor process parameters such as Spodoptera frugiperda (Sf9) cell concentration, infection timing, and cell harvest viabilities since they can have a significant influence on rAAV productivity and product quality. Herein we developed the use of dielectric spectroscopy as a process analytical technology (PAT) tool used to continuously monitor the production of rAAV in 2 L stirred tank bioreactors, achieving enhanced control over the production process. This study resulted in improved manufacturing robustness through continuous monitoring of cell culture parameters, eliminating sampling needs, increasing the accuracy of infection timing, and reliably estimating the time of harvest. To increase the accuracy of baculovirus infection timing, the cell growth/permittivity model was coupled to a feedback loop with real-time monitoring. This system was able to predict baculovirus infection timing up to 24 h in advance for greatly improved accuracy of infection and ensuring consistent high rAAV productivities. Furthermore, predictive models were developed based on the dielectric measurements of the culture. These multiple linear regression-based models resulted in correlation coefficients (Q2) of 0.89 for viable cell concentration, 0.97 for viability, and 0.92 for cell diameter. Finally, models were developed to predict rAAV titer providing the capability to distinguish in real time between high and low titer production batches.

Caenorhabditis elegans systemic RNA interference defective protein 1 enhances RNAi efficiency in a lepidopteran insect, the fall armyworm, in a tissue-specific manner

ABSTRACT RNA interference (RNAi) is an important tool for gene function studies in insects, especially in non-model insects. This technology is also being developed for pest control. However, variable RNAi efficiency among insects is limiting its use in insects. Systemic RNAi in Caenorhabditis elegans requires systemic RNA interference defective protein 1 (CeSid1). The expression of CeSid1 in insect cell lines was shown to improve RNAi. However, the mechanisms through which this double-stranded RNA (dsRNA) transporter improves RNAi efficiency in insects is not known. We stably expressed CeSid1 in two Spodoptera frugiperda cell lines, Sf9 and Sf17 cells derived from ovary and midgut, respectively. Expression of CeSid1 enhanced RNAi efficiency in ovarian Sf9 cells, but not in midgut Sf17 cells. Reduced accumulation of dsRNA in late endosomes and successful processing dsRNA to siRNA contribute to enhanced RNAi efficiency in Sf9 cells. Transgenic S. frugiperda expressing CeSid1 were produced and tested for RNAi efficiency. RNAi efficiency enhancement due to CeSid1 expression showed tissue specificity. Compared to RNAi efficiency in wild-type S. frugiperda, CeSid1 expressing transgenic S. frugiperda showed a significant improvement of RNAi in tissues such as Verson’s glands. In contrast, no improvement in RNAi was observed in tissues such as midgut. The in vitro cell-type specific and in vivo tissue-specific enhancement of RNAi efficiency by CeSid1 in S. frugiperda provides valuable information for improving RNAi in insects such as those belonging to order Lepidoptera where RNAi is variable and inefficient.

Synthesis, characterization of two matrine derivatives and their cytotoxic effect on Sf9 cell of Spodoptera frugiperda

The invasion of Spodoptera frugiperda has imposed a serious impact on global food security. Matrine is a botanical pesticide with a broad spectrum of insecticidal activity which was recommended for controlling Spodoptera frugiperda. In order to discover effective insecticide for Spodoptera frugiperda, two matrine derivatives modified with carbon disulfide and nitrogen-containing groups were systhesized. And their inhibition activities on Sf9 cell were evaluated. The structural configuration of compounds were characterized by IR, HPLC, MS, NMR and XRD, with yields of 52% and 65%, respectively. The IC50 of the two newly synthesized compounds on Sf9 cell reduced to 0.648 mmol/L and 1.13 mmol/L, respectively, compared with that of matrine (5.330 mmol/L). In addition, microscopic observation of Sf9 cell treated with the compounds showed that the number of adherent cells decreased, the cells shrunk, vacuolated and apoptotic bodies appeared. The two newly synthesized compounds exhibited better inhibitory effect on Sf9 cell than that of the parent matrine, suggesting that the positive effect of the introduction of 1-pyrrolidinecarbodithioate and diethylcarbamodithioate groups to matrine. The morphological observation of Sf9 cell induced by derivatives indicated that apoptosis induction may be a mechanism that inhibits insect cell proliferation and exerts insecticidal effect.

Uptake and imaging of glycine functionalized gold nanoclusters in Spodoptera frugiperda (Sf9) cells

Fluorescent gold nanoclusters (Au NCs) have attracted considerable interest in biological application. Here, we reported a novel Au NCs with blue fluorescence, conjugated with glycine (Gly) and rotenone (R). The structure and properties of Au–BSA–Gly–R conjugates were characterized by transmission electron microscopy (TEM), UV–visible, fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR) and Zeta potential measurement. The results of fluorescent microscopic imaging revealed that the Au NCs with different ligands were capable of being selectively uptaken by Spodoptera frugiperda (Sf9) cells. Furthermore, the gold content of Au–BSA–Gly–R in Sf9 cells was 2.2 and 2.6 times higher than that of Au–BSA and Au–BSA–R, respectively. It illustrated that nanoparticles after conjugated with Gly could gain specificity to intracellular transportation. This study indicated that the Au NCs after mediated by Gly not only had great potential for applications in insect cell imaging, but also could play an important role of amino acid for agrochemicals delivery in agricultural fields.

Production and Easy One-Step Purification of Bluetongue Recombinant VP7 from Infected Sf9 Supernatant for an Immunoenzymatic Assay (ELISA)

Bluetongue (BT) is non-contagious, vector-borne viral disease of domestic and wild ruminants, transmitted by midges (Culicoides spp.) and is caused by Bluetongue virus (BTV). BTV is the type species of the Orbivirus genus within the Reoviridae family and possesses a genome consisting of 10 double-stranded RNA segments encoding 7 structural and 4 nonstructural proteins. Viral Protein 7 (VP7) is the major sera group-specific protein and is a good antigen candidate for immunoenzymatic assays for the BT diagnosis. In our work, BTV-2 recombinant VP7 (BTV-2 recVP7), expressed in Spodoptera frugiperda (Sf9) cells using a baculovirus system, was produced and purified by affinity chromatography from the supernatant of infected cell culture. The use of the supernatant allowed us to obtain a high quantity of recombinant protein with high purity level by an easy one-step procedure, rather than the multistep purification from the pellet. RecVP7-BTV2 was detected using a MAb anti-BTV in Western blot and it was used to develop an immunoenzymatic assay.

Functional Analyses of House Fly Carboxylesterases Involved in Insecticide Resistance.

Carboxylesterase-mediated metabolism is one of major mechanisms involved in insecticide resistance. Our previous study has identified multiple carboxylesterase genes with their expression levels were significantly upregulated in pyrethroid resistant house flies. To further explore their metabolic functions, we used insect Spodoptera frugiperda (Sf9) cells to express these carboxylesterases in vitro and measure their hydrolytic activities toward esterase substrates. Our results indicated that these carboxylesterases can efficiently hydrolyze α-naphthyl acetate rather than β- naphthyl acetate. A cell based MTT cytotoxicity assay indicated that carboxylesterase-expressing cells show enhanced tolerance to permethrin, suggesting important roles of these carboxylesterases in metabolizing permethrin and thereby protecting cells from permethrin treatments. The metabolic functions of carboxylesterases were further verified by conducting in vitro metabolism studies toward permethrin and its potential metabolites 3-phenoxybenzyl alcohol and 3-phenoxybenzaldehyde, which not only suggested the potential metabolic pathway of permethrin in insects, but also important roles of these candidate carboxylesterases in metabolizing permethrin and conferring resistance in house flies. Homology modeling and docking were finally conducted to reflect interactions between permethrin ligand and carboxylesterase proteins, visually confirming the metabolic functions of carboxylesterases to insecticides in house flies.

Bioreactor-Based Antigen Production Process Using the Baculovirus Expression Vector System.

Several vaccines are already produced using the baculovirus expression vector system (BEVS). This chapter describes methods for generating recombinant baculoviral DNA (also called bacmid) for cultivating Spodoptera frugiperda Sf-9 cells and producing a baculovirus stock from the recombinant bacmid and for producing a protein-based vaccine with the BEVS in a stirred tank reactor.

Triangulation of methods using insect cell lines to investigate insecticidal mode-of-action.

This study investigated three in vitro models to assist in elucidating possible mode-of-action, which could be adopted to evaluate insecticidal activity of complex, unknown, or multi-constituent formulations. We used a combination of absorbance spectrometry, confocal scanning laser microscopy and microelectrode ion flux estimation (MIFE) to provide insight into potential target sites for insecticides. This study used two insect cell lines and evaluated three pyrethroid insecticides. We observed that the two cell lines produced distinctly different responses. Drosophila melanogaster D.mel-S2 cell line was a useful model to monitor ion flux changes, resulting from insecticides with neural toxicity; however, it was less useful to determine some metabolic pathway indicators of toxic stress. Conversely, the Spodoptera frugiperda Sf9 cell line produced acute reactive oxygen species (ROS) in response to insecticide treatments, but was not highly responsive in electrophysiological experiments. We also showed that the natural, multi-constituent botanical extract of pyrethrum elicited different Na+ , Cl- and Ca2+ ion fluxes than its synthetic, single constituent analogues, α-cypermethrin and esfenvalerate. These two methods used in combination with absorbance spectrometry measuring cell growth inhibition plus cell mortality assays shed some light on cytotoxic responses in differing model cell lines. This research highlights the importance of using multiple cell types and interdisciplinary methods to provide a better insight into mode of insecticidal action. This is especially pertinent to novel biopesticide discovery, as the underlying mechanisms for toxicity in initial screening processes are likely to be unknown.

Adenosine Receptor Modulates Permissiveness of Baculovirus (Budded Virus) Infection via Regulation of Energy Metabolism in Bombyx mori.

Although the modulation of host physiology has been interpreted as an essential process supporting baculovirus propagation, the requirement of energy supply for host antivirus reactions could not be ruled out. Our present study showed that metabolic induction upon AcMNPV (budded virus) infection of Bombyx mori stimulated virus clearance and production of the antivirus protein, gloverin. In addition, we demonstrated that adenosine receptor signaling (AdoR) played an important role in regulating such metabolic reprogramming upon baculovirus infection. By using a second lepidopteran model, Spodoptera frugiperda Sf-21 cells, we demonstrated that the glycolytic induction regulated by adenosine signaling was a conservative mechanism modulating the permissiveness of baculovirus infection. Another interesting finding in our present study is that both BmNPV and AcMNPV infection cause metabolic activation, but it appears that BmNPV infection moderates the level of ATP production, which is in contrast to a dramatic increase upon AcMNPV infection. We identified potential AdoR miRNAs induced by BmNPV infection and concluded that BmNPV may attempt to minimize metabolic activation by suppressing adenosine signaling and further decreasing the host's anti-baculovirus response. Our present study shows that activation of energy synthesis by adenosine signaling upon baculovirus infection is a host physiological response that is essential for supporting the innate immune response against infection.

Intracellular localization and cytotoxicity of Bacillus thuringiensis Vip3Aa against Spodoptera frugiperda (Sf9) cells

Vip3Aa protein is produced by Bacillus thuringiensis during vegetative growth and displays high toxicity against a wide range of lepidopteran insect larvae such as Spodoptera exigua and Spodoptera frugiperda, both important insect pests worldwide. Vip3Aa protein is synthesized as a protoxin (proVip3Aa) and becomes activated by digestion with either trypsin or insect gut proteases. The activated Vip3Aa protein (actVip3Aa) binds to a specific receptor in the brush border epithelial midgut cells, causing cell death via apoptosis, possibly induced by its pore-forming activity. Here we investigated the actVip3Aa intracellular localization to explain the molecular mechanism leading to the cytotoxicity of Vip3Aa toxin. The Spodoptera frugiperda (Sf9) cell line was incubated with fluorescently labeled Vip3Aa, namely Alexa488-actVip3Aa, and the intracellular localization was analyzed through a laser scanning confocal microscope. The Alexa488-actVip3Aa was internalized into the Sf9 cells. Immunofluorescence detection demonstrated that Alexa488-actVip3Aa did not colocalize with early endosomes which is usually implicated in clathrin-mediated endocytosis, suggesting that the actVip3Aa does not use clathrin-dependent endocytosis to transport into the cytosol. Intracellular visualization also shows that actVip3Aa does not directly target to mitochondria upon entry into the cytosol. Following internalization, actVip3Aa causes cell division disruption that subsequently could trigger cell death via apoptosis.

Preparation of Chicken Anemia Virus (CAV) Virus-Like Particles and Chicken Interleukin-12 for Vaccine Development Using a Baculovirus Expression System.

Chicken infectious anemia (CIA) is a poultry disease that causes huge economic losses in the poultry industry worldwide. Commercially available CIA vaccines are derived from wild-type chicken anemia viruses (CAVs) by serial passage in cells or chicken embryos. However, these vaccinal viruses are not completely attenuated; therefore, they can be transmitted vertically and horizontally, and may induce clinical symptoms in young birds. In this study, we sought to eliminate these issues by developing a subunit vaccine exploiting the CAV structural proteins, engineering recombinant baculovirus-infected Spodoptera frugiperda (Sf9) cells that contained both the viral protein 1 (VP1) and VP2 of CAV. Moreover, we produced single-chain chicken interleukin-12 (chIL-12) in the same system, to serve as an adjuvant. The recombinant VP1 was recognized by chicken anti-CAV polyclonal antibodies in Western blotting and immunofluorescence assays, and the bioactivity of the recombinant chIL-12 was confirmed by stimulating interferon-γ (IFN-γ) secretion in chicken splenocytes. Furthermore, the ability of the recombinant VP1 to generate self-assembling virus-like particles (VLPs) was confirmed by transmission electron microscopy. Specific pathogen-free (SPF) chickens inoculated with VLPs and co-administered the recombinant chIL-12 induced high CAV-specific antibodies and cell-mediated immunity. Taken together, the VLPs produced by the baculovirus expression system have the potential to be a safe and effective CIA vaccine. Finally, we demonstrated the utility of recombinant chIL-12 as an adjuvant for poultry vaccine development.

Four Cysteine Residues Contribute to Homodimerization of Chicken Interleukin-2.

Interleukin-2 (IL-2) is a pleiotropic cytokine regulating the immune and nervous systems. Mammalian and bird IL-2s have different protein sequences, but perform similar functions. In the current study, two bands were detected by immunoblotting using an antibody against freshly purified chicken IL-2 (chIL-2). The molecular weight of the larger band was approximately twice as much of the chIL-2 monomer, although a chIL-2 complex or homodimer has never been reported. To explain this intriguing result, several dissociation reagents were used to examine the intermolecular forces between components of the proposed chIL-2 complex. It was found that intermolecular disulphide bond promotes homodimerization of chIL-2. Subsequently, mutation of Cys residues of chIL-2 revealed that mutation of all four Cys residues disrupted homodimerization, but a single, dual, or triple Cys mutation failed to disrupt homodimerization, suggesting that all four Cys residues on chIL-2 contribute to this dimerization. Functional analysis showed that both monomeric and dimeric chIL-2 consisting of either wild type or mutant chIL-2 were able to stimulate the expansion of CD4+ T cell in vivo or in vitro, and effectively bind to chIL-2 receptor. Overall, this study revealed that the recombinant chIL-2 purified from either Escherichia coli (E. coli) or Spodoptera frugiperda (Sf9) cells could homodimerize in vitro, with all four Cys residues on each chIL-2 protein contributing to this homodimerization, and dimerization and Cys mutation not impacting chIL-2 induced stimulation of chicken CD4+ T cells.

Combined transcriptomic and proteomic analysis of harmine on Spodoptera frugiperda Sf9 cells to reveal the potential resistance mechanism

The fall armyworm, Spodoptera frugiperda, is an important invasive pest and exhibits resistance to many insecticides. Harmine, a remarkable, natural β-carboline alkaloid, exhibits a variety of bioactivities and induces programmed cell death in Sf9 cells. In the present study, transcriptomic and proteomic analyses were combined to investigate the effects of harmine on Sf9 cells. A sublethal dose, 0.05 mM, was selected and the transcriptomic analysis revealed 2463 upregulated and 689 downregulated genes after harmine treatment. The most frequently enriched pathways of differentially expressed genes (DEGs) were mainly involved in drug and xenobiotic metabolism. The proteomics analysis revealed 36 upregulated and 77 downregulated proteins, and the results showed a nonlinear relationship with mRNA expression. All the genes related to detoxification and resistance in the transcriptome and DEGs were identified and annotated. Complete open reading frames of 27 cytochrome P450s (CYPs), 27 glutathione S-transferases (GSTs), 11 carboxylesterases (CarEs), 10 UDP-glucuronosyltransferases (UGTs) and 29 heat shock proteins (HSPs) were assembled and verified using qRT-PCR. Harmine exhibited a completely different detoxification mechanism from normal pesticides. The Sigma and Delta class GSTs and UGTs might play important roles, rather than CYP6 and CYP9 clans, CarEs or HSPs. Biological significanceHarmine, a natural β-carboline alkaloid, inhibits the cell proliferation and induces programmed cell death of Sf9 cells, which derived from Spodoptera frugiperda, an important world invasive pest. In the article, the combined transcriptomic and proteomic analysis is used to explore the potential solution for its resistance management. These results supporting that harmine can be applied as a novel adjuvant or pesticide. In addition, the systematically identified resistance-related genes in fall armyworm provide the foundation for potential resistance monitoring and management.

The role of G protein-coupled receptor-related genes in cytochrome P450-mediated resistance of the house fly, Musca domestica (Diptera: Muscidae), to imidacloprid.

Ninety-four putative G protein-coupled receptors (GPCRs) were identified in the Musca domestica genome. They were annotated and compared with their homologues in Drosophila melanogaster. Phylogenetic analyses of the GPCRs from both species revealed that several family members shared a closer relationship based on the domain architecture. The expression profiles of these genes were examined by quantitative real-time PCR amongst three strains of the house fly, a near-isogenic line strain with imidacloprid resistance (N-IRS), the corresponding susceptible strain (CSS) and another strain derived from field populations with imidacloprid resistance (IRS). We found that five GPCR genes were upregulated in the N-IRS and eight GPCR genes were upregulated in the IRS strains compared to the CSS strain. The transgenic lines of D. melanogaster with the GPCR genes (LOC101899380 in the N-IRS strain and LOC101895664 in the IRS strain) exhibited significantly increased tolerance to imidacloprid, and higher expression of cytochrome P450 genes. Bioinformatic analysis of LOC101899380 was carried out based on its full-length nucleic acid sequence and putative amino acid sequence, and it was named Methuselah-like10 (Mthl10) owing to its homology with D. melanogaster Mthl10. A cell-base cell counting kit-8 toxicity assay demonstrated that the expression of the GPCR gene LOC101899380 in Spodoptera frugiperda (Sf9) cells using a baculovirus-mediated expression system can elevate the cell tolerance to imidacloprid, indirectly supporting the hypothesis that the GPCR gene LOC101899380 plays some role in imidacloprid resistance. These results should be useful for furthering understanding of the regulatory pathway by which house flies develop resistance.

Role of the G-Protein-Coupled Receptor Signaling Pathway in Insecticide Resistance

The G-protein-coupled receptor (GPCR) regulated intracellular signaling pathway is known to be involved in the development of insecticide resistance in the mosquito, Culex quinquefasciatus. To elucidate the specific role of each effector in the GPCR regulating pathway, we initially expressed a GPCR, G-protein alpha subunit (Gαs), adenylate cyclase (AC), and protein kinase A (PKA) in insect Spodoptera frugiperda (Sf9) cells and investigated their regulation function on cyclic AMP (cAMP) production and PKA activity. GPCR, Gαs, and AC individually expressed Sf9 cells showed higher cAMP production as the expression of each effector increased. All the effector-expressed cell lines showed increased PKA activity however. Moreover, Sf9 cytochrome P450 gene expression and cell tolerance to permethrin were examined. The relative expression of CYP9A32gene in Sf9 cells tested was significantly increased in all effector-expressed cell lines compared to a control cell line; these effector-expressed cell lines also showed significantly higher tolerance to permethrin. Inhibitor treatments on each effector-expressed cell line revealed that Bupivacaine HCl and H89 2HCl robustly inhibited cAMP production and PKA activity, respectively, resulting in decreased tolerance to permethrin in all cell lines. The synergistic functions of Bupivacaine HCl and H89 2HCl with permethrin were further examined in Culex mosquito larvae, providing a valuable new information for mosquito control strategies.

The microRNA pathway is involved in Spodoptera frugiperda (Sf9) cells antiviral immune defense against Autographa californica multiple nucleopolyhedrovirus infection

The microRNA (miRNA) pathway is an epigenetic mechanism that plays important roles in various biological processes including host-virus interactions by regulating gene expression of the host and/or the virus. Previously, we showed that the cellular microRNAome in Spodoptera frugiperda (Sf9) cells is modulated following Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection suggesting that miRNAs may contribute in the cellular antiviral immunity. Here, we investigated the role of core components of the miRNA pathway in Sf9-AcMNPV interaction. Gene expression analyses showed that the expression levels of Dicer-1 (Dcr1), Argonaute-1 (Ago1) and Exportin-5 (Exp5) increased following AcMNPV infection particularly at 16 h post infection (hpi). Ran expression levels, however, decreased in response to virus infection. The expression levels of cellular miRNAs, miR-184 and let-7, also diminished at the post infection times further confirming differential expression of the cellular miRNAs following AcMNPV infection. To determine the role of the miRNA pathway in the interaction, we silenced key genes in the pathway using specific dsRNAs. RNAi of Dcr1, Ago1 and Ran enhanced viral DNA replication and reduced the abundance of miR-184 and let-7 underscoring the importance of the miRNA pathway in antiviral immunity in Sf9 cells. Suppression of the miRNA pathway in mock and infected cells had no effect on Ran expression levels suggesting miRNA-independent downregulation of this gene after virus infection. In conclusion, our results suggest the antiviral role of the miRNA pathway in Sf9 cells against AcMNPV. To modulate this immune response, AcMNPV represses host miRNAs likely through downregulation of Ran to enhance its replication in the host cells.

The Spodoptera frugiperda Sf9 cell line is a heterogeneous population of rhabdovirus-infected and virus-negative cells: Isolation and characterization of cell clones containing rhabdovirus X-gene variants and virus-negative cell clones

The Sf9 cell line is broadly used for manufacturing baculovirus-expressed viral vaccines. We previously reported the presence of a novel, rhabdovirus in the Sf9 cell line, which contained a unique X gene (Sf-rhabdovirus; designated as X+ in this paper). These results were extended by other reports describing an Sf-rhabdovirus variant in Sf9 cells, which lacked 320 nucleotides encompassing the X-gene and adjacent intergenic region (designated as X− in this paper), and the development of an Sf-rhabdovirus negative cell line. Here, we report that the Sf9 cell line is a mixed-cell population, based upon isolation of cell clones with distinct phenotypes: Sf-rhabdovirus-negative, X+, and X−. We also show that Sf-rhabdovirus X+ and X− variants replicate independently in Sf-rhabdovirus-negative cells. These results shed light on the detection of different rhabdovirus variants by different laboratories using Sf9-derived cell clones and confirm that both X+ and X− viruses are infectious in rhabdovirus-negative Sf9 cells.

Characterization of the 20S proteasome of the lepidopteran, Spodoptera frugiperda

Multiple complexes of 20S proteasomes with accessory factors play an essential role in proteolysis in eukaryotic cells. In this report, several forms of 20S proteasomes from extracts of Spodoptera frugiperda (Sf9) cells were separated using electrophoresis in a native polyacrylamide gel and examined for proteolytic activity in the gel and by Western blotting. Distinct proteasome bands isolated from the gel were subjected to liquid chromatography-tandem mass spectrometry and identified as free core particles (CP) and complexes of CP with one or two dimers of assembly chaperones PAC1-PAC2 and activators PA28γ or PA200. In contrast to the activators PA28γ and PA200 that regulate the access of protein substrates to the internal proteolytic chamber of CP in an ATP-independent manner, the 19S regulatory particle (RP) in 26S proteasomes performs stepwise substrate unfolding and opens the chamber gate in an ATP-dependent manner. Electron microscopic analysis suggested that spontaneous dissociation of RP in isolated 26S proteasomes leaves CPs with different gate sizes related presumably to different stages in the gate opening. The primary structure of 20S proteasome subunits in Sf9 cells was determined by a search of databases and by sequencing. The protein sequences were confirmed by mass spectrometry and verified by 2D gel electrophoresis. The relative rates of sequence divergence in the evolution of 20S proteasome subunits, the assembly chaperones and activators were determined by using bioinformatics. The data confirmed the conservation of regular CP subunits and PA28γ, a more accelerated evolution of PAC2 and PA200, and especially high divergence rates of PAC1.