Replication of a nuclear polyhedrosis virus in an established insect cell line.
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Animals↗
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[Transfection conditions for baculovirus DNA in an insect cell line].
The following conditions were found to be necessary to achieve infection of a Spodoptera frugiperda cell line with purified DNA of nuclear polyhedrosis virus of Spodoptera exempta (NPV): (1) cell monolayers shall be prepared one day before the experiment; the number of cells added to 35-mm wells of plastic clusters (Falcon) shall be 1.0 X10(6)-1.1X10(6), and the cells shall be in the stage of logarythmic growth; DNA shall be diluted in HeBS-buffer (pH 6.95-7.05); the volume of the mixtures added per well shall be 0.3 to 0.5 ml; (2) DNA calcium phosphate mixture shall be prepared for 20-30 min at 20 degrees C; (3) the cells shall be inoculated with the DNA-calcium-phosphate mixture for 45-60 min at 28 degrees C; (4) dimethylsulphoxide (DMSO) shall be added 4 hours post-inoculation for 4 min; (5) the upper agarose layer (0.75% agarose) shall be added after 2 hours of incubation in the cell growth medium TC-100 after DMSO had been added. Under these conditions the specific activity of Spodoptera exempta NPHV DNA may achieve the value of 2.26 X 10(3) plaque-forming centres per 1 microgram.
Animals↗
Functional characterization of Bombyx mori nucleopolyhedrovirus late gene transcription and genome replication factors in the non-permissive insect cell line SF-21.
We compared the abilities of late gene transcription and DNA replication machineries of the baculoviruses Autographa californica nucleopolyhedrovirus (AcMNPV) and Bombyx mori NPV (BmNPV) in SF-21 cells, an insect-derived cell line permissive for AcMNPV infection. It has been well established that 19 AcMNPV late expression factors (lefs) stimulate substantial levels of late gene promoter activity in SF-21 cells. Thus, we constructed a set of clones containing the BmNPV homologs of the AcMNPV lefs under control of the constitutive Drosophila heat shock 70 protein promoter and tested their ability to activate an AcMNPV late promoter-reporter gene cassette in SF-21 cells. We tested the potential of individual or predicted functional groups of BmNPV lefs to successfully replace the corresponding AcMNPV gene(s) in transient late gene expression assays. We found that most, but not all, BmNPV lefs were able to either fully or partially substitute for the corresponding AcMNPV homolog in the context of the remaining AcMNPV lefs with the exception of BmNPV p143, ie-2, and p35. BmNPV p143 was unable to support late gene expression or be imported into the nucleus of cells in the presence of the AcMNPV or the BmNPV LEF-3, a P143 nuclear shuttling factor. Our results suggest that host-specific factors may affect the function of homologous proteins.
Animals↗
Function of Spodoptera exigua nucleopolyhedrovirus late gene expression factors in the insect cell line SF-21.
We used a well established transient expression assay to test the ability of the baculovirus Spodoptera exigua M nucleopolyhedrovirus (SeMNPV) homologs of Autographa californica MNPV (AcMNPV) late expression factors (lefs) to activate a late promoter-reporter gene cassette in SF-21 cells. This insect-derived cell line is fully permissive for AcMNPV infection but not for SeMNPV. In the assay, 19 AcMNPV lefs stimulate optimal levels of late gene promoter activity. SeMNPV lef-5 successfully replaced the corresponding AcMNPV gene in the context of the remaining set of AcMNPV lefs, whereas SeMNPV dnapol and 39k exhibited partial activity. When all the SeMNPV lefs were assayed together or in the presence of four lefs encoded only in AcMNPV, it resulted in background levels of late promoter-driven reporter gene activity. However, SeMNPV genomic DNA and the four AcMNPV-specific lefs stimulated low levels of reporter gene activity. Moreover, SeMNPV IE-1, but not AcMNPV IE-1, further stimulated late gene expression in the presence of SeMNPV DNA. AcMNPV IE-1 was able to mediate early gene expression cis-linked to homologous regions (hrs) derived from AcMNPV and SeMNPV. In contrast, SeMNPV IE-1 was more specific for SeMNPV-derived hr elements.
Animals↗
Heavy-metal toxicity in an insect cell line. Effects of cadmium chloride, mercuric chloride and methylmercuric chloride on cell viability and proliferation in Aedes albopictus cells.
We evaluated the toxicity of CdCl2, HgCl2, and MeHgCl on the C6/36 cell line of Aedes albopictus. This cell line proved to be a suitable tool for studying heavy-metal toxicity in insect cells. Since data on heavy-metal toxicity in invertebrate cell cultures are almost nonexistent, our results are discussed in relation to in vivo invertebrate and in vitro vertebrate studies. Viability and proliferation were assessed by dye exclusion and DNA quantification, respectively. Viability tests were carried out with and without 5% fetal calf serum in the medium. The three metal species decreased viability to different extents (MeHgCl > HgCl2 > CdCl2), and fetal calf serum had a protective effect. In serum-deprived cultures, LD50 values were 140.20, 2.51, and 2.08 mumol/L for CdCl2, HgCl2, and MeHgCl, respectively. For cultures with fetal calf serum, LD50 values were 149.71, 12.01, and 5.47 mumol/L, respectively. The viability curve for CdCl2 under serum-free conditions suggests the induction of a cell defense system. The three metal species also inhibited cell proliferation (MeHgCl > CdCl2 > HgCl2). The IC50 values were 1.75, 18.36, and 0.96 mumol/L for CdCl2, HgCl2, and MeHgCl, respectively. In summary, low MeHgCl concentrations caused both cell death and inhibition of cell proliferation; HgCl2 primarily disrupted the plasma membrane, whereas CdCl2 primarily inhibited cell proliferation.
Aedes↗
26-hydroxylation of ecdysteroids is catalyzed by a typical cytochrome P-450-dependent oxidase and related to ecdysteroid resistance in an insect cell line.
The epithelial cell line from the dipteran Chironomus tentans responds to the insect steroid hormone 20-hydroxyecdysone and the non-steroidal analogue tebufenozide by undergoing a morphogenetic and biochemical differentiation program. Long-term culture in the presence of 20-hydroxyecdysone has resulted in the selection of subclones that are resistant to the steroid but respond normally to the non-steroidal analogue. In the present study, several subclones that were resistant to the steroid hormone have been compared with steroid-sensitive subclones with respect to their capability to metabolize 20-hydroxyecdysone. Homogenates of both types of cells, when incubated with 3H-labelled steroid in the presence of NADPH, producecd 20,26-dihydroxyecdysone, which was further metabolized to two compounds, which behaved less polar than 20-hydroxyecdysone on reverse-phase HPLC. Ecdysone, a less-active hormone precursor, provided 26-hydroxyecdysone as the only product. The metabolites were identified by mass spectrometry coupled to HPLC, chromatography with authentic samples, and formation of acetonides. The structure of 20,26-dihydroxyecydsone was confirmed by 1H-NMR. The enzyme responsible for the synthesis of 20,26-dihydroxyecdysone in the Chironomus cell preparations has been characterized as a typical cytochrome P-450-dependent monooxygenase. It was a strictly microsomal enzyme, sensitive to inhibition by carbon monoxide and imidazole/triazole-based fungicides, and required NADPH for maximal activity. NADH could partly replace NADPH. The Michaelis constant (Km) for 20-hydroxyecdysone was 0.96 microM, and the maximal enzyme velocity (Vmax) was 50 pmol substrate metabolized x mg protein(-1) x min(-1). 26-Hydroxylation of 20-hydroxyecdysone was inhibited by ecdysone, an alternative substrate, and by inokosterone, a product analogue, to 50% at 1.4 microM and 0.73 microM, respectively. When various subclones were compared with respect to their in vitro rate of 20-hydroxyecdysone metabolization, those clones known to be resistant to the steroid were 'high metabolizers' (> 70% relative rate), whereas the sensitive clones were 'poor metabolizers' (< 30% relative rate). Hence, it is tempting to conclude that ecdysteroid resistance of the Chironomus cell clones is due to metabolic inactivation of the steroid hormone.
Animals↗
Action of major insecticide groups on insect cell lines of the beet armyworm, Spodoptera exigua, compared with larvicidal toxicity.
In a first series of experiments, the biological response of a continuous cell line of the beet armyworm, Spodoptera exigua, was tested with different groups of insecticides with different modes of actions: acetylcholinesterase inhibitors, acetycholine receptor agonists, inhibitors and uncouplers of oxidative phosphorylation, site I electron transport inhibitors, gamma-aminobutyric acid receptor inhibitors, chitin synthesis inhibitors, and juvenile hormone analogues. From the concentration response curves, 50% inhibition concentration (IC(50)) values were calculated. The most active compound in vitro was pyridaben with an IC(50) value of 0.0083 ppm. In a second series of experiments, the toxicity of these insecticide groups was determined on third-instar larvae of S. exigua, and lethal concentration with 50% kill (LC(50)) values were used in the evaluation of their in vivo biological activity. Toxicity bioassays showed that lufenuron was the most toxic (LC(50) = 0.098 ppm). To explain the discrepancies in biological responses in vitro with insect cells compared with in vivo conditions with whole third-instar larvae, the significance of different detoxifying enzyme systems was tested. P(450) monooxygenases, esterases, and glutathione S-transferases were measured in third-instar larvae and cells of S. exigua. Data are discussed in terms of the usefulness of insect cell cultures as tools in the screening for novel insecticide actions.
Animals↗
An insect cell line persistently infected with a baculovirus-like particle.
A persistent infection by a baculovirus-like particle was found in the established lepidopteran (Heliothis zea) cell line, IMC-HZ-1. The virus caused CPE in less than 1% of the IMC-HZ-1 cells, as measured by phase-contrast and electron microscopy. Transmission tests showed that four lepidopteran cell lines were susceptible to the persistent virus (designated as IMC-HZ-I-NOV). In inoculated TN-368 cell cultures, 90--100% infection was achieved. The ultrastructure and development of IMC-HZ-I-NOV in cell cultures were similar to known baculoviruses, and it is probable that this persistent virus is a member of the family Baculoviridae. Two lepidopterous species (Estigmene acrea and H. zea) inoculated with IMC-HZ-1-NOV by intrahemocoelic injection and/or per os feeding of larvae were not susceptible.
Cell Line↗
Characterization of Spodoptera littoralis type B nucleopolyhedrovirus infection in selected insect cell lines.
We have examined Spodoptera littoralis type B nucleopolyhedrovirus (SpliNPV) infections in CLS79, Sf9, and Se1 cells derived from lepidopteran insects of the genus Spodoptera (Family: Noctuidae), Ld652Y cells from Lymantria dispar (Family: Lymantriidae), and Md210 cells from Malacosoma disstria (Family: Lasiocampidae). CLS79, Sf9, and Se1 cells were permissive for SpliNPV infection as these cell lines supported complete viral DNA replication, virus-specific transcription, and production of viable progeny. Neither Ld652Y nor Md210 cells supported production of viable SpliNPV progeny. Ld652Y cells supported limited viral DNA replication and displayed reduced and delayed transcription of viral-specific RNAs. Md210 did not support viral DNA replication and displayed dramatically reduced transcription of viral-specific RNAs. We used transient expression assays as an indirect measure of the translation of SpliNPV early gene products in Sf9, Ld652Y, and Md210 cells. While transactivation of viral promoter-mediated luciferase expression occurred in SpliNPV-infected Ld652Y cells, little to no transactivation activity was detected in SpliNPV-infected Md210 cells. Our data indicated that the block to productive SpliNPV infection in Ld652Y and Md210 cells may be at the level of viral RNA transcription and further suggested that host factors play an important role in productive SpliNPV infection.
Animals↗
Synthesis of soluble rubella virus spike proteins in two lepidopteran insect cell lines: large scale production of the E1 protein.
The two envelope glycoproteins of rubella virus (RV), E1 of 58 kDa and E2 of 42-47 kDa, were individually expressed in lepidopteran Spodoptera frugiperda as well as in Trichoplusia ni insect cells using baculovirus vectors. The authentic signal sequences of E1 and E2 were replaced with the honeybee melittin signal sequence, allowing efficient entrance into the secretory pathway of the insect cell. In addition, the hydrophobic transmembrane anchors at the carboxyl termini of E1 and E2 proteins were removed to enable secretion rather than maintenance in the cellular membranes. Synthesis of the recombinant proteins in the absence and presence of tunicamycin revealed that both E1 and E2 were glycosylated with apparent molecular weights of 52 kDa and 37 kDa, respectively. Recombinant E2 appeared to be partially secreted, whereas E1 was essentially found inside the infected insect cell. The E1 protein was produced in large scale using a 10-1 bioreactor and serum-free medium (SFM). Purification of the recombinant protein product was performed from cytoplasmic extracts by ammonium sulphate precipitation followed by Concanavalin A affinity chromatography. This type of purified recombinant viral glycoproteins may be useful not only in diagnostic medicine or for immunization, but should enable studies designed to solve the structure of the virus particle.
Animals↗
Differential requirements of two insect cell lines for growth in serum-free medium.
The development of a serum-free medium that supports the growth of cells from a Spodoptera frugiperda and a Lymantria dispar cell line is reported. A yeast hydrolysate provided the B-vitamin complex, and a combination of a meat hydrolysate and tryptose provided most of the free amino acids required for cell growth. Supplemental cystine and methionine were required to achieve maximum cell growth. The serum or serum replacements used in earlier formulations were replaced with commercial lipid preparations and increased levels of iron salts. Although the cell growth cycle had a somewhat extended lag phase and the population doubling time of the S. frugiperda cells was longer than on serum-containing medium, the saturation densities were much higher. Spodoptera cells grown in this medium replicated the Autographa californica nuclear polyhedrosis virus well, producing 8.71 x 10(6) TCID50 extracellular virus and 4.4 x 10(6) polyhedra/ml culture. The specific activity of the polyhedra was somewhat less than that of polyhedra produced in insects.
Animals↗
Glycosylation of a recombinant protein in the Tn5B1-4 insect cell line: influence of ammonia, time of harvest, temperature, and dissolved oxygen.
Glycosylation is both cell line and protein dependent. Culture conditions can also influence the profile of glycoforms produced. To examine this possibility in the insect cell/baculovirus system, structures of N-linked oligosaccharides attached to SEAP (human secreted alkaline phosphatase), expressed under various culture conditions in BTI Tn5B1-4 cells, were characterized using FACE (fluorescence-assisted carbohydrate electrophoresis). Parameters varied were time of harvest, ammonia added during infection, dissolved oxygen, and temperature. It was found that glycosylation in the insect cell/baculovirus expression system is a robust, stable system that is less perturbed by variations in culture conditions than the level of protein expression. Addition of ammonia and low oxygen conditions affected SEAP expression, but not the oligosaccharide profile of SEAP. Time of SEAP harvest increased the amount of alpha-mannosidase resistant structures from 4.1% at 34 hours postinfection (h pi), to 5.0% at 100 h pi, and to 7.5% at 120 h pi. These structures were primarily sensitive to N-acetylhexosaminidase digest, although a small amount was insensitive to both mannosidase and N-acetyl-hexosaminidase digests. Lowering the temperature from 28 degrees C to 24 degrees C or even 20 degrees C, resulted in a twofold increase in oligosaccharides containing terminal alpha(1,3)-mannose residues. This condition did not affect the amount of mannosidase-resistant structures. However, this could result in more complete glycosylation of recombinant proteins in the BTI Tn5B1-4 cell line, because more structures with the potential for further processing would be produced.
Alkaline Phosphatase↗
Replication of baculoviruses in established insect cell lines: phenomenon of attenuation. Brief review.
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Cell Line↗
Biological activities of recombinant equine luteinizing hormone/chorionic gonadotropin (eLH/CG) expressed in Sf9 and Mimic insect cell lines.
Equine luteinizing hormone (eLH) and chorionic gonadotropin (eCG) are composed of identical alpha and beta polypeptide chains, but eCG subunits are much more heavily glycosylated and sialylated. Consequently, eCG exhibits a much longer half-life than eLH in blood. Recombinant eLH/CG, expressed in Sf9 and Mimic insect cells, were compared with one another and to the natural hormones eCG and eLH. Mimic cells are stably-transformed Sf9 cells, expressing five mammalian genes encoding glycosyltransferases involved in the synthesis of complex N-carbohydrate chains. Recombinant eLH/CG expressed in Mimic cells exhibited a higher apparent molecular weight (MW) than that expressed in Sf9 cells, suggesting that its N-glycosylation was, as expected, more complete. Nevertheless, the two recombinant eLH/CG exhibited lower MW than natural eCG from pregnant mare plasma. The two eLH/CG produced in Sf9 and Mimic cells were found to be active in in vitro LH and FSH bioassays, with potencies similar to those of eCG. By contrast, they exhibited no significant in vivo bioactivity, neither in the specific follicle-stimulating hormone (FSH) assay nor in the specific eCG assay. Although recombinant eLH/CG produced in Mimic cells bears more elaborate carbohydrate chains than recombinant eLH/CG from Sf9 cells, it exhibits no significant in vivo bioactivity, probably because of insufficient terminal sialylation of its carbohydrate chains, leading to its rapid removal from blood.
Amino Acid Sequence↗
Expression of recombinant anti-E-selectin single-chain Fv antibody fragments in stably transfected insect cell lines.
We have investigated the possibility of improving the yield of properly folded recombinant single chain Fv fragments (sFv) of an antibody by expressing the protein in stably transfected Drosophila melanogaster SC-2 cells. The DNA encoding the variable regions of the 1.2B6 anti-E-selectin antibody were used to generate a recombinant sFv. This construct was cloned into the pHEN1 vector for expression in Escherichia coli and the pRmHa-3 vector to generate stably transfected Drosophila SC-2 cell lines. Following expression in the bacterial system, and using standard refolding protocols to obtain active material, it was shown that the majority of the sFv formed non-covalent aggregates. In addition SDS-PAGE analysis indicated that even the monomeric material was heterogeneous. In contrast, expression of sFv in Drosophila SC-2 cell lines allowed purification of active sFv directly from the culture supernatant. Only a small proportion of the sFv formed aggregates, and the purified material was homogeneous as determined by SDS-PAGE. Thus the use of stably transfected insect cells has a number of potential advantages in expressing recombinant antibody fragments.
Animals↗
Effect of sublethal doses of cadmium, inorganic mercury and methylmercury on the cell morphology of an insect cell line (Aedes albopictus, C6/36).
The effect of CdCl2 (44 microM), HgCl2 (3.7 microM), and MeHgCl (2 microM) on the morphology of Aedes albopictus C6/36 cells was studied at the light microscopical level. Treatment times and metal concentrations were in the sublethal range as determined by a fluorometric dye exclusion test. The three metal species had profound effects on the cell morphology. MeHgCl treatment induced the development of a large number of short, actin-supported, tangled filopodia. Both CdCl2 and HgCl2 induced long extensions. Pretreatment with colchicine but not with cytochalasin B prevented formation of these extensions which suggests that they were supported by microtubules. This was confirmed by immunostaining for microtubules. The extensions were relatively stable towards colchicine post-treatment. To authors' knowledge, this effect has not yet been described for heavy metals. The similarity with 20-hydroxyecdysone-treated cells and the occurrence of cytoplasmic feet in insect cells is discussed.
Actins↗
Functional expression of Shaker K+ channels in a baculovirus-infected insect cell line.
We constructed a recombinant baculovirus, A. californica nuclear polyhedrosis virus, containing the Drosophila Shaker H4 K+ channel cDNA under control of the polyhedrin promoter. When infected with this recombinant baculovirus, the cell line Sf9, derived from the army-worm caterpillar S. frugiperda, expresses fully functional Shaker transient K+ currents, as assayed by whole-cell recording. K+ currents begin to appear at about 15 hr after infection, and they continue to increase over the next 3 days. Over the same period of time, a 75 kd band appears on SDS gels stained with Coomassie blue. The identity of this band as a Shaker gene product is confirmed by Western blot analysis using an anti-Shaker antiserum. The 75 kd band accounts for a substantial fraction of the membrane protein in Shaker-infected Sf9 cells. These results give hope that the baculovirus system, which has been used successfully for high-level expression of soluble proteins from higher eukaryotes, may be appropriate for producing large amounts of cloned ion channel proteins as well.
Animals↗
The influence of various insect cell lines, p10 and polyhedrin promoters in the production of secreted insulin-like growth factor-interleukin-3 chimeras in the baculovirus expression system.
A technique for the optimal synthesis of secreted fusion proteins between insulin-like growth factors (IGFs) and cytokines is described. The cDNA of BOMIGF, a fusion protein between the insect insulin-like peptide bombyxin and IGF II, has been linked to the gene of interleukin-3. The BOMIGF-interleukin 3 fusion gene was cloned downstream of the promoter regions of the p10 and polyhedrin proteins within baculovirus transfer vectors. A third, dual transfer vector was constructed with the gene inserted simultaneously behind p10 and polyhedrin promoters. Two different lepidopteran cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (BTI-TN-5B1-4) were infected with the recombinant baculoviruses obtained from the three transfer vectors. Trichoplusia ni cells produced the largest amount of recombinant protein. Although the efficiency of the three recombinant viruses was remarkably similar, the baculovirus with the gene present behind both promoters produced relatively more recombinant protein in host cells than those viruses driven with the polyhedrin or p10 promoters alone. The BOMIGF-interleukin-3 chimera was stable and continuously increased in the culture medium up to 5-6 days postinfection. Therefore the addition of a protease inhibitor was useful only at the stage of massive host cell death. Medium supplemented with copper sulfate was detrimental for the long-term production of the fusion protein.
Animals↗