Recombinant Autographa Californica Nuclear Polyhedrosis Research Articles

Quantitative analysis of cytoplasmic actin gene promoter and nuclear polyhedrosis virus immediate-early promoter activities in various tissues of silkworm <italic>Bombyx mori</italic> using recombinant <italic>Autographa californica</italic> nuclear polyhedrosis virus as vector

Cassettes harboring luciferase reporter driven by Bombyx mori cytoplasmic actin gene promoter (A3) (671 bp) and B. mori nuclear polyhedrosis virus immediate-early promoter (IE-1) (580 bp) were transferred to the bacmid AcDeltaEGT to generate the recombinant Autographa californica nuclear polyhedrosis viruses, AcNPVA3Luc and AcNPVIELuc, respectively. Recombinant baculoviruses were injected into the hemocoele of newly ecdysed 5th instar larvae. The activities of the A3 and IE-1 promoters in various tissues were measured by luciferase activity assay and normalized by the copy number of recombinant virus. Results showed that the activity of the A3 promoter was approximately 10-fold higher than the IE-1 promoter. The promoter activities of A3 and IE-1 were highest in the silk gland, followed by fat body, middle gut, malpighian tubule, and hemocyte. In silk gland, activity of the two promoters was highest in posterior silk gland, followed by middle and anterior silk glands. The difference in promoter activities reflects the growth speed of tissue in silkworm larvae. The activity of the A3 promoter remained unchanged and was not inhibited significantly by viral factors at least 3-4 d post injection of rAcNPV.

Purification and characterization of a recombinant rat prohaptoglobin expressed in baculovirus-infected Sf9 insect cells

To generate hemoglobin-free full-length haptoglobin the cDNA encoding rat haptoglobin αβ subunits was cloned into shuttle vector pVT-Bac-His and used to produce a recombinant baculovirus Autographa californica Nuclear Polyhedrosis Virus (AcNPV) as an expression vector, named HpAcNPV. Recombinant virus was used to infect Spodoptera frugiperda (Sf9) insect cells. The 50 kDa protein expressed was mostly secreted into the culture medium at relatively high titer (15 μg/mL) and was found to be rat prohaptoglobin having a vector-derived N-terminal extension of 37 amino acids, containing both a hexahistidine tag and an enterokinase recognition sequence. The protein was successfully purified by a three step procedure including nickel-linked agarose and DEAE–Sepharose chromatography steps. Hemoglobin was not detected in the purified preparations. Purified recombinant rat prohaptoglobin protein was also found to be glycosylated, and to be capable of forming a complex with rat hemoglobin in vitro.

Significant increase in recombinant protein production of a virus-infected Sf-9 insect cell culture of low MOI under low dissolved oxygen conditions

Spodoptera frugiperda Sf-9 insect cells were infected with recombinant Autographa californica nuclear polyhedrosis virus at a low multiplicity of infection (MOI) (0.1), and the effect of dissolved oxygen (DO) on the production of a polyhedrin promoter-driven recombinant protein (β-galactosidase), intrinsic proteases (carboxyl and cysteine proteases), and the virus was determined. The DO concentrations used in the present study were 45%, 25%, 5%, and 1.3% of air saturation. At 5% DO the cell growth following viral infection was greatest and β-galactosidase was about 5-fold increased in volumetric yield compared to that at 45% and 25% DO, whereas the growth at 1.3% DO was extremely poor. The virus titer in the medium at 4–8 d post-infection (dpi) was also highest at 5% DO, but the titer was significantly decreased by further increasing the culture time. This was in part attributed to the fact that baculovirus is susceptible to oxidative inactivation under aerobic conditions. The DO dependency of the specific oxygen consumption rate of virus-infected and uninfected Sf-9 cells was expressed by a Monod-type equation. A critical DO, above which the rate of oxygen utilization is not limited by DO, was estimated to be 3.5% of air saturation for virus-infected Sf-9 cells. These results indicated that for a baculovirus-infected Sf-9 insect cell culture of low MOI, the optimal DO was likely to be approximately 5% of air saturation, which is above the critical DO for the infected Sf-9 cells but sufficiently low to reduce the possibility of the oxidative inactivation of virus. For the production of carboxyl and cysteine proteases, the accumulation behavior and concentrations did not significantly vary with DO, except that a peak of cysteine protease activity was observed intracellularly only at 5% DO, coinciding with β-galactosidase production.

Persistence of an occlusion-negative recombinant nucleopolyhedrovirus in Trichoplusia ni indicates high multiplicity of cellular infection.

We use data from the serial passage of co-occluded recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) to estimate the viral multiplicity of infection of cells within infected insects. Co-occlusion, the incorporation of wild-type and mutant virus genomes in the same occlusion body, has been proposed as a strategy to deliver genetically modified viruses as insecticides in a way that contains their spread in the environment. It may also serve as a means whereby naturally occurring mutant forms of NPVs can be maintained in a stable polymorphism. Here, a recombinant strain of AcMNPV was constructed with a deletion of its polyhedrin gene, rendering it incapable of producing occlusion bodies (i.e., occlusion negative). This was co-occluded with wild-type AcMNPV and used to infect fifth-instar Trichoplusia ni larvae. The fate of both genotypes was monitored over several rounds of insect infection. Levels of the occlusion-negative virus genome declined slowly over successive rounds of infection. We applied these data to a model of NPV population genetics to derive an estimate of 4.3 +/- 0.3 viral genomes per occlusion body-producing cell.

Effects of Recombinant Baculoviruses on Three Nontarget Heliothine Predators

Abstract Genetically engineered baculoviruses, relative to their wild-type progenitors, have successfully improved the time-to-kill of these arthropod-specific biopesticides. Beneficial arthropods that prey on targeted pest insects are likely the first nontarget organisms to be adversely affected by the applications of such biopesticides. The goals of this project were to assess potential risks of the recombinant baculoviruses on Solenopsis invicta, Geocoris punctipes, and Hippodamia convergens, all of which are common predators of heliothines in Texas cotton. Four recombinant Autographa californica nuclear polyhedrosis viruses (AcNPV), one Helicoverpa zea nuclear polyhedrosis virus (HzNPV), and two corresponding wild-type NPVs were used in this risk assessment study. Risks associated with these baculoviruses were determined by possible shifts in predator life history traits (rate of food consumption, travel speed, fecundity, and survival) when fed prey infected with recombinant viruses compared to prey infected with wild-type viruses or to healthy prey. We also tested for possible transmission of these viruses by predators using the polymerase chain reaction (PCR). No significant shifts in life history characteristics were detected in predators fed Heliothis virescens larvae infected with any of the seven viruses. Viral DNA was discovered using PCR in 2.3% of fire ant workers, but not from any of the queens or eggs. In G. punctipes, 13.4% of adults and 0.5% of eggs scored positive for viruses. Twelve percent of H. convergens adults were found PCR positive. Residency in all three predators tested provides a pathway which could increase the persistence of recombinant viral particles in the environment and thus may produce an indeterminable amount of risk associated with their inadvertent movement.

Optimising fed-batch production of recombinant proteins using the baculovirus expression vector system.

Fed-batch culture can offer significant improvement in recombinant protein production compared to batch culture in the baculovirus expression vector system (BEVS), as shown by Nguyen et al. (1993) and Bedard et al. (1994) among others. However, a thorough analysis of fed-batch culture to determine its limits in improving recombinant protein production over batch culture has yet to be performed. In this work, this issue is addressed by the optimisation of single-addition fed-batch culture. This type of fed-batch culture involves the manual addition of a multi-component nutrient feed to batch culture before infection with the baculovirus. The nutrient feed consists of yeastolate ultrafiltrate, lipids, amino acids, vitamins, trace elements, and glucose, which were added to batch cultures of Spodoptera frugiperda (Sf9) cells before infection with a recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) expressing beta-galactosidase (beta-Gal). The fed-batch production of beta-Gal was optimised using response surface methods (RSM). The optimisation was performed in two stages, starting with a screening procedure to determine the most important variables and ending with a central-composite experiment to obtain a response surface model of volumetric beta-Gal production. The predicted optimum volumetric yield of beta-Gal in fed-batch culture was 2.4-fold that of the best yields in batch culture. This result was confirmed by a statistical analysis of the best fed-batch and batch data (with average beta-Gal yields of 1.2 and 0.5 g/L, respectively) obtained from this laboratory. The response surface model generated can be used to design a more economical fed-batch operation, in which nutrient feed volumes are minimised while maintaining acceptable improvements in beta-Gal yield.

Characterization of Recombinant Autographa californica Nuclear Polyhedrosis Virus (NPV) Expressing the β-Galactosidase Gene in both Sf21 and Bm5 Cells by Bombyx mori NPV p143 Helicase Gene

Characterization of Recombinant Autographa californica Nuclear Polyhedrosis Virus (NPV) Expressing the β-Galactosidase Gene in both Sf21 and Bm5 Cells by Bombyx mori NPV p143 Helicase Gene

Separation and purification of useful proteins using hydrogel ultrafiltration

The hydrogel process is a different form of ultrafiltration and has been used to separate biological molecules. In this study, the gel pore size was predicted by pulse NMR technique and neural network using a data base obtained from gel filtration chromatography and diffusion experiment. Recombinant alkaline phosphatase expressed in insect cells was concentrated 1.5 times by hydrogel ultrafiltration by swelling at 20°C and collapsing at 35°C at 53–65% separation efficiency and 78–83% enzyme recovery. Wild and recombinantAutographa californica nuclear polyhedrosis viruses (AcNPV) were also concentrated 1.4 and 1.6 times of the feed solution at 48.5 and 60.0% separation efficiency, respectively. Hydrogel ultrafiltration appears to be an attractive alternative for the concentration of AcNPV and recombinant proteins from insect cells.

Interactions of recombinant and wild-type baculoviruses with classical insecticides and pyrethroid-resistant tobacco budworm (Lepidoptera: Noctuidae).

In tests with neonate Heliothis virescens (F.), we characterized interactions of all combinations of a recombinant Autographa californica (Speyer) nuclear polyhedrosis virus (AcAaIT) that expresses an insect-selective neurotoxin (AaIT) and wild-type AcNPV when combined with low concentrations of several conventional insecticides. All combinations of the recombinant virus AcAaIT and insecticides showed a positive interaction (decrease in the median lethal time (LT50) compared with the LT50 for either component alone). A type II pyrethroid (cypermethrin, which modifies currents of sodium channels) and a carbamate (methomyl, an inhibitor of acetylcholinesterase) were synergistic in combination with AcAaIT. Other insecticides also showed a positive interaction when tested in combination with the recombinant virus, but joint activity was slightly antagonistic (i.e., less than predicted activity when combined) with wild-type AcNPV. We also characterized the effectiveness of AcAaIT against pyrethroid-resistant H. virescens larvae. Our results show that a resistant strain of H. virescens is more sensitive to the recombinant virus compared with a susceptible strain. Results of these studies should be useful in planning of future field trials to increase the effectiveness of nuclear polyhedrosis viruses and to manage resistance to pyrethroids and other insecticides.

Production of recombinant herpes simplex virus protease in 10-L stirred vessels using a baculovirus-insect cell expression system.

A gene expression system using recombinant Autographa california nuclear polyhedrosis virus (baculovirus) and Sf-9 cells has been scaled up to the 10-L tank level and shown to be capable of producing herpes simplex virus (HSV) protease in serum-free media. High densities of Spodoptera frugiperda (Sf-9) cells were achieved by modifying two 10-L Biolafitte fermenters specifically for insect cell growth. The existing Rushton impellers were replaced by marine impellers to reduce shear and the aeration system was modified to allow external addition of air/O2 mixtures at low flow rates through either the sparge line or into the head space of the fermenter. To inoculate the tanks, Sf-9 cells were adapted to grow to high cell densities (6-10 x 10(6) cells ml-1) in shake flasks in serum-free media. With these procedures, cell densities of 5 x 10(6) cells ml-1 were routinely achieved in the 10-L tanks. These cells were readily infected with recombinant baculovirus expressing the 247-amino acid catalytic domain of the HSV-1 strain 17 protease UL26 gene as a glutathione-S-transferase (GST) fusion protein (GST-247). Three days after infection at a multiplicity of infection (MOI) of 3 pfu cell-1, the GST-247 fusion protein was purified from a cytoplasmic lysate by Glutathione Sepharose 4-B affinity chromatography with reproducible yields of 11-38 mg L-1 of recombinant protein and > or = 90% purity. Maximum production of this protein was observed at a cell density of 5.0 x 10(6) cells ml-1.

Baculovirus-mediated trans-epithelial transport of proteins in infected caterpillars.

Baculovirus-mediated abnormal protein trafficking was studied in infected caterpillars by using heterologous proteins. The gene for human complement C1r was expressed in larvae of Mamestra brassicae by a recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) vector. By following the time-course of recombinant C1r distribution among various tissues, cell types and cell organelles, we concluded that the dominant site of recombinant protein synthesis was the fat body, although some production in the haemocytes and midgut was also observed. Only about 4% of the cells of the infected organs expressed recombinant C1r, which was then secreted into the haemolymph. The tracheal and integumental cuticle was rich in recombinant protein from the fourth day after infection although epidermal cells did not synthesize recombinant C1r. The morphological picture suggested that the accumulation was a consequence of a trans-epithelial transport. This transport process was checked by following the fate of the 49 kDa haemolymph protein and injected ovalbumin in AcMNPV-infected Mamestra brassicae and in Lymantria dispar nuclear polyhedrosis virus-infected Lymantria dispar larvae. Both proteins were able to pass the basal membrane of the epidermis and accumulated in the cuticle, while in control larvae neither was transported. The observed trans-epithelial transport points to the role of baculoviruses in directing recombinant, endo- and exo-genous proteins to cuticulated tissues. Based on these results we conclude that the permeability of basal membranes undergoes a characteristic change during the course of baculovirus infection.

Concentrating Autographa californica nuclear polyhedrosis virus and recombinant alkaline phosphatase from insect cells using a temperature-sensitive hydrogel.

Recombinant alkaline phosphatase expressed in insect cells was concentrated by a factor of one and half times at a separation efficiency of 54.2% using hydrogel ultrafiltration. Enzyme concentration was confirmed by SDS-PAGE as well as by spectrophotometric measurement. Wild and recombinant Autographa californica nuclear polyhedrosis viruses (AcNPV) were concentrated 1.4 and 1.6 times of the feed solution at 48.5 and 60.0% separation efficiency, respectively. Hydrogel ultrafiltration appears to be an attractive alternative for the concentration of AcNPV and recombinant proteins from insect cells.

Effects of Recombinant Baculoviruses on a Nontarget Endoparasitoid ofHeliothis virescens

Abstract Our study evaluated the effect of wild-type and recombinant Autographa californica nuclear polyhedrosis viruses (AcNPVs) on an endoparasitoid of Heliothis virescens. The development time of the parasitoid Microplitis croceipes was shortened in AcNPV-infected hosts. However, parasitoid survival in hosts treated with recombinant NPVs did not significantly differ from survival rates in hosts infected with wild-type NPV or uninfected controls. Earlier emergence was recorded for parasitoid larvae developing in hosts infected with recombinant NPVs than parasitoid larvae from hosts infected with wild-type NPV. Adult parasitoids which had developed as larvae in hosts infected with recombinant NPVs were significantly smaller in size, suggesting a cost associated with more rapid development rates. PCR primers for AcNPV produced a predicted product in approximately 40% of F1 adults developing from NPV-infected hosts.

Baculovirus-driven expression and purification of glycine receptor α1 homo-oligomers

The glycine receptor is a ligand-gated anion channel protein of postsynaptic membranes. We expressed a homo-oligomeric receptor composed of human α1 subunits in Spodoptera frugiperda cells by infection with a recombinant Autographa californica nuclear polyhedrosis virus. A substantial fraction of the recombinant receptor was incorporated as a functional channel protein into the cell's plasma membrane at expression levels 4-to 30-fold higher than in other eukaryotic heterologous expression systems or native rat spinal cord membranes, respectively. Upon detergent solubilization, the al receptor was found to exist in a predominantly monodisperse state and could be affinity-purified to near homogeneity. This preparation is a potential starting point for future crystallisation studies.

Maturation of the dengue-2 virus NS1 protein in insect cells: effects of downstream NS2A sequences on baculovirus-expressed gene constructs.

A series of recombinant baculoviruses was constructed in order to study the influence of downstream NS2A sequences on the processing of the dengue virus NS1 glycoprotein in insect cells. NS1 alone was expressed at a high level in its native dimeric form and processed efficiently through the Spodoptera frugiperda (Sf) cell secretory pathway. Recombinant NS1 was found associated with the plasma membrane and was also secreted into the extracellular medium. Although both intra- and extracellular NS1 were processed to an endo H-resistant form in Sf cells, Triton X-114 phase separation analysis further suggested that some modifications in addition to dimerization account for the hydrophobic properties of NS1, and that N-glycosylation was therefore not the only difference between the cell-associated and secreted forms. Cleavage at the NS1-NS2A junction of these recombinants demonstrated that as few as 26 amino acids from the N terminus of NS2A provide a sufficient, but not optimal, recognition sequence for a functional cleavage mediated by a protease present in Sf cells infected with recombinant Autographa californica nuclear polyhedrosis virus expressing NS1.

Expression of Semliki Forest virus nsP1-specific methyltransferase in insect cells and in Escherichia coli

We have expressed the Semliki Forest virus (SFV)-specific nonstructural protein nsP1 both in insect cells and in Escherichia coli in the absence of other viral proteins. A substantial amount of nsP1 was synthesized in Sf9 cells infected with the recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) AcNPV-nsP1. These cells had a high level of guanine-7-methyltransferase activity compared with that of wild-type AcNPV-infected cells. The methyltransferase activity and nsP1 were mostly in the mitochondrial pellet fraction (P15). The enzymatic activity was increased by treatment with deoxycholate (DOC), as in the case of SFV-infected BHK cells. The material released by DOC treatment from P15 of the AcNPV-nsP1-infected cells was analyzed by gel filtration and sucrose gradient centrifugation. Both the methyltransferase activity and nsP1 were in aggregates. nsP1 expressed in E. coli at 37 degrees C sedimented at 15,000 x g, whereas after expression at 15 degrees C, both nsP1 and methyltransferase activity were in the supernatant fraction. Paradoxically, the activity from E. coli was completely inhibited by Triton X-100 and DOC. Sucrose gradient analysis showed that even the "soluble" nsP1-methyltransferase was in aggregates. The methyltransferase activities in the P15 fractions of SFV-infected BHK cells and AcNPV-nsP1-infected Sf9 cells and in E. coli catalyzed linear incorporation of the [3H]methyl group from S-adenosylmethionine to GTP for a 60-min period. The enzymes from the three sources had similar substrate specificities and Km values for S-adenosylmethionine. In addition to GTP, they all methylated dGTP and GpppG, but not m7GTP or GpppA, or in vitro-transcribed RNAs with GpppA and GpppG caps. The unique properties of SFV-specific nsP1 methyltransferase are discussed.

Specificity of Baculovirus p10 Functions

Three functional domains in baculovirus p10 proteins have been postulated for aggregation, nuclear disintegration, and fibrillar structure formation (Van Oers et al., J. Gen. Virol. 74, 563-574, 1993). To study the specificity of these functions, a recombinant Autographa californica nuclear polyhedrosis virus (AcCR1) was constructed in which the coding sequence of the p10 gene was replaced with the p10 sequence of the distantly related Spodoptera exigua (Se) MNPV. In AcCR1-infected cells the SeMNPV p10 protein was produced at similarly high levels as AcMNPV p10 in wild type (wt) AcMNPV infections. Formation of fibrillar structures occurred in a similar fashion in SeMNPV and AcCR1-infected cells. Hence, the SeMNPV p10 protein retained the ability to associate into fibrillar structures when expressed in an otherwise AcMNPV context. Mixed infection with wt AcMNPV and AcCR1 indicated that only p10 proteins of the same species aggregate and that these aggregates associate into fibrillar structures. In contrast to S. exigua cells infected with AcMNPV or SeMNPV, S. exigua cells infected with AcCR1 failed to release polyhedra. This result indicated that interaction of p10 with at least one virus-specific factor is required for nuclear disintegration.

Nuclear export of recombinant baculovirus nucleocapsids through small pore or nuclear-pore-like structure in Sf9 cells.

Translocation of baculovirus nucleocapsids (45 nm in diameter, approximate length of 280-300 nm) from nucleoplasm to cytoplasm was studied morphologically using cryofixation and gold labeled wheat germ agglutinin (WGA-gold) during recombinant Autographa californica nuclear polyhedrosis virus infection in Sf9 cells. Nucleocapsids formed in the nucleoplasm migrated into protrusions of the nuclear envelope, but not into nuclear pore complexes. We found cross-like membranous structures. Small pores seemed to be in the protruding nuclear double membranes. The middle piece of a nucleocapsid was located within the small pore whereas the upper part was in the cytoplasm. Other nucleocapsids were situated within pores without colocalization of WGA-gold in the nuclear envelope. These results suggest that baculovirus nucleocapsids use small pores in the nuclear-derived membranes or incomplete nuclear pores in the nuclear envelope to migrate from the nucleoplasm to the cytoplasm, but not complete nuclear pore complexes proper.

Functional expression and purification of a homomeric human alpha 1 glycine receptor in baculovirus-infected insect cells.

The human alpha 1 glycine receptor (GlyR) was expressed in Sf9 insect cells infected with a recombinant Autographa californica nuclear polyhedrosis baculovirus. Previous studies had indicated that transient expression of this subunit in Xenopus oocytes or human kidney cell lines is sufficient to form active agonist-gated chloride channels. Expression of the alpha 1 GlyR protein resulted in functional channels present on the cell surface of infected Sf9 cells as evidenced by whole-cell patch-clamping and single-channel recordings. These channels were gated by glycine, but not in the presence of strychnine. An immunoreactive 48-kDa protein could be easily visualized on Coomassie-stained sodium dodecyl sulfate-polyacrylamide gels of whole-cell lysates with maximal expression 3 days postinfection. The alpha 1 GlyR protein was solubilized from a membrane fraction of infected Sf9 cells in 1% digitonin and 0.1% deoxycholate and purified by affinity chromatography using aminostrychnine agarose, yielding 0.33 mg/liter of cells. Given the low natural abundance of the native channel, the development of this expression system now provides sufficient purified channel protein for future biochemical and biophysical characterization. Since the glycine receptor shares sequence and structural homology with other members of a ligand-gated channel superfamily, further characterization may establish general rules governing the structure and mechanism of these membrane protein channels.

Production of recombinant proteins in high‐density insect cell cultures

The effect of the growth phase of Spodoptera frugiperda (Sf9) cells on the production of recombinant proteins (beta-galactosidase and glucocerebrosidase) was investigated. Cells infected with the recombinant Autographa californica nuclear polyhedrosis virus at the late exponential and stationary phases yielded low quantities of expressed protein. Highest enzyme yields were obtained using Sf9 cells from the early exponential phase (0.9 mg beta-galactosidase/10(6) cells and 1.7 microg glucocerebrosidase/10(6) cells). Infection of resuspension of cells collected from various phases of growth in fresh medium resulted in 75% restoration of maximal expression levels. This finding suggested either nutrient limitation or waste product accumulation as the cause of the decrease in productivity at the latter phases of growth. Further experiments revealed that the highest productivity levels could be obtained with cultures of Sf9 cells grown in a fermentor to a cell concentration of 4 x 10(6) mL(-1). The medium needed to be replaced prior to infection with the recombinant virus and supplemented with a mixture of glucose, L-glutamine, and yeastolate ultrafiltrate.