Baculovirus Autographa Californica Nuclear Polyhedrosis Research Articles

Expression of the baculovirus p35 gene inhibits mammalian neural cell death.

Expression of the apoptosis suppressor gene p35, derived from the baculovirus Autographa californica nuclear polyhedrosis virus, markedly inhibited the cell death of stably transfected mammalian neural cells whether the cell death was induced by glucose withdrawal, calcium ionophore, or serum withdrawal. The p35 protein, which is required to block virus-induced apoptosis of cultured insect cells, is only the second gene product shown to block mammalian neural cell death, with Bcl-2 being the first. Because there is no apparent homology between p35 and Bcl-2, the existence of a cellular death program that may be modulated at multiple points is suggested. Furthermore, these findings demonstrate that the putative cellular death program is conserved across species and cell types.

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.

Expression of Full-Length and Truncated Forms of Crystal Protein Genes from Bacillus thuringiensis subsp. kurstaki in a Baculovirus and Pathogenicity of the Recombinant Viruses

Full-length and truncated forms of the crystal protein gene cryIA (b) derived from Bacillus thuringiensis subsp. kurstaki HD-1 and full-length cryIA(c) gene of B. thuringiensis subsp. kurstaki HD-73 were introduced into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus, in place of the polyhedrin gene. All gene constructs were expressed at high levels in insect cells and insects upon infection with the recombinant viruses. The protein products were shown to be biologically and immunologically similar to the natural crystal protein. The expressed proteins formed crystals (in insects) up to 10 times bigger (in length) than their bacterial counterpart. The LT50 values for recombinant viruses were not significantly shorter than wild-type virus.

Apoptosis reduces both the in vitro replication and the in vivo infectivity of a baculovirus

Apoptotic programmed cell death occurs when the insect cell line SF-21, derived from Spodoptera frugiperda, is infected with mutants of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) which lack a functional p35 gene. However, infection of the Trichoplusia ni TN-368 cell line with p35 mutants does not result in apoptosis (R. Clem, M. Fechheimer, and L. Miller, Science 254:1388-1390, 1991). We have examined the effect of apoptosis on AcMNPV infections in cell lines and larvae of these two insect species. Production of viral progeny was significantly lower in SF-21 cells infected with p35 mutants than in cells infected with wild-type (wt) or revertant viruses. Viral gene expression was abnormal in SF-21 cells infected with p35 mutants; there was a delay in the transcription and translation of early and late viral genes, a lack of expression of very late genes, and a total cessation of protein synthesis late in the apoptotic process. In vivo analysis revealed that the dose of budded virus required for 50% lethality in S. frugiperda larvae was approximately 1,000-fold higher for p35 mutants than for wt or revertant viruses. In contrast, the replication and infectivity of p35 mutant viruses was equivalent to that of wt AcMNPV during infection of both TN-368 cells and T. ni larvae. Thus, the data indicate that a host apoptotic response provides protection against viral infection at the organismal level and that the p35 gene constitutes a host range determinant for AcMNPV infection.

Efficient secretion of attacin from insect fat-body cells requires proper processing of the prosequence.

The attacins constitute a group of immune proteins induced after bacterial infection in the moth Hyalophora cecropia. They are synthesized as preproproteins and undergo post-translational modification during transport to the hemolymph. The processing and transport rates of attacin were studied in its natural host as a response to infection. Monensin totally inhibited the processing from proattacin to attacin and the radiolabeled proattacin remained intracellular. This observation indicated that the prosequence is removed at or after the trans-Golgi compartment. It is also suggested that the processing of the prosequence does not occur in acidic vesicles, as the process was not inhibited by the weak base chloroquine. To study prosequence function, the attacin gene and genes with mutations in the prosequence were cloned into the baculovirus Autographa californica nuclear polyhedrosis virus. The processing of proattacin and the transport of attacin were studied by pulse-chase experiments with fat body isolated from Trichoplusia ni larvae. The rate of secretion from fat body was lowest for proattacin, which could not be processed to attacin, intermediate for attacins lacking the prosequence and highest for natural attacin. We could not detect any biological activity for proattacin.

Nucleotide Sequence and Transcriptional Analysis of a Gene Encoding gp41, a Structural Glycoprotein of the Baculovirus Autographa californica Nuclear Polyhedrosis Virus

Nucleotide Sequence and Transcriptional Analysis of a Gene Encoding gp41, a Structural Glycoprotein of the Baculovirus <i>Autographa californica</i> Nuclear Polyhedrosis Virus

A structured dynamic model for the baculovirus infection process in insect‐cell reactor configurations

A mathematical description of the infection of insect cells with baculovirus in a continuously operated reactor configuration is presented. The reactor configuration consists of one bioreactor in which insect cells (Spodoptera frugiperda) are grown followed by one or two bioreactors in which cells are infected by a baculovirus (Autographa californica nuclear polyhedrosis virus). It was demonstrated that the so-called passage effect is responsible for the observed difference in run time between a configuration with one or with two infection vessels. Furthermore, a model is presented based on the hypothesis that the limited run time of series of continuously operated bioreactors is associated with the occurrence of a virus particle (so-called virion) that is defective and has interfering properties. With the assumption that not all nonoccluded virions are capable of establishing a correct infection leading to new virus production, infection levels in continuously operated reactor configurations could be described well with the model.

Nucleotide sequence and transcriptional analysis of a gene encoding gp41, a structural glycoprotein of the baculovirus Autographa californica nuclear polyhedrosis virus.

We have identified and sequenced a region of the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome encoding the major polyhedron-derived virus structural glycoprotein gp41. The open reading frame is located entirely within the AcMNPV SstII-M fragment. The protein sequence does not have hydrophobic regions characteristic of integral membrane proteins and contains a putative O-linked GlcNAc glycosylation site. gp41 is expressed as a late gene, with transcripts starting within two consensus late transcription start sites (TAAG) located immediately upstream of the first methionine codon. A major transcription termination signal is bypassed, possibly generating a bicistronic message. Finally, the nucleotide and protein sequences of AcMNPV and Bombyx mori nuclear polyhedrosis virus are highly conserved.

A structural polypeptide of the baculovirus Autographa californica nuclear polyhedrosis virus contains O-linked N-acetylglucosamine.

A structural glycopeptide, gp41, derived from the occluded virus of the baculovirus Autographa californica nuclear polyhedrosis virus was characterized. The peptide specifically bound wheat germ agglutinin but was not recognized by a panel of seven other lectins. Reactivity with wheat germ agglutinin was eliminated by treatment of gp41 with beta-N-acetylglucosaminidase, indicating that N-acetylglucosamine (GlcNAc) was present as terminal residues. gp41 was efficiently galactosylated by galactosyltransferase only in the presence of Nonidet P-40, suggesting that GlcNAc residues are not exposed on the surface of the virion. Metabolic labelling of gp41 with [3H]GlcNAc occurred in the presence of tunicamycin. The carbohydrate was released by alkaline borohydride treatment and comigrated with N-acetylglucosaminitol in descending paper chromatography. The data indicate that gp41 contains single residues of GlcNAc O glycosidically linked to the polypeptide chain. Evidence suggesting that gp41 is located in the region between the envelope membrane and the capsid (defined here as the tegument) of the occluded virus is also presented.

Alteration of ecdysteroid metabolism due to baculovirus infection of the fall armyworm Spodoptera frugiperda: Host ecdysteroids are conjugated with galactose

The egt gene of the baculovirus Autographa californica nuclear polyhedrosis virus enables the virus to regulate the development of its infected host insect. This gene encodes an enzyme known as ecdysteroid UDP-glucosyltransferase that catalyzes the sugar conjugation of ecdysteroids. In this study, we have extented our characterization of this transferase and investigated the effects of virus infection on ecdysteroid metabolism in fall armyworm Spodoptera frugiperda (J.E. Smith) larvae. We show that the enzyme has a cleavable signal sequence at its amino-terminus and is rapidly secreted into the hemolymph of infected insects. In vitro studies indicate that ecdysteroid UDP-glucosyltransferase can catalyze both the glucose and galactose conjugation of ecdysteroids. Several lines of evidence indicate that, in vivo, the ecdysteroids are conjugated with galactose. Steady-state levels of ecdysteroids in the hemolymph of infected insects remain at low levels throughout infection.

Purification and characterization of human salivary-gland prokallikrein from recombinant baculovirus-infected insect cells.

A full-length cDNA encoding human salivary-gland preprokallikrein was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. The gene was expressed in transfected Spodoptera frugiperda cells and the recombinant product secreted into the culture medium. By alternating anion-exchange chromatography and gel-filtration steps, twice repeated, prokallikrein was purified to homogeneity, which was confirmed by amino acid analysis and N-terminal sequence determination. The prepropeptide was processed correctly, including the removal of the signal peptide. The resulting proenzyme was found to be glycosylated, had a molecular mass of 35 kDa and an isoelectric point of 4.6. The yield of purified recombinant protein reached a level of 5 mg/l insect cell culture. After trypsin digestion of prokallikrein, the biological activity of the released kallikrein was demonstrated by its specific amidase, esterase and kininogenase activity. The expression and purification of prokallikrein, as described here, offers the opportunity to study the proenzyme activation through protein engineering techniques in detail.

Transcription analysis of the EcoRI D region of the baculovirus Autographa californica nuclear polyhedrosis virus identifies an early 4-kilobase RNA encoding the essential p143 gene

We have investigated the transcriptional activity of the 60.1- to 68.3-map-unit region of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV). Twelve transcripts mapping to this region were expressed at various times during infection. An early 4.0-kb transcript, potentially coding for a 143-kDa peptide essential for viral DNA replication, was maximally abundant at 6 h postinfection (p.i.). Transcripts of 0.5, 1.1, 1.4, 2.1, and 3.1 kb were most abundant at 12 h p.i., while two large transcripts of 5.2 and 6.8 kb were expressed maximally at 24 h p.i. In the presence of cycloheximide, and in ts8-infected cells at the nonpermissive temperature, only the 4.0-kb RNA was expressed. Northern (RNA) blot analysis using DNA subfragments from the EcoRI D fragment as probes suggested that many of the transcripts overlapped. Strand-specific cRNA probes revealed that the majority of the RNAs were transcribed in the counterclockwise direction. S1 nuclease and primer extension analysis were used to map the 5' ends of transcripts coded within the 60.1- to 64.8-map-unit region. Mapping of the 3' ends of the 1.1-, 4.0-, 5.2-, and 6.8-kb transcripts suggested that these RNAs were all coterminal at their 3' ends. A minicistron was found between the early 4.0-kb transcription start site and the predicted ATG start codon of the p143 gene. Several similar sequence motifs were identified in the promoter regions of the p143 gene and the AcMNPV DNA polymerase gene.

Diapausing pupae of Hyalophora cecropia: An alternative host for baculovirus mediated expression

The insect baculovirus Autographa californica nuclear polyhedrosis virus was found to propagate in the North America silk moth Hyalophora cecropia after injection. Wild tyoe virus and four recombinant viruses were used to infect pupae of H. cecropia, larvae of Trichoplusia ni and Spodoptera frugiperda cells in culture. The expression of recombinant proteins were compared in the different hosts and it was found that H. cecropia is equal to or in some cases better than T. ni as a host for heterologous expression in vivo. The expression of recombinant proteins in H. cecropia also has several practical advantages to that of expression in T. ni.

Expression of foreign epitopes on recombinant occlusion bodies of baculoviruses

Recombinant polyhedrin proteins of the baculovirus Autographa californica nuclear polyhedrosis virus were constructed to serve as immunologic carriers of foreign epitopes. Several recombinants containing an influenza haemagglutinin epitope were obtained. Three of the five recombinants formed occlusion bodies (OBs) and two did not. All of the recombinant polyhedrin proteins reacted with a monoclonal antibody (mAb) specific for an influenza epitope in Western blots. Presentation of the foreign epitope on the surface of recombinant OBs was demonstrated by specific immunoprecipitation of the anti-influenza mAb with recombinant OBs. The recombinant polyhedrin-influenza epitope fusion protein stimulated an influenza-specific immune response in rabbits.

Improvement of a Baculovirus Pesticide by Deletion of the EGT Gene

The egt gene of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes the enzyme ecdysteroid UDP–glucosyltransferase. Expression of egt enables the virus to inhibit molting of its infected host. In this study, we have compared the growth and development of insects infected by viruses with and without a functional egt gene. Our data reveal that, since insects normally stop feeding for both larval–larval and larval–pupal molts, expression of egt prevents the insect from experiencing this feeding arrest. Thus, egt functions to prolong the length of time the insect feeds after infection, with a resultant increase in the weight gain of the insect. We show that deletion of the egt gene significantly improves the pesticide characteristics of AcMNPV. Larvae infected with an egt deletion mutant display considerably reduced feeding and earlier mortality than wild–type AcMNPV–infected larvae.

Expression of the tobacco mosaic virus movement protein using a baculovirus expression vector

A cDNA clone of the tobacco mosaic virus 30K movement protein (MP) gene was constructed and introduced into an Autographa californica nuclear polyhedrosis baculovirus expression vector. Infection of Spodoptera frugiperda cells with the vector resulted in the synthesis of low levels of MP, which was detected by anti-MP serum as two closely related species of Mr approximately 34K and a third species of 32K. The authenticity of the recombinant MP was confirmed by comparison of the protein, on the basis of migration during SDS-PAGE, with authentic MP from several sources. It appeared that the recombinant MP was not modified by N-linked glycosylation, but was phosphorylated. The recombinant MP was produced in both a phosphorylated and an unphosphorylated state and the former species was shown to comigrate with plant-expressed MP during SDS-PAGE.

Secretion of single-chain urokinase-type plasminogen activator from insect cells

A cDNA encoding human urokinase-type plasminogen activator was inserted downstream from the polyhedrin promoter of the baculovirus Autographa californica nuclear polyhedrosis virus. A protein of similar M r to urokinase (UK) was synthesized and approx. 90% was secreted from recombinant virus-infected Spodoptera frugiperda cells. Zymography and Western blotting analysis of the insect-derived protein demonstrated that it was comprised solely of the high- M r ,- form of UK. No low- M r - UK was detected. Amidolytic activity assays showed that 96 % of the insect cell-derived UK was in the single-chain proenzyme form. The yield of UK from insect cells was 1986 international units/ml/106 infected cells.

.alpha.-Mannosidase-catalyzed trimming of high-mannose glycans in noninfected and baculovirus-infected Spodoptera frugiperda cells (IPLB-SF-21AE). A possible contributing regulatory mechanism for assembly of complex-type oligosaccharides in infected cells

Incubation of a Spodoptera frugiperda (IPLB-SF-21AE) cell extract with the oligosaccharide Man9GlcNAc2, the aglucosyl derivative of the glycan that is normally transferred from the dolichol carrier to the relevant Asn residue in the nascent protein, results in its trimming to Man6GlcNAc2, an intermediate that is relatively stable to further alpha-D-mannosidase action in these cells. On the other hand, incubation of a similar extract from cells that had been infected for various times with a wild-type baculovirus (Autographa californica nuclear polyhedrosis virus) or a recombinant baculovirus (r-BAC)/human plasminogen (HPg) construct employed for expression of HPg led to rapid trimming of Man6GlcNAc2 to Man5GlcNAc2 and Man3GlcNAc2. These latter reactions displayed temporal effects, in that an enhancement of this latter trimming process occurred as a function of the time of infection of the cells with the wild-type and recombinant viral constructs. We have previously demonstrated that the nature of the oligosaccharide assembled on Asn289 of HPg expressed in several lepidopteran insect cell lines was dependent on the time of infection of the cells with r-BAC/HPg and that the amount of complex glycan found on this recombinant protein increased with an increase in infection times [Davidson, D. J., & Castellino, F. J. (1991) Biochemistry 30, 6167-6174].(ABSTRACT TRUNCATED AT 250 WORDS)

A baculovirus homolog of aCu/Znsuperoxide dismutase gene

A gene with greater than 50% amino acid sequence identity to eukaryotic Cu/Zn superoxide dismutase genes ( sod) was found at 19 map units in the genome of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV). This gene was transcribed into 1.4- and 1.5-kb RNAs late in virus infection. The two late RNAs have coterminal 3′ ends but initiate from two different start points, both of which map to the central adenine of the sequence motif ATTAAG. The late gene product was found to be a low abundance 20-kDa polypeptide which was observed only upon partial purification by chloroform/ethanol extraction. Viruses with mutations in sodwere able to replicate normally both in cell culture and in insect larvae. Viruses lacking an intact sod showed no evidence of a replication disadvantage even in the presence of paraquat, an inducer of superoxide anions. A viral-encoded superoxide dismutase (SOD) activity was not detected although endogenous insect SODs appeared to be induced in insect hemolymph and in other tissues following virus infection. The function of sod in the baculovirus infection process remains unclear.

Identification of upstream promoter elements mediating early transcription from the 35,000-molecular-weight protein gene of Autographa californica nuclear polyhedrosis virus

Site-directed mutagenesis was used to examine the organization of cis-acting regulatory elements that comprise the promoter of the early 35,000-molecular-weight protein gene (35K protein gene) encoded by the EcoRI-S region of the baculovirus Autographa californica nuclear polyhedrosis virus. The promoter fragment, extending from positions -226 to +12 relative to the early RNA start site (position +1), was fused to the reporter gene encoding chloramphenicol acetyltransferase (CAT) and then inserted into the genome of recombinant viruses (3.96 map units) in order to ascertain the role of regulatory elements in the context of a normal infection. A combination of deletions and linker insertions revealed that early transcription was mediated by a basal (minimum) promoter, consisting of the TATA element (positions -30 to -25), that was in turn responsive to an upstream activating region located between -90 and -30. The TATA element exerted the single greatest influence on the level of early promoter activity and contained all information necessary to direct transcription from a site located 30 nucleotides downstream. The upstream activating region provided a 10- to 15-fold stimulation of transcription from the early +1 start site that was mediated by distinct DNA elements. These regulatory elements included two GC motifs (centered at positions -81 and -54, respectively), composed of alternating G and C residues, and a CGT motif (position -40) that contained the core sequence A(A/T)CGT(G/T). Each motif was required for full promoter activity during the early phase of infection. This organization that employs diverse cis-acting stimulatory elements is typical of promoters responsive to RNA polymerase II and may facilitate increased expression of A. californica nuclear polyhedrosis virus genes early in infection when the level of viral DNA for transcription is critically low.