Identification and characterization of a baculovirus occlusion body glycoprotein which resembles spheroidin, an entomopoxvirus protein.

A 37-kDa polypeptide specified by Autographa californica nuclear polyhedrosis virus was found to share significant homology with Choristoneura biennis entomopoxyvirus spheroidin protein, which is the major component of entomopoxvirus occlusion bodies. Antibodies raised against spheroidin cross-reacted with the 37-kDa protein and confirmed its expression in the late phase of wild-type baculovirus infection. Immunoblot analysis and fluorescence microscopy demonstrated that the protein was associated with purified A. californica nuclear polyhedrosis virus occlusion bodies and was absent in purified virions. Immunofluorescence studies localized the protein to the periphery of occlusion bodies and the internal membranes of cells infected with wild-type baculovirus. The open reading frame encoding this spheroidinlike protein was inserted into a baculovirus expression vector, and recombinant protein was synthesized under control of the polyhedrin promoter. Studies of the recombinant protein demonstrated that it was heterogeneous in molecular mass as a result of N-linked glycosylation. Tunicamycin inhibited carbohydrate addition and yielded proteins of 34 and 33 kDa.     

Localization of juvenile hormone esterase during development in normal and in recombinant baculovirus-infected larvae of the moth Trichoplusia ni.

The pathogenesis and cellular localization of juvenile hormone esterase (JHE) was examined in larvae of the moth Trichoplusia ni, infected with a recombinant baculovirus (Autographa californica nuclear polyhedrosis virus: AcNPV) engineered to produce high levels of JHE (JHE virus). The course of JHE localization in the recombinant virus infected larvae was compared with that of both wild type AcNPV infected, and uninfected larvae, using immunogold electron microscopy. In the JHE virus infected insects, high levels of JHE were observed in the endoplasmic reticulum of all cells showing evidence of viral structures in the nucleus, except for gut cells which showed only background JHE levels. Tracheole cells and haemocytes appeared to play a role in the dissemination of infection. In uninfected larvae, fat body and epidermis were the major tissues staining for JHE, which was only detectable at peak times of JHE activity during the fifth instar: lower levels at other times could not be distinguished from background. JHE was also present in lysosomes of granular haemocytes: these lysosomes increased in number in the fifth instar compared to the fourth instar. Similar lysosome-like granules in the pericardial cells did not become highly positive for JHE antigen until the fifth instar.     

An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif.

Spodoptera frugiperda SF-21 cells infected with Autographa californica nuclear polyhedrosis virus mutants which lack a functional p35 gene undergo apoptosis, a type of programmed cell death. To identify p35-homologous genes in other baculoviruses, A. californica nuclear polyhedrosis virus DNA containing a deletion in p35 was cotransfected into SF-21 cells along with genomic DNAs from other baculoviruses. One of the viral DNAs which were able to rescue wild-type infection was from Cydia pomonella granulosis virus (CpGV). The CpGV gene responsible for the effect was mapped to a 1.6-kb SalI-SstI subclone of the SalI B fragment of CpGV. The sequence of the SalI-SstI subclone revealed an open reading frame capable of encoding a polypeptide of 31 kDa which was sufficient to rescue wild-type infection; this gene was thus called iap (inhibitor of apoptosis). The predicted sequence of the IAP polypeptide exhibited no significant homology to P35 but contained a zinc finger-like motif which is also found in other genes with the potential to regulate apoptosis, including several mammalian proto-oncogenes and two insect genes involved in embryonic development. In the context of the viral genome, both iap and p35 were able to block apoptosis induced by actinomycin D, indicating that these genes act by blocking cellular apoptosis rather than by preventing viral stimulation of apoptosis. Several independent recombinant viruses derived from cotransfections with either the entire CpGV genome or the 1.6-kb subclone were characterized.     

Expression of adenovirus type 2 DNA polymerase in insect cells infected with a recombinant baculovirus.

Sequences encoding adenovirus type 2 DNA polymerase were placed under control of the polyhedrin promoter and inserted into the baculovirus Autographa californica nuclear polyhedrosis virus by homologous recombination. Insect cells infected with the recombinant virus produced substantial amounts of the adenovirus type 2 DNA polymerase protein which was functional in both DNA polymerase and replication initiation reactions. Thus, the baculovirus expression system can provide active adenovirus type 2 DNA polymerase that is produced in quantities suitable for biochemical and structural analysis.     

Expression of biologically active recombinant equine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae

The full-length equine interferon-gamma (eIFN-gamma) cDNA, including the secretion signal peptide coding region, was recloned into baculovirus transfer vector pAcYM1. This vector was co-transfected with Autographa californica nuclear polyhedrosis virus DNA or hybrid nuclear polyhedrosis virus DNA into Spodoptera frugiperda cells. The recombinant viruses, named AcEIFN-gamma and HyEIFN-gamma, were then recovered. Recombinant eIFN-gamma (reIFN-gamma) was accumulated in the culture fluid of the AcEIFN-gamma or HyEIFN-gamma infected Tricoplusia ni -derived cell line, BTI TN 5B1-4, and hemolymph of HyEIFN-gamma infected silkworm larvae. These reIFN-gamma forms were shown to be 14, 16, 18 and 20kDa proteins, and glycosylated as confirmed by SDS-PAGE and tunicamycin treatment. Both reIFN-gamma proteins, showed high-level biological activities to vesicular stomatitis virus by cytopathic effect reduction assay, and MHC class II antigen induction on the equine fetal kidney-78 cell line.     

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.     

Expression of largest RNA segment and synthesis of VP1 protein of bluetongue virus in insect cells by recombinant baculovirus: association of VP1 protein with RNA polymerase activity.

The bluetongue virus core particles have been shown to contain an RNA-directed RNA polymerase (1). To identify the protein responsible for the virion RNA polymerase activity, the complete 3.9 Kb DNA clone representing the largest RNA segment 1 (L1) of bluetongue virus (BTV-10) was placed under control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). The derived recombinant virus was used to infect Spodoptera frugiperda cells. As demonstrated by stained polyacrylamide gel electrophoresis and by the use of bluetongue virus antibody, infected insect cells synthesized the largest protein of BTV-10 (VP1, 150 k Da). Antibody raised in rabbit to recombinant VP1 protein recognized bluetongue virus VP1 protein. The recombinant virus infected cell lysate had significantly inducible levels of RNA polymerase enzymatic activity as determined by a poly (U)-oligo (A) polymerase assay. The availability of enzymatically active bluetongue virus RNA polymerase provides a system in which we can precisely delineate the role this protein plays in the regulation of bluetongue replication.     

Strong and regulated expression of Escherichia coli beta-galactosidase in insect cells with a baculovirus vector.

The N-terminal region of the gene encoding polyhedrin, the major occlusion protein of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), has been fused to DNA encoding Escherichia coli beta-galactosidase. The fused gene was inserted into the AcNPV DNA genome by cotransfection of insect cells with recombinant plasmid DNA and wild-type AcNPV genomic DNA. Recombinant viruses were selected as blue plaques in the presence of a beta-galactosidase indicator, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Studies of one such virus, L1GP-gal3, indicated that the synthesis of beta-galactosidase is temporally controlled beginning late (20 h) in infection after the release of infectious virus particles from the cell. By 48 h postinfection, a remarkably high level of expression is achieved. On the basis of these results, AcNPV should be a useful vector for the stable propagation and expression of passenger genes in a lepidopteran cell background. A generalized transplacement vector that facilitates the construction and selection of recombinant viruses carrying passenger genes under their own promoter control has also been developed.     

Bluetongue virus tubules made in insect cells by recombinant baculoviruses: expression of the NS1 gene of bluetongue virus serotype 10.

Bluetongue virus (BTV) forms tubules in mammalian cells. These tubules appear to be composed of only one type of protein, NS1, a major nonstructural protein of the virus. To obtain direct evidence for the origin of the tubules, the complete M6 gene of BTV serotype 10 was inserted into the baculovirus transfer vector pAcYM1, so that it was under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus. After cotransfection of Spodoptera frugiperda cells with wild-type A. californica nuclear polyhedrosis virus DNA in the presence of recombinant transfer vector DNA, polyhedrin-negative baculoviruses were recovered. When S. frugiperda cells were infected with one of the derived recombinant viruses, a protein similar in size and antigenic properties to the authentic BTV NS1 protein was made (representing ca. 50% of the stained cellular proteins). The protein reacted with BTV antibody and formed numerous tubular structures in the cytoplasm of S. frugiperda cells. The tubular structures have been purified to homogeneity from infected-cell extracts by gradient centrifugation. By enzyme-linked immunosorbent assay, the recombinant virus antigen has been used to identify antibodies to five United States BTV serotypes in infected sheep sera, indicating the potentiality of the expressed protein as a group-reactive antigen in the diagnosis of BTV infections.     

Baculovirus interaction with nontarget organisms: a virus-borne reporter gene is not expressed in two mammalian cell lines

The safety of baculoviruses with respect to mammalian species was studied by using a genetically engineered recombinant of Autographa californica nuclear polyhedrosis virus. This recombinant contains the chloramphenicol acetyltransferase (CAT) gene under the control of a mammalian-active promoter and expresses substantial levels of CAT activity on infection of permissive and nonpermissive insect cells (L.F. Carbonell, M.J. Klowden, and L.K. Miller, J. Virol. 56:153-160, 1985). Extremely low levels of CAT activity were detected in mouse and human cell lines that were continuously exposed to the A. californica nuclear polyhedrosis virus recombinant. The appearance of CAT was not inhibited by cycloheximide. Isopycnic centrifugation of purified inoculum showed that a low level of CAT activity was associated with the insect-derived viral particles. Thus, the observed CAT activity is carried into the cells with the virus inoculum, and active expression of the baculovirus-borne CAT gene is not observed in either cell line. The inability of the CAT gene to be expressed in these cell lines with this model system provides additional assurance of the safety of insect baculoviruses with respect to mammalian species.     

Baculovirus interaction with nontarget organisms: a virus-borne reporter gene is not expressed in two mammalian cell lines.

The safety of baculoviruses with respect to mammalian species was studied by using a genetically engineered recombinant of Autographa californica nuclear polyhedrosis virus. This recombinant contains the chloramphenicol acetyltransferase (CAT) gene under the control of a mammalian-active promoter and expresses substantial levels of CAT activity on infection of permissive and nonpermissive insect cells (L.F. Carbonell, M.J. Klowden, and L.K. Miller, J. Virol. 56:153-160, 1985). Extremely low levels of CAT activity were detected in mouse and human cell lines that were continuously exposed to the A. californica nuclear polyhedrosis virus recombinant. The appearance of CAT was not inhibited by cycloheximide. Isopycnic centrifugation of purified inoculum showed that a low level of CAT activity was associated with the insect-derived viral particles. Thus, the observed CAT activity is carried into the cells with the virus inoculum, and active expression of the baculovirus-borne CAT gene is not observed in either cell line. The inability of the CAT gene to be expressed in these cell lines with this model system provides additional assurance of the safety of insect baculoviruses with respect to mammalian species.     

Functional mapping of a trans-activating gene required for expression of a baculovirus delayed-early gene

The temporal regulation of an early gene of the baculovirus Autographa californica nuclear polyhedrosis virus was examined. We constructed a plasmid (plasmid 39CAT) in which the bacterial gene for chloramphenicol acetyltransferase was placed under the control of the promoter for the gene for a A. californica nuclear polyhedrosis virus 39,000-dalton protein (39K). A transient expression assay of plasmid 39CAT revealed that the 39K gene was expressed in infected cells but not in uninfected cells, indicating that the 39K gene should be classified as a delayed-early gene. The 39K promoter also efficiently directed the synthesis of chloramphenicol acetyltransferase when the plasmid was cotransfected with viral DNA which had been restricted with several restriction enzymes. To map the location of the gene(s) required for the synthesis of 39K, plasmid 39CAT was cotransfected with purified restriction fragments of A. californica nuclear polyhedrosis virus DNA. Fragments which mapped between 90.7 and 100.8 map units induced plasmid 39CAT. Plasmid pEcoRI-B, containing EcoRI fragment B (90 to 100 map units), activated plasmid 39CAT. Functional mapping of plasmid pEcoRI-B indicated that the essential region was located between 95.0 and 97.5 map units. The 5' end of this gene was mapped, and the chloramphenicol acetyltransferase gene was inserted under the control of its promoter. Transient assay experiments indicated that the trans-acting regulatory gene was expressed in uninfected cells and is therefore an immediate-early gene. This gene was named IE-1.     

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)     

Enzymatic activities of overexpressed herpes simplex virus DNA polymerase purified from recombinant baculovirus-infected insect cells.

Biochemical characterization of the herpes simplex virus (HSV) DNA polymerase, a model DNA polymerase and an important target for antiviral drugs, has been limited by a lack of pure enzyme in sufficient quantity. To overcome this limitation, the HSV DNA polymerase gene was introduced into the baculovirus, Autographa californica nuclear polyhedrosis virus, under the control of the polyhedrin promoter to give rise to a recombinant baculovirus, BP58. BP58-infected Spodoptera frugiperda insect cells expressed a polypeptide that was indistinguishable from authentic polymerase by several immunological and biochemical properties, at levels approximately ten-fold higher per infected cell than found in HSV-infected Vero cells. The DNA polymerase was purified to apparent homogeneity from BP58-infected insect cells. Using activated DNA as primer-template, the purified enzyme exhibited specific activity similar to that of enzyme isolated from HSV-infected Vero cells, indicating that additional polymerase-associated proteins from HSV-infected cells are not critical for activity with this primer-template. 3'-5' exonuclease activity co-purified with the BP58-expressed HSV DNA polymerase, demonstrating that this activity is intrinsic to the polymerase polypeptide. The purified enzyme also exhibited RNAse H activity. The recombinant baculovirus should permit detailed biochemical and biophysical studies of this enzyme.     

Substrate limitation in the baculovirus expression vector system

The inability to infect insect cell cultures at the highest achievable cell densities has imposed major limitations to both the fundamental understanding of the Baculovirus Expression Vector System (BEVS) as well as full exploitation of its potential productive capacity for recombinant (beta-galAcNPV) products. The current literature does not characterize and identify the exact nature of the observed limitations, which therefore has become the major objective and contribution of the following study. Critical densities for infection of Spodoptera frugiperda (Sf9) cells with nuclear polyhedrosis virus expressing beta-galactosidase (Autographa californica) grown in media both containing fetal calf serum (FCS) and free of serum were found to be at 2 x 10(6) and 5 x 10(6) cells/ml respectively. Medium exchange was found to completely reverse the effect if renewed up to 24 hours post-infection (HPI). The inevitable arrest of uninfected cell growth and decreased production of recombinant products at high cell densities of infection were both correlated to nutrient depletion. Cystine was found to be depleted in uninfected insect cell cultures at the onset of the stationary phase and in serum-free insect cell cultures infected with baculovirus above a cell density of 5 x 10(6) cells/ml. Neither glucose depletion nor accumulation of possible inhibitory metabolites such as alanine, ammonia, or lactate could be correlated to growth arrest or decreased recombinant product yields. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 32-44, 1997.     

Expression and one-step purification of bovine interleukin-21 (IL-21) in silkworms using a hybrid baculovirus expression system

A hybrid baculovirus, a hybrid of the Autographa californica nuclear polyhedrosis virus and the Bombyx mori nuclear polyhedrosis virus, was used for the large-scale production of bovine interleukin-21 (IL-21) in silkworms. A recombinant hybrid baculovirus containing the full length of the cDNA of bovine interleukin-21 was constructed and used to infect silkworm larvae or silkmoth pupae. After the infection of the virus, bovine mature IL-21 was produced in the haemolymph or pupal cell lysates. A one-step purification of bovine mature IL-21 from haemolymph using a cation exchange column gave 0.5 mg. IL-21 from 30 ml haemolymph. The bovine IL-21 produced by silkworms strongly induced NK cell proliferation using a human NK cell-line, NK0, and enhanced the lymphokine activated killer (LAK) activity of bovine peripheral blood mononuclear cells.     

Production of human c-myc protein in insect cells infected with a baculovirus expression vector.

A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells. By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera. The insect-derived proteins were compared with recombinant human c-myc-encoded proteins synthesized in Escherichia coli and Saccharomyces cerevisiae cells. The c-myc gene product was found predominantly in the nucleus by subcellular fractionation of infected insect cells.