Baculovirus transfer vectors.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
R D Possee
Publications and source records attributed to R D Possee.
The complete DNA sequence of Autographa californica nuclear polyhedrosis virus.
The complete nucleotide sequence of the genome of clone 6 of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has been determined. The molecule comprises 133,894 base pairs and has an overall A + T content of 59%. Our analysis suggests that the virus encodes some 154 methionine-initiated, and potentially expressed, open reading frames (ORFs) of 150 nucleotides or greater. These ORFs are distributed evenly throughout the virus genome on either strand. The ORFs are arranged as adjacent, nonoverlapping reading frames separated by short intergenic regions. Based on the primary nucleotide sequence, predictions have been made concerning the functions of certain genes, the sites for initiation of viral DNA replication, the regulation of early and late gene transcription, and factors that may affect the AcNPV gene translational efficiency. The genome sequence data confirm, with minor differences, the information obtained for other AcNPV clones. It is proposed that clone C6 is considered the archetype AcNPV for comparison purposes.
Base Sequence↗
Quantification of latent Mamestra brassicae nuclear polyhedrosis virus in M. brassicae insects using a PCR-scintillation proximity assay.
A laboratory culture of Mamestra brassicae insects (MbLC) was found to harbour a latent baculovirus infection. The copy number of the occult MbNPV genome in both the MbLC larvae, and in a cell line derived from the fat body of MbLC was determined by the use of a rapid and convenient PCR-scintillation proximity assay (SPA). The SPA system relies on the use of fluomicrospheres (SPA beads) coated with acceptor molecules which are capable of binding radiolabelled ligands in solution. In the assay described, a biotinylated PCR primer is used and [3H]dNTPs are incorporated into the amplified DNA. The SPA beads are coated with streptavidin, and after binding the biotinylated primer, any amplified, radiolabelled DNA will activate the fluor. The amount of amplified DNA from the target sequence can then be directly quantified using a scintillation counter. The number of MbNPV genomes present in a persistently infected M. brassicae cell, as proposed by SPA, suggest between 13 and 20 copies of the viral genome may be present in individual fat body cells.
Animals↗
Superior expression of juvenile hormone esterase and beta-galactosidase from the basic protein promoter of Autographa californica nuclear polyhedrosis virus compared to the p10 protein and polyhedrin promoters.
The expression characteristics of the p10, polyhedrin and basic protein promoters of Autographa californica nuclear polyhedrosis virus were compared using two reporter enzymes, juvenile hormone esterase (JHE) and beta-galactosidase. In these systems, JHE is exported from the cell and beta-galactosidase is localized to the cytosol. Expression of JHE from the basic, p10 and polyhedrin promoters was first detected in the medium at 13, 19 and 27 h post-infection respectively. The basic protein promoter yielded the highest expression of the three promoters tested for both enzymes, as determined by protein and enzyme activity assays. In addition, yields of beta-galactosidase and JHE under control of the p10 promoter are higher relative to expression under control of the polyhedrin promoter. These data highlight the importance of investigation of viral promoters other than the polyhedrin promoter for high yield protein expression in vitro, and for insecticidal use of recombinant baculoviruses requiring high levels of expression. The results support revision of the current concept that very late viral promoters are always optimal for high yield recombinant protein expression.
Carboxylic Ester Hydrolases↗
Recombination between genetically modified and unmodified Autographa californica nuclear polyhedrosis virus in Trichoplusia ni larvae.
Trichoplusia ni larvae have been injected with a mixture of wild-type Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and a mutant derivative, AcRP8.UW1.lacZ, which lacks the polyhedrin gene, and has the p10 gene replaced by the Escherichia coli beta-galactosidase gene. Following plaque assay of the haemolymph and subsequent staining for beta-galactosidase activity and scoring for polyhedra, recombinant plaques were identified and the recombination frequency estimated as 6.6%.
Animals↗
Activation and detection of a latent baculovirus resembling Mamestra brassicae nuclear polyhedrosis virus in M. brassicae insects.
A laboratory culture of Mamestra brassicae insects (MbLC) has been found to harbor a latent baculovirus infection. The latent virus was activated by feeding the M. brassicae larvae with either the closely related Panolis flammea nuclear polyhedrosis virus (NPV), or the distantly related Autographa californica NPV. Restriction fragment profiles of the activated virus DNA showed that it is very closely related, if not identical, to M. brassicae NPV. Polymerase chain reaction amplification of polyhedrin gene sequences demonstrated that the latent virus was present throughout the life cycle of the insect; eggs, larvae, pupae, and adults. We failed to detect the presence of a latent virus in a second culture of M. brassicae insects, obtained from the environment and only recently adapted to growth in laboratory conditions, and thus these insects acted as an effective negative control in all the PCR and activation experiments. Using PCR analysis of DNA isolated from dissected tissues of fourth instar MbLC larvae, latent virus sequences were only detected in the fat body. Cell lines established from the isolated MbLC fat body tissue were also shown to harbor the latent virus sequences and should prove useful in further studies to elucidate the mechanisms of latency and virus activation.
Animals↗
A method for producing recombinant baculovirus expression vectors at high frequency.
A system has been developed that can generate recombinant baculovirus expression vectors at frequencies approaching 100%. This system provides a selection for recombinant viruses by using the essential gene downstream of the Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin expression locus. Two AcMNPV derivatives were constructed in which the expression locus and part of the downstream gene are flanked by restriction sites. The parental viruses are viable; however, restriction of the viral DNAs removes an essential piece of the viral genome. Transfer vectors carry a copy of the missing sequences downstream from the site into which foreign genes are inserted for expression; hence, recombination between a transfer vector and the restricted viral DNA can restore the integrity of the essential gene. Such recombination events also transfer any foreign gene present in the expression locus of the transfer vector to the viral genome. Recombinant viruses therefore have a selective advantage over nonrecombinant viral DNAs. Consequently, a high proportion of the viruses obtained by co-transfecting transfer vector DNA and restricted viral DNA of one of these new viruses expresses the target gene from the transfer vector. This system greatly reduces the time needed to make recombinant baculovirus expression vectors.
Animals↗
Assembly of functional GABAA receptors in insect cells using baculovirus expression vectors.
We have constructed recombinant baculoviruses containing cDNAs encoding either the alpha 1- or the beta 1-subunit of the bovine GABAA receptor. In Spodoptera frugiperda (IPLB-Sf-21) cells infected with recombinant virus expressing either the alpha 1- or beta 1-subunit, or in cells co-infected with both viruses, functional GABAA receptors were detected by whole-cell electrophysiological recordings. The threshold for the responses mediated by the homo-oligomeric channels (alpha- or beta-) was 2-3 x 10(-6) M GABA, and for the co-infected cells was 8 x 10(-8) M GABA, suggesting that hetero-oligomeric channels formed in these cells. All GABA-induced currents were found to be inhibited by bicuculline and picrotoxin, potentiated by pentobarbital but were insensitive to benzodiazepines.
Animals↗
Dissimilar expression of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus polyhedrin and p10 genes.
The temporal expression of the Autographa californica multiple nucleocapsid nuclear polyhedrosis virus polyhedrin and p10 genes in Spodoptera frugiperda cells was studied using virus recombinants in which either gene was replaced by the juvenile hormone esterase (JHE) gene of Heliothis virescens. The JHE served as a highly specific and sensitive reporter for gene expression. Activation of the p10 gene followed a pattern different to that of polyhedrin. The p10 gene was activated a few hours earlier than the polyhedrin gene, but its expression reached a lower maximum level. Northern blot analysis complemented and confirmed the results obtained from the JHE assays. Co-infection of sense recombinants and those containing an antisense copy of the JHE gene in place of the polyhedrin or p10 gene resulted in reduced levels of JHE gene expression. These experiments independently supported the hypothesis that the p10 gene promoter is more active at earlier times post-infection than that of the polyhedrin gene. The results also highlight the potential of the antisense strategy as an experimental approach for the study of baculovirus gene regulation and possibly insect metabolism.
Animals↗
Characterization of two variants of Panolis flammea multiple nucleocapsid nuclear polyhedrosis virus.
The baculovirus Panolis flammea multiple nucleocapsid nuclear polyhedrosis virus (PfMNPV) was originally isolated from a natural virus epizootic and shown to consist of a mixture of variants. Two subclasses of variants (PfMNPV A and B) were identified by Southern blot hybridization, their polyhedrin genes being located on different restriction fragments. The proportion of the A and B variants changed according to the larval host in which the virus was propagated; PfMNPV(B) predominated in P. flammea but PfMNPV(A) was predominant in Mamestra brassicae. Bioassays of the two pure virus variants in M. brassicae larvae have shown the LD50 values to be 4610 polyhedron inclusion bodies (pibs) for PfMNPV(A) and 5937 pibs for PfMNPV(B). Genomic DNA from the two variants was compared using restriction endonuclease analysis, and dot blot and Southern blot hybridization. Reciprocal quantitative dot blot hybridization analysis in 50% formamide showed PfMNPV(A) and PfMNPV(B) to be only distantly related to Autographa californica MNPV (less than 1%) and more closely related to M. brassicae MNPV (21 to 26%). The two PfMNPV variants exhibited a very high degree of identity to each other (nearly 100%) and therefore are very closely related. This was confirmed by physical mapping of the virus genomes. The nucleotide sequence of the polyhedrin gene of PfMNPV(B) was determined and compared with the published DNA sequences of other polyhedrin genes.
Animals↗
Construction of an improved baculovirus insecticide containing an insect-specific toxin gene.
Baculoviruses provide alternatives to chemicals for controlling insect pests and can be applied by spraying. Baculoviruses have a limited host range, but work relatively slowly. They are dissolved in the midgut of insect larvae to release infectious virions which enter gut epithelial cells and begin to replicate. Replication in other organs causes extensive tissue damage and eventually death. This process can take 4-5 days, but in the field may last for more than a week, allowing the larvae to feed for longer and thereby damaging the host plant. Baculovirus expression vectors expressing foreign genes, such as those for insect-specific toxins, hormones or enzymes, might alleviate this problem. We have now constructed a recombinant baculovirus derived from Autographa californica nuclear polyhedrosis virus containing an insect-specific neurotoxin from the venom of the North African (Algerian) scorpion, Androctonus australis Hector. The neurotoxin acts by causing specific modifications to the Na+ conductance of neurons, producing a presynaptic excitatory effect leading to paralysis and death; it has no effect in mice. Expression of the neurotoxin by the virus causes a reduction in the time required to kill the host insect.
Amino Acid Sequence↗
Nucleotide sequence of the Autographa californica nuclear polyhedrosis 9.4 kbp EcoRI-I and -R (polyhedrin gene) region.
The nucleotide sequence of a 9.4-kbp region including the polyhedrin gene of the C6 strain of the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome was determined. These data provide a complete description of the EcoRI-I fragment, which is used to produce transfer vectors for inserting foreign genes into the AcMNPV. Ten potential open reading frames (ORFs) were identified in the complete sequence, on either strand of DNA. The largest of these was 1629 nucleotides in length and was located downstream from the polyhedrin coding sequences, but on the opposite strand of DNA. Northern blot hybridization analysis of ORF 8 (1629) identified an RNA of 2000 nucleotides which was produced in infected cells from 12 hr p.i. and remained until at least 48 hr p.i. S1 nuclease mapping and analysis of cDNA clones located the 3' end of the mRNA at a site 16 nucleotides downstream of the polyhedrin coding sequences. The 5' end of the mRNA was mapped using primer extension analysis of polyadenylated RNA. The mRNA start site was positioned within a late/very late consensus transcription initiation motif (ATAAG), 428 nucleotides upstream from the potential ATG translation initiation codon. The biological significance of the putative gene product was assessed by inserting a synthetic oligonucleotide in the carboxyl terminal coding sequences of ORF 8 (1629) to prematurely terminate translation. Recombinant viruses containing this mutation were not isolated, suggesting that the ORF 1629 gene product is essential for virus replication.
Amino Acid Sequence↗
A baculovirus dual expression vector derived from the Autographa californica nuclear polyhedrosis virus polyhedrin and p10 promoters: co-expression of two influenza virus genes in insect cells.
A baculovirus transfer vector, pAcUW3, was developed to facilitate the insertion of two influenza virus genes, those encoding the haemagglutinin (HA) and neuraminidase (NA) membrane glycoproteins, into the Autographa californica nuclear polyhedrosis virus genome in a single cotransfection experiment. The NA gene was inserted in place of the polyhedrin coding sequences under the control of the polyhedrin promoter, whereas the HA gene was placed under the control of a copy of the p10 promoter at a site upstream of and in opposite orientation to the polyhedrin promoter. After infection of Spodoptera frugiperda cells with the recombinant virus, AcUW3HANA, both HA and NA were expressed in the very late phase of infection and were shown to be functional in appropriate assays. Immunofluorescence assays demonstrated their localization at the surface of infected insect cells. The expression of both foreign genes in the recombinant virus was found to be stable for at least 12 passages in cell culture.
Animals↗
Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors.
Engineered derivatives of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) possessing a unique restriction site provide a source of viral DNA that can be linearized by digestion with a specific endonuclease. Circular or linearized DNA from two such viruses were compared in terms of their infectivity and recombinogenic activities. The linear forms were 15- to 150-fold less infectious than the corresponding circular forms, when transfected into Spodoptera frugiperda cells using the calcium phosphate method. Linear viral DNA was, however, proficient at recombination on co-transfection with an appropriate transfer vector. Up to 30% of the progeny viruses were recombinant, a 10-fold higher fraction of recombinants than was obtained from co-transfections with circular AcMNPV DNA. The isolation of a recombinant baculovirus expression vector from any of the AcMNPV transfer vectors currently in use can thus be facilitated by linearization of the viral DNA at the appropriate location.
Animals↗
Functional analysis of a 603 nucleotide open reading frame upstream of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus.
The sequence of the 2000 nucleotides immediately upstream of the polyhedrin gene of the Autographa californica multiple nucleocapsid nuclear polyhedrosis virus has been determined. Comparative analysis of the data identified a 603 nucleotide open reading frame (ORF) separated from the polyhedrin gene coding sequences by 156 nucleotides and in the opposite strand of DNA. Northern hybridization analysis of polyadenylated RNA from infected cells highlighted a 3.7 kb species produced maximally at 12 h post-infection, but not in the presence of cycloheximide. Preliminary nuclease S1 analysis of the 5' end of this RNA suggested that it initiated at a position very close to that of the polyhedrin mRNA start site. Deletion of a portion of the ORF 603 from viruses containing the normal polyhedrin gene and the lacZ gene in lieu of polyhedrin did not affect replication in cell culture or the production of beta-galactosidase protein. A virus which lacked the ORF 603 gene but produced polyhedrin had similar infectivity in Trichoplusia ni larvae compared to the wild-type virus. The chloramphenicol acetyltransferase (CAT) gene was also inserted in lieu of the ORF 603 in a virus containing the lacZ gene instead of the polyhedrin (Ac.CAT.lacZ). Analysis of CAT expression revealed that a maximum level was reached at 16 h p.i. and that transcription was initiated in Ac.CAT.lacZ at the same site as for the normal gene.
Amino Acid Sequence↗
A baculovirus expression vector derived from the basic protein promoter of Autographa californica nuclear polyhedrosis virus.
The basic protein of Autographa californica nuclear polyhedrosis virus (AcMNPV) is associated with virus DNA in virion nucleocapsids and is produced in infected cells during the late phase of gene expression. A transfer vector was constructed containing the beta-galactosidase gene, under the control of a copy of the putative basic protein promoter, in place of the polyhedrin gene within the AcMNPV EcoRI fragment I. After cotransfection of Spodoptera frugiperda cells with the transfer vector and infectious AcMNPV DNA, polyhedrin-negative recombinant viruses were selected which expressed high levels of beta-galactosidase. Radiolabelling of infected cell proteins showed that beta-galactosidase was expressed at the same time as the viral basic protein, between 8 to 24 h post-infection, with a peak synthesis at 12 to 15 h. These results demonstrated that the temporal regulation of the basic protein promoter was not affected by its position within the virus genome. Furthermore, a new baculovirus vector system is now available for high level expression of foreign genes at earlier times in infected cells.
Animals↗
Analysis of very late gene expression by Autographa californica nuclear polyhedrosis virus and the further development of multiple expression vectors.
The consequences of locating the polyhedrin gene coding sequences and the p10 promoter at heterologous positions within the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome were investigated. Positioning the polyhedrin or beta-galactosidase coding sequences under the control of the p10 gene promoter via the use of the new transfer vector, pAcUW1, resulted in viable recombinant viruses able to produce high levels of each non-fused gene product at the appropriate time. Polyhedra were also produced by the virus with the p10 promoter-polyhedrin hybrid gene and appeared normal in thin sections. Therefore the combination of polyhedrin promoter and coding sequences is evidently not essential for efficient expression of this protein. The p10 promoter can serve this function equally well. Viruses with the p10 promoter and beta-galactosidase coding sequences placed upstream from the polyhedrin gene in either orientation produced large amounts of beta-galactosidase protein in infected cells, thus demonstrating that the p10 promoter can function at an alternative position within the virus genome. A second transfer vector, pAcUW2B, was constructed, with a copy of the p10 gene promoter placed upstream and in opposition to the polyhedrin gene. This mediates the insertion of any foreign gene under the control of the p10 promoter while preserving normal p10 gene expression. The advantages of these constructs over the conventional vectors presently used to express foreign genes in insect cell systems and their utilization in the production of virus insecticides are discussed.
Animals↗
Construction of genetically engineered baculovirus insecticides containing the Bacillus thuringiensis subsp. kurstaki HD-73 delta endotoxin.
The delta-endotoxin gene from Bacillus thuringiensis subsp. kurstaki HD-73 was inserted into Autographa californica nuclear polyhedrosis virus (AcMNPV) using two transfer vector systems. In the first, the delta-endotoxin gene was placed under the control of the polyhedrin gene promoter in lieu of the polyhedrin coding sequences, thus deriving a polyhedrin-negative virus. In the second, it was inserted under the control of a copy of the AcMNPV p10 promoter positioned upstream of the polyhedrin gene to produce a polyhedrin-positive virus. Analysis of infected cell extracts showed that the delta-endotoxin was expressed in insect cells as 130K, 62K and 44K proteins, with peak syntheses at 18 h post-infection. Each of these products reacted with antisera specific for the complete protoxin and the cleaved, active form. When extracts from the cells infected with the polyhedrin-negative virus were fed to Trichoplusia ni larvae, feeding by the insects was inhibited and deaths occurred that were inconsistent with virus infection. This effect was also observed after the inoculum had been treated with detergents to inactivate virus particles prior to feeding to the larvae. These data indicate that the expression of the B. thuringiensis delta-endotoxin gene by a baculovirus in insect cells produces material with insecticidal activity. The biological activities of the two recombinant viruses were assessed in conventional bioassay tests by feeding virus particles or occlusion bodies to the insects. The polyhedrin-negative virus preparation appeared to be contaminated with endotoxin which inhibited feeding of the insects and prevented determination of the LD50 value. The polyhedrin-positive virus had an LD50 value about twofold higher than that of unmodified AcMNPV. The significance of these data for the genetic engineering of virus insecticides is discussed.
Animals↗