High frequency recombination between homologous baculoviruses in cell culture.

The frequency of recombination between homologous baculoviruses was investigated in cell culture upon coinfection with two Autographa californica nucleopolyhedrovirus (AcMNPV) recombinants. These parental recombinants differed at two loci, separated by 20 kb, each carrying a different marker. The progeny recombinants generated by homologous recombination were easily distinguishable by the presence or absence of these markers. The mean percentage of the newly generated recombinants relative to a single locus varied between 23 and 41%, depending on the multiplicity of infection used for coinfection. These results provide further evidence that homologous recombination occurring during baculovirus replication is a highly frequent event.

Translational properties of the untranslated regions of the p10 messenger RNA of Autographa californica multicapsid nucleopolyhedrovirus.

Protein yields in the baculovirus expression system do not always correlate with the presence of abundant amounts of corresponding mRNAs. Therefore, a novel aspect of the baculovirus expression system was studied: initiation of translation of very late mRNAs of Autographa californica multicapsid nucleopolyhedrovirus. The untranslated regions (UTRs) of the p10 mRNA of this baculovirus were studied by in vitro translation and after transfection into Spodoptera frugiperda insect cells. Lysates from insect cells were optimized for translation of in vitro transcripts containing p10 sequences. The lysates were used to measure the effects of various deletions in either the 5' or 3'UTR on protein synthesis. Transcripts containing the p10 5'UTR were translated efficiently. Large deletions in the 5'UTR severely decreased this efficiency. Deletions in the 3'UTR negatively affected expression of the reporter gene in vivo; however, no effect on translational efficiency in the insect-cell lysates was measured. The translational properties of the p10 transcripts were very similar in lysates made from either uninfected or baculovirus-infected insect cells. Determination of optimal salt conditions for either uncapped or capped transcripts showed that the p10 5'UTR was used very efficiently for translation initiation in vitro, even in the absence of a cap-structure at its 5' end. Addition of cap-analogue to the in vitro translation assays did not inhibit p10 5'UTR-driven translation, while translation of a cap-dependent mRNA was severely inhibited. These data suggest that the very late mRNAs of baculovirus are translated in a cap-independent manner.

Lysosomal protective protein/cathepsin A. Role of the "linker" domain in catalytic activation.

Lysosomal protective protein/cathepsin A is a serine carboxypeptidase that forms a complex with beta-galactosidase and neuraminidase. The enzyme is synthesized as a 54-kDa precursor/zymogen and processed into a catalytically active 32- and 20-kDa two-chain form. We have expressed in baculovirus-infected insect cells the human one-chain precursor as well as the two separate subunits in order to establish the mode of catalytic activation of the zymogen and the assembly and activation of the two subunits. Infected insect cells synthesize large quantities of the exogenous proteins, which are glycosylated and secreted but not processed. Co-expression of the two subunits results in their assembly into a two-chain form of 34- and 20-kDa with negligible enzymatic activity. Limited proteolysis with trypsin of the 54-kDa precursor and the reconstituted 34- and 20-kDa form gives rise to a fully active 32- and 20-kDa product. These results enabled us to map the sites of proteolytic cleavage needed for full activation of the cathepsin A zymogen. They further indicate that the 34- and 20-kDa form is a transient processing intermediate that is converted into a mature and active enzyme by removal of a 2-kDa "linker" peptide from the COOH terminus of the 34-kDa subunit.

Production of native creatine kinase B in insect cells using a baculovirus expression vector.

A full-length human creatine kinase B (B-CK) cDNA was used to produce a recombinant baculovirus (AcDZ1-BCK). Sf9 cells infected with this recombinant expressed a homodimeric protein composed of 43 kDa subunits which, under optimal conditions, formed up to 30% of the total soluble cellular protein. Upon analysis by PAGE, zymogram assay and gel filtration chromatography the recombinant protein behaved like authentic dimeric human BB-CK protein. Studies with a newly produced monoclonal antibody (CK-BYK/21E10) directed against an epitope in the N-terminus of the protein confirmed the identity of the product. The recombinant BB-CK protein was purified to over 99% homogeneity from the total protein extract of AcDZ1-CKB infected cells in one single step involving anion exchange column chromatography on MonoQ in FPLC. Dialysed protein had a specific activity of 239 U/mg protein.

Biological activity of cauliflower mosaic virus aphid transmission factor expressed in a heterologous system.

Aphid transmission factor (ATF) activity of cauliflower mosaic virus (CaMV) gene II product was recovered after expression of the gene by a baculovirus recombinant. The expression product, when first acquired by aphids through parafilm membrane, was able to mediate the transmission of two aphid nontransmissible isolates (CM1841, CM4-184) providing the first direct evidence that the product of the gene II is the CaMV ATF. The CaMV ATF in its active conformation has a strong tendency to aggregate and all attempts at solubilizing it resulted in the loss of the ATF activity. The CaMV ATF was also expressed in Escherichia coli, using the pGEX 3X plasmid vector, as a fusion protein to glutathione S-transferase (GST) and was purified. The fusion product (GST-P18), whether purified or not, was not able to complement the transmission of transmission-defective isolates. However, when GST-P18 was added to some extracts from a plant infected with an aphid-transmissible isolate (Cabb B-JI), the transmission was inhibited. This suggests that it could be possible to block the in vitro transmission of CaMV using a molecule analogous to the ATF.

Biophysical and biochemical properties of baculovirus-expressed CaMV P1 protein.

Cauliflower mosaic virus (CaMV) gene I encodes a protein (P1) that has been implicated in the control of virus movement in infected plants. To assist in the characterization of the mechanism of action of P1, gene I has been expressed efficiently in Spodoptera frugiperda (Sf) cells using recombinant baculovirus. Control of the expression of CaMV gene I by the polyhedrin late promoter in the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) resulted in very high levels of P1 accumulation late in the infection cycle. This was predominantly as insoluble inclusion bodies within the cytoplasm of infected Sf cells, and not extracellularly. Evidence from anomalous gel migration and sequence homology with an analogous viral protein (tobacco mosaic virus 30K) indicated that P1 may be post-translationally processed. However, neither phosphorylation nor glycosylation of P1 occurred in this system, suggesting a functional distinction between P1 and TMV 30K. P1 from insect cells and native P1 from infected plants were immunologically related, allowing the expressed product to be used in the preparation of anti-P1 serum for detecting P1 in plant extracts. The full-size (46 kD) P1 product from insect cells, from plants, and from in vitro translations of in vitro gene I transcripts all showed similar behavior on two-dimensional protein gels, with a major pI of 7.0. Using a combination of 4 M urea, 1 M NaCl, and high temperature, P1 was solubilized. Approximately 5% of the starting material remained in solution after dialysis and remained stable to freeze/thawing. This preparation should enable us to identify the biochemical function of P1 and to resolve its role in controlling virus spread.

Expression of cauliflower mosaic virus ORF II in a baculovirus system.

The cauliflower mosaic virus ORF II encoding the aphid transmission factor (ATF) was mutagenized to introduce a BamHI restriction site upstream from the initiation codon and then cloned into an eukaryotic viral expression vector (Autographa californica nuclear polyhedrosis virus). All recombinant viruses tested in Spodoptera frugiperda (SF21) cells expressed a protein of about 18 kD which comigrated in PAGE with ATF from infected plants. Western blotting using an oligopeptide antiserum to ATF confirmed the identity of the 18-kD protein from infected cells as the product of the ORF II sequences (P18). Subcellular fractionation of cells infected with the recombinant AcMNPV demonstrated that the expressed P18 accumulated intracellularly in an insoluble form. Antiserum was produced in rabbit against the partially purified P18 expressed in SF21 cells. When used to immunogold label ultrathin sections of cauliflower mosaic virus (CaMV)-infected turnip tissue, this antiserum was shown to be highly specific, labelling only the electronlucent inclusion bodies (containing P18) and not other plant cellular components.

Expression of plant virus genes in animal cells: high-level synthesis of cowpea mosaic virus B-RNA-encoded proteins with baculovirus expression vectors.

The baculovirus expression system has been used to produce non-structural proteins encoded by bottom-component RNA (B-RNA) of cowpea mosaic virus (CPMV). For this, cDNAs containing the 60K, 87K, 110K and 170K protein coding sequences were each provided with an ATG start codon and the cDNA containing the 60K coding sequence with a TAA stop codon immediately downstream of the coding sequence. Recombinant baculoviruses were retrieved which harboured the modified B-cDNA sequences under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Upon infection of Spodoptera frugiperda cells with these recombinant baculoviruses, proteins were produced which were indistinguishable from the viral proteins found in CPMV-infected plants as judged by their migration in polyacrylamide gels and their reactivity with CPMV-specific antisera. Specific processing of CPMV polyproteins in cells infected with the 110K- and 170K-encoding baculovirus recombinants proved that the CPMV-encoded 24K protease activity contained in these polyproteins is active in these cells. Approximately 10% of the 110K protein was processed into 87K and 24K proteins and the 170K protein almost completely into the 110K, 87K, 84K, 60K and 24K polypeptides. In S. frugiperda cells infected by recombinant AcNPVs harbouring the 87K or 110K coding sequences, the CPMV-specific proteins amounted to 10 to 20% of the total cellular protein content, whereas in cells infected by recombinants encoding the 60K and 170K polypeptides the amounts of CPMV-specific proteins synthesized were much lower. Northern blot analysis indicated that the low-level synthesis of the 60K and 170K polypeptides was not due to inferior transcription of the cloned genes but was probably the result of inefficient translation of the RNAs derived from these constructs. It is concluded that plant virus genes can be efficiently expressed in an animal cell expression system to yield proteins that are structurally and, in at least one case (24K protein), functionally identical to the authentic plant virus proteins.

Expression of cauliflower mosaic virus gene I using a baculovirus vector based upon the p10 gene and a novel selection method.

A new baculovirus expression vector based upon the p10 gene of Autographa californica nuclear polyhedrosis virus (AcNPV) and a novel system for the screening of p10 recombinants have been developed. The insertion of a cassette containing the lacZ gene under the control of a heat-shock promoter of Drosophila melanogaster downstream from the cloning site in p10 transfer vectors allows the convenient identification of putative recombinants by virtue of their expression of beta-galactosidase. Using this p10 transfer vector an AcNPV recombinant was engineered with a cDNA copy of gene I of cauliflower mosaic virus (CaMV) in place of the p10 coding sequence. This p10 recombinant expressed CaMV gene I at levels equivalent to those of p10 and polyhedrin, and was shown to be as effective in producing this protein as recombinants exploiting the polyhedrin promoter. CaMV gene I protein formed large numbers of hollow fiber-like structures in the cytoplasm of infected cells. Because the polyhedrin gene remains intact, these p10 expression vectors may be exploited for the expression of heterologous proteins in insects infected per os and for the enhancement of baculovirus pathogenicity for insect control.

Expression of cauliflower mosaic virus gene I in insect cells using a novel polyhedrin-based baculovirus expression vector.

An improved polyhedrin-based baculovirus expression vector was constructed to expedite distinguishing infections by putative baculovirus recombinants from infections by wild-type (wt) baculovirus. The vector utilizes the Escherichia coli beta-galactosidase gene (lacZ) as a genetic marker for positive recombination between wt Autographa californica nuclear polyhedrosis virus and the baculovirus transfer vector. The marker gene/expression cassette was constructed so that lacZ and the deleted polyhedrin gene were transcribed in opposite orientations, both terminating in a simian virus 40 DNA fragment which acts as a bidirectional terminator. In the constructed vector, lacZ is transcribed from the Drosophila melanogaster heat-shock promoter (hsp 70), which is constitutively expressed in baculovirus-infected Spodoptera frugiperda (Sf) cells, thereby making the site of the deleted polyhedrin gene available for the insertion and expression of foreign genes under the control of the polyhedrin promoter. Recombinant baculoviruses are readily selected in plaque assays by the development of a blue colour upon the addition of X-Gal. The colour selection renders the retrieval of recombinants less dependent on a high frequency of recombination between the transfer vector and wt baculovirus DNA. The usefulness of this new vector was illustrated by expressing gene I of cauliflower mosaic virus, which encodes a protein of Mr 46,000. Expression of gene I was at the same level as in cells infected with a conventional polyhedrin-based expression vector. Gene I protein formed large hollow fibre-like structures in the cytoplasm of infected Sf cells. This is the first plant virus protein to be expressed in insect cells by a recombinant baculovirus.

Functional studies on the p10 gene of Autographa californica nuclear polyhedrosis virus using a recombinant expressing a p10-beta-galactosidase fusion gene.

The beta-galactosidase gene (lacZ) of Escherichia coli was inserted in phase with the coding sequence of the Autographa californica nuclear polyhedrosis virus (AcMNPV) late-expressed Mr 10,000 (p10) gene. The fusion gene was inserted into the AcMNPV genome by cotransfection of a recombinant plasmid pAcR159Z, consisting of the EcoRI P fragment-containing pBR325-derived plasmid pAcR159 and the lacZ insert in the p10 gene, and wild-type AcMNPVDNA. Infection of Spodoptera frugiperda cells by the resulting recombinant AcMNPV/p10Z-2 showed high level expression of a p10-lacZ fusion protein, but no synthesis of p10. Therefore, the p10 gene is dispensable for virus replication and the p10 promoter is effective in driving the expression of foreign genes. Cells infected with AcMNPV/p10Z recombinants resembled those infected with wild-type AcMNPV in the amounts of polyhedrin synthesized and polyhedra formed, although p10 was absent. The nucleus and cytoplasm of AcMNPV/p10Z-2-infected cells lacked the fibrous structures that are associated with p10 in wild-type AcMNPV-infected cells. Instead, large granular structures were observed that were found by immunogold labelling to contain the lacZ gene product. The electron-dense 'spacers', thought to be precursors of the polyhedron membrane, were absent from cells infected by the recombinant virus and the polyhedra did not have a membrane. The recombinant AcMNPV/p10Z-2 was at least twice as virulent for second instar S. exigua larvae than was wild-type AcMNPV. The increased virulence of the recombinant is an important property for the control of insects.