Autographa California Nuclear Polyhedrosis Virus Research Articles

Interleukin 12-containing influenza virus-like-particle vaccine elevate its protective activity against heterotypic influenza virus infection

To produce monovalent and bivalent influenza vaccines composed of virus-like particles (VLPs) containing hemagglutinin (HA), we generated four recombinant Baculoviruses derived from Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa california nuclear polyhedrosis virus (AcNPV). Monovalent Fukushima (A/tufted duck/Fukushima/16/2011 [H5N1]) (FkH5) and Anhui (A/Anhui/1/2013 [H7N9]) (AnH7) VLP influenza vaccines were produced in silkworm pupae infected with FkH5-BmNPV or AnH7-BmNPV. To produce a bivalent FkH5 and AnH7 vaccine, the pupae were simultaneously inoculated with FkH5-BmNPV and AnH7-BmNPV. Then, interleukin (IL)-containing bivalent vaccines were produced by Eri silkworm pupae following triple infection with FkH5-AcNPV, AnH7-AcNPV, and IL-12-AcNPV. Fluorescent antibody tests in Sf9 cells triple-infected with FkH5-AcNPV, AnH7-AcNPV, and IL-12-AcNPV showed coexpression of FkH5, AnH7, and IL-12 antigens, suggesting the presence of VLPs containing all three antigens. We then performed competitive hemagglutination inhibition (CHI) tests to calculate the VLP vaccine constituents. Inoculation with two recombinant viruses led to the production of bivalent vaccines containing very similar amounts of the H5 and H7 antigens, suggesting that our dual infection system can be used to produce bivalent VLP vaccines. Immunisation of mice with our developed monovalent and bivalent VLP vaccines induced the production of HI antibody, which protected against a sublethal dose of influenza virus. These IL-12-containing vaccines tended to display increased protection against hetero-subtype influenza viruses.

A new set of versatile vectors for the heterologous expression of foreign genes using the baculovirus system

A new set of versatile advanced baculovirus (BV) vectors for the production of fused proteins in insect cells, under the control of the strong polyhedrin promoter of the Autographa california nuclear polyhedrosis virus (AcMNPV), has been constructed. The vectors contain peptide tags which allow immunological detection, as well as purification of recombinant protein produced via the BV expression system

Properties of a baculovirus mutant defective in the protein phosphatase gene

Autographa california nuclear polyhedrosis virus (AcMNPV) contains a gene, ptp, encoding a protein tyrosine/serine phosphatase, BV-PTP. To investigate the biological function of ptp in the baculoviral replication cycle, we constructed a recombinant baculovirus, vPTPdel, in which the catalytically active site of BV-PTP was deleted. Although the vPTPdel mutant was viable in cell culture, it was partially defective in occluded virus production in SF-21 but not TN-368 cell lines. SF-21 cells infected with vPTPdel were heterogeneous in their ability to support occluded virus production. These results suggest that BV-PTP functions in a cell line-specific and possibly a cell cycle-specific fashion. The yield of occlusion bodies, infectivity (concentration of virus causing 50% mortality) and virulence (the time at which 50% of the cells died) of vPTPdel appeared to be normal in insect larvae. We identified a 35-kDa phosphoprotein as a potential target of the BV-PTP in SF-21 cells.

Overexpression, purification, and characterization of third component of complement

The third component of complement (C3) plays a critical role in both pathways of complement activation by interacting with numerous other complement proteins. To elucidate the molecular features of C3 that relate to the functional activities of the molecule, we expressed the cDNA of human complement component C3 in cultured insect cells using a baculovirus expression vector system derived from the baculovirus Autographa california nuclear polyhedrosis virus (AcNPV). The expression of C3 was controlled by the promoter of the polyhedrin gene and, when recombinant baculovirus infected insect cells were cultured in serum-free medium, C3 was detected at a level of 10 μg/ml of culture medium. Characterization of the recombinant C3 (rC3) by SDS-PAGE revealed that the C3 gene product was translated as a 188 kDa protein comprised of two chains of 115 kDa and 73 kDa analogous to the α and β chains of serum-derived human C3 (sC3). An analysis of the glycosylation pattern of purified rC3 revealed that, whereas both the α and β chains were glycosylated as in sC3, the proC3 moiety of rC3 also was glycosylated. When rC3 was produced in the High Five cell line of insect cells and evaluated for reactivity with a panel of anti-C3 monoclonal antibodies (MoAb), the results suggested that the conformation of the baculovirus expressed C3 was similar to that of native C3. When the rC3 was purified by anion exchange column chromatography, it was able to react with several C3-binding proteins (CR1, P and H), reconstitute C3-deficient serum and support the activation of both complement pathways thus demonstrating that a baculovirus-expressed C3 can participate in the formation of and can be cleaved by both the classical and alternative pathway convertases. Incubation of rC3 with factor I and H revealed that both C3 and proC3 are susceptible to cleavage by factor I.

Expression of the Newcastle Disease Virus (NDV) Fusion Glycoprotein and Vaccination against NDV Challenge with a Recombinant Baculovirus

The hybrid baculovirus constructed from Autographa california nuclear polyhedrosis virus (NPV) and Bombyx mori (silkworm) NPV was used for expression of fusion glycoprotein (F) of Newcastle disease virus (NDV) strain D26. The gene encoding F protein was introduced into the improved baculovirus expression vector derived from the host-range-expanded baculovirus. In Spodoptera frugiperda (fall armyworm) cells infected with a recombinant baculovirus, HyF121, the expressed F protein was properly located onto the cell surface. After silkworm pupae were infected with HyF121, a subunit vaccine against NDV was prepared from the HyF121-infected pupae. Chickens inoculated with the subunit vaccine were protected against virulent NDV challenge.

Expression of bovine vitamin K-dependent carboxylase activity in baculovirus-infected insect cells.

A vitamin K-dependent carboxylase has recently been purified from bovine liver microsomes and candidate cDNA clones have been isolated. Definitive identification of the carboxylase remains circumstantial since expression of candidate carboxylase cDNAs in mammalian cells is confounded by the presence of endogenous carboxylase activity. To overcome this problem, a recombinant strain of baculovirus (Autographa california nuclear polyhedrosis virus, AcMNPV) encoding a putative carboxylase (vbCbx/AcMNPV) was used to infect Sf9 insect cells, which we demonstrate have no endogenous carboxylase activity. Infection with vbCbx/AcMNPV conferred vitamin K-dependent carboxylase activity to Sf9 insect cells. Carboxylase activity was demonstrated to peak 2-3 days after infection with vbCbx/AcMNPV. Metabolic radiolabeling with L-[35S]methionine revealed that the 90-kDa recombinant protein is the major protein synthesized at the time of peak activity after infection. An anti-peptide antibody directed against residues 86-99 reacted with bovine liver carboxylase on Western blot analysis and immunoprecipitated recombinant carboxylase from infected Sf9 microsomal protein preparations. Since Sf9 insect cells lack endogenous vitamin K-dependent carboxylase activity, expression of carboxylase activity in Sf9 insect cells with recombinant baculovirus demonstrates that the protein encoded by this cDNA is a vitamin K-dependent gamma-glutamyl carboxylase.

Expression of human IL-2 receptor alpha- and beta-chains using the baculovirus expression system.

The genes encoding the alpha- and beta-chains of the human interleukin-2 receptor were expressed in lepidopteran insect cells using the baculovirus expression vector system. The corresponding genes were inserted under the polyhedrin promoter of the Autographa california nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line during viral infection. The recombinant receptor proteins were identified in the insect cell lysates by using protein dot blot and ELISA techniques. At 36 h post infection the corresponding proteins were clearly detected using anti-IL-2 alpha- and beta-receptor-specific antibodies. A large amount of the alpha-chain was also found in the supernatant culture media at 72 h post infection and metabolic labelling with [35S]-methionine indicated that it was proteolytically cleaved into a 32 kDa soluble form. A similar soluble or secreted form of the beta-chain was, however, not observed. Both receptor proteins were expressed on the surface of the insect cells as determined by flow cytometry analysis. Studies performed with the different IL-2 receptor forms (alpha- and beta-chains alone or in combination) in the presence or absence of rIL-2 suggest that the receptor proteins when expressed in infected insect cells are non-functional with respect to tyrosine phosphorylation.

Expression and characterization of biologically active human hepatocyte growth factor (HGF) by insect cells infected with HGF-recombinant baculovirus.

A cDNA containing the entire coding sequence of human hepatocyte growth factor (HGF) [also known as scatter factor (SF)] was inserted into the genome of Autographa california nuclear polyhedrosis virus (baculovirus) adjacent to the polyhedrin promoter by homologous recombination. Insect cells (Spodoptera frugiperda) infected with the recombinant virus secrete relatively high levels (3-8 mg/L) of biologically active HGF into the culture medium. The recombinant HGF induces pronounced morphological changes and scattering of primary cultures of rat, mouse, and human hepatocytes within 24 h after plating and stimulates DNA synthesis in these cells with the same magnitude as native HGF derived from human placenta or rabbit serum. The human recombinant HGF produced by the insect cells is N-glycosylated, binds to heparin like native HGF, and is recognized by polyclonal antiserums raised against human or rabbit HGF as assessed by immunoblot, ELISA, and immunoneutralization experiments. Metabolic radiolabeling with L-[35S]methionine (pulse-chase experiments) as well as Western blot analysis indicates that the recombinant HGF is synthesized and secreted by the infected insect cells as the unprocessed single-chain form (pro-HGF) when the cells are cultured in serum-free medium. However, when the infected insect cells are cultured in insect culture medium (Grace's medium) containing fetal bovine serum, the secreted HGF is present mainly in the mature heterodimeric form. Addition of serum to the baculovirus-expressed single-chain [125I]HGF in a cell-free system results in conversion to the heterodimeric two-chain form, and the activation is prevented by the serine protease inhibitor PMSF. Incubation of 125I-labeled pro-HGF with rat liver or spleen extracts resulted in conversion of pro-HGF to the heterodimeric two-chain form. A truncated form of HGF containing the N-terminal portion of HGF (kringles 1-3) was also produced in the same expression system. This deleted HGF, by itself, did not have any detectable biological activity; however, it abrogated the stimulatory effects of full-length HGF on hepatocytes. This is the first successful production of bioactive recombinant HGF in large quantities, which will allow purification on the milligram scale of pro-HGF and will permit future studies to elucidate pathways involved in HGF activation by its target tissues.

Non-homologous recombination between adenovirus and AcNPV DNA fragments in cell-free extracts from insect Spodoptera frugiperda nuclei

In previous work, we have developed a cell-free system from nuclear extracts of hamster cells to study the mechanism of integrative recombination between adenovirus type 12 (Ad12) DNA and hamster cell DNA (Jessberger et al., 1989; Tatzelt et al., 1992). We have also demonstrated that in insect cells the left terminal fragment of Ad2 DNA can insert by non-homologous recombination into the 32.6 to 34.5 map unit ( EcoRI-O) fragment and into other segments of Autographa california nuclear polyhedrosis virus (AcNPV) DNA (Xiong et al., 1991). We have now imitated this recombination event in vitro by incubating the E1 fragment of Ad2 DNA and the EcoRI-O fragment of AcNPV DNA, both in the plasmid-cloned circular forms, with partly purified nuclear extracts from Spodoptera frugiperda insect cells. Proteins from these extracts have been fractionated by gel filtration. After the reextraction of DNA from the incubation mixture, recombinants generated in this cell-free system have been identified directly with the polymerase chain reaction (PCR) by using Taq polymerase and appropriate primers which are unique to either of the two reaction partners. The recombinants identified are all different. The results of control experiments argue against the possibility that unspecific reaction products might have been generated during PCR. Nucleotide sequence determinations in some of the recombinants localize the sites of genetic exchange between the two partners and assess the non-homologous nature of the reaction. The recombinants are characterized by the presence of short patch. homoogies at or close to the sites of linkage between the reaction partners, as described earlier in the hamster cell system (Tatzelt et al., 1992). The occurrence of recombinants in the cell-free system can also be demonstrated by a biological test in which potential recombinants are isolated by transfection into recA − strains of Escherichia coli.

Baculovirus gene ME53, which contains a putative zinc finger motif, is one of the major early-transcribed genes

We have been investigating the first viral genes to be transcribed in Spodoptera frugiperda cells after the infection with Autographa california nuclear polyhedrosis virus in order to identify regulatory proteins required for the activation of early and/or late gene transcription. By screening a cDNA library from polyadenylated RNA transcribed at 1 h postinfection, we identified a gene which is designated ME53. This newly identified gene is located upstream of the late p74 gene and forms a divergent promoter unit with the immediate-early gene IE0. The determination of the nucleotide sequence of ME53 has revealed an open reading frame encoding a gene product of 53 kDa. Furthermore, the sequence data suggest a putative DNA binding motif, a zinc finger, whose functional significance has yet to be shown. Transcription of ME53 does not require previous viral protein synthesis, and during infection, the level of promoter activity seems to be independent of trans-acting viral factors, as suggested by transient expression studies.

Potential role of monocyte chemoattractant protein 1/JE in monocyte/macrophage-dependent IgA immune complex alveolitis in the rat.

We have examined the role of monocyte chemoattractant protein 1 (MCP 1) in the pathogenesis of monocyte/macrophage-dependent IgA immune complex alveolitis in the rat. Rat MCP 1 was cloned and expressed in order to facilitate analysis of its function in rat models of human disease. A cDNA library was constructed from rat pulmonary artery endothelial cells stimulated with TNF-alpha. The cDNA library was screened with synthetic oligonucleotide probes based on the recently published rat MCP 1 cDNA sequence. Among numerous MCP 1-positive clones, four full length (approximately 480 bp) cDNA were rescued, amplified by polymerase chain reaction, and ligated into a pJVETLZ baculovirus transfer vector. Spodoptera frugiperda insect cells (Sf-21) infected with baculovirus recombinants (Auto-grapha california nuclear polyhedrosis virus) bearing properly oriented MCP 1 cDNA (AcMCP 1) directed the expression of unique peptides of 18, 21, and 23 kDa. Treatment of AcMCP 1-infected Sf-21 cells with tunicamycin resulted in reduced production of the 21- and 23-kDa proteins and an increase in 16- to 18-kDa products, the predicted size range of uncleaved and nonglycosylated rat MCP 1. Denatured and refolded 23-kDa and 21-kDa rat MCP 1 species exhibited dose-dependent monocyte-specific chemotactic activity at concentrations as low as 10(-10) M whereas the 18-kDa species exhibited negligible activity. Antibodies that react with the immunoblot, block rat rMCP 1-directed monocyte chemotaxis, and neutralize monocyte-specific chemotactic activity secreted by TNF-stimulated rat endothelial cells were raised in rabbits immunized with the 23-kDa MCP 1 species. Intravenous administration of anti-MCP 1 antibodies upon initiation of IgA immune complex lung injury resulted in a marked reduction in lung injury as measured by pulmonary vascular permeability, alveolar hemorrhage, and pulmonary monocyte/macrophage recruitment and pulmonary monocyte/macrophage recruitment. These data suggest that MCP 1 may play an important role in the pathogenesis of monocyte/macrophage-dependent IgA immune complex alveolitis in the rat.

Purification and functional characterization of the human β 2-adrenergic receptor produced in baculovirus-infected insect cells

A human cDNA fragment bearing the complete coding region for the β 2-adrenergic receptor was introduced into the genome of Autographa california nuclear polyhedrosis virus under the control of the polyhedrin promoter. Binding studies using [ 125I]iodocynnopindolol showed that Sf9 insect cells infected with the recombinant virus expressed ≈ 1 × 10 4 β 2-adrenergic receptors on their cell surface. Photoaffinity labeling of whole cells and membraines revealed a molecular weight of ≈ 46000 for the expressed receptor. The receptor produced in insect cells is glycosylated but the extent and pattern differ from that of the receptor from human tissue. The heterologously expressed receptor was purified by alprenolol affinity chromatography, and was able to activate isolated Gs-protein.

Immunoselection of recombinant baculoviruses expressing high levels of biologically active herpes simplex virus type 1 glycoprotein D.

The DNA sequence encoding the complete herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) was inserted into a baculovirus transfer vector under control of the polyhedrin gene promoter of the baculovirus Autographa california nuclear polyhedrosis virus (AcNPV). After co-transfection of Spodoptera frugiperda (Sf9) insect cells with wild-type AcNPV DNA and the recombinant transfer vector DNA, polyhedrin-negative recombinants that expressed high levels of HSV-1 gD were isolated using immunoaffinity selection with antibody coated magnetic particles followed by plaque purification. These recombinant baculoviruses expressed a protein that was slightly smaller than virion HSV-1 gD made in Vero cells. This recombinant protein was expressed at high levels. The expressed protein was glycosylated, was found on the membrane of Sf9 cells, and reacted with gD specific antibodies. Antibodies raised in mice to the recombinant gD neutralized HSV-1 as measured by plaque reduction assays. Mice inoculated with the recombinant baculovirus were completely protected from lethal challenge with HSV-1.

Molecular analysis of the transcriptional regulatory region of an early baculovirus gene

Transcription of the gene encoding a 35,000-molecular-weight protein (35K protein) from the EcoRI-S region (86.8 to 87.8 map units) of Autographa california nuclear polyhedrosis virus (AcMNPV) occurs early in infection and declines later. The region promoting the gene for the 35K protein, extending from 426 base pairs (bp) upstream to 12 bp downstream from the RNA start site, was linked to the bacterial chloramphenicol acetyltransferase gene (CAT) for analysis. CAT expression was monitored in cells that were transfected with plasmids containing the promoter-CAT fusion as well as cells infected with recombinant viruses containing the chimeric gene inserted into the AcMNPV genome. Mapping of the 5' ends of CAT-specific RNAs indicated that transcription initiated from the proper sites in both assays; moreover, the promoter fragment retained its early activity, despite an alternate location in the viral genome. The 5' boundary of upstream regulatory sequences was determined by constructing deletions of the promoter fragment extending toward the early RNA start site (position +1). In transient assays, a gradual reduction in CAT expression occurred as sequences from positions -426 to -31 were removed. In contrast, promoter deletions from positions -426 to -155 in recombinant viruses exhibited no effect on CAT expression, whereas deletions to position -55 abolished early expression but had no effect on late expression. Late CAT expression was eliminated when deletions to position -4 removed part of the late RNA start site. DNA signals potentiating early transcription were therefore located upstream (between positions -155 and -55) from those involved in late transcription of the gene encoding the 35K protein. Potential consensus sequences for early and late regulatory elements were identified.

Functional mapping of Autographa california nuclear polyhedrosis virus genes required for late gene expression.

A plasmid containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of an Autographa california nuclear polyhedrosis virus (AcNPV) late gene promoter was constructed. This plasmid (pL2cat) also contained the AcNPV hr5 enhancer element. Transient-expression assay experiments indicated that the late promoter was active in Spodoptera frugiperda cells cotransfected with pL2cat and AcNPV DNA but not when pL2cat was transfected alone. Low levels of CAT activity were observed in cells cotransfected with pL2cat and pIE-1 DNAs. However, CAT activity was not induced in a similar plasmid which lacked the cis-linked enhancer element, indicating that the enhancer was required for expression of the late gene. Cotransfection mapping of pPstI clones of AcNPV DNA indicated that the pPstI-G clone of viral DNA contained a factor which further stimulated late gene expression 3- to 10-fold. Transient-expression assay analysis of subclones of pPstI-G localized the trans-active factor to a 3.0-kilobase XbaI fragment. The nucleotide sequence of this fragment was determined and found to contain three potential open reading frames. A computer-assisted search of a protein database revealed no closely related proteins. One of the predicted amino acid sequences contained potential metal-binding domains similar to those found in nucleic acid-binding proteins. Subcloning and subsequent CAT assay indicated that two of the open reading frames were required for the activation of pL2cat. Nuclease S1 mapping of infected and transfected RNAs indicated that the two open reading frames were transcribed as delayed-early genes. Quantitative nuclease S1 analysis and differential DNA digestion of recovered plasmids indicated that the activation of pL2cat was not due to an increase in steady-state levels of mRNA replication of the viral DNA.

Baculovirus induction of a DNA polymerase.

The baculovirus, Autographa california nuclear polyhedrosis virus, induced a new aphidicolin-sensitive, alpha-like, DNA polymerase upon infection of the lepidopteran noctuid, Trichoplusia ni. The new virus-induced DNA polymerase could be separated from the host alpha-like polymerase by phosphocellulose chromatography. The two polymerases differed in their sensitivities to heat inactivation, high salt concentrations, and 0.1 M phosphate buffer.