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efficient transfection of insect cells with baculovirus dna using electroporation.efficient transfection of spodoptera frugiperda cells with autographa californica nuclear polyhedrosis virus (acnpv) dna has been carried out using the technique of electroporation. the efficiency of transfection was monitored by assaying the extracellular virus in cell culture supernatants 3 days post-electroporation. maximum titres of 2 x 10(9) p.f.u./ml acnpv were obtained when using a pulse length of 7.7 ms and a field strength of 500 v/cm. this compared with a titre of 2 x 10(6) p.f.u./ml a ...19892691634
[use of baculovector for the expression of hbsag gene in insect cells].based on the information of molecular biology of autographa californica nuclear polyhedrosis virus (acnpv), a recombinant transfer plasmid pacmv was constructed by molecular procedures included using two synthetic localized probes, which provided an inserted position linked with bamhi sequences nearly at polyhedrin initiating atg codon. then an expression vector pacmv-hbsag was reconstructed, it contained hbsag gene from subclone pypss-1 derived from adwserotype of hbv. the recombinant virus con ...19892800542
alginate concentration: a key factor in growth of temperature-sensitive baculovirus-infected insect cells in microcapsules.the desire to increase cell density and product concentration has been the primary driving force for the development of better animal cell culture processes. in the technique used in our laboratory-microencapsulation-insect cells (spodoptera frugiperda), infected with a temperature-sensitive mutant of the autographa californica nuclear polyhedrosis virus (acnpv), were cultured in multiple membrane alginate-polylysine (pll) microcapsules which had a controlled membrane molecular-weight cutoff and ...198918588202
co-occlusion and persistence of a baculovirus mutant lacking the polyhedrin gene.a co-occlusion process was evaluated as a commercially and ecologically acceptable strategy for the development of genetically improved baculovirus insecticides. coinfection of spodoptera frugiperda (iplb-sf-21) tissue culture cells with autographa californica nuclear polyhedrosis virus (acmnpv) and an acmnpv mutant (ac-e10) lacking the polyhedrin gene resulted in occlusion of both virus types within polyhedra. the amount of occluded ac-e10 virions in progeny polyhedra populations during serial ...199016348313
temporal expression of hiv-1 envelope proteins in baculovirus-infected insect cells: implications for glycosylation and cd4 binding.three different human immunodeficiency virus type i (hiv-1) envelope derived recombinant proteins and the full length human cd4 polypeptide were expressed in spodoptera frugiperda (sf9) cells. dna constructs encoding cd4, gp120, gp160, and gp160 delta (full length gp160 minus the transmembrane and cytoplasmic region of gp41) were cloned into the baculovirus expression vector pvl941 or a derivative and used to generate recombinant viruses in a cotransfection with dna from autographa californica n ...19902076345
expression of the bacillus anthracis protective antigen gene by baculovirus and vaccinia virus recombinants.the gene encoding bacillus anthracis protective antigen (pa) was modified by site-directed mutagenesis, subcloned into baculovirus and vaccinia virus plasmid transfer vectors (pacym1 and psc-11, respectively), and inserted via homologous recombinations into baculovirus autographa californica nuclear polyhedrosis virus or vaccinia virus (strains wr and connaught). expression of pa was detected in both systems by immunofluorescence assays with antisera from rabbits immunized with b. anthracis pa. ...19902105271
identifying the rna polymerases that synthesize specific transcripts of the autographa californica nuclear polyhedrosis virus.nuclear run-on assays carried out in the presence and absence of the rna polymerase ii inhibitor, alpha-amanitin, were used to determine the exact timing of the switch from inhibitor-sensitive transcription catalysed by host rna polymerase ii, to inhibitor-resistant transcription catalysed by the baculovirus-induced rna polymerase. these studies revealed that the onset of alpha-amanitin-resistant transcription is just after 6 h post-infection, simultaneous with the beginning of the late phase of ...19902106003
regulation of expression of a baculovirus ecdysteroid udpglucosyltransferase gene.the autographa californica nuclear polyhedrosis virus egt gene encodes an ecdysteroid udpglucosyltransferase which catalyzes the transfer of glucose from udpglucose to ecdysteroid insect molting hormones. expression of this gene allows the virus to block molting and pupation of infected insect larvae. in this study, we present the nucleotide sequence of the a. californica nuclear polyhedrosis virus egt gene and characterize egt gene expression at the transcriptional and translational levels. egt ...19902106039
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 ...19902109042
localization of the 34 kda polyhedron envelope protein in spodoptera frugiperda cells infected with autographa californica nuclear polyhedrosis virus.using immuno-electron microscopy the 34 kda polyhedron envelope (pe) phosphoprotein (pp 34) was localized in cells infected with autographa californica multiple-nucleocapsid nuclear polyhedrosis virus (acmnpv). in wild-type acmnpv-infected cells this protein was found associated with electron-dense "spacers" and the polyhedron envelopes demonstrating their structural relationship. in these cells pp 34 was also found associated with fibrillar structures present in the nucleus and cytoplasm of inf ...19902109589
alpha 1----3-galactosyltransferase: the use of recombinant enzyme for the synthesis of alpha-galactosylated glycoconjugates.we have reported the isolation and characterization of a bovine cdna clone containing the complete coding sequence for udp-gal:gal beta 1----4glcnac alpha 1----3-galactosyltransferase [joziasse, d. h., shaper, j. h., van den eijnden, d. h., van tunen, a. j. & shaper, n. l. (1989) j. biol. chem. 264, 14290-14297]. insertion of this cdna clone into the genome of autographa californica nuclear polyhedrosis virus (acnpv) and subsequent infection of spodoptera frugiperda (sf9) insect cells with recom ...19902116309
expression and purification of p24, the core protein of hiv, using a baculovirus-insect cell expression system.the baculovirus autographa californica nuclear polyhedrosis virus (acnpv) has been genetically manipulated to yield a recombinant virus capable of expressing p24, the major core protein of hiv-1, in insect cell culture. the expressed product is a p24 protein flanked by short regions of p17 at the amino terminus and p12 at the carboxy terminus. it has been identified and characterized using monoclonal antibodies on western blots and by amino-terminal sequence analysis. the presence of p24 in the ...19902126440
co-expression of the human hla-b27 class i antigen and the e3/19k protein of adenovirus-2 in insect cells using a baculovirus vector.we have expressed the human mhc class i hla-b27 antigen, human beta 2-microglobulin, and the e3/19k protein of adenovirus-2 in spodoptera frugiperda insect cells (sf9) by using the autographa californica nuclear polyhedrosis virus. all genes were inserted under the strong polyhedrin promoter in the vector pvl941. the proteins were expressed at high levels, ranging from 1 to 8 mg protein per 3 x 10(9) cells. both a full-length and a truncated form of hla-b27 were expressed. the latter was termina ...19902150332
identification of bluetongue virus vp6 protein as a nucleic acid-binding protein and the localization of vp6 in virus-infected vertebrate cells.recently the insect baculovirus autographa californica nuclear polyhedrosis virus (acnpv) has been effectively adapted as a highly efficient vector in insect cells for the expression of various genes. a cdna sequence of rna segment 9 of bluetongue virus serotype 10 (btv-10, an orbivirus member of the reoviridae family) encoding a minor core protein (vp6) has been inserted into the bamhi site of the pacym1 transfer vector derived from acnpv. spodoptera frugiperda cells were cotransfected with the ...19902152806
identification of a viral gene encoding a ubiquitin-like protein.the baculovirus autographa californica nuclear polyhedrosis virus (acmnpv, which is representative of the mnpv subtype in which the virions may contain many nucleocapsids within a single viral envelope) encodes a protein, v-ubi, that has 76% identity with the eukaryotic protein ubiquitin. transcriptional mapping indicated that the gene for v-ubi was transcribed during the late phase of viral infection. two transcriptional start sites potentially encoding v-ubi were identified. both sites were co ...19902153300
baculovirus diversity and molecular biology.with the identification and characterization of a number of structural and nonstructural protein genes, advances have been made in our understanding of baculovirus structure, regulation of gene expression, and replication. since less than 30% of the acmnpv genome has been sequenced and characterized, the continued identification and assignment of function to baculovirus genes is perhaps the most crucial of enterprises now facing baculovirologists and is critical to the development of our underst ...19902154158
sequence comparison of cellular and viral copies of host cell dna insertions found in autographa californica nuclear polyhedrosis virus.the nucleotide sequence and structural characteristics of two nonhomologous host dna insertions of spodoptera frugiperda (fall armyworm) origin isolated from few polyhedra mutants of the baculovirus autographa californica nuclear polyhedrosis virus (acmnpv) have been determined. neither of the host insertions contain long open reading frames suggesting that cellular genes were not introduced into the viral genome. one of the host dna insertions, ifp1.6, terminated in short imperfect inverted rep ...19902154879
purification and characterization of the recombinant extracellular domain of human nerve growth factor receptor expressed in a baculovirus system.to obtain the large quantities of the extracellular domain of the nerve growth factor receptor (ngf-r) necessary for structural analyses, we produced this protein in the baculovirus expression system. a cdna coding for the extracellular domain of the human ngf-r was first introduced into transfer vector pvl941. recombinant baculovirus was produced by cotransfecting spodoptera frugiperda cells with the transfer vector and dna of autographa californica nuclear polyhedrosis virus. recombinant viral ...19902155239
primary structure of the s peplomer gene of bovine coronavirus and surface expression in insect cells.the nucleotide sequence of the s peplomer gene of bovine coronavirus (bcv) has been determined. a single open reading frame of 4089 nucleotides encodes a polypeptide of 150k with 20 potential sites for addition of n-linked oligosaccharides. expression of the cloned bcv s gene by a recombinant of autographa californica nuclear polyhedrosis virus resulted in production of a 180k glycosylated polypeptide which was transported to the surface of the cell. comparison of the bcv s gene with the analogo ...19902155283
synthesis of bluetongue virus (btv) corelike particles by a recombinant baculovirus expressing the two major structural core proteins of btv.the l3 and m7 genes of bluetongue virus (btv), which encode the two major core proteins of the virus (vp3 and vp7, respectively), were inserted into a baculovirus dual-expression transfer vector and a recombinant baculovirus expressing both foreign genes isolated following in vivo recombination with wild-type autographa californica nuclear polyhedrosis virus dna. spodoptera frugiperda insect cells infected with the recombinant synthesized large amounts of btv corelike particles. these particles ...19902157041
an insertion of insect cell dna in the 81-map-unit segment of autographa californica nuclear polyhedrosis virus dna.in this report, a transposonlike insertion of spodoptera frugiperda insect cell dna was analyzed in single-plaque isolate e of the insect baculovirus autographa californica nuclear polyhedrosis virus (acnpv). the 634-base-pair insertion is characterized by an 18-base-pair terminal inverted repeat and carries an ecori site. this additional ecori site in the 81-map-unit segment of the dna of plaque isolate e of acnpv explains the difference between the ecori restriction map of the dna from this is ...19902157067
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 ...19902157192
identification of epstein-barr virus terminal protein 1 (tp1) in extracts of four lymphoid cell lines, expression in insect cells, and detection of antibodies in human sera.the terminal proteins tp1 and tp2 are putative products of epstein-barr virus (ebv) genes expressed during the latent cycle of the virus. they are predicted to code for 53- and 40-kilodalton integral membrane proteins. we used the baculovirus autographa californica nuclear polyhedrosis virus as an expression vector to produce tp1 in large amounts in insect cells. the dna sequences used to express tp1 originated from a tp1 cdna derived from an m-aba/cbl1 cdna library. rabbit antisera raised again ...19902159542
high level expression of the two outer capsid proteins of bluetongue virus serotype 10: their relationship with the neutralization of virus infection.dna representing rna segments 2 and 5 of bluetongue virus (btv) serotype 10, corresponding to the genes that code for the outer capsid proteins vp2 and vp5, have been inserted into a baculovirus transfer vector in lieu of the coding region of the polyhedrin gene of autographa californica nuclear polyhedrosis virus (acnpv). after co-transfection of spodoptera frugiperda cells with wild-type acnpv dna in the presence of the recombinant transfer vector dnas polyhedrin-negative recombinant baculovir ...19902160763
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 ge ...19902165136
synthesis of foot-and-mouth disease virus capsid proteins in insect cells using baculovirus expression vectors.foot-and-mouth disease virus (fmdv) cdna cassettes containing sequences encoding the capsid precursor p1-2a with and without those encoding the proteases l and 3c were introduced into autographa californica nuclear polyhedrosis virus (acmnpv) expression vectors. procapsid proteins 1ab, 1c and 1d were produced in cells infected with recombinant baculoviruses, when l and 3c were present in the constructs, indicating that these fmdv proteases were active in insect cells. unlike p1 processing in pol ...19902167924
biophysical studies on the morphology of baculovirus-expressed bluetongue virus tubules.bluetongue virus tubules were purified from spodoptera frugiperda cells infected with a recombinant baculovirus containing the ns1 gene from bluetongue virus serotype 10, and expressed under control of the autographa californica nuclear polyhedrosis virus polyhedrin promoter. these tubules were subjected to a variety of chemical and physical treatments and the resulting effects on tubule morphology were examined by electron microscopy. a number of morphological similarities were noted between bl ...19902167940
synthesis of bluetongue virus-encoded phosphoprotein and formation of inclusion bodies by recombinant baculovirus in insect cells: it binds the single-stranded rna species.a dna clone of rna segment 8 (s8) of bluetongue virus type 10 (btv-10), an orbivirus member of the reoviridae family has been expressed to high levels (20 mg/1 x 10(9) cells) using an autographa californica nuclear polyhedrosis virus expression vector (pa-cym1). the expressed protein is similar to the authentic btv phosphoprotein ns2, in its size, antigenicity, and also the manner of phosphorylation (e.g. same peptides and residues). both mammalian and insect cell-derived ns2 proteins are phosph ...19902170574
expression of avian leukaemia virus env-gp85 in spodoptera frugiperda cells by use of a baculovirus expression vector.we studied the genetic expression of gp85 of avian leukaemia virus (alv) subgroup a in a baculovirus/insect cell system. 5'terminal sequences of the gag gene were added to precede the alv gp85 sequence and a stop codon was introduced at the boundary of gp85 and gp37. the resulting construct was then cloned into the baculovirus transfer vector pacym1, which contains the polyhedrin promoter of autographa californica nuclear polyhedrosis virus (acnpv). cells of the insect spodoptera frugiperda (sf9 ...19902174957
general analysis of receptor-mediated viral attachment to cell surfaces.viruses are multivalent particles that attach to cells through one or more bonds between viral attachment proteins (vap) and specific cellular receptors. three modes of virus binding are presented that can explain the diversity in binding data observed among viruses. they are based on multivalency of attachment and spatial versus receptor saturation effects which are easily distinguished based upon simple criteria. mode 1 involves only monovalent virus/receptor binding. modes 2 and 3 involve mul ...19902177356
the p6.5 gene region of a nuclear polyhedrosis virus of orgyia pseudotsugata: dna sequence and transcriptional analysis of four late genes.the gene encoding the basic dna-binding protein (p6.5) of the multicapsid nuclear polyhedrosis virus of orgyia pseudotsugata (opmnpv) was localized by southern blot analysis using a cdna probe containing the autographa californica virus (acmnpv) p6.9 gene. the opmnpv p6.5 gene was mapped to the hindiii g fragment at map unit 67. nucleotide sequence and transcriptional analysis of a 3.26 kb region encompassing this area revealed four open reading frames (orfs 1 to 4) oriented in the same directio ...19902179466
expression and secretion of the bovine coronavirus hemagglutinin-esterase glycoprotein by insect cells infected with recombinant baculoviruses.a cdna fragment representing the hemagglutinin-esterase (he) gene of bovine coronavirus (bcv) was inserted into the genome of autographa californica nuclear polyhedrosis virus. infection of insect cells with the recombinant virus resulted in the production of a 120-kilodalton disulfide-linked dimeric form of the bcv he polypeptide. deletion of the carboxy-terminal hydrophobic domain from the he polypeptide resulted in secretion of a dimeric form of the truncated he polypeptide. the acetylesteras ...19902181154
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 ...19902181402
characterization of the da26 gene in a hypervariable region of the autographa californica nuclear polyhedrosis virus genome.a region of the baculovirus autographa californica nuclear polyhedrosis virus genome that is frequently found to be altered after serial passage of the virus in cell culture was characterized. sequence analysis of this region of the genome in wild-type and mutant viruses revealed that some of the mutations affected a 675 bp open reading frame, designated da26. the da26 gene was disrupted both by deletion and by insertion of sequences that resembled transposable elements. northern blot analysis o ...19902189022
rabies diagnostic reagents prepared from a rabies n gene recombinant expressed in baculovirus.a gene encoding the nucleoprotein (n) of rabies virus was inserted into the genome of the baculovirus autographa californica nuclear polyhedrosis virus. recombinant gene expression was controlled by the strong polyhedrin gene promoter. insect cells (spodoptera frugiperda) infected by a baculovirus recombinant containing the rabies virus n gene produced abundant amounts of a novel 55-kilodalton protein of a size comparable to that of the rabies virus n protein, as demonstrated by polyacrylamide g ...19902191008
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 w ...19902197369
expression of influenza a and b virus nucleoprotein antigens in baculovirus.full-length cdna clones of the nucleoprotein (np) genes of influenza a/ann arbor/6/60 and b/ann arbor/1/86 viruses were constructed from virion rna and subsequently expressed in spodoptera frugiperda (sf9) cells using the baculovirus vector, autographa californica nuclear polyhedrosis virus. western blot analysis of lysates prepared from sf9 cells infected with the recombinant viruses confirmed that the baculovirus-expressed np antigens were reactive with monoclonal antibodies specific for eithe ...19902197370
human acidic fibroblast growth factor overexpressed in insect cells is not secreted into the medium.we have overexpressed human acidic fibroblast growth factor (afgf) in spodoptera frugiperda sf9 cells from a cdna clone under the control of the promoter of the polyhedrin gene of the baculovirus autographa californica nuclear polyhedrosis virus. a 16.5-kd product was made in recombinant virus-infected cells that specifically reacted in immunoblots with various antibodies prepared against afgf. recombinant afgf was mitogenic for bhk21 cells and its activity was stimulated by heparin. the mechani ...19902200451
expression of the gene encoding firefly luciferase in insect cells using a baculovirus vector.a cdna encoding the firefly luciferase [photinus luciferin: oxygen 4-oxidoreductase (decarboxylating, atp-hydrolyzing), ec 1.13.12.7] was cloned downstream from the polyhedrin gene promoter of autographa californica nuclear polyhedrosis virus and expressed in spodoptera frugiperda clone-9 cells. synthesis of luciferase (luc) was accurately measured in insect cells growing in a 96-well plate, by a simple, rapid, nonradioactive, inexpensive and sensitive method based on fogging of x-ray film. luc ...19902205537
differential gene expression mediated by late, very late and hybrid baculovirus promoters.a series of recombinant viruses was constructed to study the regulation of expression from the promoter (pcap) for the major capsid-protein-encoding gene (vp39) of the baculovirus, autographa californica nuclear polyhedrosis virus. each virus of this series contains the cat reporter gene under the control of pcap or portions thereof. pcap regulation of cat was determined at the rna and enzyme activity levels, and was compared to cat gene expression driven by ppolh, the promoter of the polyhedrin ...19902205543
the expression of the autographa californica nuclear polyhedrosis virus genome in insect cells.this report presents a synopsis of recently published work in our laboratory on the molecular biology of the insect baculovirus autographa californica nuclear polyhedrosis virus (acnpv). the following studies have been summarized. (1) on the mode of transcription of the acnpv genome in insect cells. (2) translation of proteins encoded in the 81.2 to 85.0 map unit segment of acnpv. (3) inserts of insect cell dna in the acnpv genome. (4) expression of influenza (fowl plague) virus haemagglutinin i ...19902205973
expression and characterization of the chick nicotinic acetylcholine receptor alpha-subunit in insect cells using a baculovirus vector.a baculovirus transfer vector was constructed containing an entire cdna copy of the chick nicotinic acetylcholine receptor (nachr) alpha-subunit under control of the autographa californica nuclear polyhedrosis virus (acnpv) polyhedrin gene promoter. recombinant baculovirus was obtained by co-transfection of spodoptera frugiperda cells with infectious, wild-type acnpv dna and the transfer vector. polyhedrin-negative, recombinant viruses were identified which expressed the nachr alpha-subunit. the ...19902209600
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 ...19902216760
nucleotide sequence and characterization of the 39k gene region of autographa californica nuclear polyhedrosis virus.the complete nucleotide sequence of the pstl-k fragment of the autographa californica nuclear polyhedrosis virus (acmnpv) genome was determined. this region of the genome contains the delayed early 39k gene and v-ubi, a viral protein with homology to ubiquitine. in addition, pstl-k potentially encodes five other proteins. hybrid-select translation mapped nine viral-specific proteins to this fragment. three proteins were observed with both early and late rna; two of them comigrated with proteins ...19902219715
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 ...19902219726
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 polyh ...19902230725
[purification of autographa californica nuclear polyhedrosis virus dna and cloning of the polyhedrin gene].autographa califonica nuclear polyhedrosis virus (acnpv) dna from larvae of lepidopteran infected by acnpv has been isolated. the ecori fragments of acnpv dna have been cloned using plasmid pbr325 as a vector. the polyhedrin gene fragment containing recombinant plasmid pac-18 was isolated through in situ hybridization. for the convenience of further construction the ecori insertion fragment was cloned into puc-8. thus plasmid puac-2 was obtained. the results of restriction mapping of pac-18 and ...19902242284
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 associat ...19902243377
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 o ...19902254749
nucleotide sequence and transcriptional analysis of the hindiii p region of a temperature-sensitive mutant of autographa californica nuclear polyhedrosis virus.dna sequence analysis of the hindiii p region of a temperature-sensitive mutant of autographa californica nuclear polyhedrosis virus confirmed the specific amplification of 1.4 kb of viral dna from this region of the genome. the sequenced region included an open reading frame, translated in a counterclockwise direction, which would potentially encode a 74k protein. the amplified dna was contained within this open reading frame, resulting in in-frame amplifications of a domain within the protein. ...19902273394
expression of human granulocyte-macrophage colony-stimulating factor gene in insect cells by a baculovirus vector.a plasmid pac373gm-csf was constructed and co-transfected into spodoptera frugiperda (sf9) cells with wild-type autographa californica nuclear polyhedrosis virus (acnpv) dna. the recombinant virus vac373gm-csf was identified and purified by several rounds of plaque hybridization. by assaying the culture medium, we demonstrated recombinant virus infected sf9 cells expressing hgm-csf. recombinant hgm-csfs with apparent molecular masses of 14.5, 15.5 and 16.5 kda were detected by the western blot m ...19902403525
rate of increase of autographa californica nuclear polyhedrosis virus in trichoplusia ni larvae determined by dna:dna hybridization.the rate of increase and doubling time of the hob clone of autographa californica nuclear polyhedrosis virus (acmnpv-hob) in neonate trichoplusia ni larvae was determined by measuring the increase in viral dna through time following inoculation with average doses of 50 or 17,400 occlusion bodies per larva. changes in total dna and viral dna through time were followed by fluorescence spectroscopy and quantitative slot-blot dna:dna hybridization, respectively. total dna content (i.e., larval dna a ...19902405064
structure of preproattacin and its processing in insect cells infected with a recombinant baculovirus.from a cdna library made from fatbody of immunized hyalophora cecropia pupae, a full-length clone corresponding to basic attacin was isolated. the corresponding amino acid sequence suggests that basic attacin is synthesized as a 233-residue preproprotein. this was confirmed by cloning the cdna fragment encoding the basic attacin in the baculovirus autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter and expressing the protein in spodoptera frugiperda (sf9) cell ...19902406140
expression of the lassa virus nucleocapsid protein in insect cells infected with a recombinant baculovirus: application to diagnostic assays for lassa virus infection.the coding region of the gene for the nucleocapsid protein of lassa virus has been inserted into the genome of autographa californica nuclear polyhedrosis virus (acnpv) using the transfer vector pacym1, so that expression of the foreign dna is under the control of the promoter of the acnpv polyhedrin gene. infection of cultured spodoptera frugiperda cells with recombinant virus resulted in the synthesis of high levels of a protein that was indistinguishable from the authentic lassa virus protein ...19902406367
overlapping divergent transcripts mapping to the hindiii f region of the autographa californica nuclear polyhedrosis virus.we have mapped a set of transcripts that cross the xhoi site in the hindiii f fragment of the genome of the autographa californica nuclear polyhedrosis virus. these transcripts overlap at their 5' ends by about 550 bases and run in opposite directions. we have tentatively identified two open reading frames corresponding to the leftward transcript. the rightward transcript is present from 8 h post-infection (p.i.) to 24 h p.i.; the leftward transcripts are present from 2 h p.i. to 24 h p.i. the e ...19902406368
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 ...19902407804
autographa californica m nuclear polyhedrosis virus: microtubules and replication.progressive reorganization and depolymerization of microtubules corresponded with virus-induced rounding of autographa californica m nuclear polyhedrosis virus (acmnpv)-infected spodoptera frugiperda iplb-sf-21 cells, suggesting that microtubules were instrumental in maintaining the normal shape of these cells. depolymerization of all cortical and most of the paranuclear microtubules with colchicine also resulted in cell rounding, confirming this hypothesis. studies with aphidicolin and cyclohex ...19902408230
isolation of temperature sensitive mutants of autographa californica nuclear polyhedrosis virus: phenotype characterization of baculovirus mutants defective in very late gene expression.temperature sensitive mutants of autographa californica nuclear polyhedrosis virus were isolated following exposure of parental replicating virus to 5-bromo-2'-deoxyuridine. two mutants designated ts317 and ts538 were characterized in detail. both mutants synthesized viral dna but were defective in infectious extracellular virus production at the nonpermissive temperature suggesting defects in late gene regulation. pulse-labeling experiments indicated that a switch to late protein synthesis occu ...19902408231
continuous production of baculovirus in a cascade of insect-cell reactors.insect cells (spodoptera frugiperda) were cultured in a continuous stirred-tank reactor. the effluent was led to a cascade of another two reactors, each containing half the volume of the cell-growth reactor, where the cells were infected with autographa californica nuclear polyhedrosis virus. for about 10 days production of 10(7) polyhedra (virus particles embedded in a protein capsule) per cm3 was achieved. this short production time compared to previous experiments involving an analogous syste ...19901366563
multistage production of autographa californica nuclear polyhedrosis virus in insect cell cultures.the aim of our study was to establish an efficient system for the in vitro production of the insect pathogenic autographa californica nuclear polyhedrosis virus in a spodoptera frugiperda cell line. we optimized cultivation conditions for cell proliferation as well as for virus replication in a 1.5 litre stirred tank bioreactor. cell and virus propagation were found to be optimal at a constant oxygen tension of 40%. in order to provide sufficient nutrients during virus synthesis filtration and p ...19901366992
dna distribution and respiratory activity of spodoptera frugiperda populations infected with wild-type and recombinant autographa californica nuclear polyhedrosis virus.spodoptera frugiperda cells were infected with a wild-type autographa californica nuclear polyhedrosis virus and with a recombinant autographa californica nuclear polyhedrosis virus. the recombinant virus was derived from the wild-type virus and produced beta-galactosidase instead of polyhedrin. the changes in cell size, cell growth, viability, dna distribution, and respiratory activity were followed through the time course of the infection. the dna content as measured by flow cytometry of infec ...19901367465
transcription of the baculovirus polyhedrin gene reduces the levels of an antisense transcript initiated downstream.a late 3.2-kilobase (kb) rna initiated approximately 2 kb downstream of the 3' end of the autographa californica nuclear polyhedrosis virus polyhedrin-coding sequence and traversed the polyhedrin gene in an antisense direction. this rna was sense rna for the two open reading frames flanking the polyhedrin gene. a mutant virus, vxpoly, which differs from wild-type virus only at the essential rna initiation site in the polyhedrin promoter, exhibited higher levels of the 3.2-kb rna than did wild-ty ...19901692351
gene organization and transcription of ted, a lepidopteran retrotransposon integrated within the baculovirus genome.a single copy of the retrotransposon ted, from the moth trichoplusia ni (a lepidopteran noctuid), was identified within the dna genome of the baculovirus autographa californica nuclear polyhedrosis virus. determination of the complete nucleotide sequence (7,510 base pairs) of the integrated copy indicated that ted belongs to the family of retrotransposons that includes drosophila melanogaster elements 17.6 and gypsy and thus represents the first nondipteran member of this invertebrate group to b ...19901692964
categorizing some early and late transcripts directed by the autographa californica nuclear polyhedrosis virus.using an s1 mapping assay on rna from spodoptera frugiperda cells infected by the autographa californica nuclear polyhedrosis virus in the presence and absence of cycloheximide and aphidicolin, we can distinguish three classes of transcripts. first, there are those whose synthesis is blocked by the dna synthesis inhibitor aphidicolin and which are therefore late transcripts. these include the late transcript of the 39k gene and a late leftward transcript across the xhoi site in the hindiii-f reg ...19901698929
characterization of virus-like particles produced by a recombinant baculovirus containing the gag gene of the bovine immunodeficiency-like virus.the entire gag gene of the bovine immunodeficiency-like virus (biv) was inserted behind the strong polyhedron promoter of autographa californica nuclear polyhedrosis virus (acnpv). the resultant recombinant baculovirus (acnpv-bivgag) was used to infect insect cells in order to overexpress and characterize biv gag gene products. the infection resulted in the high-level expression of a protein similar in size to the predicted biv gag precursor (pr53gag). biv pr53gag was detected in acnpv-bivgag-in ...19901699349
expression of rat tyrosine hydroxylase in insect tissue culture cells and purification and characterization of the cloned enzyme.rat tyrosine hydroxylase has been expressed at high levels in spodoptera frugiperda cells using a baculovirus expression system. a cdna containing the coding region for pc12 tyrosine hydroxylase was inserted into the unique ecori site of the transfer vector pljc8 to yield the recombinant vector pljc9. spodoptera frugiperda cells were then co-infected with pljc9 and wild type autographa californica nuclear polyhedrosis virus. recombinant virus particles containing the cdna for tyrosine hydroxylas ...19901967606
expression of the human multidrug transporter in insect cells by a recombinant baculovirus.the plasma membrane associated human multidrug resistance (mdr1) gene product, known as the 170-kda p-glycoprotein or the multidrug transporter, acts as an atp-dependent efflux pump for various cytotoxic agents. we expressed recombinant human multidrug transporter in a baculovirus expression system to obtain large quantities and further investigate its structure and mechanism of action. mdr1 cdna was inserted into the genome of the autographa californica nuclear polyhedrosis virus under the cont ...19901970935
processing, secretion, and immunoreactivity of carboxy terminally truncated dengue-2 virus envelope proteins expressed in insect cells by recombinant baculoviruses.two recombinant baculoviruses were constructed by inserting via the transfer vector pacym1 the genes coding for the structural proteins of dengue (den)-2 virus downstream from the polyhedrin promoter of autographa californica nuclear polyhedrosis virus. the two recombinants differed in truncation of 26 and 71 amino acids, respectively, in the carboxy-terminal sequence of den-specific envelope (e) glycoprotein. recombinant den-2 e glycoproteins were processed and transported to the surface of spo ...19911984665
proteins encoded in the 81.2- to 85.0-map-unit fragment of autographa californica nuclear polyhedrosis virus dna can be translated in vitro and in spodoptera frugiperda cells.we have previously demonstrated that five open reading frames exist in the nucleotide sequence of the 81.2- to 85.0-map-unit (m.u.) segment of plaque isolate e of autographa californica nuclear polyhedrosis virus (acnpv) dna. the corresponding polypeptides are 9.8, 12.1, 36.6, 25.0, and 48.2 kda in size (c. oellig, b. happ, t. müller, and w. doerfler, j. virol. 63:1494, 1989), and we have investigated whether these proteins can be translated in infected cells. on subfragments of this viral dna s ...19911985220
identification of the very early transcribed baculovirus gene pe-38.we have started to identify early viral rnas that are transcribed at 1 h after inoculation to investigate the mechanism involved in the regulation of early gene expression of autographa californica nuclear polyhedrosis virus (acnpv). cloned viral dna fragments were hybridized to northern (rna) blots of polyadenylated rna isolated from spodoptera frugiperda cells at 1, 2, and 6 h postinfection to localize very early transcripts. subsequently we prepared a cdna library of polyadenylated rna transc ...19911987375
transient expression of the autographa californica nuclear polyhedrosis virus immediate-early gene, ie-n, is regulated by three viral elements.autographa californica nuclear polyhedrosis virus (acmnpv) is a double-stranded dna virus that expresses several immediate-early genes under the control of different promoters. the expression of one of these transcription units, ie-n, is shown here, by a transient expression assay, to be regulated by both cis- and trans-acting viral elements. the steady-state levels of ie-n mrna were very abundant soon after infection but were nearly undetectable during the late phase of the viral life cycle. an ...19911987380
identification and characterization of the ie-1 gene of orgyia pseudotsugata multicapsid nuclear polyhedrosis virus.the ie-1 gene of orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (opmnpv) was mapped between 95.7 and 97.1 map units on the viral genome. sequence analysis of the opmnpv ie-1 gene (opie-1) identified an open reading frame that coded for a predicted protein of 560 amino acids with a molecular weight of 64,775. transcriptional analysis of opmnpv-infected lymantria dispar (ld652y) cells identified two rnas homologous to the opie-1 open reading frame that were 1.7 and 1.9 kb in size. the ...19911989381
nucleotide sequence and transcript mapping of the hindiii f region of the autographa californica nuclear polyhedrosis virus genome.the organization of genes in the 2.95 kb ecori-sali fragment (0.7 to 3.0 map units) located within the hindiii f region of the autographa californica nuclear polyhedrosis virus genome was studied by a combination of dna sequencing, northern blot analysis, s1 mapping and primer extension analysis. in addition to the two divergent overlapping transcripts [leftward early (es1) and rightward late (es2)] previously reported, a third transcript which is present at 18 h p.i. and which also runs leftwar ...19911993873
nucleotide sequence of a gene essential for viral dna replication in the baculovirus autographa californica nuclear polyhedrosis virus.the nucleotide sequence of the 60.1- to 65.5-m.u. region of autographa californica nuclear polyhedrosis virus (acmnpv) was determined. seven large open reading frames were identified. two open reading frames potentially encoding gene products of 143 and 38 kda were found in the counterclockwise direction upstream of the p6.9 gene. four additional open reading frames were found in the opposite direction. analysis of the predicted amino acid sequence of the 143-kda gene revealed a potential leucin ...19911994581
characterization and purification of a functional rat glucocorticoid receptor overexpressed in a baculovirus system.the structure-function relationship of the oligomeric unactivated glucocorticoid receptor is not fully understood. an essential step in the process of understanding such a relationship involves the production of large quantities of the receptor. using a baculovirus expression system we have been able to overproduce a recombinant rat glucocorticoid receptor (rgr). a cdna coding for the entire rgr was introduced into the genome of the wild type baculovirus, autographa californica nuclear polyhedro ...19911995640
sequential rearrangement and nuclear polymerization of actin in baculovirus-infected spodoptera frugiperda cells.proper assembly of nucleocapsids of the baculovirus autographa californica nuclear polyhedrosis virus is prevented by cytochalasin d, a drug that interferes with actin microfilament function. to investigate the involvement of microfilaments in a. californica nuclear polyhedrosis virus replication, a fluorescence microscopy study was conducted that correlated changes in distribution of microfilaments with events in the life cycle of the virus. tetramethylrhodamine isothiocyanate-labeled phalloidi ...19911995943
the influence of antisense rna on transcriptional mapping of the 5' terminus of a baculovirus rna.s1 nuclease protection and primer extension analyses were used to determine the 5' end of the autographa californica nuclear polyhedrosis virus 603 open reading frame (orf) transcript upstream of and on the opposite strand to the polyhedrin gene. these analyses suggested that the 5' end of the 603 orf was located near the initiation site for polyhedrin gene transcription. primer extension products of reverse transcription of 603 rna were dependent on the presence of the taag sequence, an essenti ...19912005431
enhanced secretion from insect cells of a foreign protein fused to the honeybee melittin signal peptide.the baculovirus/insect cell system has been remarkably successful in yielding high levels of synthesis of many proteins which have been difficult to synthesize in other host/vector systems. the system is also capable of secreting heterologous proteins, but with generally low efficiency. we have increased the efficiency of secretion of the system by using signal peptides of insect origin to direct the secretion of a foreign protein. the precursor of the plant cysteine protease papain (propapain) ...19912016060
baculovirus-directed high level expression of the hepatitis delta antigen in spodoptera frugiperda cells.the hepatitis delta antigen (hdag) is a multifunctional protein. it forms the core-like structure of the hepatitis delta virus (hdv) but also enhances replication of hdv in the nucleus of the hepatocyte. a cdna fragment encoding hdag was inserted adjacent to the polyhedrin promoter of autographa californica nuclear polyhedrosis virus present in the baculovirus transfer vector pvl941. after transfection of spodoptera frugiperda (sf9) cells a recombinant baculovirus ac delta 1 was isolated and pur ...19912016595
molecular analysis of a baculovirus regulatory gene.to better understand the structure and function of a baculovirus regulatory gene, the nucleotide sequence of ie-n expressed by autographa californica nuclear polyhedrosis virus was determined. the 2.0-kb psti-ecorv restriction fragment (97.5 to 98.9 mu) encodes the upstream regulatory sequences, open reading frame, and downstream sequences of the immediate early ie-n gene. using a convenient restriction site, the 285-bp promoter of ie-n was divided into two functional regions as defined by trans ...19912024466
autographa californica m nuclear polyhedrosis virus and cytochalasin d: antagonists in the regulation of protein synthesis.during infection by the baculovirus autographa californica multiple nuclear polyhedrosis virus (acmnpv), synthesis of host proteins in iplb-sf-21 cells is inhibited. we report here that cytochalasin d (cd), a drug that specifically interacts with actin, behaved as an antagonist in the virus-mediated shutdown of host proteins actin and tubulin. in uninfected cells, cd caused an increase in actin synthesis but had no apparent effect on tubulin synthesis. in infected cells, cd similarly enhanced ac ...19912024491
expression and cell membrane localization of rat m3 muscarinic acetylcholine receptor produced in sf9 insect cells using the baculovirus system.a recombinant baculovirus bearing the cdna coding for the rat muscarinic acetylcholine receptor subtype m3 placed under the control of the autographa californica nuclear polyhedrosis virus polyhedrin gene promoter, was constructed. polymerase chain reaction screening was used to identify the recombinant baculovirus. northern blot analysis of total rna from insect cells infected with the recombinant baculovirus indicated that the transcripts were abundant. binding assays carried out with the musc ...19912037073
host range expansion by recombination of the baculoviruses bombyx mori nuclear polyhedrosis virus and autographa californica nuclear polyhedrosis virus.the mechanisms of host specificity of nuclear polyhedrosis viruses (npvs) (baculoviridae) were analyzed after coinfection of bombyx mori npv (bmnpv) and one of four distinct groups of spodoptera litura npv (slnpv), including an autographa californica npv (acnpv) variant (s. maeda, y. mukohara, and a. kondo, j. gen. virol. 71:2631-2639, 1990), into various lepidopteran cell lines. replication of bmnpv in nonpermissive cells (tn-386, sf-21, and cls-79) was induced by coinfection with acnpv but not ...19912041087
expression of an enhancer-binding protein in insect cells transfected with the autographa californica nuclear polyhedrosis virus ie1 gene.the baculovirus autographa californica nuclear polyhedrosis virus contains an element known as homologous region 5 (hr5) which is an enhancer of delayed-early viral gene expression. to begin to identify proteins that interact with hr5, dna-protein interactions were analyzed by using extracts from spodoptera frugiperda cells and a fragment of dna containing the left half of the hr5 enhancer. this 252-bp dna fragment contains two copies of a 30-bp direct repeat (dr30) and two copies of a 24-bp imp ...19912041088
baculovirus vectors for multiple gene expression and for occluded virus production.the nature and properties of a variety of plasmids are described that facilitate the construction of baculovirus vectors for expression of one or more heterologous genes. the plasmids are designed for use with autographa californica nuclear polyhedrosis virus, acmnpv, as a vector for protein production in insect cells and/or insect larvae. several plasmids described here facilitate the simultaneous insertion and expression of two different genes. some vector systems allow high and equal levels o ...19912055465
expression of the x protein of hepatitis b virus in insect cells using recombinant baculoviruses.the baculovirus system was used to express the x protein of human hepatitis b virus (hbv). the x open reading frames (x orfs) from cloned viral dna of the hbv subtypes ayw and adr were introduced into the genome of autographa californica nuclear polyhedrosis virus (acnpv). the hbv-dna of subtype adr derived from a hepatocellular carcinoma contains an x orf and a 5' extended prex/x orf, which were both used to construct x recombinant baculoviruses. infection of sf9 insect cells with these recombi ...19912058090
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 f ...19912062383
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 insert ...19912072443
changes in macromolecular synthesis of gypsy moth cell line iplb-ld652y induced by autographa californica nuclear polyhedrosis virus infection.the aberrant replication of the autographa californica multiple-enveloped nuclear polyhedrosis virus (acmnpv) in the lymantria dispar cell line iplb-ld652y was used as a model system for the investigation of factors regulating baculovirus host specificity. a previous study of this system indicates that viral gene expression in infected cells is extremely attenuated and subsequently all cellular and viral protein synthesis is inhibited. in the present study, infection of iplb-ld652y cells with ac ...19911709681
enzyme activities in four different forms of human immunodeficiency virus 1 pol gene products.five cassettes of the pol gene of human immunodeficiency virus 1 were constructed and inserted under the control of the polyhedrin gene promoter of autographa californica nuclear polyhedrosis virus by homologous recombination. the first cassette polf contains the full-length pol open reading frame; the second cassette pol100 starts with the first aug codon of the pol gene and deletes 103 amino acids from the amino terminus of the pol gene product; the third cassette pol97 deletes the entire prot ...19911711203
expression of post-translational processing of preprocecropin a using a baculovirus vector.a cdna fragment encoding preprocecropin a was inserted into the baculovirus autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. the gene was expressed in recombinant-infected last instar larvae of trichoplusia ni and in diapausing pupae of hyalophora cecropia. the identity of the recombinant product was established by electrophoresis with detection of antibacterial activity and mass spectrometry. the prepropeptide had been correctly processed including remova ...19911712729
production of recombinant proteins by baculovirus-infected gypsy moth cells.an experimental study was undertaken to evaluate alternative insect cell lines to sf9 [from spodoptera frugiperda (fall armyworm)] for the production of recombinant proteins. insect cell lines from two different organisms were considered: iplb-ldeita (ldeita) from lymantria dispar (gypsy moth) and iplb-hvt1 (hvt1) from heliothis virescens (tobacco budworm). both ldeita and hvt1 produced higher total activity levels of recombinant beta-galactosidase in monolayer culture than sf9 after infection w ...19911367995
secretion of biologically active leech hirudin from baculovirus-infected insect cells.the thrombin inhibitor, hirudin, from the leech hirudo medicinalis, is the most powerful natural anticoagulant known. it has been characterized as a polypeptide of 65 amino acids which exhibits its anticoagulant properties by binding tightly and specifically to alpha-thrombin. the potency and specificity of hirudin have generated interest on its possible use in the treatment or prophylaxis of various thrombotic diseases. we have used the baculovirus expression system to efficiently produce activ ...19911647362
high level expression of dna polymerases from herpesviruses.the dna polymerase genes of human cytomegalovirus (hcmv) and varicella-zoster virus (vzv) were inserted separately into the polyhedrin gene of autographa californica nuclear polyhedrosis virus (acnpv) by cotransfection of spodoptera frugiperda (sf9) cells with baculovirus transfer vectors carrying the genes and acnpv infectious dna. infection of sf9 cells with the recombinant viruses resulted in expression from the polyhedrin promoter of proteins of the expected mrs. these proteins possessed dna ...19911649906
functional expression of the human growth factor activatable na+/h+ antiporter (nhe-1) in baculovirus-infected cells.we constructed a recombinant baculovirus, based on autographa californica nuclear polyhedrosis virus, containing the human na+/h+ antiporter cdna under control of the polyhedrin promoter. when infected with this recombinant baculovirus, the sf9 cell line, derived from spodoptera frugiperda, expresses a fully functional na+/h+ antiporter as measured by the generation of an amiloride-sensitive na+ influx in response to an acid load. the na+/h(+)-exchange activity, not detectable in sf9 cells, emer ...19911654101
processing and antigenicity of entire and anchor-free spike glycoprotein s of coronavirus tgev expressed by recombinant baculovirus.the s gene of transmissible gastroenteritis virus (tgev) was inserted into the genome of autographa californica nuclear polyhedrosis virus (acnpv) using the transfer plasmid pvl941. infection of sf9 insect cells with the recombinant virus resulted in the synthesis of a 175k polypeptide which was able to trimerize and was transported to the cell surface as is the authentic tgev s protein. despite the lack of complete carbohydrate processing, the recombinant s protein exhibited antigenic propertie ...19911660201
a functional hepatitis b virus x protein produced in insect cells.we have constructed a recombinant baculovirus from autographa californica nuclear polyhedrosis virus, called acx, that expresses the gene encoding the hepatitis b virus x protein in infected spodoptera frugiperda (sf21ae) insect cells. a 16.5-kda monomer and a 33-kda dimer of the x protein were detected in extracts from acx-infected cells on immunoblots using a polyclonal anti-x antibody. the biological activity of the insect cell-produced x protein was assayed by fusing acx-infected sf21ae cell ...19911660211
temporal analysis and spatial mapping of lymantria dispar nuclear polyhedrosis virus transcripts and in vitro translation polypeptides.genomic expression of the lymantria dispar multinucleocapsid nuclear polyhedrosis virus (ldmnpv) was studied. viral specific transcripts expressed in cell culture at various times from 2 through 72 h postinfection were identified and their genomic origins mapped through northern analysis. sixty-five distinct transcripts were identified in this analysis. most viral transcripts were expressed late in infection, and originated from throughout the viral genome. viral polypeptides expressed in infect ...19911685048
expression and characterization of recombinant mouse beta 2-microglobulin type a in insect cells infected with recombinant baculoviruses.the murine beta 2-microglobulina cdna was cloned into pac373 and pvl941 transfer vectors and introduced via homologous recombination into the genome of autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. both types of recombinant baculoviruses were isolated and used to infect spodoptera frugiperda (sf9) lepidopteran cells. beta 2m was synthesized at a substantially higher rate in cells infected with the pvl941-derived virus than when the pac373-based virus wa ...19911754712
effect of an autographa californica nuclear polyhedrosis virion component(s) on dna synthesis and growth in several insect cell lines.thirteen different insect cell lines representing three different orders were infected with autographa californica nuclear polyhedrosis virus (acmnpv) whose genome had been inactivated in situ by photochemical means or by short wave uv irradiation. changes in rates of cellular dna synthesis, as measured by [3h]thymidine incorporation, and cell growth were subsequently measured at various times post infection. seven cell lines exhibited a significant decline in [3h]thymidine incorporation (compar ...19911759911
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