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At least 811 records · Page 45Linked to original sources
PubMed2001

Production of functional antibodies generated in a nonlytic insect cell expression system.

A monoclonal antibody directed against the type 2 adenovirus (Ad2) penton base protein was cloned and expressed in Spodoptera frugiperda (Sf9) cells using a nonlytic vector system. The coding sequences for the immunoglobulin light and heavy chains were placed under the control of the Orgyia pseudotsugata multicapsid nucleopolyhedrosis virus immediate-early 2 (OpIE2) promoter. Transfected Sf9 cells continuously secreted the antibody which retained the ability to recognize both native and recombinant Ad2 penton base proteins. Bifunctional penton base antibodies were also generated by fusing a gene for a growth factor or a cytokine at the 3' end of the Ig constant heavy chain domain. The quantity and activity of recombinant antibodies generated in the nonlytic insect cell system could be determined relatively quickly compared to other expression systems. Moreover, these recombinant proteins were not subjected to proteolytic degradation as frequently occurs during baculovirus-mediated cell lysis and the levels of recombinant antibodies produced in the nonlytic system were comparable to those reported for cytolytic baculovirus vectors.

Adenoviridae↗
PubMed2001

Expression of C1 esterase inhibitor by the baculovirus expression vector system: preparation, purification, and characterization.

C1 esterase inhibitor (C1INH) is an important regulator of the classical complement pathway. Hereditary deficiency of C1INH causes angioedema of the skin, gut, and respiratory tissues that may be fatal. C1INH replacement therapy may be lifesaving for patients with this disorder. The objective of this study was to evaluate the use of the baculovirus expression vector system for mass producing biologically active human recombinant (rC1INH). A recombinant baculovirus was constructed coding the human native (nC1INH) sequence under control of the polyhedrin promoter. Spodoptera frugiperda Sf-9 insect cells were infected with this recombinant baculovirus in a medium-scale (10-L) bioreactor to produce rC1INH with a specific activity of 45 U/mg. Purification of rC1INH from the culture harvested at 60 h postinfection yielded 5.9 microg rC1INH/mL supernatant of a 75-kDa product with a specific activity of 31,000 U/mg purified rC1INH compared to 71,000 U/mg purified nC1INH from human serum using the same procedure. This rC1INH was about 25 kDa smaller than nC1INH, suggesting that Sf-9 cells express underglycosylated rC1INH. Glycan analysis showed that both N-glycan and O-glycan chains were present in rC1INH. The N-glycan chains, released using PNGaseF and fluorescently labeled, were analyzed using exoglycosidase treatment and capillary electrophoresis. Their high-mannose structure was consistent with the known failure of the insect cell glycosylation pathway to afford the fully elaborated biantennary structures found on human native nC1INH.

Animals↗
PubMed2001

Expression and purification of Manduca sexta prophenoloxidase-activating proteinase precursor (proPAP) from baculovirus-infected insect cells.

Analogous to human thrombin, prophenoloxidase-activating proteinase (PAP) is a terminal enzyme of a serine proteinase cascade in the tobacco hornworm Manduca sexta. In order to purify and study the activating enzyme for PAP from this insect, we produced the zymogen of PAP (proPAP) in a bacterial expression system. The affinity-purified protein was then used as an antigen to generate a specific rabbit antiserum. Immunoblot analysis indicated that the proPAP was present at a low level in Manduca larval hemolymph, but was induced by six- to eightfold in larvae that had been injected with Escherichia coli or Micrococcus lysodeikticus. To produce the native proenzyme for functional analyses, we constructed a recombinant baculovirus to infect Spodoptera frugiperda Sf21 cells. ProPAP was secreted into the medium at a low concentration of approximately 0.37 mg/liter under the optimal conditions. We then developed a simple, efficient scheme to enrich and purify this protein, which involves two lectin affinity and one HPLC ion-exchange chromatographic steps. Immunoblot analysis following SDS-polyacrylamide gel electrophoresis indicated that the recombinant proPAP is nearly identical in mobility to the zymogen from Manduca hemolymph. After the purified proPAP was added to the larval hemolymph, it was readily activated by an unknown proteinase in the presence of M. lysodeikticus.

Animals↗
PubMed1995

Proteolysis of p6.9 induced by cytochalasin D in Autographa californica M nuclear polyhedrosis virus-infected cells.

Cytochalasin D, a fungus-derived compound that interferes with actin polymerization, inhibits Autographa californica M nuclear polyhedrosis virus production in infected Spodoptera frugiperda (IPLB-Sf-21) cells. Cytochalasin D appears to inhibit nucleocapsid morphogenesis by interfering with nucleoprotein packaging. We were interested in determining, therefore, whether the drug affected the synthesis or processing of p6.9, the major core protein involved in nucleoprotein packaging. We found that cytochalasin D had no effect on the synthesis, phosphorylation, or dephosphorylation of p6.9, but that it induced the proteolysis of p6.9, an effect which could account for the inhibition of nucleocapsid morphogenesis. We also determined that the cytochalasin D-induced proteolysis of p6.9 was reversible upon removal of the drug, even in the absence of protein synthesis.

Actins↗
PubMed1995

Identification and characterization of a second putative origin of DNA replication in a baculovirus of Orgyia pseudotsugata.

A 7.5-kb region (96.8-2.5 m.u.), called Op5, of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome that contains an origin of DNA replication was characterized. This region replicates several times more efficiently and is unrelated to the previously identified putative origin of replication located on the viral HindIII-N fragment. In contrast to HindIII-N, the origin on Op5 contains repeated sequences with limited sequence identity to the homologous regions from AcMNPV. In isolation, these repeated sequences were not sufficient for origin activity in OpMNPV-infected Lymantria dispar cells, but required an additional 1.3 kb of sequences located to the left of the repeats. Four regions of the OpMNPV genome that crosshybridize with the repeated region were also found to replicate in our infection-dependent DNA replication assay. A deletion clone of Op5 that replicates efficiently in OpMNPV-infected L. dispar cells, was found to replicate at less than 2% the replication level of AcMNPV hr2 in AcMNPV-infected Spodoptera frugiperda cells.

Animals↗
PubMed1995

The Autographa californica nuclear polyhedrosis virus homologous region 1a: identical sequences are essential for DNA replication activity and transcriptional enhancer function.

Hr1a consists of two 30-bp imperfect palindrome sequences separated by 58 bp and each palindrome contains a naturally occurring EcoRI site at its core. Plasmid subclones of the hr1a-containing AcMNPV HindIII-N fragment were examined for their ability to replicate in virus-infected (Spodoptera frugiperda) Sf9 cells, and to stimulate transcription when linked in cis with a 39K gene promoter-beta-glucuronidase fusion and cotransfected into cells along with a plasmid (ple-1) containing the gene encoding the trans-acting factor IE-1. Only those plasmids containing hr1a underwent infection-dependent replication and were able to stimulate transcription. Sequences mapping to the left of hr1a were required for maximal levels of replication. A single palindrome from hr1a was sufficient for supporting plasmid replication and for stimulating transcription, although both activities were more efficient when both palindromes were present. Plasmids containing only one-half of a palindrome or disruptions of the central EcoRI core either did not replicate, or replicated very poorly, and did not exhibit enhanced transcriptional activity from the 39K gene promoter. Gel retardation experiments showed that labeled hr1a-containing DNA fragments had retarded migration after incubation with extracts from cells transfected with ple-1. Supershift experiments using polyclonal antibodies to IE-1 indicated that IE-1 is a component of the protein complex bound to hr1a. Fragments containing disruptions of the EcoRI-core still bound IE-1, as shown by gel retardation assays, indicating that IE-1 binding alone is not sufficient to allow replication and transcriptional enhancement.

Animals↗
PubMed1995

Cloning and expression of the nucleoprotein of peste des petits ruminants virus in baculovirus for use in serological diagnosis.

Peste des petits ruminants (PPR) is a viral disease of goats and sheep characterized by erosive stomatitis, enteritis, and pneumonia. The virus is a member of the family Paramyxoviridae and the genus Morbillivirus. The disease has high morbidity and mortality rates and has a substantial economic impact in developing countries. We have cloned and sequenced the cDNA of the nucleocapsid (N) gene of the Nigeria 75/1 strain of PPR virus (PPRV). A comparison of its nucleotide and deduced amino acid sequence with those of the N gene of the tissue culture-attenuated strain of PPRV was performed. A divergence of 8.9 and 5.0% was found at the nucleotide and amino acid level, respectively. A recombinant baculovirus that expresses the N protein in insect cells and larvae (Spodoptera frugiperda) was generated. The recombinant protein, characterized by Western blot analysis, was shown to have a molecular weight of 58 kDa and was recognized by anti-PPRV antibodies. The recombinant protein was used successfully as a coating antigen in an enzyme-linked immunosorbent assay for the serological diagnosis of PPRV.

Amino Acid Sequence↗
PubMed1995

Identification and analysis of a putative origin of DNA replication in the Choristoneura fumiferana multinucleocapsid nuclear polyhedrosis virus genome.

A recombinant plasmid containing the Choristoneura fumiferana multinucleocapsid nuclear polyhedrosis virus (CfMNPV) HindIII R fragment (m.u. 2.2-3.9) was shown to undergo CfMNPV infection-dependent DNA replication in Cf-124T cells. Replication of this DNA sequence was detectable by 24 hr p.i. and did not appear to have resulted as a consequence of recombination with the virus genome. Replication was inhibited by mimosine, an inhibitor of eukaryotic DNA replication. These data suggest that HindIII R of CfMNPV DNA contains an origin of DNA replication which we call ori1. HindIII R contains five GC-rich and three AT-rich regions and a 0.9-kb homologous repeat region 1 (hr1). Two short 440- and 740-bp contiguous sequences at the right end of the HindIII R fragment separately exhibited limited ori function. HindIII R subfragments with optimal ori activity contained a cluster of repeated and inverted sequences including nine copies of a 50-bp homologous repeat sequence (hr1a to hr1i) within hr1. The CfMNPV hr1 sequence was somewhat homologous with the homologous repeat (hr) of the putative Autographa californica MNPV (AcMNPV) replication origins. HindIII Y, another CfMNPV DNA fragment containing an hr sequence, hr3, also supported infection-dependent DNA replication, suggesting that it too contains an ori. Although replication of a putative AcMNPV origin (HindIII Q) was detectable in CfMNPV-infected Cf-124T cells, replication of CfMNPV HindIII R was not detectable in AcMNPV-infected Spodoptera frugiperda cells.

Animals↗
PubMed1995

Replication of Orgyia pseudotsugata baculovirus DNA: lef-2 and ie-1 are essential and ie-2, p34, and Op-iap are stimulatory genes.

A transient DNA replication assay was used to identify genes located within m.u. 90.5-7.0 of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome that influenced replication of a reporter plasmid containing an OpMNPV origin of replication, when cotransfected into uninfected Lymantria dispar cells. The viral transactivator ie-1 and a 2.4-kb subclone were found to be essential for replication. The 2.4-kb region was sequenced and open reading frames were identified. Replication assays using subclones from this region identified a gene called late expression factor 2 (lef-2), as the essential replication gene. The OpMNPV lef-2 gene encodes a protein with a predicted molecular weight of 22.7 kDa (204 amino acids) and exhibits 54.7% amino acid sequence identity with its homolog in the genome of the Autographa californica MNPV. Transcriptional mapping using both Northern blot and S1 nuclease protection assays demonstrated that OpMNPV lef-2 was expressed at both early and late times postinfection as a transcript of about 1.6 kb. The early transcript initiated approximately 30 nt downstream of a TAATA box, whereas the late transcript initiated from within a late promoter consensus motif. In addition, we identified three genes stimulatory for DNA replication including two OpMNPV transcriptional activators (ie-2 and p34) and Op-iap, which is the functional analog of the AcMNPV p35 gene that inhibits apoptosis in AcMNPV-infected Spodoptera frugiperda cells.

Amino Acid Sequence↗
PubMed1995

Immune protection conferred by the baculovirus-related glycoprotein of Thogoto virus (Orthomyxoviridae).

The coding region of segment 4 of Thogoto (THO) virus, a tick-borne member of the Orthomyxoviridae, was expressed in a baculovirus system under the control of the polyhedrin promoter. This construct expressed authentic envelope glycoprotein as determined by size and antigenic reactivity with a panel of monoclonal antibodies (MAbs). Immunization of hamsters with Spodoptera frugiperda (Sf21) cells infected with the recombinant baculovirus induced neutralizing and protective antibodies against virus challenge; control hamsters developed clinical disease with high-titer viremia 3 days postchallenge. In contrast to hamsters, guinea pigs are comparatively resistant to THO virus infection but support nonviremic transmission between cofeeding infected and uninfected ticks. However, when uninfected ticks fed on guinea pigs immunized with the baculovirus recombinant, only 2% became infected following virus challenge of the animals compared to 26% of ticks on control nonimmune guinea pigs. Furthermore, neutralizing MAbs specific for THO viral glycoprotein protected mice against lethal challenge with THO virus; nonneutralizing MAbs specific for the glycoprotein, which inhibit THO viral agglutinating activity, did not induce a protective response. Thus at least in the murine model, protective immunity is conferred by antibodies directed against the neutralizing epitope(s) of the baculovirus-related glycoprotein of THO virus.

Animals↗
PubMed1996

Sequence, function, and phylogenetic analysis of an ascovirus DNA polymerase gene.

We have sequenced a 5.5-kb region of the DNA genome of the Spodoptera Ascovirus (SAV) containing a DNA polymerase gene. The gene codes for a 1104-amino-acid polypeptide with seven motifs characteristic of DNA polymerases and three additional motifs associated with polymerases possessing 3' to 5' exonuclease activity. The SAV DNA polymerase gene was able to functionally substitute for a baculovirus DNA polymerase gene in a transient assay that relies on origin-specific reporter plasmid DNA replication. Analysis of the predicted DNA polymerase sequence using neighbor-joining and protein parsimony algorithms indicated that this gene was only distantly related to other known viral and cellular DNA polymerases. The SAV DNA polymerase gene is the first ascovirus gene to be identified and sequenced. The molecular phylogenetic analyses of this gene supports the placement of insect ascoviruses in a separate virus family.

Amino Acid Sequence↗
PubMed1996

Actin binding and proteolysis by the baculovirus AcMNPV: the role of virion-associated V-CATH.

Infection of larvae by Autographa californica M nuclear polyhedrosis virus (AcMNPV) results in liquefaction of susceptible hosts, presumably due to the breakdown of cells and extracellular matrices. In Spodoptera frugiperda tissue culture cells, infection leads to dramatic rearrangement and eventual destruction of the actin cytoskeleton. The first of these rearrangements is the formation of actin cables in the cytoplasm of the cell. Cable formation requires release of the budded virus (BV) nucleocapsid from the endosome, but does not require new protein synthesis, suggesting that the nucleocapsid contains the activity necessary to induce cable formation. We have identified two distinct BV-associated actin-targeting activities. The first, a nucleocapsid-associated actin-binding activity, enabled actin copelleting and may also induce actin polymerization and cable formation. The second activity, associated with the nucleocapsid and envelope fractions of BV, was a protease that specifically degraded actin. This protease was identified as V-CATH, a cathepsin L-like protease that is a product of the AcMNPV v-cath gene.

Actins↗
PubMed1996

Trans-activation of a cell housekeeping gene promoter by the IE1 gene product of baculoviruses.

Protein IE1 is the product of a baculovirus gene, ie1, that is activated immediately upon entrance of the viral genome into the cell nucleus. This protein was previously shown to be a trans-regulator of viral genes whose products are required for initiation of the infectious cycle including viral DNA replication. To test whether the IE1 protein is also capable of trans-regulating nuclear genes of the host in vitro and in vivo, we transfected the ie1 gene of Bombyx mori nuclear polyhedrosis virus (BmNPV) into silkworm Bm5 tissue culture cells together with expression cassettes directing expression of chloramphenicol acetyl transferase or juvenile hormone esterase under the control of the cytoplasmic actin A3 gene promoter of B. mori. Cotransfection with the ie1 gene resulted in a dramatic increase in the amount of the two enzymes expressed in the transfected cells. The increased enzyme activities correlate with an increased accumulation of the corresponding mRNAs, and the latter is caused by an increase in the rate of transcription directed by the cytoplasmic actin gene promoter. The chromosomal cytoplasmic actin gene of Bm5 cells is also upregulated upon transfection of the cells with the ie1 gene. However, infection of cells with BmNPV does not cause an increase in the level of expression of the endogenous cytoplasmic actin gene. Thus, the effect of IE1 on the transcriptional properties of the cytoplasmic actin gene vary depending on whether IE1 is expressed in isolation or in the context of a viral infection. The trans-activating effects of BmNPV ie1 gene expression on the silkmoth actin promoter are also evident in Spodoptera frugiperda Sf21 and Choristoneura fumiferana Cf1 tissue culture cells. Finally, the ie1 gene of Autographa californica nuclear polyhedrosis virus can substitute for its BmNPV counterpart in all cell lines tested.

Actins↗
PubMed1996

CfMNPV blocks AcMNPV-induced apoptosis in a continuous midgut cell line.

Morphological and molecular changes produced by Autographa californica nuclear polyhedrosis virus (AcMNPV) infection in a permissive cell line, IPLB-SF-21AE (SF-21), of Spodoptera frugiperda and a nonpermissive cell line, FPMI-CF-203 (CF-203), of Choristoneura fumiferana are described. CF-203 cells inoculated with AcMNPV showed a DNA ladder and morphological changes such as plasma membrane granulation, blebbing, and nuclear fragmentation, which are characteristic of apoptosis. Typical virus replication and occlusion body (OB) production were seen in SF-21 cells inoculated with AcMNPV and no apoptosis-like symptoms were observed. mRNA for the apoptosis suppressor gene p35 was detected 9 hr later in AcMNPV-inoculated CF-203 cells than in SF-21 cells. Only a trace amount of mRNA for the AcMNPV-inhibitor of apoptosis homologue (Ac-iap) gene and no mRNAs for the late genes, AcMNPV-polyhedrin (Ac-polh) and AcMNPV-p10 (Ac-p10), were detected in AcMNPV-inoculated CF-203 cells. Inoculation of CF-203 cells with CfMNPV at least 12 hr prior to inoculation with AcMNPV prevented apoptosis-like cell death, and mRNAs for Ac-iap, Ac-polh, and Ac-p10 genes were expressed resulting in successful virus replication and OB production.

Animals↗
PubMed1997

Characterization of P91, a protein associated with virions of an Orgyia pseudotsugata baculovirus.

Polyclonal antiserum produced against preoccluded virions from the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) was used to screen an OpMNPV lambda gt11 expression library. The insert from one of the immunoreactive phage isolates hybridized to OpMNPV orf86 (p91), a 2460-bp (819 amino acids) open reading frame that encodes a predicted protein of 91 kDa. Antibodies generated against a maltose binding protein-P91 fusion detected a band of approximately 91 kDa on Western blots of extracts of OpMNPV-infected Lymantria dispar cells. This band was first observed at 18 hr p.i. and was present at all later time points. Similar results using this antiserum were seen with a time course of Autographa californica-infected Spodoptera frugiperda cells. Localization of P91 by confocal immunofluorescence microscopy showed that the protein was concentrated near the nuclear membrane and at late times p.i. was most concentrated near polyhedra. Immunoelectron microscopy indicated that P91 was present in both the capsid and envelope surrounding the capsid of occlusion-derived virions. Fractionation studies employing NP-40 and Western blot analysis indicated that P91 was associated with the capsid structure.

Amino Acid Sequence↗
PubMed1998

Characterization of the acidic domain of the IE1 regulatory protein from Orgyia pseudotsugata multicapsid nucleopolyhedrovirus.

This study presents a detailed analysis of the acidic N-terminal region of the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) transactivator IE1. The N-terminal region of IE1 is rich in acidic amino acids and has been hypothesized to be an acidic activation domain. Removal of the N-terminal 126 amino acids containing the acidic domain of IE1 resulted in complete loss of transactivation activity, indicating that this region is essential for transactivation. The OpMNPV acidic domain was replaced with the archetype acidic activation domain from VP16 and the acid-rich region of Autographa californica multicapsid NPV (AcMNPV) IE1. These chimeric constructs were fully capable of transactivation in transient assays. The chimeric OpMNPV IE1s containing the herpes simplex virus VP16 and AcMNPV IE1 acidic activation domains consistently transactivated a reporter gene to higher levels than the OpMNPV IE1 acidic activation domain. Transactivation by the chimeric constructs is enhanced synergistically when cotransfected with IE2 into Lymantria dispar and Spodoptera frugiperda cells. Both N- to C-terminal and C- to N-terminal deletions of the OpMNPV acidic activation domain were constructed to define functional domains within the OpMNPV IE1 acidic activation domain. At least two potential activation domains were identified. Within each of these domains, two core regions at amino acids 28-43 and amino acids 113-124 were identified that were similar to core regions of VP16 and GAL4, which contain predominately acidic and bulky hydrophobic amino acids.

Amino Acid Sequence↗
PubMed1999

Identification of additional genes that influence baculovirus late gene expression.

We were unable to confirm transient late gene expression using constructs of 18 genes that had been reported to support Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) late gene expression when transfected into Spodoptera frugiperda cells [Lu, A., and Miller, L. K. (1995). J. Virol. 69, 975-982]. Three genes (orf66, orf68, and orf41) were included, all or in part, in the constructs used in that study, but they had not been independently tested. Therefore we investigated these and neighboring orfs for their influence on late gene expression. We found that orf41 was required for late gene expression and that sequences within orf45 appeared to be required for the expression of orf41. Although orf66 and orf68 did not appear to affect late gene expression, orf69 stimulated expression. orf69 was found to have high homology to recent entries in GenBank from a variety of organisms. In addition, it was found that orf121, which was shown to be involved in early gene expression, and the viral homolog of pcna did not influence late gene expression.

Animals↗
PubMed1999

Host cell receptor binding by baculovirus GP64 and kinetics of virion entry.

GP64 is the major envelope glycoprotein from budded virions of the baculoviruses Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). To examine the potential role of GP64 as a viral attachment protein in host cell receptor binding, we generated, overexpressed, and characterized a soluble form of the OpMNPV GP64 protein, GP64solOp. Assays for trimerization, sensitivity to proteinase K, and reduction by dithiothreitol suggested that GP64solOp was indistinguishable from the ectodomain of the wild-type OpMNPV GP64 protein. Virion binding to host cells was analyzed by incubating virions with cells at 4 degrees C in the presence or absence of competitors, using a single-cell infectivity assay to measure virion binding. Purified soluble GP64 (GP64solOp) competed with a recombinant AcMNPV marker virus for binding to host cells, similar to control competition with psoralen-inactivated wild-type AcMNPV and OpMNPV virions. A nonspecific competitor protein did not similarly inhibit virion binding. Thus specific competition by GP64solOp for virion binding suggests that the GP64 protein is a host cell receptor-binding protein. We also examined the kinetics of virion internalization into endosomes and virion release from endosomes by acid-triggered membrane fusion. Using a protease sensitivity assay to measure internalization of bound virions, we found that virions entered Spodoptera frugiperda Sf9 cells between 10 and 20 min after binding, with a half-time of approximately 12.5 min. We used the lysosomotropic reagent ammonium chloride to examine the kinetics of membrane fusion and nucleocapsid release from endosomes after membrane fusion. Ammonium chloride inhibition assays indicated that AcMNPV nucleocapsids were released from endosomes between 15 and 30 min after binding, with a half-time of approximately 25 min.

Animals↗