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At least 595 records · Page 33Linked to original sources
PubMed1990

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

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

Animals↗
PubMed1996

Studies of Choristoneura fumiferana nuclear polyhedrosis virus gene expression in insect cells.

To investigate the mechanisms regulating baculovirus virulence and host range we have begun to study Choristoneura fumiferana nuclear polyhedrosis virus (CfMNPV) and its gene expression in permissive and nonpermissive cells. We have identified and mapped three genes on the CfMNPV genome. The polyhedrin gene is located from 0.0 to 2.0 m.u. and two other genes, dnapol and p143, both of which are essential for baculovirus DNA replication, are located from 35.3 to 40.9 m.u. and 55.5 to 63.4 m.u., respectively. To gain insight into the expression of CfMNPV genes in permissive C. fumiferana and nonpermissive Spodoptera frugiperda cells, we constructed three expression plasmids in which the promoter region of the dnapol, the p143, and polyhedrin genes were placed in front of a chloramphenicol acetyltransferase reporter gene. All three CfMNPV promoters were active in nonpermissive cells in the presence of Autographa californica nuclear polyhedrosis virus (AcMNPV) DNA, but no activity was detected in permissive cells either in the presence of CfMNPV DNA or AcMNPV DNA. This lack of promoter activity was not due to failure of viral or plasmid DNA to enter the cell nucleus. It was possible that the reporter plasmids were inefficient templates for transcriptional transactivation so we developed a CfMNPV transfer vector and generated a recombinant virus in which the polyhedrin promoter driving CAT gene cassette was integrated into the CfMNPV genome. In this case, the CfMNPV polyhedrin promoter was highly active in the permissive cells.

Animals↗
PubMed1976

Comparative ultrastructural studies of insect granulosis and nuclear polyhedrosis viruses.

Optical diffraction studies indicated that the periodic lattice structure in electron micrographs of the capsids of two granulosis and two nuclear polyhedrosis viruses were indistinguishable. The capsid is composed of stacked rings spaced 4-5 nm apart. Comparison of the intracellular forms of Bombyx mori nuclear polyhedrosis virus with negatively stained virus particles leads to encourage the more general use of the term 'capsid' instead of 'intimate membrane' and the term 'virus membrane' in place of 'developmental membrane'. These terms are consistent with those currently used for most animal and plant viruses.

Animals↗
PubMed1996

Production of human papillomavirus type 45 virus-like particles in insect cells using a recombinant baculovirus.

The L1 major capsid protein of human papillomavirus type 45 was expressed in insect cells using recombinant baculovirus technology. Human papillomavirus type 45 L1 major capsid protein self-assembled into empty virus-like particles (VLPs). These 50-60 nm diameter particles were present in the nuclei of recombinant baculovirus-infected insect cells and had a density of 1.29 - 1.30 g/cm3 in cesium chloride. The expressed human papillomavirus type 45 L1 protein sequence is identical to the reference human papillomavirus type 45 strain except for one amino acid located at position 49 of the human papillomavirus type 45 L1 protein. It must be noted that the quantity of purified human papillomavirus type 45 virus-like particles is at a lower level than that previously observed with human papillomavirus type 16. Nevertheless, the ability to generate preparative amounts of human papillomavirus type 45 virus-like particles is of great importance for the production of an anti-human papillomavirus vaccine.

Animals↗
PubMed2000

Molecular phylogenetics and the classification of honey bee viruses.

We present the phylogenetic relationships of several picorna-like RNA viruses found in honey bees, with respect to 13 additional plant and animal positive-strand RNA viruses. Most of the honey bee viruses fall into an unnamed family of insect RNA viruses typified by the Drosophila C virus. Different bee viruses are broadly distributed within this group, suggesting either that the ability to infect honey bees has evolved multiple times, or that these viruses are generalistic in their abilities to infect insect hosts. At least one major change in gene order has occurred among the bee viruses, based on their phylogenetic affiliations. At the amino-acid level, the bee viruses differed by 15-28% at three conserved loci. Most differed by greater than 50% at the RNA level, indicating that sequence-based methods for bee virus identification must be tailored to at least three different virus clades independently.

Amino Acid Sequence↗
PubMed2004

Role of an arbovirus nonstructural protein in cellular pathogenesis and virus release.

The insect-borne Bluetongue virus (BTV) is considered the prototypic Orbivirus, a member of the Reovirus family. One of the hallmarks of Orbivirus infection is the production of large numbers of intracellular tubular structures of unknown function. For BTV these structures are formed as the polymerization product of a single 64-kDa nonstructural protein, NS1, encoded by the viral double-stranded RNA genome segment 6. Although the NS1 protein is the most abundant viral protein synthesized in infected cells, its function has yet to be determined. One possibility is that NS1 tubules may be involved in the translocation of newly formed viral particles to the plasma membrane, and NS1-specific monoclonal antibodies have been shown to react with viral particles leaving infected cells. In the present study we generated a mammalian cell line that expresses a recombinant single-chain antibody fragment (scFv) derived from an NS1-specific monoclonal antibody (10B1) and analyzed the effect that this intracellular antibody has on BTV replication. Normally, BTV infection of mammalian cells in culture results in a severe cytopathic effect within 24 to 48 h postinfection manifested by cell rounding, apoptosis, and lytic release of virions into the culture medium. However, infection of scFv-expressing cells results in a marked reduction in the stability of NS1 and formation of NS1 tubules, a decrease in cytopathic effect, an increased release of infectious virus into the culture medium, and budding of virions from the plasma membrane. These results suggest that NS1 tubules play a direct role in the cellular pathogenesis and morphogenesis of BTV.

Animals↗
PubMed1989

Expression of two related nonstructural proteins of bluetongue virus (BTV) type 10 in insect cells by a recombinant baculovirus: production of polyclonal ascitic fluid and characterization of the gene product in BTV-infected BHK cells.

In vitro translation of bluetongue virus (BTV) double-stranded RNA in the rabbit reticulocyte lysate system has shown segment 10 (S10) to code for two related proteins, NS3 and NS3A. The presence of both products in vivo, however, has remained unconfirmed owing to the very low level of synthesis of the S10 gene product(s) in BTV-infected BHK cells. In the present work, a cDNA copy of BTV type 10 (BTV-10) S10 RNA was inserted into Autographa californica nuclear polyhedrosis baculovirus (AcNPV) in lieu of the 5' coding region of the AcNPV polyhedrin gene. Spodoptera frugiperda cells infected with the recombinant baculovirus synthesized two polypeptides, which were shown to represent NS3 and NS3A by Western blot (immunoblot) and peptide map analysis. Antibodies raised to the expressed NS3 by immunization of mice detected both NS3 and NS3A in BTV-10-infected BHK cells but not in purified BTV-10 virus particles. In contrast to in vitro translation of BTV S10 RNA in which NS3 and NS3A are synthesized in equimolar amounts, NS3 was the principle product both in the baculovirus expression system and in vivo in BTV-infected cells. The results indicate the caution which should be exercised when using the rabbit reticulocyte lysate system to predict the pattern of protein synthesis from a gene with alternative start codons. The expressed NS3 and NS3A proteins reacted strongly with sera from sheep infected with homologous and heterologous BTV serotypes, suggesting that the S10 gene products are highly conserved group-specific antigens.

Amino Acid Sequence↗
PubMed1990

Altered phosphorylation pattern of simian virus 40 T antigen expressed in insect cells by using a baculovirus vector.

The phosphorylation pattern of simian virus 40 (SV40) large tumor (T) antigen purified from insect cells infected with a recombinant baculovirus was compared with that reported previously for T antigen from SV40-infected monkey cells. The specific activity of metabolic phosphate labeling of baculovirus T antigen was reduced, and the phosphopeptide map of the baculovirus protein, while qualitatively similar to that of lytic T, revealed several quantitative differences. The most striking difference was the prominence in the baculovirus map of peptides containing phosphothreonine 124. These peptides are known to arise from other phosphopeptides upon dephosphorylation of neighboring serines, suggesting that baculovirus T may be underphosphorylated at these serines and perhaps other sites. Functional assays used to further investigate the phosphorylation state of the baculovirus protein included SV40 DNA binding after enzymatic dephosphorylation with alkaline phosphatase and after phosphorylation by a murine homolog of cdc2 protein kinase. The results imply that baculovirus T antigen is underphosphorylated, in particular at those serine residues whose phosphorylation is responsible for down regulation of DNA-binding activity at site II in the core origin of DNA replication. In contrast, no evidence for a functionally significant underphosphorylation at threonine 124 could be found.

Animals↗
PubMed1998

Localization of a baculovirus-induced chitinase in the insect cell endoplasmic reticulum.

Confocal immunofluorescence microscopy was used to demonstrate that the Autographa californica nucleopolyhedrovirus (AcMNPV) chitinase was localized within the endoplasmic reticulum (ER) of virus-infected insect cells. This was consistent with removal of the signal peptide from the chitinase and an ER localization motif (KDEL) at the carboxyl end of the protein. Chitinase release from cells, a prerequisite for liquefaction of virus-infected insect larvae, appears to be aided by synthesis of the p10 protein. Deletion of p10 from the AcMNPV genome delayed the appearance of chitinase activity in the medium of virus-infected cells by 24 h and also delayed liquefaction of virus-infected Trichoplusia ni larvae by the same period.

Amino Acid Sequence↗
PubMed1998

Hepatitis C virus structural proteins assemble into viruslike particles in insect cells.

Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. The study of HCV has been hampered by the low level of viral particles in infected individuals, the inability to propagate efficiently the virus in cultured cells, and the lack of a convenient animal model. Due to these obstacles, neither the structure of the virus nor the prerequisites for its assembly have been clearly defined. In this report, we describe a model for the production and purification of HCV-like particles in insect cells using a recombinant baculovirus containing the cDNA of the HCV structural proteins. In insect cells, expressed HCV structural proteins assembled into enveloped viruslike particles (40 to 60 nm in diameter) in large cytoplasmic cisternae, presumably derived from the endoplasmic reticulum. Biophysical characterization of viruslike particles by CsCl and sucrose gradient centrifugation revealed biophysical properties similar to those of putative virions isolated from infected humans. The results suggested that HCV core and envelope proteins without p7 were sufficient for viral particle formation. Analysis of particle-associated nucleic acids demonstrated that HCV RNAs were selectively incorporated into the particles over non-HCV transcripts. The synthesis of HCV-like particles in insect cells may provide an important tool to determine the structural requirements for HCV particle assembly as well as to study viral genome encapsidation and virus-host interactions. The described system may also represent a potential approach toward vaccine development.

Animals↗
PubMed1993

Release of bluetongue virus-like particles from insect cells is mediated by BTV nonstructural protein NS3/NS3A.

Recombinant baculoviruses and immunoelectron microscopy have been used to demonstrate the association of virus-like particles (VLPs) of bluetongue virus (i.e., particles composed of BTV, VP2, VP3, VP5, and VP7 proteins) and the cell cytoskeleton, as well as the release of such particles from infected cells in the presence of BTV NS3/NS3A, but not when BTV NS1 protein was coexpressed. Examination of cells infected with recombinants that express core-like particles (CLPs) composed of VP3 and VP7 showed that CLPs were present within the soluble fraction of the cells and not associated with the cell cytoskeleton. The simultaneous expression of the nonstructural NS1 or NS3/NS3A proteins with CLPs did not lead to the association of such particles with the cytoskeleton, nor to their release from cells. The failure of CLPs synthesized in the presence of VP2 or VP5 to attach to the cytoskeleton indicated that both outer coat proteins are required for a stable virus-cytoskeleton interaction. The ultrastructural, immunoelectron microscopical, and biochemical examinations, showed conclusively that the presence of NS3/NS3A is required for the budding and subsequent release of VLPs from infected cells.

Animals↗
PubMed1996

Assembly of virus-like particles in insect cells infected with a baculovirus containing a modified coat protein gene of potato leafroll luteovirus.

DNA encoding the coat protein (P3) of a Scottish isolate of potato leafroll virus (PLRV) was inserted into the genome of Autographa californica nucleopolyhedrovirus (AcNPV) such that the coat protein was expressed either in an unmodified form or with the addition of the amino acid sequence MHHHHHHGDDDDKDAMG at the N terminus (P3-6H). Insect cells infected with these recombinant baculoviruses accumulated substantial amounts of P3 and P3-6H. P3 could not be recovered from cell extracts unless it was denatured in SDS but a proportion of the P3-6H was recoverable in a soluble form in non-denaturing conditions. Immunogold labelling of sections of infected cells showed that P3 accumulated in nuclei in large amorphous bodies. In contrast, although much of the P3-6H also accumulated in nuclei, it formed virus-like particles (VLP) which were often grouped in close-packed, almost cystalline arrays. When electron microscope grids coated with antibodies to PLRV were floated on cell extracts containing P3-6H, VLP were trapped which were indistinguishable from PLRV particles trapped from extracts of PLRV-infected plants. The VLP co- sedimented in sucrose gradients with PLRV particles which suggests that the VLP contained RNA. VLP collected from sucrose density gradient fractions contained protein which reacted with nickel chelated to nitrilotriacetic acid, a histidine-specific reagent. Cells infected with either recombinant baculovirus also synthesized a protein, with an Mr of about 17000, which was shown to be the translation product of the P4 gene which is in the +1 reading frame within the coat protein gene. This protein was also found in the nuclear fraction of infected cells but was more readily soluble than was P3.

Amino Acid Sequence↗