Host Insect Cells Research Articles

Immunogold Localization of Rice Stripe Virus Particle Antigen in Thin Sections of Insect Host Cells.

Immunogold Localization of Rice Stripe Virus Particle Antigen in Thin Sections of Insect Host Cells.

Host-dependent variation of Bluetongue virus neutralization

To determine if neutralizing epitopes of Bluetongue virus (BTV) 17 are host dependent, e.g., that monoclonal antibodies (mAb) to Bluetongue virus 17 (BTV 17) differ in their ability to neutralize BTV infectivity in insect versus mammalian cells, a panel of neutralizing mAb was developed. The relative neutralizing titer of eight mAb for BTV 17 infectivity in mammalian versus insect target cells was determined. Four mAb differed in their relative neutralization titer when assayed on mammalian target cells as compared to insect target cells. These findings suggest that different epitopes involved in neutralization might be important in virus infectivity and neutralization in mammalian versus insect target cells. To determine which viral protein(s) these mAb bind, the specificity of the mAb was determined by radioimmunoprecipitations. Five BTV 17 neutralizing mAb bound to the major outer coat protein P2, a 98-kDa protein, whereas the BTV protein(s) bound by the other three neutralizing mAb could not be determined. The potential role of the two BTV outer coat proteins in infection of mammalian and insect host cells is discussed.

Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the beta-galactosidase gene

An improved baculovirus expression vector was developed to expedite screening and facilitate oligonucleotide-directed mutagenesis. This vector contained twin promoters derived from the P10 and polyhedrin genes of Autographica californica nuclear polyhedrosis virus. The P10 promoter directed the synthesis of beta-galactosidase, whereas the polyhedrin promoter controlled the synthesis of foreign gene products. These two genes recombined with wild-type virus genome to yield recombinants which were polyhedrin negative, produced the foreign gene product, and formed blue plaques when beta-galactosidase indicator was present in the agarose overlay. An origin of replication derived from M13 or f1 bacteriophage was also included in the plasmid to permit the synthesis of single-stranded DNA. This template DNA was used to introduce or delete sequences through the process of site-specific mutagenesis. The measles virus virion possesses a membrane envelope which contains two glycoproteins: the hemagglutinin (H) and membrane fusion (F) proteins. The H polypeptide has receptor-binding and hemagglutinating activity, whereas the F protein mediates virus penetration of the host cell, formation of syncytia, and hemolysis of erythrocytes. Genes for these two glycoproteins were inserted into the NheI cloning site of the modified expression vector described above. The vector and purified wild-type viral DNA were introduced into Sf9 insect cells by calcium phosphate precipitation. A mixture of wild-type and recombinant virus was generated and used to infect Sf9 cells, which were subsequently overlaid with agarose. After 3 days, 0.1 to 1% of the plaques became blue in the presence of beta-galactosidase indicator. At least 70% of these blue viral colonies contained the foreign gene of interest as determined by dot blot analysis. Recombinant virus was separated from contaminating wild-type virus through several rounds of plaque purification. Insect cells were then infected with the purified recombinants, and synthesis of H and F proteins were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot detection and Coomassie blue staining. Glycosylation of the proteins appeared to be impaired somewhat, and the precursor to the F protein was not completely cleaved by the proteases present in insect host cells. On the other hand, both proteins appeared to be active in hemagglutination, hemolysis, and cell fusion assays. Levels of synthesis were in the order of 50 to 150 mg of protein per 10(8) cells.

Regulation of Host RNA Levels during Baculovirus Infection

During infection of the permissive host insect cell line Spodoptera frugiperda IPLB-SF-21 by the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV), the levels of host actin, histone, and heat shock 70 (hsp70) RNAs are reduced substantially. Reduction of the host RNA levels occurs primarily during a narrow window of the replication process, from approximately 12 to 18 hr postinfection (p.i.), corresponding to the phase in which the extracellular form of the virus buds into the media. A late viral protein appears to be required for this reduction since cycloheximide, an inhibitor of cytosolic protein synthesis, and aphidicolin, an inhibitor of host and viral DNA polymerases, inhibit the reduction of actin and histone RNA levels. A cDNA corresponding to the car☐yl half of the S. frugiperda mitochondrial cytochrome c oxidase subunit III (COIII) gene was isolated, sequenced, and characterized. Two differentially regulated mitochondrial transcripts of this gene are observed. The level of the larger of these transcripts, which is dependent on active cytosolic protein synthesis, is reduced during virus infection in a fashion similar to that of the nuclear host genes. The smaller COIII transcript is stable until at least 24 hr p.i. but the level of this RNA eventually declines by 48 hr p.i.

Production of polypeptides in virally infected insect host cells — vector construction, application for e.g. hepatitis B virus surface antigen preparation for vaccine development

Production of polypeptides in virally infected insect host cells — vector construction, application for e.g. hepatitis B virus surface antigen preparation for vaccine development

Phosphorylation of a ribosomal protein in invertebrate cells infected with ultraviolet-inactivated iridovirus type 6

Summary Chilo iridescent virus (CIV) infection induces a rapid shut-off of macromolecular synthesis in insect host cells. We have previously shown that CIV infection induced the appearance of new phosphoproteins in permissive cells of Choristoneura fumiferana (Cf-124) [15]. Among them, phosphorylation of a 31 Kd ribosomal protein (very similar to the S6 protein) was observed 3 h after infection of Cf-124 cells with UV-inactivated CIV. However, such phosphorylation could not be detected in cells infected native CIV in the same conditions. As previously postulated [15], these results could be interpreted either as an absence of phosphorylation of this ribosomal protein, or as a dephosphorylation step occurring very rapidly during the early stages of the replication cycle, in cells infected with native CIV, as reported for the infection of BHK21 cells by vaccinia virus [1].

Electron microscopy of wound tumor virus assembly sites in insect vectors and plants

Thin sections were prepared for electron microscopy from Agallia constricta insect vectors and from Trifolium incarnatum and Melilotus alba plants infected with wound tumor virus. Insects acquired the virus by feeding on wound tumor diseased plants. Electron microscopy of thin sections of various organs of viruliferous insects indicated that the virus is assembled in accumulations of electron-dense aggregates, designated viroplasms, formed in the cytoplasm of fat body, muscle, midgut, Malpighian tubules, trachea, salivary gland, and epidermal cells. The presence of viroplasms in the cells of the mycetome was not ascertained. The viroplasms are considered the sites of virus assembly. Careful examination of infected plants during early stages of disease revealed the formation of viroplasms in the root tumor cell cytoplasm and in the cytoplasm of cells of enlarged leaf veinlets. In plant cell sections prepared shortly after the appearance of first signs of the wound tumor disease, mature particles were found first at the periphery of the viroplasmic zone. A few days later virions were detected in increasing number inside the viroplasmic area. These morphological findings indicate that wound tumor virus is assembled in plant and insect host cells in viroplasms, formed within the cytoplasmic matrix proper. The similarity of wound tumor viroplasms with those of several viruses infecting higher animals, and the significance of the observations in the elucidation of the precise foci of virus assembly in cells and organs of plant and invertebrate animal hosts of the virus are discussed.