pactamycin and Cell-Transformation--Viral

Published ultraviolet (uv) inactivation data and in vitro transcription studies have suggested that vesicular stomatitis virus (VSV) leader RNA was solely responsible for the inhibition of host cell RNA synthesis by this virus. Since no protein product is encoded in leader RNA, this conclusion implied that no protein synthesis should be required for this effect. Therefore, the inhibitory activity of VSV was examined in the presence of the protein synthesis inhibitors, cycloheximide, pactamycin, and emetine. Protein synthesis inhibitors are known not to interfere with VSV primary transcription, but in their presence viral replication and amplification of transcription do not take place. Although at 39 degrees the VSV mutant tsG22 could undergo only primary transcription, maximum inhibition of host cell RNA synthesis took place. However, in the presence of the protein synthesis inhibitors the VSV mutant was no longer able to interfere with host cell RNA synthesis. These results could not be explained by a change in the concentration of intracellular leader RNA which remained unaltered by the drugs. Similar results were also obtained with wild-type VSV in the presence of cycloheximide. Upon removal of the drug, inhibition of host cell RNA synthesis was reestablished in parallel with the restoration of protein synthesis. It is concluded that protein synthesis is required for the inhibitory activity of VSV, presumably because the active inhibitory complex is a nucleoprotein containing leader RNA and either a cellular protein or the viral N protein. The cellular protein would have to be in limiting supply since de novo protein synthesis was required for the inhibition to take place.

Topics: Animals; Cell Line; Cell Transformation, Viral; Cricetinae; Cycloheximide; Emetine; Kidney; Kinetics; Mutation; Pactamycin; Protein Biosynthesis; RNA; RNA, Viral; Serotyping; Transcription, Genetic; Vesicular stomatitis Indiana virus; Virion

Epstein-Barr virus-transformed human B cells (EBV-B cells), but not resting B cells or B cells activated by T cell-derived factors, have been shown to support the proliferation of tetanus toxoid (TT)-specific autologous T cell clones in response to TT antigen. The accessory cell function of EBV-B cells was compared to that of monocytes with regard to antigen uptake and processing. After an 18-h incubation period with 125I-labeled TT, the amount of radioactivity associated with the cells (approximately 50 ng/10(7) cells) and the percentage of cells containing radiolabeled material (approximately 50%) were equivalent for EBV-B cells and monocytes. Like with monocytes, EBV-B cells pulsed with TT for 18 h or more were equivalent in their capacity to induce T cell proliferation to EBV-B cells to which soluble TT was added for the duration of the culture period. The requirements for antigen uptake and presentation to T cells were similar for both EBV-B cells and monocytes. Both processes were energy dependent, inhibited by cold (4 degrees C), 2-deoxyglucose, and azide, and both required no de novo protein synthesis as they were not affected by pretreatment of the cells with the irreversible protein inhibitor pactamycin . Trypsin treatment of antigen-pulsed EBV-B cells and monocytes followed by fixation for 1 min in 0.03% paraformaldehyde completely abolished the capacity of both cell types to induce T cell proliferation. In both EBV-B cells and monocytes, antigen presentation, but not antigen uptake, was inhibited by the addition of the lysosomotropic agent chloroquine during the antigen-pulse period suggesting that the mechanisms of antigen processing are similar for both cell types. Vacuoles positive for acid phosphatase with an electron microscopic structure similar to that of lysosomes were found in EBV-B cells but not in resting B cells or B cells activated by T cell-derived factors. The present observations indicate that EBV-B cells take up antigen and process it in a fashion similar to monocytes. The presence of lysosomes appears to correlate with the capacity of B cells to present antigen.

Topics: Azides; B-Lymphocytes; Cell Transformation, Viral; Chloroquine; Clone Cells; Deoxyglucose; Diphtheria Toxoid; Formaldehyde; Herpesvirus 4, Human; Humans; Kinetics; Lymphocyte Activation; Monocytes; Pactamycin; Polymers; T-Lymphocytes; Tetanus Toxoid; Trypsin

Topics: Animals; Antigens, Neoplasm; Antigens, Viral; Avian Sarcoma Viruses; Cell Transformation, Viral; Cells, Cultured; Chick Embryo; Clone Cells; Cycloheximide; Mutation; Pactamycin; Protein Biosynthesis; Puromycin; Temperature

Topics: Cell Transformation, Viral; Cycloheximide; Galactose; Galactosyltransferases; Glucosyltransferases; Glycolipids; HeLa Cells; Humans; Kinetics; Measles virus; Neoplasm Proteins; Pactamycin; Palmitic Acids; Poliovirus; Protein Biosynthesis; Serine; Vesicular stomatitis Indiana virus