Gebhard Koch

Selective and reversible inhibition of initiation of protein synthesis in mammalian cells

Abstract Hypertonicity effected by elevation of NaCl concentration in the growth medium results in a rapid cessation of protein synthesis in HeLa cells accompanied by a complete breakdown of polyribosomes. Since elongation and termination of polypeptide synthesis proceeds normally, it is concluded that elevated NaCl concentration in the medium selectively inhibits the initiation of peptide chain formation. Pulse-labeling of poliovirus-infected cells at different times after incubation of the cells in hypertonic medium can be used to map the poliovirus genome. Upon restoration of isotonicity re-initiation of protein synthesis occurs only at the proper initiation site and synthesis proceeds at a normal rate. This synchronized re-initiation of protein synthesis observed upon restoration of isotonicity, in turn, allowed us to determine the gene sequence of poliovirus RNA in a novel way.

Synthesis and processing of viral proteins in Friend erythroleukemia cell lines

Abstract The synthesis and processing of viral proteins in a number of Friend erythroleukemia cell lines were characterized by the use of monospecific antisera to purified viral structural proteins. In all cell lines studied, a major intracellular species precipitated with anti-gp69/71 serum was a protein of 55,000 MW. This 55,000 MW protein was detectable after a 5-min pulse, along with the 85,000 MW glycoprotein precursor to gp69/71, suggesting that it might be a primary translation product. The major glycoprotein gp69/71 was only observed after a 30-min chase period. Analysis of pulse-labeled cell extracts with monospecific antisera to the core proteins p12, p15, and p30 revealed the presence of viral specific proteins intermediate in size between the gag precursor polyprotein and the mature viral proteins. These intermediate species probably represent processing intermediates, and an analysis of their antigenic specificities was in agreement with the core protein arrangement, p15-p12-p30-p10, within the gag polyprotein precursor. Under conditions where more membrane-associated material was extracted, a 180,000 MW species precipitable with antiserum against p30 as well as anti-reverse transcriptase serum was observed. Alterations in viral precursor processing were observed in the Friend cell lines during the chemically induced differentiation process.

Interaction of Poliovirus-Specific RNAs with HeLa Cells and E. coli

General aspects of nucleic acid uptake by mammalian cells have been the subject of several reviews during the last few years (Pagano, 1970; Bhargava and Shanmugam, 1971; Dubes, 1971; Ryser, 1967). These reviews covered methods used for the infection of cells by viral nucleic acids as well as interaction of mammalian cells with non-viral nucleic acids.

Sensitization of HeLa cells for viral RNA infection by cytochalasin B.

Abstract Incubation of HeLa cells in medium containing 5 μg cytochalasin B/ml for 5–10 min at 37° results in: (a) 30- to 80-fold increase in the sensitivity of cells for infection by isolated poliovirus RNA, (b) reduced viral RNA adsorption and penetration, and (c) immediate inhibition of incorporation of labeled amino acids into proteins. Protein synthesis in poliovirus-infected cells, however, continues unabated for 15 min in the presence of a ten-fold higher concentration of cytochalasin B (50 μg/ml). Apparently, viral RNA can efficiently direct protein synthesis under conditions where host RNA translation is inhibited.

Control of Peptide Chain Initiation in Uninfected and Virus Infected Cells by Membrane Mediated Events

Initiation of protein synthesis in tissue culture cells is rapidly inhibited or blocked by addition of either DMSO, ethanol, TPCK, cytochalasin B, or sucrose to the growth medium. In contrast, these agents do not interfere with the initiation of protein synthesis in cell-free extracts to a comparable extent. These results support the hypothesis that protein synthesis in tissue culture cells can be influenced by membrane mediated events. Translation of viral mRNA in RNA virus infected cells is resistant to a number of these inhibitors of peptide chain initiation and proceeds under conditions where translation of host mRNA is almost completely suppressed. It appears that viral mRNA possesses a greater ability than host mRNA to form mRNA-ribosome initiation complexes when the overall rate of peptide chain initiation is reduced. This observation has led to a number of predictions concerning the strategy of virus directed suppression of host mRNA translation. Under optimal growth conditions protein synthesis appears to be regulated mainly, but not exclusively, by the amount of the mRNA available for translation. However, when cellular growth and/or the overall rate of peptide chain initiation is restricted, control of protein synthesis at the translational level becomes decisive with the translation of each mRNA species proceeding with its own characteristic efficiency most probably as a result of inherent differential affinities of individual mRNA species for ribosomes.

Inhibition of protein synthesis by HeLa cell surface peptides

Summary Peptides are removed from the surface of HeLa S3 cells or from purified plasma membranes by mild proteolytic treatment. The crude peptide preparation decreases overall protein synthesis in vivo in HeLa S3 and MPC-11 cells within 10 to 20 min after addition. In cell-free translation systems derived from HeLa cells or rabbit reticulocytes, protein synthesis is also inhibited. The HeLa surface peptides enter the cells, and may interact directly with monosomes and polysomes.

Initiation of protein synthesis in vivo in poliovirus-lnfected HeLa cells

Abstract Initiation of protein synthesis in vivo in poliovirus-infected HeLa cells has been studied. When these cells are synchronized for initiation by fluoride treatment and then double labeled with [ 35 S]methionine and either tritiated proline, phenylalanine, or valine for short pulses, the percentage of N-terminal methionine incorporated in the nascent peptides compared to total incorporation is significantly higher than that of the tritiated amino acids tested. The data indicate that methionine is the initiator amino acid for the synthesis of poliovirus-specific proteins.

Partial characterizatio and proposed mode of action of inhibitory heLa cell surface polypeptides

Polypeptides removed from the HeLa cell surface by mild pronase treatment rapidly inhibit protein synthesis when added to HeLa cells or cell-free translation systems derived from HeLa cells. The inhibitory activity is heat stable. Protein and carbohydrate components of these polypeptides are required for inhibition of protein synthesis in vivo and in vitro. Two peaks of activity can be recovered from polyacrylamide gels, corresponding to polypeptides with molecular weights of approximately 29 000 and 41 000. Inhibition of protein synthesis in cell-free translation systems appears to be primarily an effect on elongation of polypeptide chains, whereas in the intact cell the primary target may be polypeptide chain initiation.