Rapid ephemeral cell sensitization as the mechanism of histone-induced and protamine-induced enhancement of transfection by poliovirus RNA

Summary

The mechanism of transfection enhancement by protamine and histone was investigated, using poliovirus RNA and sheet cultures of the CLI line of chimpanzee liver cells. The transfection obtained using inocula prepared by premixing the viral RNA with a large excess of basic protein could be quantitatively accounted for by direct sensitization of the cells by the excess free basic protein postinoculation. Such direct sensitization to transfection was very rapid; at 25 °C with protamine chloride or histone at 1 mg/ml peak sensitivity was reached in about 16 and 25 seconds, respectively. Sensitivity then rapidly decreased, but could be partially restored later by a second pretreatment with fresh basic protein. The original sensitization, desensitization, and resensitization phases of the cell sensitivity curves are transfectionspecific, since ordinary infection with intact poliovirions was not influenced by the pretreatments with basic protein, except that prolonged pretreatment with histone decreased sensitivity to intact virus. The viral RNA transfectivity in inocula premixed with protamine chloride at 1 mg/ml and held at 0 °C was labile, with an initial half-life of only 5 minutes; the transfectivity of RNA similarly held in buffer alone or with histone was completely stable at 0 °C for at least 1.5 to 2 hours. With the best basic protein transfection method (cell sensitization by protamine), the specific transfectivity of RNA isolated from purified poliovirions was 130,000 plaque-forming units/μg RNA.