Sequential suppression by actinomycin D of interferon production and cellular resistance induced by Poly I:C.

Cellular RNA and protein synthesis are required both for interferon synthesis (Heller, 1963; Wagner, 1963) and the expression of the antivirus activity of interferon (Taylor, 1964). The suppression of the antivirus activity of interferon by metabolic inhibitors has been interpreted to mean that interferon acts as a de-repressor for the synthesis of a cellular antivirus protein. While direct proof for the interferon-induced antivirus protein is still lacking, recent circumstantial evidence supports this interpretation (Dianzani, Buckler & Baron, 1968; Stewart & Lockart, 197o). In a recent publication, Kjeldsberg & Flikke (I97I) reported that polyinosinic-poly-cytidylic acid (poly I:C) inhibited poliovirus RNA synthesis in cells whose DNA-dependent RNA synthesis had been suppressed by actinomycin D. They suggested that, rather than acting through interferon induction, low concentrations of poly I:C may directly interfere with virus replication by binding to the virus-specific RNA polymerase. Our experiments were done in rabbit kidney cell cultures prepared as previously described (Vil6ek et al. I968). Poly I:C (double-stranded sodium salt, lyophilized) was purchased from P-L Biochemicals, Milwaukee, Wisconsin. The methods of titration of vesicular stomatitis virus (VSV) in cultures of chick embryo cells (Vil6ek & Freer, ~966) and of interferon titration by a plaque-reduction test with VSV in secondary rabbit kidney cell cultures (Vil6ek & Ng, 197I) have been described previously. Cultures in 6o mm. Petri dishes were incubated with o'5 #g./ml. of actinomycin D or control medium for 45 min. They were then treated with various concentrations of poly I : C or with rabbit kidney cell interferon. Six hr later all cultures were thoroughly washed and inoculated with VSV at a multiplicity of 3 p.f.u./cell. The virus was adsorbed for I hr. The cultures were then washed to remove extracellular virus and replenished with medium. The virus yield was titrated from culture fluids collected ~6 hr after inoculation. The inhibitory effect of three different concentrations of poly I:C on growth of VSV was abolished by previous exposure of cells to actinomycin D. The antivirus action of interferon was also suppressed by actinomycin D (Fig. I). This experiment indicated the necessity of a cellular function for the expression of the antivirus action of poly I : C. However, it did not demonstrate that interferon was a necessary intermediate in this reaction. It could not be ruled out that poly I: C served as a direct stimulus for the synthesis of the cellular antivirus protein and that, strictly speaking, …