Mammalian Cell‐Free Protein Synthesis Directed by Viral Ribonucleic Acid

A cell‐free system from mouse Krebs II ascites cells is described which responds by increased amino acid incorporation into protein on addition of encephalomyocarditis virus (EMC‐RNA). RNA from other sources does not produce this response. The stimulation by EMC‐RNA occurs over a narrow range of Mg 2+ concentrations and is maximal at 5 mM, which is optimal for the endogenous incorporation and lower than that required in the presence of poly(U). The EMC‐RNA‐directed product is distinguished from the endogenous products by its size and composition. After an initial lag of 4 min, during which the nascent chains are too short to be acid‐insoluble, EMC‐RNA‐directed protein synthesis is active for more than 1 h. EMC‐RNA‐directed synthesis is particularly sensitive to the inhibitors cycloheximide and dextran sulphate. Using the latter, it has been shown that the initiation of protein synthesis is completed during the first 15 min of incubation. Only 20–30% of the product of the cell‐free system, whether endogenous or EMC‐RNA‐directed, is released from the ribosomes. Despite the size of the viral RNA, it did not appear to promote the formation of exceptionally large polysomes. It is concluded that the EMC‐RNA is acting as a message for viral proteins in the ascites cell‐free system.