Binding of ATP to eukaryotic initiation factor 2. Differential modulation of mRNA-binding activity and GTP-dependent binding of methionyl-tRNAMetf.

Eukaryotic initiation factor 2 (eIF-2) is shown to bind ATP with high affinity. Binding of ATP to eIF-2 induces loss of the ability to form a ternary complex with Met-tRNAf and GTP, while still allowing, and even stimulating, the binding of mRNA. Ternary complex formation between eIF-2, GTP, and Met-tRNAf is inhibited effectively by ATP, but not by CTP or UTP. Hydrolysis of ATP is not required for inhibition, for adenyl-5'-yl imidodiphosphate (AMP-PNP), a nonhydrolyzable analogue of ATP, is as active an inhibitor; adenosine 5'-O-(thiotriphosphate) (ATP gamma S) inhibits far more weakly. Ternary complex formation is inhibited effectively by ATP, dATP, or ADP, but not by AMP and adenosine. Hence, the gamma-phosphate of ATP and its 3'-OH group are not required for inhibition, but the beta-phosphate is indispensible. Specific complex formation between ATP and eIF-2 is shown 1) by effective retention of Met-tRNAf- and mRNA-binding activities on ATP-agarose and by the ability of free ATP, but not GTP, CTP, or UTP, to effect elution of eIF-2 from this substrate; 2) by eIF-2-dependent retention of [alpha-32P]ATP or dATP on nitrocellulose filters and its inhibition by excess ATP, but not by GTP, CTP, or UTP. Upon elution from ATP-agarose by high salt concentrations, eIF-2 recovers its ability to form a ternary complex with Met-tRNAf and GTP. ATP-induced inhibition of ternary complex formation is relieved by excess Met-tRNAf, but not by excess GTP or guanyl-5'-yl imidodiphosphate (GMP-PNP). Thus, ATP does not act by inhibiting binding of GTP to eIF-2. Instead, ATP causes Met-tRNAf in ternary complex to dissociate from eIF-2. Conversely, affinity of eIF-2 for ATP is high in the absence of GTP and Met-tRNAf (Kd less than or equal to 10(-12) M), but decreases greatly in conditions of ternary complex formation. These results support the concept that eIF-2 assumes distinct conformations for ternary complex formation and for binding of mRNA, and that these are affected differently by ATP. Interaction of ATP with an eIF-2 molecule in ternary complex with Met-tRNAf and GTP promotes displacement of Met-tRNAf from eIF-2, inducing a state favorable for binding of mRNA. ATP may thus regulate the dual binding activities of eIF-2 during initiation of translation.

Superinduction of the human gene encoding immune interferon.

Mitogen-induced interferon-gamma (IFN-gamma) gene expression was analyzed in human tonsil cells by titration of IFN-gamma activity and by quantitation of IFN-gamma mRNA. Expression of the IFN-gamma gene can be superinduced extensively by two distinct methods: exposure to various inhibitors of translation, or to low doses of gamma-irradiation. gamma-Irradiated cells produce, after exposure to cycloheximide, up to 12-fold greater amounts of IFN-gamma activity. Within as little as 4 h after the addition of translation inhibitors, IFN-gamma mRNA levels rise 3- to 5-fold. Superinduction acts to increase the size of the wave of IFN-gamma mRNA. Primary transcription of the IFN-gamma gene does not increase in cells superinduced by cycloheximide, nor can superinduction be explained by stabilization of IFN-gamma mRNA sequences. These findings show that, during normal induction, a labile protein acts post-transcriptionally to repress the accumulation of mature IFN-gamma mRNA sequences. The superinductive effects of cycloheximide and gamma-irradiation on levels of IFN-gamma are additive, suggesting that they affect different aspects of IFN-gamma gene expression. Superinduction by gamma-irradiation also has a post-transcriptional basis and is consistent with the possibility that expression of the IFN-gamma gene is normally controlled by the action of suppressor T cells. Even though the genes for human IFN-gamma and for interleukin-2 are both superinducible, a striking difference in the regulation of expression of these lymphokine genes is observed. Superinduction of IFN-gamma mRNA is not due to superinduction of interleukin-2.

Inactivation of foot-and-mouth disease virus vaccine strains by activation of virus-associated endonuclease.

A new inactivation process for foot-and-mouth disease virus (FMDV) has been developed. This process is based on the activation of the FMDV endonuclease by incubation of unfractionated viral suspension or purified virions at 37 degrees C in the presence of high concentrations of monovalent cations such as K+, Cs+ or NH4+ at pH 8.5. This procedure completely inactivated several FMDV vaccine strains yielding preparations having similar amounts of 140S particles to untreated controls. The inactivation followed first-order kinetics and the rate of inactivation was faster than that achieved with other agents, e.g. binary ethyleneimine. Testing in suckling mice or tissue culture revealed no residual infectivity after inactivation. Virus particles purified from inactivated preparations showed (i) the same sedimentation coefficient as non-inactivated preparations, (ii) electrophoretic patterns of their viral capsid proteins identical to those derived from non-inactivated preparations, and (iii) extensive degradation of the 35S viral RNA. This method is safer than inactivation with aziridines because only innocuous chemicals are used in the process.