Eukaryotic protein synthesis initiation factor 4B (eIF‐4B) is an 80,000 dalton polypeptide which is essential for the binding of mRNA to ribosomes. A highly purified preparation of eIF‐4B from HeLa cells was subjected to enzymatic cleavage and amino‐terminal amino acid sequence analysis. Degenerate oligonucleotide probes were used to isolate a 3851 bp cDNA encoding eIF‐4B from a human cDNA library. The DNA encodes a protein comprising 611 residues with a mass of 69,843 daltons. The amino‐terminal domain of eIF‐4B contains a consensus RNA binding domain present in a number of other RNA binding proteins. Expression of eIF‐4B in transfected COS‐1 cells yielded a polypeptide which reacted with anti‐eIF‐4B antiserum and comigrated with purified eIF‐4B. Expression of eIF‐4B in COS‐1 cells resulted in a general inhibition of translation, possibly due to a 50‐fold eIF‐4B overproduction.

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Five initiation factors, eIF-2, eIF-3, eIF-4A, eIF-4B, and eIF-5, were purified from human HeLa cells. Methods of protein fractionation and assays for initiation factors which had been developed for rabbit reticulocytes were found to be suitable for HeLa factors. The initiation factors from HeLa cells are similar to or indistinguishable from the corresponding rabbit reticulocyte factors with respect to specific activities, molecular weights as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subunit structure. The molecular weight of eIF-3 particles from both species is about 410000 as determined by equilibrium sedimentation analytical centrifugation. The partial protease fragmentation patterns of corresponding proteins also are similar and indicate that the primary sequences of the factors are related in the two species. Antisera raised in goats against rabbit eIF-3 and human eIF-2, eIF-4A, and eIF-4B cross-react with the cognate factors from both species. On the basis of immunoblotting techniques, eIF-4A is highly conserved, eIF-2 alpha, eIF-3, and eIF-4B are somewhat less conserved, and eIF-2 beta is the least conserved of the proteins examined. The functional, structural, and immunological results are all consistent with the view that initiation factors from different mammalian cells are very similar.

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A cDNA clone of the influenza virus NS (non-structural protein) gene in a vector carrying a bacteriophage T7 RNA polymerase promoter was manipulated so as to reiterate the initiation site to give two in-frame AUG codons 57 nucleotide residues apart. Each initiation site was in either a preferred context (. . . AUAAUGG.. .) or a less favourable context (. . .UUU-G.. .) and the four possible permutations were constructed. When capped mRNA transcripts of these clones were translated in the rabbit reticulocyte lysate system, products from initiation at both AUG codons werc observed. At low RNA concentrations the frequency of initiation at the 5'-proximal AUG codon rather than the second was higher when the first AUG codon was in the preferred context, in qualitative agreement with the scanning ribosome model. However, a completely unexpected finding was that the ratio of initiation at the first A U C codon to initiation at the second decreased with increasing mRNA concentration, irrespective of the particular context involved. Several lines of evidence indicated that the increased frequency of initiation at the second AUG codon was not due solely to the lower density of ribosome loading per mRNA at high RNA concentrations, and may therefore be the result of high RNA concentrations out-titring the capacity of endogenous reticulocyte factors responsible for preferential initiation at the 5'-proximal AUG codon. The effect of supplementing the system with purified initiation factors was examined. Only eIF-2 was capable of decreasing the frequency of initiation at the second AUG codon and promoting use of the first AUG at high mRNA concentrations; eIF-3,4A, 4B, 4 C + 4 D , 4 F and 5 were inactive.

Protein synthesis initiation factor preparations from poliovirus-infected HeLa cells have reduced ability to initiate translation on capped mRNA. The defect in initiation factors has been variously attributed to inactivation of eucaryotic initiation factor 3 (eIF3), eIF4B, or a cap-binding protein (CBP) complex. We have developed a series of in vitro protein synthesis assays to show that eIF3 is active but a CBP complex activity is inactivated after poliovirus infection. eIF3 activity, when determined in the presence of purified CBP complex, is present in sucrose gradients of factors from both infected and uninfected cells. CBP complex activity, determined in the presence of eIF3 from poliovirus-infected cells, is present in uninfected cells only and comigrates on sucrose gradient with an activity which restores the ability of crude initiation factors from infected cells to translate capped globin mRNA. This is the first demonstration by a fractionated translation system that an activity which is attributable to CBP complex is inactivated in poliovirus-infected cells. The results also indicate that eIF3 is undetectable or has greatly reduced activity in the absence of CBP complex.