Paul J. Farrell

Phosphorylation of initiation factor eIF-2 and the control of reticulocyte protein synthesis

When rabbit reticulocyte lysates are incubated in the absence of hemin or in the presence of low concentrations of double-stranded RNA, the rate of initiation of protein synthesis is severely reduced after a lag period in which control rates are observed. This reduced initiation rate is due to inhibition of the binding of Methionyl-tRNAf to native 40S ribosomal subunits and is caused by a macromolecular inhibitor which is activated under these conditions. This paper shows that the inhibitors activated in these two situations appear to be different entities, but that in both cases, the inhibitor has an associated protein kinase activity which is highly selective for the small subunit of elF-2, the initiation factor which catalyzes binding of Methionyl-tRNAf to 40S subunits. We present several lines of evidence in support of the hypothesis that the phosphorylation of elF-2 by these kinases is basis of the control of initiation in lysates incubated under these conditions.

Interferon, double-stranded RNA and RNA degradation. Fractionation of the endonucleaseINT system into two macromolecular components; Role of a small molecule in nuclease activation

Abstract We reported earlier that a) the incubation of an extract from interferon-treated Ehrlich ascites tumor cells with double-straded RNA and ATP results in the activation of an endonuclease and b) after the activation the double-stranded RNA and ATP can be degraded without impairing the activity of the endonuclease. We report now the separation and partial purification of two macromolecular components (DE1 INT and DE2 INT ) involved in the process. Upon incubation with double-stranded RNA and ATP component DE1 INT generates a heat-stable product of low molecular weight (designated as nuclease activator). On incubation with the nuclease activator a latent nuclease in component DE2 INT is activated.

Extracts of interferon-treated cells can inhibit reticulocyte lysate protein synthesis.

Summary Cytoplasmic extracts of interferon-treated L929 cells are capable of forming an inhibitor of reticulocyte lysate protein synthesis when incubated with double stranded RNA and ATP. It appears that this inhibition arises through phosphorylation of the methionyl-tRNA f binding factor, causing a defect in polypeptide chain initiation.

Novel nuclear antigens recognized by human sera in lymphocytes latently infected by Epstein-Barr virus.

We have used three latently infected cell lines, X50-7, JC-5, and Raji, to identify two new nuclear antigen complexes by Western immunoblotting with human anti-EBNA (Epstein-Barr Virus Nuclear Antigen) sera. One antigen complex, termed EBNA III, is composed of a group of high molecular weight proteins between 130 and 160 kDa and the other antigen complex, termed EBNA IV, is a size-related group of polypeptides between 28 and 62 kDa. Both the EBNA III and EBNA IV groups of proteins display variation in size among the different strains of EBV. Cell fractionation of X50-7, JC-5, Raji, and C16, a cell clone of P3HR1, showed that both new antigen complexes were completely recovered from the nuclei of latently infected lymphocytes as were previously described EBNA I and II. Because these new antigens are only detected by anti-EBNA sera in EBV infected cells, it seems likely that they may be encoded by the viral genome and play some role in the immortalization of lymphocytes by the virus.

INTERFERON‐INDUCED mRNAs AND PROTEINS IN EHRLICH ASCITES TUMOR AND HeLa CELLS

We are interested in the regulation and activation of various cytoplasmic enzymes in mouse Ehrlich ascites tumor (EAT) and human HeLa S3 cells by interferon [IF) and have looked at changes which occur in the protein and mRNA populations of these cells after IF treatment. In EAT cells we previously found’ one mRNA to be clearly induced by highly purified mouse IF; this mRNA is translated in vitro into a protein of MW 14,500 and PI 7.5. This protein is found in increased amounts in EAT cells after IF treatment and its synthesis is maximal after 8 hr of IF treatment. More recently we have examined proteins and mRNAs induced in HeLa cells by human interferon. HeLa cell monolayers were treated with 500 U/ml of partially purified human leukocyte IF (specific activity 5 x lo6 U/mg; a kind gift of Dr. K. Cantell) in the presence or absence of 2 Mg/ml actinomycin D, or were not treated with IF (control cells). At various times after IF treatment, the cells were pulse labeled with [%]methionine for 1 hour, lysed, and the extracts analyzed by SDS-PAGE. One protein band (MW 53,000) was enhanced in the IF-treated cells. Synthesis of this protein occurred maximally 7-8 hr after IF treatment. To determine whether this change was mirrored by a change in the mRNA population of the cells, poly A’ mRNA was extracted from either cells treated with 500 U/ml IF for 8 hr, cells treated with 500 U/ml IF and 2 pg/ml actinomycin D for 8 hr or control cells. When the products of translation of these mRNAs in a wheat germ system were analyzed by SDS-PAGE. the mRNA from IF-treated cells yielded increased amounts of the 53,000 dalton protein compared with the mRNA from control cells. The mRNA from the actinomycin-treated cells gave only the control amount of the 53,000 dalton protein. To examine more closely the populations of mRNAs and proteins, we analyzed the cell extracts and mRNA translation products by two dimensional gel electrophoresis.’ Compared with control cells, two proteins (both MW 50,000 and PI approx 7) and one protein (MW 53.000, PI approx 6.5) accumulated in increased amounts in HeLa cells pulse-labeled for 1 hr with [35S]methionine after 7 hr of IF treatment. The same three proteins were found to be enhanced in the in vitro translation products of mRNA from IF-treated HeLa cells as compared with those derived from control cell mRNA. Further analyses were done by nonequilibrium pH gradient electroph~resis.~ One additional enhanced protein was observed in the translation products of mRNA from IF-treated HeLa cells. However, this protein was not observed in IF-treated HeLa cells by this method of analysis so is of doubtful significance. No additional enhanced proteins or mRNAs were detected by this method in the EAT cells treated with mouse interferon. We have not yet been able to correlate any of these induced proteins with the enzyme activities known to be induced in IF-treated cells. Although we have purified preparations of the pppA(X’pYA), synthetase and the eIF-2 protein