The double-stranded RNA-activated protein kinase PKR is dispensable for regulation of translation initiation in response to either calcium mobilization from the endoplasmic reticulum or essential amino acid starvation.

The alpha-subunit of eukaryotic initiation factor eIF2 is a preferred substrate for the double-stranded RNA-activated protein kinase, PKR. Phosphorylation of eIF2alpha converts the factor from a substrate into a competitive inhibitor of the guanine nucleotide exchange factor, eIF2B, leading to a decline in mRNA translation. Early studies provided evidence implicating PKR as the kinase that phosphorylates eIF2alpha under conditions of cell stress such as the accumulation of misfolded proteins in the lumen of the endoplasmic reticulum, i.e., the unfolded protein response (UPR). However, the recent identification of a trans-microsomal membrane eIF2alpha kinase, termed PEK or PERK, suggests that this kinase, and not PKR, might be the kinase that is activated by misfolded protein accumulation. Similarly, genetic studies in yeast provide compelling evidence that a kinase termed GCN2 phosphorylates eIF2alpha in response to amino acid deprivation. However, no direct evidence showing activation of the mammalian homologue of GCN2 by amino acid deprivation has been reported. In the present study, we find that in fibroblasts treated with agents that promote the UPR, protein synthesis is inhibited as a result of a decrease in eIF2B activity. Furthermore, the reduction in eIF2B activity is associated with enhanced phosphorylation of eIF2alpha. Importantly, the magnitude of the change in each parameter is identical in wildtype cells and in fibroblasts containing a chromosomal deletion in the PKR gene (PKR-KO cells). In a similar manner, we find that during amino acid deprivation the inhibition of protein synthesis and extent of increase in eIF2alpha phosphorylation are identical in wildtype and PKR-KO cells. Overall, the results show that PKR is not required for increased eIF2alpha phosphorylation or inhibition of protein synthesis under conditions promoting the UPR or in response to amino acid deprivation.

Regulation of the double-stranded RNA-dependent protein kinase PKR by RNAs encoded by a repeated sequence in the Epstein-Barr virus genome.

During the initial infection of B lymphocytes by Epstein-Barr virus (EBV) only a few viral genes are expressed, six of which encode the EBV nuclear antigens, EBNAs 1-6. The majority of EBNA mRNAs share common 5'-ends containing a variable number of two alternating and repeated exons transcribed from the BamHI W major internal repeats of the viral DNA. These sequences can also exist as independent small RNA species in some EBV-infected cell types. We present evidence that transcripts from these W repeat regions can exert a trans-acting effect on protein synthesis, through their ability to activate the dsRNA-dependent protein kinase PKR. UV cross-linking and filter binding assays have demonstrated that the W transcripts bind specifically to PKR and can compete with another EBV-encoded small RNA, EBER-1, which was shown previously to bind this kinase. In the reticulocyte lysate system the W RNAs shut off protein synthesis through an ability to activate PKR. In contrast to EBER-1, the W RNAs are unable to block the dsRNA-dependent activation of PKR. Using a purified preparation of the protein kinase we have shown that the W transcripts directly activate PKR in vitro. The results suggest that EBV has the ability both to activate and to inhibit PKR through the actions of different products of viral transcription.

Regulation of the interferon-inducible eIF-2 alpha protein kinase by small RNAs.

This review describes the structure and function of the double-stranded RNA-dependent protein kinase (PKR) and its interaction with RNA activators and inhibitors. The abilities of small virally-encoded RNAs such as VAI RNA of adenovirus, the Epstein-Barr virus encoded (EBER) RNAs and the Tat-responsive region RNA of HIV-1 to bind to and regulate PKR are reviewed, and the physiological implications of such regulation for the control of viral replication and cell growth are discussed. The potential effects on the activity of PKR of other proteins that bind double-stranded RNA and/or small viral and cellular RNAs are also considered.

Relationship of cellular oncogene expression to inhibition of growth and induction of differentiation of Daudi cells by interferons or TPA.

Human alpha or beta interferons inhibit the proliferation of Daudi Burkitt lymphoma cells and induce the differentiation of these cells towards a mature plasma cell phenotype. Similar responses are seen when Daudi cells are treated with the phorbol ester, TPA. Both interferons and TPA down-regulate expression of the c-myc oncogene in these cells. Although TPA can mimic the effect of interferon on cell differentiation, it does not induce 2'5' oligoadenylate synthetase or the interferon-sensitive mRNAs, 6-16 or 9-27. Thus chronic stimulation of protein kinase C by TPA cannot mimic all of the effects of interferon treatment on gene expression. Inhibition of ADP-ribosyl transferase activity by 3-methoxybenzamide impairs interferon- or TPA-induced differentiation of Daudi cells. This agent induces a higher level of c-myc mRNA in the cells and stimulates the incorporation of [3H]thymidine into DNA; although these effects are partially counteracted by interferon or TPA treatment, the elevated expression of the c-myc gene may be sufficient to prevent terminal differentiation and allow cell proliferation to continue.

Inhibition of polypeptide chain initiation in Daudi cells by interferons. Evidence that activity of initiation factor eIF-2 and availability of mRNA are unimpaired.

The accompanying paper [McNurlan & Clemens (1986) Biochem. J. 237, 871-876] shows that the inhibition of proliferation of Daudi cells by human interferons is associated with impairment of the overall rate of protein synthesis. We have examined whether two of the mechanisms which are believed to control translation in interferon-treated virus-infected cells may be responsible for the inhibition of protein synthesis during the antiproliferative response in these uninfected cells. Although the rate of polypeptide chain initiation is lower in interferon-treated Daudi cells, as indicated by the disaggregation of polysomes, there is no significant inhibition of activity of initiation factor eIF-2 or of [40 S . Met-tRNAf] initiation complex formation in cell extracts. The phosphorylation state of the alpha subunit of eIF-2 remains unaltered. There is no major decrease in mRNA content as a proportion of total RNA up to 4 days of interferon treatment, as judged by poly(A) content, although the amount of total mRNA/10(6) cells eventually declines. The mRNA present in extracts from interferon-treated cells remains translatable when added to an mRNA-dependent reticulocyte lysate system. We conclude that neither the interferon-inducible eIF-2 protein kinase pathway nor the 2',5'-oligo(adenylate)-ribonuclease L pathway are responsible for the inhibition of polypeptide chain initiation. Rather, the data suggest impairment at the level of formation of [80 S ribosome X mRNA] initiation complexes.

Regulation of protein synthesis in lymphoblastoid cells during inhibition of cell proliferation by human interferons.

Treatment of human lymphoblastoid (Daudi) cells with interferons inhibits cell proliferation in culture within 24 h. The failure of cell growth has been shown to be associated with impaired processing and decreased stability of newly replicated DNA. Because there is a close relationship between DNA replication and protein synthesis we have measured protein synthesis in intact Daudi cells. Protein synthesis declined steadily between 24 and 96 h after interferon treatment to a value which is only 20-30% of the rate in control cells. The enzyme 2',5'-oligo(A) synthetase is induced but our data do not support a role for the 2',5'-oligo(A)-activated ribonuclease in the control of translation in this system.

Inhibition of cell proliferation by interferons. 1. Effects on cell division and DNA synthesis in human lymphoblastoid (Daudi) cells.

Treatment of Daudi cells with human lymphoblastoid interferons for up to 5 days progressively inhibits cell proliferation. For the first 3 days cells continue to grow but with prolonged doubling times; subsequently, net proliferation ceases and is accompanied by a loss of cell viability. We have investigated the changes in labelling of DNA with radioactive precursors which occur during the first phase of the response to interferon treatment. We have shown previously [Gewert et al. (1981) Eur. J. Biochem. 116, 487-492] that inhibition of incorporation of [3H]thymidine into DNA can be accounted for by impairment of thymidine transport and thymidine kinase activity. In spite of this inhibition, the total intracellular dTTP pool is larger in interferon-treated than in control cells. Because of these changes it has been necessary to use other methods to determine whether interferon treatment inhibits the overall rate of DNA synthesis. The results of experiments employing (a) moderately high thymidine concentrations or (b) incorporation of radioactivity from deoxynucleoside triphosphates into DNA in detergent-lysed or permeabilised cell systems indicate that there is in fact relatively little inhibition of the overall rate of DNA synthesis in cells exposed to up to 100 units/ml of interferons for at least 48 h. Furthermore, a similar proportion of cells incorporate [3H]thymidine in control and interferon-treated cultures and there is only a small decrease in the number of cells in S phase after interferon treatment, as revealed by fluorescence-activated cell sorting. These results indicate that cell proliferation may be regulated in this system by a mechanism in which there is a loss of coordination between the initiation of DNA synthesis and the subsequent events required for cell division.