Asher Zilberstein

Specific phosphorylation in vitro of a protein associated with ribosomes of interferon-treated mouse L cells.

Cultures of L cells treated with mouse interferon yield cell extracts with a reduced activity to translate exogenously added mRNAs into proteins [l-5] . There is a strong correlation between the interferoninduced antiviral state in intact cells and the development of the in vitro translational inhibition [3] . The selectivity of interferon’s effect against viral, but not cellular, protein synthesis in intact cells [6-81 is, however, partially lost in the cell-free systems at high doses of interferon [3 ] . The reduced translational activity in vitro results from a dominant inhibitor(s), loosely associated with the ribosomes, whose activity increases with the dose of interferon used to treat the cells [3], but which has been only partially purified and characterized [3-5,9]. The inhibition affects both initiation and elongation of the polypeptide chains [9--l 11, and can be reduced by supplementing the extracts with excess amounts of some tRNA species [ II151. In contrast, the addition of double stranded RNA (ds RNA), as poly I: C [ 161 or the replicative form of Mengo RNA [ 111, increases the translation inhibition seen in extracts of interferon-treated cells. Recent work with reticulocyte lysates [ 17191 has indicated that the inhibitory activity of ds RNA on translation may be mediated by protein kinase activities. This observation prompted us to examine whether the increased sensitivity of extracts from interferon treated cells to ds RNA, is also mediated by protein phosphorylation. We show, here, that in the subcellular fraction which contains the interferoninduced inhibitor(s) of translation, there is strong

Interferon action: isolation of nuclease F, a translation inhibitor activated by interferon-induced (2'-5') oligo-isoadenylate.

Cell cultures treated by interferon become unable to support viral replication. At least one of interferon’s antiviral effects is to inhibit viral protein synthesis [l-3]. Interferon induces several biochemical mechanisms which could mediate this inhibition: (i) A double-stranded RNA (dsRNA) and ATP-dependent phosphorylation of initiation factor eIF2 and possibly other ribosome-associated proteins [4-71; (ii) The dsRNA ATP-dependent synthesis of an unusual oligo-adenylate isomer with 2’-5 phosphodiester bonds [7-91; (iii) A mechanism which affects polypeptide chain elongation, that does not require dsRNA and is reversed by tRNAs [lo-l 21. We have reported [3,9] the separation and isolation, from extracts of interferon-treated L cells, of the dsRNA ATP-activated protein kinase PKi, and of the (2’-5’) oligo-isoadenylate synthetase E. Here, we demonstrate that the oligonucleotide produced by E requires, to inhibit mRNA translation, an additional protein F already present constitutively in untreated cells. The purified F is shown to be an oligo-isoadenylate-dependent ribonuclease which degrades the mRNA template. Our data show that the dsRNA ATP-dependent nuclease activity reported [ 13,141 to be increased in extracts of interferon-treated cells, can be explained as the activation of a constitutive ribonuclease, F, by the product of the interferon-induced and dsRNA-dependent oligo-isoadenylate synthetase E. This is in

Control of messenger RNA translation by minor species of leucyl-transfer RNA in extracts from interferon-treated L cells

Mammalian cells contain a large number of iso-acceptor transfer RNAs, some of which are present in very small amounts as compared to the major species, and until now the function of these minor tRNA species has not been clarified. We have found that extracts from mouse cells treated by interferon require specifically the addition of some minor species of tRNA to allow proper translation of exogenous messenger RNA. By a series of chromatographic steps, we have purified minor Leu-tRNA species required for Mengo virus RNA translation from the other Leu-tRNAs, and identified their cognate codons. Globin mRNA and Mengo RNA require different minor Leu-tRNA species. This represents the first system in which translation of different mRNA is shown to be completely dependent on the addition of minor iso-acceptor tRNA which cannot be replaced by major species, even if these recognize the same codons.

Mechanism of the interferon‐induced block of mRNA translation in mouse L cells: Reversal of the block by transfer RNA

Addition of mouse interferon to L cells makes these cells unable to support the multiplication of viruses by blocking viral gene expression [ 1 ] . Interferon is also known to reduce the rate of cell division and the intracellular multiplication of some parasites [2]. To determine the changes induced by interferon in the cell, studies of gene expression in cell-free systems have been undertaken in several laboratories. Evidence for an effect of interferon treatment on mRNA translation is now well established in vitro [3-5,6,7]. Our work [3] has shown that cell-free extracts from interferon treated mouse L cells have lost their ability to translate into protein exogenously added natural mRNAs as Mengo RNA or Hemoglobin mRNA. This block in translation appears in uninfected L cell extracts, only under conditions in which interferon induces the antiviral state [4]. Endogenous protein synthesis and poly U translation are not significantly inhibited in interferon treated cell-extracts. We have previously shown that an inhibitor of translation, which appears to be a protein associated with the ribosomes, accumulates in extracts of interferon treated L cells [4,.5]. We show here that this inhibition of translation can be eliminated by the addition of purified fractions of mammalian transfer RNA. The requirement for tRNA in interferon treated cell extracts is caused by the ribosome associated inhibitor. The tRNA species which restore the translation of Mengo RNA and Hemoglobin mRNA in interferon treated cell

Reversal of the interferon-induced block of protein synthesis by purified preparations of eucaryotic initiation factor 2

Abstract The translation of Mengo virus RNA in extracts of interferon-treated mouse L cells in the presence of double-stranded RNA is inhibited about 80% when compared to translation in control cell extracts. Addition of eucaryotic initiation factor 2 (eIF-2) purified from rabbit reticulocytes leads to effective reversal of the interferon-induced block. This finding suggests that in interferon-treated cell extracts, inactivation of eIF-2 is a major mechanism of translational control.

INTERFERON-INDUCED TRANSLATIONAL REGULATION

Publisher Summary This chapter elaborates the interferon-induced translational regulation. Interferon treatment induces, thus, a translational inhibitory activity in SV40 infected BSC-1 cells, similar to that observed consistently in several laboratories for interferon-treated uninfected mouse L cells, or Ehrlich ascites tumor cells. Extracts of these interferon-treated cells, high-speed supernatant, or the fraction washed-off ribosomes by high salt, inhibit the translation of various mRNAs when added to cell-free protein synthesis systems derived from nontreated mouse cells. In these systems, the translational inhibitory activity was not specific against viral mRNAs, and affected also cellular mRNAs. Over the past few years, a strong correlation has been established between the induction of the antiviral state by interferon, and formation of the translational inhibitory activity. Highly purified preparations of mouse interferon induce the translational inhibitor, but even crude interferons from other animal species, which do not induce the antiviral effect in mouse cells, were inactive.

Biological Activities of Human Interferon-β2 Produced by cDNA Expression in Hamster Cells and Possible Autocrine Functions of this Cytokine-Induced IFN

Human fibroblasts induced by poly (rI)(rC) and sequential cycloheximide/Actinomycin D treatment produce, in addition to IFN-β1 and its 0.9 kb mRNA, another mRNA of 1.3 kb encoding IFN activity neutralized by anti-IFN-β antibodies and hence designated IFN-β 2 (1,2). An IFN-β2 cDNA was cloned (1) and used to screen a human genomic library from which two intron-containing genes IFA-2 (IFN-β2a) and IFA-11 (IFN-β2b) were identified (3). The IFN-β2a gene is 4.8 kb long, and was mapped on human chromosome 7 (4). Both genomic clones have been expressed in rodent cells and produced human IFN antiviral activities (5). The recombinant IFN-β2 induces (2′–5′) oligo A synthetase mRNA and HLA mRNAs in the presence of cycloheximide (3,6) and has antiviral activity on mouse-human hybrid cells containing human chromosome 21 (but not 9), excluding the possibility that IFN-32 acts through IFN-β1 induction (3,6). By immunocompetition with the in vitro translation product (23–26 Kd) of IFN-β2 mRNA, the native form of IFN-β2 secreted by human cells, was identified as a 21–22 Kd glycoprotein (3). Native IFN-β2 can be separated from IFN-β1 because it is not retained on Blue-Sepharose and elutes from DEAE-cellulose pH 7.4 at lower salt (150 mM NaCl) than IFN-β1.

The Interferon System: Studies on the Molecular Mechanism of Interferon Action

Exposure of sensitive cells to purified interferon, from a compatible species, induces a series of biological effects, the best known among these being the antiviral state and the decrease in the rate of cell proliferation. At least one of interferon’s antiviral effects is to inhibit viral protein synthesis (l-3). This inhibition is not immediate but requires several hours of active cellular RNA and protein synthesis before it fully develops. In the past year, interferon was shown to induce, in the treated cells, several new enzymatic activities which regulate protein biosynthesis and produce the translational inhibition observed. These biochemical mechanisms induced by interferon are reviewed here, and tentatively shown as three “pathways” in Figure 1.

THE REGULATION OF PROTEIN SYNTHESIS BY INTERFERON

Publisher Summary Cell cultures treated by interferon become unable to support viral replication. At least one of interferons antiviral effects is to inhibit viral protein synthesis. Interferon has been shown to induce several translation control mechanisms which could mediate this inhibition. The multiple antiviral effects by which interferon inhibit viral protein synthesis could each have its function in the cells. The tRNA sensitive inhibition plays its role mainly in the noninfected cell, where dsRNA is very low or absent. The protein kinase pathway is activated at low dsRNA concentrations but at higher dsRNA levels, it is switched off and the iso-adenylate-nuclease system may act. This multiphase antiviral state could insure that the interferon induced control of gene expression is appropriate to the state of the cell.

Interferon-type and Other Activities of IFN-ß-2/BSF-2/HSF

The multiple activities of IFN-β-2 (BSF-2, IL-6, 26 Kd, HGF, HSF) suggest that this multifunctional cytokine plays a role in the various aspects of inflammation resulting from pathogenic infections.