Interferon translational inhibition

Mittnacht S, Straub P, Kirchner H, Jacobsen H (1988) Interferon treatment inhibits onset of herpes simplex virus immediate- early transcription. Virology 164 201-210 Mohr I, Gluzman Y (1996) A herpesvirus genetic element which affects translation in the absence of the viral GADD34 function. Embo J 15 4759-4766... 

This inhibition is not immediate but requires several hoiors 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. 

Analysis of the cell-free translation system by sucrose gradient centrifugation, shows that PK-i causes a marked decrease in the amount of (55S-met-tRNA 4OS ribosome) complexes (12). Exogenous eIF-2 overcomes part of the dsRNA-dependent translational inhibition of mengo or globin mRNA in extracts from interferon treated cells (13) ... 

In mouse L cells, addition of one or two minor species of mammalian leucyl-tRNA were shown to be sufficient to restore translation (27). A similar conclusion was reached with yeast tRNA (28), even with poly(U,C) as template. The type of tRNA to be added appears to depend on the mRNA used (25, 27) and also on the extract Friend cells were reported to require lysyl-tRNA (29). These tRNA species were, however, not absent from interferon-treated cell extracts (24, 27, 50), l> u.t had to be added in excess over what is normally needed. A decreased charging of leu-tRNA or an increased deacylation were reported (5I, 32). Prolonged pre-incubation for 1-2 h of the extracts can increase the interferon-induced elongation block reversed by tRNA (55)-have recently observed that ATP is required to activate the translational inhibition seen in interferon-treated cell extracts without dsRNA. This could be shown by comparing extracts freed of endogenous mRNA activity either by micrococcal nuclease (54) or by pre-incubation with ATP. Table 5 shows that tRNA reversible-inhibition developed when ATP was present. It is not clear yet whether the protein kinase, or E and F may play a role in this inhibition without dsRNA. We also observed an inhibitor of leu-tRNA charging in ribosomal extracts of interferon-treated cells (Schmidt, A. and Revel, M., impublished). 

Most of the cell-free extracts used in these studies had been pre-incubated to reduce the translation of endogenous mRNA. Since different preparations of lysates exhibited varying degrees of interferon-induced inhibition, we studied the influence of the length of time of pre-incubation on the interferon-induced inhibition of vitro protein synthesis. e could establish that the ability of cell-free extracts to translate mengo virus RNA was a function of the time of pre-incubation. The ability of an extract from interferon-treated (int. S-10) cells to translate mengo virus RNA was not impaired when compared with an extract from control cells until between 60 and 90 of pre-incubation. At this point, although the activity of the control extract declined somewhat, the activity of the Int. S-10 declined much more rapidly. 

In marked contrast with mengo virus RNA, poly (lJ,C) translation was essentially unaffected by the addition of dsRNA to cell-free extracts from interferon-treated cells and in the presence of dsRNA, tRNA was still able to reverse the interferon-induced inhibition apparent in extracts pre-incubated for 75 (Table 4) Thus, with two doses of poly(l) poly(C), which gave 80% inhibition of mengo RNA translation in interferon-treated cell extracts pre-incubated for 15 min, only a slight effect (12%) on poly (U,C) translation was observed. After pre-incubation for 75 min, translation of poly (lJ,C) by the interferon-treated cell extract was... 

PKR, a widely-expressed serine/theonine protein kinase, is activated by dsRNA, interferon, and other agents.48 PKR was first described to inhibit translation via phos-... 

Interferons (IFN) are glycoproteins that, among other products, are released from virus-infected cells. In neighboring cells, interferon stimulates the production of "antiviral proteins." These inhibit the synthesis of viral proteins by (preferential) destruction of viral DNA or by suppressing its translation. Interferons are not directed against a specific virus, but have a broad spectrum of antiviral action that is, however, species-specific. Thus, interferon for use in humans must be obtained from cells of human origin, such as leukocytes (IFN-a), fibroblasts (IFN-P), or lymphocytes (IFN-y). Interferons are also used to treat certain malignancies and autoimmune disorders (e.g., IFN-a for chronic hepatitis C and hairy cell leukemia IFN-p for severe herpes virus infections and multiple sclerosis). 

Interference with assembly of viral coat proteins and viral RNA into new virus particles. Interferons may induce in the ribosomes of the host cells production of enzymes that inhibit translation of viral proteins. Avarol and avarine are thought to interfere with cytoskeletal assembly of virus particles. PROTEASE inhibitors can prevent the release of reverse transcriptase, and HIV-1 proteinase (e.g. saquinavir) are under development or in trial application. 

Another group of cytokines produced by many cell types, including macrophages, in response to viral challenge is the interferons (IFN). In mammalian cells, IFN induces the synthesis of Mx proteins that can inhibit the translation of viral mRNA. Induction of Mx proteins has been used as an indirect measure of IFN activity in fish40,68. Recently, Altmann et aV cloned, sequenced and characterized the first teleost interferon gene from the zebrafish, Danio rerio, so it should soon become feasible to quantify IFN production directly as a measure of anti-viral response. 

FIGURE 49-3 Interferon-mediated antiviral activity occurs via mub le mechanisms. The binding of IFN to specific cell surface receptor molecules signals die cell to produce a series of antiviral proteins. The stages of viral replication diat are inhibited by various IFN-induced antiviral proteins are shown. Most of these act to inhibit the translation of viral proteins (mechanism 2), but odier steps in viral replication also are affected (mechanisms 1,3, and 4). The roles of diese mechanisms in the other actions of IFNs are under study. Key IFN = interferon mRNA = messenger RNA Mx = specific cellular protein tRNA = transfer RNA RNase L = latent cellular endoribonuclease 2 5 A = 2 -5 -oligoadeny-lates eIF-2a = protein synthesis initiation factor. 

Although interferons are mediators of immune response, different mechanisms for the antiviral action of interferon have been proposed. Interferon-a possesses broad-spectrum antiviral activity and acts on virus-infected cells by binding to specific cell surface receptors. It inhibits the transcription and translation of mRNA into viral nuoleic acid and protein. Studies in cell-free systems have shown that the addition of adenosine triphosphate and double-stranded RNA to extracts of interferon-treated cells activates cellular RNA proteins and a oellular endonuclease. This activation causes the formation of translation inhibitory protein, which terminates production of viral enzyme, nucleic acid, and structural proteins (28). Interferon also may act by blocking synthesis of a cleaving enzyme required for viral release. 

Le Dinh T, Freneaux E, Labbe G, Letteron P, Degott C, Geneve J, Berson A, Larrey D, Pessayre D (1988) Amineptine, a tricyclic antidepressant, inhibits the mitochondrial oxidation of fatty acids and produces microvesicular steatosis of the liver in mice. J Pharmacol Exp Ther 247 745-750 Le Roy F, Bisbal C, Silhol M, Martinand C, Lebleu B, Salehzada T (2001) The 2-5A/RNase L/RNase inhibitor (RLl) pathway regulates mitochondrial mRNAs stability in interferon-a-treated H9 cells. J Biol Chem 276 48473 8482 Le Roy F, Silhol M, Salehzada T, Bisbal C (2007) Regulation of mitochondrial mRNA stability by RNase L is translation-dependent and controls IFNalpha-induced apoptosis. Cell Death Differ 14 1406-1413... 

Cell sap contains a specific phosphoprotein phosphatase (P) which dephosphoiylates protein 67, eIF-2 and several other dsRNA-dependent phosphorylated proteins (5). The protein phosphatase P is separated from PK-i on DEAE-cellulose and elutes at 25O1DM KCl. As shown in Figure 5> P stimulates mengo RNA translation in interferon-treated cell extracts (SlO-int) supplemented with dsRNA, but not in control cell extracts (SIO-cont). This suggests a regulatory mechanism which controls the level of PK-i dependent inhibition of protein synthesis. 

Factor P was first detected as an activity which inhibits translation only when added to extracts from interferon-treated cells in the presence of dsRNA, but not in control cell extracts or without dsRNA. Figure 5 shows that P elutes from DEAE-cellulose at 120mM KCl. It is- not well separated at this stage from a stimulator of protein synthesis, but could be purified on phosphocellulose pH 7 9 from which P eluted at 200-500mM KCl. Overall purification was 120-fold (I8). 

Addition of dsENA to extracts prepared from interferon-treated cells caused 80-90% inhibition of mengo virus EEA translation, regardless of the time of pre-incubation (Table 5) The same dose of dsEEA caused an inhibition of only 8-15% in control extracts. 

The ribonuclease activity was found in a sedimentable fraction known to contain the replication complex where synthesis of virus-coded MA takes place. Therefore, the antiviral action of interferon may be explained in this particular system by the ability of such a nuclease to degrade viral mRNA. Although the nuclease is not only active on mengo virus ENA its location within the cell would limit its action to the virus-coded single stranded ENA, the final consequence being some discrimination between viral and host cell messengers. This finding does not exclude the possibility that the inhibition of protein synthesis also occurs by other mechanisms such as impaired initiation of translation. 

Exposure of animal cells to interferon results in the establishment of an antiviral state manifested by the inhibition of virus replication (l). In interferon-treated cells transcription and translation of viral templates are specifically inhibited by the activation of cellular defence mechanisms, the molecular basis of which is still unclear (2). 

Translation is more sensitive to inhibition by dsRNA in extracts from interferon-treated cells than in those from control cells (Kerr et al., 1974). As in reticulocyte lysates, dsRNA causes eIF-2 to be phos-phorylated to a significant extent in interferon-treated cell extracts, but much less in extracts of uninfected cells (Lebleu et al., 1976 Roberts et al., 1976 Zilberstein et al., 1978). The translational block observed in the presence of dsRNA in extracts of interferon-treated mouse L cells can be overcome completely by the addition of eIF-2 (Kaempfer et al., 1979b)... 

These observations can account for the greater sensitivity of translation to dsRNA in extracts from interferon-treated cells. Apparently, interferon treatment induces a rise in the level of dsRNA-dependent eIF-2-kinase, but since the enzyme is inactive in the absence of sufficient dsRNA, protein synthesis in interferon treated, uninfected cells continues normally. During infection, however, virus-generated dsRNA potentiates the kinase and the resulting extensive phosphorylation of eIF-2 leads to a general inhibition of initiation of translation. As noted for reticulocyte lysates, in extracts of interferon-treated cells, too, there is no good correlation between the extent of phosphorylation of eIF-2 and inhibition of translation (Jacobsen et al., 1983). 

One question that deserves attention is the fact that, at least in the case of some virus-cell systems, viral RNA translation is inhibited preferentially over host mRNA translation, for example, in reovirus-infected L cells (Gupta et al., 1974). An interesting possibility was raised by Nilsen and Baglioni (1979). They showed that, in extracts of interferon-treated cells, VSV mRNA hybridized with poly (U) at its poly (A) tail or EMC RNA hybridized with poly (I) at its poly (C) tract are more rapidly degraded than the corresponding control mRNAs. They proposed that, in infected, interferon-treated cells, activation of the endoribonuclease takes place near the replicative intermediate of RNA viruses, because the dsRNA moiety therein promotes the formation of (2 -5 )oligo (A) in its vicinity. As a result, the viral mRNA portion in the replicative intermediate may be more sensitive to degradation than host mRNA. 

Gupta, S. L., Sopori, M. L., and Lengyel, P., 1974, Release of the inhibition of mRNA translation in extracts of interferon-treated Ehrlich ascites tumor cells by added tRNA, Biochem. Biophys. Res. Commun. 57 763. 

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