Protein synthesis interferon-induced inhibition

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. 

Yamazaki and Wagner (1970) found that pretreatment of primary rabbit kidney cells with highly purified rabbit interferon greatly reduced VSV replication and inhibited synthesis of virus-specific proteins despite this inhibition of viral protein synthesis, interferon did not prevent the switch-off by VSV of cellular protein synthesis. This observation was interpreted, in retrospect erroneously, to be due to an input virion component rather than a newly synthesized viral product. It was originally thought that primary transcription of VSV is inhibited in interferon-treated cells (Marcus et al., 1971 Manders et al., 1972), but it now seems clear that only secondary transcription and not primary transcription is inhibited in interferon-pretreated cells (Repik et al., 1974). There is now quite convincing evidence that the interferon-induced inhibitor of VSV replication functions at the translational level (Sen, 1982). These circumstantial data point to the hypothesis that a VSV transcription product, alone or in association with another factor, is the inhibitor of cellular protein synthesis. 

Administration of cycloheximide (60 mg/kg) 1 hr prior to tilorone administration inhibited the interferon response43,44. The inhibition by cycloheximide suggested that protein synthesis was involved in the appearance of interferon in the serum. For a more complete discussion of interferon induction, see the review on synthetic interferon inducers written by DeClercq45. ... 

Aronica SM, Mantel C, Gonin R, et al. Interferon-inducible protein 10 and macrophage inflammatory protein-1 alpha inhibit growth factor stimulation of Raf-1 kinase activity and protein synthesis in a human growth factor-dependent hematopoietic cell line. J Biol Chem 1995 270 21998-2007. 

In reticulocytes, elF-2 is subject to phosphorylation by the heme-regulated elF-2-kinase (HRI) which is regulated via the heme concentration. Another protein kinase that can phosphorylate and regulate elF-2 is the RNA-dependent elF2a-kinase (PKR). The latter is induced by interferons and activated by double stranded RNA. Stress influences activate the protein kinases PRPK and GCN2 allowing for inhibition of protein synthesis via elF-2 phosphorylation too. 

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. 

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-5) ... 

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. 

The association of ( S) methionyl-tRRA with the 4O-S subunit was strongly inhibited by poly(l) poly(c) in extracts of interferon-treated cells. In extracts of control cells poly(l) poly(c) had no significant effect upon the formation of methionyl-tRHA 40-S subunit complexes. Addition of crude rabbit reticulocyte initiation factors greatly reduced the poly(l) poly(c)-induced inhibition of (55s) methionyl-tRRA 4O-S subunit complex formation. The effect was most marked when the initiation factors were added during the incubation with dsRRA, strongly suggesting that in interferon-treated cell extracts dsRHA acts at the level of a protein synthesis initiation factor which binds methionyl-tRNA to native 4O-S ribosomal subunits, a situation analogous to that of the rabbit reticulocyte lysate (20-22 and chapter 10 of this volume). 

To summarize, it seems that dsRRA inhibits protein synthesis in extracts of interferon-treated cells by preventing initiation and by inducing the destruction of mRRA. The mechanism by which dsRNA produces these effects remains to be demonstrated, although it seems likely that activation of a specific protein kinase is involved in the initiation defect. The formation of a small oligonucleotide which inhibits protein synthesis has been reported in interferon-treated cell extracts incubated with dsRNA (l 6). 

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). 

The molecular basis by which interferons promote their characteristic effects, in particular antiviral activity, is understood at least in part. Interferon stimulation of the JAK-STAT pathway induces synthesis of at least 30 different gene products, many of which cooperate to inhibit viral replication. These antiviral gene products are generally enzymes, the most important of which are 2 -5 oligoadenylate synthetase (2,5-A synthetase) and the eIF-2a protein kinase. 

Other cytokines synthesised by activated neutrophils (but not bloodstream neutrophils) include interferon-a G-CSF and M-CSF. Interferon-a expression is stimulated by exposure of blood neutrophils to G-CSF (incubation times >3 h are required), but expression of this cytokine is not activated by exposure to either LPS or fMet-Leu-Phe. G-CSF induces 0.95- and 1.2-kb mRNA molecules, the latter transcript containing the message plus a 3 noncoding region. Synthesis of interferon-a protein (as determined by a radioimmunoassay) requires incubation times in excess of 6 h, and the functions of this cytokine include inhibition of neutrophil-colony formation and platelet formation in vitro. 

Interferon. Any of a family of glycoproteins that exert virus-nonspecific but host-specific antiviral activity by inducing the transcription of cellular genes coding for antiviral proteins that selectively inhibit the synthesis of viral RNA and proteins. Interferons have immunoregulatory functions and can inhibit the growth of nonviral intracellular parasites. 

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. 

Promotes chemotaxis and granule release by neutrophils, basophils, T cells Mitogenic for myeloid cells, supports antigen-independent growth of helper T cells Inhibits interferon secretion, induces thymocyte proliferation in presence of other IL s, inhibits Thl cytokine production Induction of synthesis of acute phase proteins, enhances stimulation of hematopoietic cell proliferation by IL-3 and T-cell development of B cells Activates NK cells, stimulates production of IFN-a by Thl cells... 

It was quickly established that interferon was not single protein, but rather, a large family of proteins. These are now classified as a-interferon (more than 24 species) and p-interferons, which are produced by most types of cells when invaded by a virus, and y-interferon, which is produced by infected T-lymphocytes. Once released, the interferons attach to specific receptors within other cells, and induce the synthesis of more than two dozen different kinds of proteins that provide resistance to viral attack. This is effected in a variety of ways that include inhibition of viral uptake and uncoating inhibition of viral mRNA production and thence of viral proteins and also inhibition of... 

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