Inhibition of Protein Synthesis in Cell-Free Systems from Interferon-Treated, Infected Cells: Further Characterization and Effect of Formylmethionyl-tRNA(F)

The translation of encephalomyocarditis virus (EMC) RNA is markedly inhibited in cell-free systems from interferon-treated, vaccinia virus-infected L-cells (10, 11). The polypeptide products synthesized in response to EMC RNA in cell-free systems from these and untreated infected cells have been analyzed by electrophoresis on polyacrylamide gels. Qualitatively, the same EMC-specific polypeptides were synthesized throughout. In experiments using preincubated microsomes from normal Krebs cells to assay cell sap from L-cells which had been exposed to interferon prior to infection, only the amount of the EMC-specific polypeptide products was reduced. This result suggests that there is an inhibition very early in translation in interferon-treated, infected cells. Initiation seems a priori the more attractive site for this inhibition, but an effect shortly after initiation cannot be excluded. With unfractionated cell-free systems from interferon-treated infected L-cells, however, there appeared to be an additional minor inhibitory effect on polypeptide chain elongation, in that the EMC-specific polypeptides synthesized showed not only a reduction in amount but also a bias towards lower molecular weight. The formylated methionyl initiator tRNA (Fmet-tRNA_F) was used as a further probe into the apparent effect on intiation. With this reagent we have confirmed that there is one major initiation site for the translation of EMC RNA in these cell-free systems. In addition, the results have shown that EMC-specific polypeptide chains initiated with Fmet escape the major interferon-mediated inhibition at or shortly after initiation.     

Protein Synthesis in Cell-Free Systems: an Effect of Interferon

The activity of ribosome and cell-sap fractions from interferon-treated and control chick embryo fibroblasts was compared in mixed chick-mouse and purely chick cell-free systems capable of the synthesis of viral polypeptide(s) in response to viral ribonucleic acid (RNA). Interferon treatment of cells did not affect the intrinsic amino acid incorporation activity of these systems or their response to polyuridylic acid. With encephalomyocarditis (EMC) virus RNA as messenger, however, a fraction of the ribosomes from interferon-treated cells appeared less active than parallel controls. The results obtained with the corresponding cell-sap fractions were variable. Although competition between endogenous and added messengers cannot be excluded in these systems, a reduced level of translation of EMC RNA with interferon-treated cell ribosomes was also suggested by the results of analyses of tryptic digests of the products formed in response to the RNA. In addition, these analyses showed that this reduced activity must reflect a reduction in the rate or frequency of translation rather than a decrease in the length of the EMC RNA translated, for the same polypeptides were synthesized in response to the RNA with material from interferon-treated and control cells. Interferon added directly to the cell-free system was without effect. Although suggestive, these results do not provide definitive evidence for or against the hypothesis that virus protein synthesis is inhibited at the translational level in the interferon-treated cell. Possible alternative interpretations of the data are discussed.     

Analogue inhibitor of 2-5A action: effect on the interferon-mediated inhibition of encephalomyocarditis virus replication.

Chemically synthesised CH3Sp(A2'p)2A2'pp3'OCH3 has been used to assess the importance of the ppp(A2'p)nA (n greater than or equal to 2: 2-5A) system in the antiviral action of interferon against encephalomyocarditis virus (EMC). It inhibits activation of the 2-5A-dependent RNase by 2-5A in intact mouse L929 cells and cell-free systems. In interferon-treated, EMC-infected L929 cells it inhibits 2-5A-mediated rRNA cleavage and partially restores EMC RNA synthesis and virus yield. Activation of the 2-5A-dependent RNase must, therefore, play some part in interferon action against the growth of EMC virus in such cells.     

Interferon treatment of Ehrlich ascites tumor cells: effects on exogenous mRNA translation and tRNA inactivation in the cell extract.

We reported earlier that in cell extracts that were prepared from interferon-treated Ehrlich ascites tumor cells and preincubated and passed through Sephadex G-25 (S60INT), the translation of exogenous mRNA (viral and host) was impaired and the impairment could be overcome to a large extent by adding a crude tRNA preparation from Ehrlich ascites tumor cells but not from Escherichia coli. We find now that the rate of inactivation of some tRNA's (especially those specific for leucine, lysine, and serine) but not those of many others is faster in S30INT than in corresponding extracts from control cells. This increased rate of tRNA inactivation may perhaps account for the need for added RNA to overcome at least partially the impairment of translation in S30INT. The relationship of the increased rate of tRNA inactivation to the antiviral effect of interferon is unclear. So far no significant difference has been detected in the amount of tRNA needed to overcome the impairment of encephalomyocarditis virus RNA translation in S30INT between tRNA from interferon-treated cells and tRNA from control cells. Futhermore, no difference was found in the rate of inactivation in S30INT between leucine-specific tRNA's from interferon-treated and from control cells. tRNA's specific for leucine and lysine were not inactivated (unless very slowly) during incubation under out conditions in an extract from interferon-treated (or from control) cells unless the extract had been passed through Sephadex G-25 or dialyzed. The translation fo exogenous mRNA was, however, impaired in an extract from interferon-treated cells that had not been passed through Sephadex G-25. This impairment was apparently not overcome by added tRNA.     

Interferon Treatment of Ehrlich Ascites Tumor Cells: Effects on Exogenous mRNA Translation and tRNA Inactivation in the Cell Extract

We reported earlier that in cell extracts that were prepared from interferon-treated Ehrlich ascites tumor cells and preincubated and passed through Sephadex G-25 (S30_INT), the translation of exogenous mRNA (viral and host) was impaired and the impairment could be overcome to a large extent by adding a crude tRNA preparation from Ehrlich ascites tumor cells but not from Escherichia coli. We find now that the rate of inactivation of some tRNA's (especially those specific for leucine, lysine, and serine) but not those of many others is faster in S30_INT than in corresponding extracts from control cells. This increased rate of tRNA inactivation may perhaps account for the need for added RNA to overcome at least partially the impairment of translation in S30_INT. The relationship of the increased rate of tRNA inactivation to the antiviral effect of interferon is unclear. So far no significant difference has been detected in the amount of tRNA needed to overcome the impairment of encephalomyocarditis virus RNA translation in S30_INT between tRNA from interferon-treated cells and tRNA from control cells. Furthermore, no difference was found in the rate of inactivation in S30_INT between leucine-specific tRNA's from interferon-treated and from control cells. tRNA's specific for leucine and lysine were not inactivated (unless very slowly) during incubation under our conditions in an extract from interferon-treated (or from control) cells unless the extract had been passed through Sephadex G-25 or dialyzed. The translation of exogenous mRNA was, however, impaired in an extract from interferon-treated cells that had not been passed through Sephadex G-25. This impairment was apparently not overcome by added tRNA.     

Adenovirus VAI RNA antagonizes the antiviral action of interferon by preventing activation of the interferon-induced eIF-2 alpha kinase

The VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for efficient translation of host cell and viral mRNAs late after infection. The growth of a viral mutant that is unable to produce the RNA is inhibited by interferon, while wild-type virus is not affected. VAI RNA prevents activation of the interferon-induced P1/eIF-2 alpha kinase. This inhibition can be reproduced in extracts of interferon-treated cells where purified VAI RNA prevents activation of latent kinase by double-stranded RNA.     

Studies on the Mechanism of Interferon Action

Interferon does not inactivate viruses or viral RNA. Virus growth is inhibited in interferon-treated cells, but apart from conferring resistance to virus growth, no other effect of interferon on cells has been definitely shown to take place. Interferon binds to cells even in the cold, but a period of incubation at 37°C is required for development of antiviral activity. Cytoplasmic uptake of interferon has not been unequivocally demonstrated. Studies with antimetabolites indicate that the antiviral action of interferon requires host RNA and protein synthesis. Experiments with 2-mercapto-1(β-4-pyridethyl) benzimidazole (MPB) suggest that an additional step is required between the binding and the synthesis of macromolecules. Interferon does not affect the adsorption, penetration, or uncoating of RNA or DNA viruses, but viral RNA synthesis is inhibited in cells infected with RNA viruses. The main action of interferon appears to be the inhibition of the translation of virus genetic information probably by inhibiting the initiation of virus protein synthesis.     

Synthesis of an oligonucleotide inhibitor of protein synthesis in rabbit reticulocyte lysates analogous to that formed in extracts from interferon-treated cells.

A heat-stable, low-molecular-weight inhibitor of protein synthesis is formed on incubation of haemin-supplemented rabbit reticulocyte lysates with ATP and double-stranded RNA (dsRNA). It inhibits the translation of both added encephalomyocarditis virus RNA (EMC RNA) and endogeneous messenger RNA in reticulocyte lysates and mouse L-cell extracts. The enzyme responsible for the synthesis of the inhibitor binds to dsRNA and can be purified on a column of poly(I).poly (C) bound to an inert support. The highly purified enzyme in its stable column-bound state can be conveniently employed to synthesise the inhibitor and to label it with [3H]ATP, or [alpha-32P]ATP or [gamma-32P]ATP as substrate. The radioactive inhibitor synthesised in this way with material from rabbit reticulocyte lysates shows the same spectrum of resistance and sensitivity to alkali and a variety of enzymes as corresponding material similarly synthesised with extracts from interferon-treated mouse L-cells. The inhibitors from the two systems have comparable absorbance spectra, are chromatographically and electrophoretically indistinguishable and are apparently identical in specific activity in the inhibition of protein synthesis in the cell-free system. The inhibitor is also formed on inhibition of protein synthesis by dsRNA in reticulocyte lysates. On comparison of the spectrum of polypeptide products synthesised in response to EMC RNA in the reticulocyte lysate, the effects of the inhibitor or dsRNA were similar: a distinctly different effect was obtained with the haemin-controlled repressor, a known inhibitor of initiation. The significance of these results with respect to the mechanism of action of the inhibitor and its role in the inhibition observed in response to dsRNA is discussed.     

Different mRNAs induced by interferon in cells from inbred mouse strains A/J and A2G

Treatment of cells from inbred mouse strains A/J and A2G with interferon resulted in the development of different antiviral states for influenza viruses. A2G mice-derived cells that carry the resistance gene Mx were efficiently protected by interferon against influenza viruses, whereas the interferon protection against the same viruses in wild-type A/J mice-derived cells was only marginal. The two cell types, however, were equally protected by interferon against vesicular stomatitis virus and other non-orthomyxoviruses. The interferon-induced mRNAs of mouse embryonic fibroblast cells that carried either homozygous wild-type alleles or homozygous Mx alleles were compared. The isolated polysome-bound mRNAs from A/J (+/+) and A2G (Mx/Mx) cells were translated in a cell-free translation system, and the translation products were analyzed after two-dimensional gel electrophoresis. New mRNAs coding for at least eight proteins with molecular weights (MW) ranging from 30,000 to 80,000 were found in interferon-treated cells but not in control cells. Differences in the interferon-induced mRNAs from A/J and A2G cells were also found. An mRNA coding for a 72,000-MW protein was found in interferon-treated A2G cells but not in interferon-treated A/J cells. Interferon-treated A/J cells, on the other hand, contained an mRNA coding for a 65,000-MW protein that was not found in interferon-treated A2G cells. The in vitro-synthesized 65,000-MW protein efficiently bound to GMP. Cytoplasmic extracts prepared from interferon-treated A/J cells also contained a GMP-binding 65,000-MW protein that was undetectable in similarly treated A2G cells.     

Characterization of the Polypeptides Formed in Response to Encephalomyocarditis Virus Ribonucleic Acid in a Cell-Free System from Mouse Ascites Tumor Cells

The polypeptide products synthesized at different times in a cell-free system from Krebs mouse ascites tumor cells in response to the addition of encephalomyocarditis (EMC) virus ribonucleic acid (RNA) were characterized by electrophoresis on polyacrylamide gels and fingerprint analysis of their tryptic peptides. Translation of the EMC RNA genome with time occurred in a nonrandom fashion in these systems, to yield products containing sequences characteristic of both virion capsid polypeptides and EMC-specific polypeptides present only in the infected cell. The molecular weights of the products fell in a series from 20,000 to 140,000 daltons, although occasionally traces of larger polypeptides were also observed. All of the major polypeptides appeared to arise from partial or complete translation of about 60% of the EMC RNA genome. They were not formed by cleavage of a large precursor molecule. It is suggested that they are artifacts generated by premature "termination" of nascent polypeptide chains at preferred sites.     

Virus Protein Synthesis in Animal Cell-Free Systems: Nature of the Products Synthesized in Response to Ribonucleic Acid of Encephalomyocarditis Virus

Ribonucleic acid (RNA) from encephalomyocarditis (EMC) virus stimulates the incorporation of amino acids into protein in cell-free protein-synthetic systems derived from Krebs mouse ascites tumor cells and chick embryo fibroblasts; the mouse system is the more responsive to the viral RNA. The greater part of this difference in activity can be ascribed to the cell sap, but the origin of the ribosomes also has a marked effect. The nature of the polypeptides formed in these cell-free systems was investigated by electrophoresis on polyacrylamide gels and by fingerprint analysis of tryptic digests. The same product in part appears to be synthesized in response to the EMC RNA in both systems. It was not detected if the EMC RNA was partly degraded (相似文献   

Indiscriminate degradation of RNAs in interferon-treated, vaccinia virus-infected mouse L cells.

In this report we used Northern blot hybridization analysis to characterize the fate of several species of viral RNA transcribed from internal and terminal regions of vaccinia DNA in interferon-treated, infected mouse L cells grown in suspension. All species of viral RNAs were expressed but were reduced in amount. Larger-sized RNAs were reduced more than smaller-sized RNAs. This reduction appears to be related to the activation of the interferon-mediated double-stranded RNA-dependent 2-5A synthetase-endoribonuclease system, as the rRNA cleavage pattern characteristic of this system was observed early in infection and in cell extracts in response to exogenous 2-5A. Thus, in interferon-treated, vaccinia-infected mouse L cells in suspension, there is indiscriminate degradation of viral and cellular RNAs, and this RNA breakdown might play a role in the interferon-mediated inhibition of protein synthesis.     

Synthesis of Capsid and Noncapsid Viral Proteins in Response to Encephalomyocarditis Virus Ribonucleic Acid in Animal Cell-Free Systems

The polypeptide products formed in two cell-free protein-synthetic systems programmed with encephalomyocarditis (EMC) virus ribonucleic acid (RNA) have been compared with the virus-specific proteins found in EMC-infected cells and with the capsid proteins of the purified virion. Tryptic peptides of (35)S-methioninelabeled proteins from these three sources were compared by co-chromatography and electrophoresis and by isoelectric focussing. Fifty-two methionine-containing peptides were resolved in digests of material from infected cells, of which about one-third were also clearly present in digests of the virion capsid proteins. The product formed in response to EMC RNA in cell-free systems from Krebs mouse ascites tumor cells yielded 26 to 29 such peptides. Most of these peptides were shown to behave identically with virus-specific peptides from infected cells, whereas just under half of them appeared to be identical with peptides from the virion capsid proteins. The product formed in response to EMC RNA in the L-cell cell-free system was similar, whereas six additional EMC-specific peptides were detected in mixed Krebs L-cell systems. The results indicate that the EMC RNA genome is partially translated in the mouse cell-free systems used to yield products containing both virion capsid and virus-specific noncapsid polypeptides.     

Induction of interferon in HeLa cells of a protein kinase activated by double-stranded RNA

Treatment of HeLa cells with human fibroblast interferon results in increased levels of latent protein kinase activity that can be activated by double-stranded RNA (dsRNA). The protein kinase activity in extracts of interferon-treated cells is assayed by two methods: (a) inhibition of protein synthesis in rabbit reticulocyte lysates and (b) phosphorylation of two polypeptides of Mr 72000 (P1) and 38000 (the eIF-2 alpha subunit of initiation factor 2). When extracts of interferon-treated cells are fractionated by centrifugation at 150000 x g, the protein kinase activity is found in the pellet fraction. The kinase is maximally activated by 0.1 micrograms/ml poly(I) . poly(C). An increase in protein kinase activity is detected after 8 h of treatment with 100 units interferon/ml or after a 17-h treatment with 12.5 units/ml or greater interferon concentrations. Therefore, the kinase activity increases as a function of both time of treatment and interferon concentration. Addition of actinomycin D to cells during interferon treatment prevents this increase. The protein kinase activity decreases gradually over three days when interferon-treated cells are subsequently grown in the absence of interferon.     

Correlation Between the Antiviral Effect of Interferon Treatment and the Inhibition of In Vitro mRNA Translation in Noninfected L cells

When noninfected L-cell suspension cultures are treated with interferon (specific activities superior to 10(6) reference units per mg of protein), the cell-free cytoplasmic extracts obtained are inactive for the translation of exogenous natural mRNAs. The dose-response curve shows that comparable amounts of interferon are required to produce a 50% reduction of Mengo virus multiplication in vivo and Mengo RNA translation in vitro. With higher doses of interferon, Mengo RNA translation is completely abolished, while poly U translation and endogenous protein synthesis are only slightly affected. The inactivation of Mengo RNA translation is reversible; after removal of interferon, normal translation activity is regained together with the ability to support Mengo virus multiplication. Fractionation of the cell-free extracts shows that the effect is localized in the fraction which can be washed off the ribosomes by high salt. These results establish that interferon induces a block in genetic translation in noninfected L cells.     

The interaction of wheat germ tyrosyl-tRNA synthetase and the tRNA-like end of brome mosaic virus RNA has no effect on in vitro viral protein synthesis and on in vitro encapsidation.

The effect of aminoacylation of the tRNA-like end of brome mosaic virus RNA during in vitro protein synthesis and in vitro viral encapsidation was investigated. The components of the homologous system were: BMV RNA, wheat germ cell-free protein synthesizing system and pure tyrosyl-tRNA synthetase from wheat germ. During in vitro protein synthesis directed with tyrosylated as well as non-tyrosylated BMV RNA, no differences were observed in the amount and in the class of polypeptides formed neither in the velocity of the translation reaction. Excess active TyrRS was added during in vitro translation, without modifying the translation efficiency. BMV RNA and active TyrRS were preincubated prior to translation in order to interact without the translation system components and then subjected to translation in vitro. Similar results were obtained when BMV RNA was preincubated with inactive TyrRS or BSA. These results indicate that the aminoacylation of BMV RNA has no pronounced effect on viral protein synthesis in vitro. During BMV RNA encapsidation either tyrosylated or non-tyrosylated BMV RNA 4 could be encapsidated in a similar way.     

Inhibition of Arbovirus Protein Synthesis by Interferon

Infection of cells treated with guanidine and actinomycin D and then washed to remove the guanidine inhibition of virus growth had no effect on antiviral activity already established by interferon. Protein synthesis in interferon-treated cells infected under these conditions was decreased as compared to control cells similarly treated but not exposed to interferon. In these control cells, analysis by polyacrylamide gel electrophoresis indicated that six proteins were produced during the first hour after guanidine reversal. Five of these proteins have been previously identified as probably being viral in origin. In interferon-treated cells, only a single major protein was produced. Ribonucleic acid (RNA) synthesis by Semliki Forest virus during the first hour after guanidine reversal was markedly depressed by incubation at 42 C, but no inhibition of total virus protein synthesis was seen; this finding suggested that much of the virus protein produced in the first hour after guanidine reversal was carried out by input virus RNA. Interferon was fully active in cells incubated at 42 C. The results suggested that interferon inhibits the production of Semliki Forest virus proteins ordinarily produced under the direction of the virus genome.     

Protein synthesis in extracts from interferon-treated Mengo virus infected cells

We previously showed in intact L cells that interferon treatment did not modify the shut-off of cellular RNA and protein synthesis induced by infection with Mengo virus although viral replication is inhibited (1,2). We have also demonstrated that inhibition of host protein synthesis was not due to degradation of messengers since cellular mRNA could be extracted from interferon-treated infected cells and efficiently translated in a reticulocyte lysate(2). Cellular mRNA was not degraded although 2–5A was present as reported here. We prepared cell-free systems from such cells at a time when cellular shut-off was fully established. The undegraded messengers remained untranslated under cell-free protein synthesis conditions and almost no polysomes were detected. The decreased amount of [³⁵S]Met-tRNA-40S complex observed in these lysates might account for the inhibition of protein synthesis at the level of initiation.     

Differential antiviral effects of interferon in three murine cell lines.

Infectious leukemia virus production by two chronically infected NIH/MOL lines was strongly inhibited by interferon treatment of the cells. The corresponding degree of inhibition in JLSV-11 cells was much lower. Multiplication of encephalomyocarditis virus in all three cell lines was barely affected by interferon treatment. Replication of vesicular stomatitis virus, on the other hand, was highly sensitive to interferon in the JLSV-11 line and in one NIH/MOL line but was practically insensitive in the other NIH/MOL line. Anticellular actions of interferon were more pronounced in the JLSV-11 line than in the others. In response to interferon treatment, 2',5'-oligoadenylate synthetase activity was induced to a high level in JLSV-11 cells and to lower levels in the NIH/MOL lines. We failed to detect any 2',5'-oligoadenylate-dependent endonuclease activity in extracts of these cells. Double-stranded RNA-dependent protein kinase activity was present in extracts of interferon-treated NIH/MOL cells, but it was barely detectable in extracts of interferon-treated JLSV-11 cells. The above studies demonstrated that interferon could differentially affect the replication of three different viruses in three different cell lines, including two seemingly identical NIH/MOL lines, and that certain tentative conclusions can be drawn regarding the roles of different interferon-inducible enzyme markers in the different antiviral actions of interferons.     

Sindbis virus translation is inhibited by a PKR/RNase L-independent effector induced by alpha/beta interferon priming of dendritic cells

The tropism of Sindbis virus (SB) for cells of the dendritic cell (DC) lineage and the virulence of SB in vivo are largely determined by the efficacy of alpha/beta interferon (IFN-alpha/beta)-mediated antiviral responses. These responses are essentially intact in the absence of PKR and/or RNase L (K. D. Ryman, L. J. White, R. E. Johnston, and W. B. Klimstra, Viral Immunol. 15:53-76, 2002). In the present studies, we investigated the nature of antiviral effects and identity of antiviral effectors primed by IFN-alpha/beta treatment of bone marrow-derived DCs (BMDCs) generated from mice deficient in PKR and RNase L (TD). IFN-alpha/beta priming exerted significant antiviral activity at very early stages of SB replication and most likely inhibited the initial translation of infecting genomes. The early effect targeted cap-dependent translation as protein synthesis from an SB-like and a simple RNA were inhibited by interferon treatment, but an encephalomyocarditis virus internal ribosome entry site-driven element exhibited no inhibition. Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 was defective after virus infection of TD cells, suggesting other mechanisms of translation inhibition. To identify components of these alternative antiviral pathway(s), we have compared global gene regulation in BMDCs derived from normal 129 Sv/Ev, IFNAR1-/-, and TD mice following infection with SB or treatment with IFN-alpha/beta. Candidate effectors of alternative antiviral pathways were those genes induced by virus infection or IFN-alpha/beta treatment in 129 Sv/Ev and TD-derived BMDC but not in virus-infected or IFN-alpha/beta-treated IFNAR1-/- cells. Statistical analyses of gene array data identified 44 genes that met these criteria which are discussed.