The interferon-induced protein kinase PK-i from mouse L cells.

Interferon-treated L cells are characterized by an increased protein kinase activity that can selectively phosphorylate the small subunit of eukaryotic initiation factor 2. This protein kinase, PK-i, has been extensively purified and shown to be a potent inhibitor of mRNA translation. The purified PK-i contains the endogenously phosphorylated 67,000 Mr protein characteristic of interferon-treated cell extracts. PK-i can also phosphorylate arginine-rich histones. Purified PK-i can be activated by preincubation with ATP (but not adenylyl imidodiphosphate) and low concentrations of double-stranded RNA. The activation results in an increase in the first rate of eIF-2 phosphorylation. Activated PK-i becomes resistant to high concentrations of double-stranded RNA and more thermostable. A stimulator of PK-i activity, factor A, was isolated, as well as a specific phosphoprotein phosphatase that dephosphorylates the 67,000 Mr protein and eIF-2. These two factors, which are present in untreated L cells, may regulate the translation inhibitory activity of the interferon-induced and double-stranded RNA-activated protein kinase PK-i.     

Involvement of the 24-kDa cap-binding protein in regulation of protein synthesis in mitosis

The rate of protein synthesis in metaphase-arrested cells is reduced as compared to interphase cells. The reduction occurs at the translation initiation step. Here, we show that, whereas poliovirus RNA translation is not affected by the mitotic translational block, the translation of vesicular stomatitis virus mRNAs is. In an attempt to elucidate the mechanism by which initiation of protein synthesis is reduced in mitotic cells, we found that the interaction of the mRNA 24-kDa cap-binding protein (CBP) with the mRNA 5' cap structure is reduced in mitotic cell extracts, consistent with their lower translational efficiency. Addition of cap-binding protein complex stimulated the translation of endogenous mRNA in extracts from mitotic but not interphase cells. In addition, we found that the 24-kDa CBP from mitotic cells was metabolically labeled with 32P to a lesser extent than the protein purified from interphase cells. These results are consistent with a hypothesis that the 24-kDa CBP is implicated in the inhibition of protein synthesis in metaphase-arrested cells. Possible mechanisms for this inhibition are offered.     

Translational control by influenza virus: suppression of the kinase that phosphorylates the alpha subunit of initiation factor eIF-2 and selective translation of influenza viral mRNAs.

Selective translation of influenza viral mRNAs occurs after influenza virus superinfection of cells infected with the VAI RNA-negative adenovirus mutant dl331 (M. G. Katze, Y.-T. Chen, and R. M. Krug, Cell 37:483-490, 1984). Cell extracts from these doubly infected cells catalyze the initiation of essentially only influenza viral protein synthesis, reproducing the in vivo situation. This selective translation is correlated with a 5- to 10-fold suppression of the dl331-induced kinase that phosphorylates the alpha subunit of eucaryotic initiation factor eIF-2. This strongly suggests that influenza virus encodes a gene product that, analogous to the adenoviral VAI RNA, prevents the shutdown of overall protein synthesis caused by an eIF-2 alpha kinase turned on by viral infection. Adenoviral mRNA translation was restored to the extract from the doubly infected cells by the addition of the guanine nucleotide exchange factor eIF-2B, which is responsible for the normal recycling of eIF-2 during protein synthesis. This indicates that the residual kinase in the doubly infected cells leads to a limitation in functional (nonsequestered) eIF-2B and hence functional (GTP-containing) eIF-2 and that under these conditions influenza viral mRNAs are selectively translated over adenoviral mRNAs. Addition of double-stranded RNA to the extracts from these cells restored the eIF-2 alpha kinase to a level approaching that seen in extracts from cells infected with dl331 alone and caused the inhibition of influenza viral mRNA translation. This suggests that the putative influenza viral gene product acts against the double-stranded RNA activation of the kinase and indicates that influenza viral mRNA translation is also linked to the level of functional eIF-2. Our results thus indicate that a limitation in functional eIF-2 which causes a nonspecific reduction in the rate of initiation of protein synthesis results in the preferential translation of the better mRNAs (influenza viral mRNAs) at the expense of the poorer mRNAs (adenoviral mRNAs).     

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.     

The effects of haem on translational control of protein synthesis in cell-free extracts from fed and lysine-derived Ehrlich ascites tumour cells

1. Addition of haem to cell-free extracts from Ehrlich ascites tumour cells stimulates protein synthesis only in extracts from cells previously incubated in nutritionally complete conditions. Extracts from amino-acid-deprived cells do not respond to haem. The stimulation of protein synthesis in fed cell extracts is due to increased initiation on endogenous mRNA mediated by an increase in the levels of 40-S-subunit X Met-tRNA initiation complexes. Extracts from starved cells exhibit a defect in 40-S initiation complex formation which cannot be overcome by haem. 2. Experiments to test for the presence of an inhibitor of initiation in Ehrlich cell extracts by monitoring effects on translation in haem-supplemented reticulocyte lysates have revealed that extracts from both fed and starved cells contain one or more inhibitory activities which shut off protein synthesis, dissagregate polysomes and reduce the level of 40-S initiation complexes in the lysate. Extracts from starved cells are more inhibitory for protein synthesis than those from fed cells. 3. Initiation factor eIF-2 is phosphorylated by an endogenous Ehrlich cell protein kinase in vitro, but this occurs to the same extent in extracts from fed and starved cells. 4. We propose a possible model for the role of eIF-2 in the control of protein synthesis by amino acid supply in Ehrlich cells.     

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.     

Mechanism of interferon action: evidence for intermolecular autophosphorylation and autoactivation of the interferon-induced, RNA-dependent protein kinase PKR.

The interferon-induced RNA-dependent protein kinase (PKR) is postulated to have an important regulatory role in the synthesis of viral and cellular proteins. Activation of the enzyme requires the presence of a suitable activator RNA and is accompanied by an autophosphorylation of PKR. Active PKR phosphorylates the alpha subunit of protein synthesis eukaryotic initiation factor 2, resulting in an inhibition of translation initiation. The mechanism of autophosphorylation is not well understood. Here we present evidence that the autophosphorylation of human PKR can involve intermolecular phosphorylation events, i.e., one PKR protein molecule phosphorylating a second PKR molecule. Both wild-type PKR and the point mutant PKR(K296R) synthesized in vitro were phosphorylated, even though PKR(K296R) was deficient in kinase catalytic activity. Phosphorylation of both wild-type PKR and PKR(K296R) was inhibited in the presence of 2-aminopurine. Furthermore, purified human recombinant PKR(K296R) was a substrate for the purified wild-type human PKR kinase. This intermolecular phosphorylation of mutant PKR(K296R) by wild-type PKR was dependent on double-stranded RNA and was inhibited by 2-aminopurine. Finally, PKR mRNA was capable of mediating an autoactivation of wild-type PKR kinase autophosphorylation in vitro.     

Characterization and regulation of the 58,000-dalton cellular inhibitor of the interferon-induced, dsRNA-activated protein kinase.

The P68 protein kinase is a serine/threonine kinase induced by interferon treatment and activated by double-stranded RNAs (dsRNAs). Once activated, the kinase phosphorylates its natural substrate, the alpha subunit of eukaryotic initiation factor 2 (eIF-2) leading to potential limitations in functional eIF-2 and decreases in protein synthesis initiation. We have recently purified from influenza virus-infected cells a P68 kinase inhibitor, found to be a 58-kDa cellular protein. We have now investigated the mechanisms by which the 58-kDa inhibitor regulates P68 kinase activity and how the inhibitor itself is controlled. The 58-kDa inhibitor did not function by degrading or sequestering the dsRNA activator of P68 but could repress phosphorylation of eIF-2 alpha by an already activated protein kinase. Utilizing antibody prepared against a 58-kDa-specific peptide, we showed that the 58-kDa proteins from infected and uninfected cells were present in equivalent amounts. Although kinase inhibitory activity could not be detected in crude uninfected cell extracts, ammonium sulfate treatment unmasked this activity and allowed purification of the cellular inhibitor with identical chromatographic properties as that from influenza virus-infected cells. Finally, we have identified and partially purified a specific inhibitor of the 58-kDa protein which we refer to as an "anti-inhibitor." Based on these data, we present a model depicting the complex regulation of the interferon-induced protein kinase in eukaryotic cells.     

Expression and Purification of the α-Subunit of Eukaryotic Initiation Factor eIF2: Use as a Kinase Substrate

The α-subunit of eukaryotic initiation factor eIF2 (eIF2α) plays an important role in the regulation of mRNA translation through modulation of the interaction of eIF2 and a second initiation factor, eIF2B. The interaction of the two proteins is regulatedin vivoby phosphorylation of eIF2α at Ser⁵¹. In the present study, rat eIF2α was expressed in Sf21 cells using the baculovirus expression system. The recombinant protein was purified to >90% homogeneity in a single immunoaffinity chromatographic step. The protein was free of endogenous eIF2α kinase activity and was rapidly phosphorylated by the eIF2α kinases HCR and PKR. A variant of eIF2α in which the phosphorylation site was changed to Ala was also expressed and purified. The variant eIF2α was not phosphorylated by either HCR or PKR, demonstrating that the kinases specifically phosphorylate the correct site in the recombinant protein even in the absence of the other two subunits of the protein. In summary, a rapid and inexpensive method for obtaining eIF2α has been developed. Use of the wildtype and variant forms of eIF2α to measure eIF2α kinase activity in cell and tissue extracts should greatly facilitate examination of the regulation of mRNA translation under a variety of conditions.     

Regulation of mRNA translation and cellular signaling by hepatitis C virus nonstructural protein NS5A

The NS5A nonstructural protein of hepatitis C virus (HCV) has been shown to inhibit the cellular interferon (IFN)-induced protein kinase R (PKR). PKR mediates the host IFN-induced antiviral response at least in part by inhibiting mRNA translation initiation through phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). We thus examined the effect of NS5A inhibition of PKR on mRNA translation within the context of virus infection by using a recombinant vaccinia virus (VV)-based assay. The VV E3L protein is a potent inhibitor of PKR. Accordingly, infection of IFN-pretreated HeLa S3 cells with an E3L-deficient VV (VVDeltaE3L) resulted in increased phosphorylation levels of both PKR and eIF2alpha. IFN-pretreated cells infected with VV in which the E3L locus was replaced with the NS5A gene (VVNS5A) displayed diminished phosphorylation of PKR and eIF2alpha in a transient manner. We also observed an increase in activation of p38 mitogen-activated protein kinase in IFN-pretreated cells infected with VVDeltaE3L, consistent with reports that p38 lies downstream of the PKR pathway. Furthermore, these cells exhibited increased phosphorylation of the cap-binding initiation factor 4E (eIF4E), which is downstream of the p38 pathway. Importantly, these effects were reduced in cells infected with VVNS5A. NS5A was also found to inhibit activation of the p38-eIF4E pathway in epidermal growth factor-treated cells stably expressing NS5A. NS5A-induced inhibition of eIF2alpha and eIF4E phosphorylation may exert counteracting effects on mRNA translation. Indeed, IFN-pretreated cells infected with VVNS5A exhibited a partial and transient restoration of cellular and viral mRNA translation compared with IFN-pretreated cells infected with VVDeltaE3L. Taken together, these results support the role of NS5A as a PKR inhibitor and suggest a potential mechanism by which HCV might maintain global mRNA translation rate during early virus infection while favoring cap-independent translation of HCV mRNA during late infection.     

Partial purification and characterization of phorbol ester-regulated translational inhibitor(s) in human HL-60 leukemic cells

Addition of nanomolar concentration of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) to the human promyelocytic HL-60 leukemia cells is associated with a cessation of cellular proliferation and a subsequent differentiation into macrophage-like cells. Because the growth rate of mammalian cells is tightly coupled to the functions of the protein synthetic machinery, we have examined whether TPA induces a change in HL-60 translational functions. Addition of control HL-60 cell extracts to rabbit reticulocyte lysates results in a pronounced inhibition of protein synthesis, while TPA-treated HL-60 cell extracts are significantly less inhibitory. The reduction in TPA-induced translational inhibitory activity can be observed after a 3-6-h treatment and reaches a maximum after 24 h. Fractionation of control cell extracts on DEAE-cellulose columns reveals two inhibitory activities, eluting at 100 and 350 mM KCl, respectively. The DEAE-100 inhibitor(s) is further resolved into two activities by heparin-agarose column chromatography (HEP-100 and HEP-250). TPA treatment of HL-60 cells for 48 h completely eliminates the HEP-250 inhibitory activity and reduces the HEP-100 and the DEAE-350 inhibitory activities by 50 and 25%. Inhibition of protein synthesis in rabbit reticulocyte lysates by DEAE-100 inhibitory activities can be partially reversed by the addition of globin mRNA while translational inhibition by DEAE-350 inhibitor(s) can be reversed by addition of eukaryotic initiation factor (eIF) 2 or fructose 6-phosphate. The DEAE-100 inhibitor(s) causes extensive degradation of radioactive polynucleotides while the DEAE-350 inhibitor(s) is capable of phosphorylating both the alpha- and the beta-subunits of the highly purified rabbit reticulocyte initiation factor eIF-2. These data show that the DEAE-100 inhibitor(s) contains a nuclease while the DEAE-350 inhibitor(s) is associated with eIF-2 alpha and eIF-2 beta protein kinases.     

An adenovirus mutant unable to express VAI RNA displays different growth responses and sensitivity to interferon in various host cell lines.

The VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for the efficient translation of host cell and viral mRNAs late after infection. VAI RNA prevented activation of the interferon-induced P1/eIF-2 alpha kinase. In its absence the kinase was activated, eIF-2 alpha was phosphorylated, and translational initiation was inhibited. H5dl331 (dl331), a mutant which cannot express VAI RNA, grew poorly in 293 cells but generated wild-type yields in KB cells. The growth phenotype of the mutant appeared to correlate with the kinetics of kinase induction and activation. Active kinase appeared more rapidly in cell extracts prepared from infected 293 cells, in which dl331 grew poorly, than in extracts of KB cells, in which the mutant grew well. However, when kinase was induced in KB cells by interferon treatment and then activated subsequent to dl331 infection, viral protein synthesis was less severely inhibited than in interferon-treated 293 cells. Thus, activated kinase per se is insufficient to severely inhibit dl331 protein synthesis in KB cells.     

Stability of poliovirus RNA in cell-free translation systems utilizing two initiation sites

The stability of purified poliovirus RNA in cell-free translation systems prepared from HeLa cells or rabbit reticulocytes has been examined. Degradation of the RNA occurs with a t1/2 of approximately 35 min at 30 degrees C under conditions used for in vitro translation. Degradation is due in part to activity in the cell lysate, and in part to contaminants in the commercial preparations of creatine phosphokinase used in the energy-regenerating system. Addition of crude preparations of initiation factors significantly slows degradation, presumably as a result of protein-RNA interactions which confer resistance to nuclease action. Prior treatment of RNA with methylmercury hydroxide has no effect on degradation rates. On the other hand, endogenous mRNA, present as a messenger ribonucleoprotein particle in extracts from poliovirus-infected HeLa cells, remains completely intact during in vitro translation. These infected cell extracts synthesize the normal complement of viral proteins and utilize two different initiation sites for translation. Treatment of the infected cell extract with micrococcal nuclease destroys the endogenous mRNA. Subsequent addition of exogenous RNA to the same extract results in the formation of a protein-associated RNA particle with sedimentation properties slightly different from the endogenous messenger ribonucleoprotein, and the added RNA is unstable. We conclude that two initiation sites can be utilized on intact poliovirus mRNA, and fragmentation of the RNA is not prerequisite for generation of a second site in this RNA.     

Protein synthesis and endoplasmic reticulum stress can be modulated by the hepatitis C virus envelope protein E2 through the eukaryotic initiation factor 2alpha kinase PERK

The hepatitis C virus envelope protein, E2, is an endoplasmic reticulum (ER)-bound protein that contains a region of sequence homology with the double-stranded RNA-activated protein kinase PKR and its substrate, the eukaryotic translation initiation factor 2 (eIF2). We previously reported that E2 modulates global translation through inhibition of the interferon-induced antiviral protein PKR through its PKR-eIF2alpha phosphorylation site homology domain (PePHD). Here we show that the PKR-like ER-resident kinase (PERK) binds to and is also inhibited by E2. At low expression levels, E2 induced ER stress, but at high expression levels, and in vitro, E2 inhibited PERK kinase activity. Mammalian cells that stably express E2 were refractory to the translation-inhibitory effects of ER stress inducers, and E2 relieved general translation inhibition induced by PERK. The PePHD of E2 was required for the rescue of translation that was inhibited by activated PERK, similar to our previous findings with PKR. Here we report the inhibition of a second eIF2alpha kinase by E2, and these results are consistent with a pseudosubstrate mechanism of inhibition of eIF2alpha kinases. These findings may also explain how the virus promotes persistent infection by overcoming the cellular ER stress response.     

Oligonucleotides antisense to catalytic subunit of cyclic AMP-dependent protein kinase inhibit mouse mammary epithelial cell DNA synthesis

Mouse mammary epithelial cells were plated onto 24-well culture plates (50,000 per well), allowed to attach and serum starved for 24 h. Following serum starvation, DNA synthesis was induced by the addition of 10% fetal calf serum and determined by a 1-h pulse with [3H]thymidine from 17 to 18 h after serum addition. Addition of oligonucleotides antisense to the translation start region of cyclic AMP-dependent protein kinase (kinase A) mRNA inhibited thymidine incorporation into DNA (total or percentage of cells incorporating thymidine, as measured by autoradiography). This inhibition was apparent whether compared to controls with no oligonucleotide addition, sense oligonucleotides, or mismatch oligonucleotides. Enzymatic assays indicated that the antisense oligonucleotides lowered kinase A activity in cells. Time course studies indicated that the inhibition in DNA synthesis was not an artifact of the time at which DNA synthesis was estimated. Long-term (4 day) cultures indicated that effects on induction of DNA synthesis were reflected in long-term cell proliferation.     

Translational regulation of cyclin D1 by 15-deoxy-delta(12,14)-prostaglandin J(2).

The D-group cyclins play a key role in the progression of cells through the G(1) phase of the cell cycle. Treatment of MCF-7 breast cancer cells with the cyclopentenone prostaglandin 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) results in rapid down-regulation of cyclin D1 protein expression and growth arrest in the G(0)/G(1) phase of the cell cycle. 15d-PGJ(2) also down-regulates the expression of cyclin D1 mRNA; however, this effect is delayed relative to the effect on cyclin D1 protein levels, suggesting that the regulation of cyclin D1 occurs at least partly at the level of translation or protein turnover. Treatment of MCF-7 cells with 15d-PGJ(2) leads to a rapid increase in the phosphorylation of protein synthesis initiation factor eukaryotic initiation factor 2alpha (eIF-2alpha) and a shift of cyclin D1 mRNA from the polysome-associated to free mRNA fraction, indicating that 15d-PGJ(2) inhibits the initiation of cyclin D1 mRNA translation. The selective rapid decrease in cyclin D1 protein accumulation is facilitated by its rapid turnover (t(1/2) = 34 min) after inhibition of cyclin D1 protein synthesis. The half-life of cyclin D1 protein is not significantly altered in cells treated with 15d-PGJ(2). Treatment of cells with 15d-PGJ(2) results in strong induction of heat shock protein 70 (HSP70) gene expression, suggesting that 15d-PGJ(2) might activate protein kinase R (PKR), an eIF-2alpha kinase shown previously to be responsive to agents that induce stress. 15d-PGJ(2) strongly stimulates eIF-2alpha phosphorylation and down-regulates cyclin D1 expression in a cell line derived from wild-type mouse embryo fibroblasts but has an attenuated effect in PKR-null cells, providing evidence that PKR is involved in mediating the effect of 15d-PGJ(2) on eIF-2alpha phosphorylation and cyclin D1 expression. In summary, treatment of MCF-7 cells with 15d-PGJ(2) results in increased phosphorylation of eIF-2alpha and inhibition of cyclin D1 mRNA translation initiation. At later time points, repression of cyclin D1 mRNA expression may also contribute to the decrease in cyclin D1 protein.     

Heat shock impairs the interaction of cap-binding protein complex with 5' mRNA cap

Cell-free protein synthesizing systems prepared from heat-shocked Ehrlich cells retain the inhibition of translation that is seen at the cellular level. Recently, we showed that a highly purified cap-binding protein complex composed of the p220 and p28 subunits of eukaryotic initiation factor 4F, in a 1:1 molar ratio, restores protein synthesis in these cell-free translation systems (Lamphear, B.J., and Panniers, R. (1990) J. Biol. Chem. 265, 5333-5336). Here we have estimated the amount of cap-binding complex in cell extracts that can restore protein synthesis in heat-shocked cells. We find reduced restoring activity in heat-shocked cell extracts. Further, less cap-binding complex can be purified by 7-methyl-guanosine triphosphate Sepharose affinity chromatography from heat-shocked cell extracts, and we conclude that heat shock impairs the binding of complex to 5' mRNA cap. We have ruled out proteolysis and competitive inhibitors as mediators of this impairment. However we cannot distinguish between two possible explanations: (i) reduced association of p220 with p28 or (ii) a non-competitive inhibitor blocks complex binding to cap. We have also examined the affect of heat shock on the phosphorylation state of two forms of p28, p220.p28 complex and p28 free of p220. Both forms have reduced levels of phosphorylation during heat shock. The significance of these changes is discussed.     

Catalytic utilization of eIF-2 and mRNA binding proteins are limiting in lysates from vesicular stomatitis virus infected L cells

Infection of mouse L cells by vesicular stomatitis virus results in the inhibition of cellular protein synthesis. Lysates prepared from these infected cells are impaired in their ability to translate endogenous or exogenous cellular and viral mRNAs. The ability of initiation factors from rabbit reticulocytes to stimulate protein synthesis in these lysates was examined. Preparations of eukaryotic initiation factor 2 (eIF-2) and the guanine nucleotide exchange factor (GEF) stimulated protein synthesis strongly in L cell lysates from infected cells but only slightly in lysates from mock-infected cells. Maximal stimulation was obtained when a fraction containing eukaryotic initiation factors 4B (eIF-4B) and 4F (eIF-4F) was also present. In lysates from infected cells, these initiation factors increased endogenous cellular mRNA translation on the average 2-fold. In contrast, endogenous viral mRNA translation was increased to a much greater extent: the M protein was stimulated 8-fold, NS 5-fold, N 2.5-fold, and G 12-fold. When fractions containing eIF-4B, eIF-4F, or eIF-4A were added to these lysates in the presence of eIF-2, all three stimulated translation. Fractions containing rabbit reticulocyte initiation factors eIF-3 and eIF-6 had no effect on translation in either lysate. The results suggest that lysates from infected L cells are defective in the catalytic utilization of eIF-2 and deficient in mRNA binding protein activity.     

Inhibition of cell-free protein synthesis by pppA2'p5'A2'p5'A: a novel oligonucleotide synthesized by interferon-treated L cell extracts

The oligonucleotide pppA^2′ p^5′ A^2′ p^5′ A is synthesized by extracts from interferon-treated mouse L cells in the presence of double-stranded RNA. This compound is a potent inhibitor of protein synthesis in cell-free systems prepared from L cells or rabbit reticulocytes.After an initial lag, rates of protein synthesis in vitro are severely depressed in the presence of the oligonucleotide, and polysomes become disaggregated. In the presence of high concentrations of emetine, an inhibitor of chain elongation, reticulocyte polysomes containing an average of 4–6 ribosomes per mRNA are partially degraded to structures containing 1–4 ribosomes after incubation with the oligonucleotide. The level of association of exogenous ³⁵S-Met-tRNA_f with initiation complexes is not decreased, and under some conditions is even increased, by the oligonucleotide.When RNA is extracted from control and inhibited reticulocyte lysates and assayed for active mRNA content by retranslation in a fresh mRNA-dependent system, the results show extensive loss of template activity in the material obtained from the incubations containing pppA^2′ p^5′ A^2′ p^5′ A. The data are consistent with a mechanism in which this inhibitor activates a nuclease which prevents mRNA from being utilized for protein synthesis. This mechanism is contrasted with that of the heme-controlled repressor, another potent inhibitor of translation, which causes extensive inhibition of Met-tRNA_f binding to initiation complexes, has no effect on polysome size in the presence of emetine and does not inactivate mRNA.