Subunit Of Initiation Factor eIF-2 Research Articles

Response of Three Different Viruses to Interferon Priming and Dithiothreitol Treatment of Avian Cells.

We have previously shown that the replication of avian reovirus (ARV) in chicken cells is much more resistant to interferon (IFN) than the replication of vesicular stomatitis virus (VSV) or vaccinia virus (VV). In this study, we have investigated the role that the double-stranded RNA (dsRNA)-activated protein kinase (PKR) plays in the sensitivity of these three viruses toward the antiviral action of chicken interferon. Our data suggest that while interferon priming of avian cells blocks vaccinia virus replication by promoting PKR activation, the replication of vesicular stomatitis virus appears to be blocked at a pretranslational step. Our data further suggest that the replication of avian reovirus in chicken cells is quite resistant to interferon priming because this virus uses strategies to downregulate PKR activation and also because translation of avian reovirus mRNAs is more resistant to phosphorylation of the alpha subunit of initiation factor eIF2 than translation of their cellular counterparts. Our results further reveal that the avian reovirus protein sigmaA is able to prevent PKR activation and that this function is dependent on its double-stranded RNA-binding activity. Finally, this study demonstrates that vaccinia virus and avian reovirus, but not vesicular stomatitis virus, express/induce factors that counteract the ability of dithiothreitol to promote eIF2 phosphorylation. Our data demonstrate that each of the three different viruses used in this study elicits distinct responses to interferon and to dithiothreitol-induced eIF2 phosphorylation when infecting avian cells. Type I interferons constitute the first barrier of defense against viral infections, and one of the best characterized antiviral strategies is mediated by the double-stranded RNA-activated protein kinase R (PKR). The results of this study revealed that IFN priming of avian cells has little effect on avian reovirus (ARV) replication but drastically diminishes the replication of vaccinia virus (VV) and vesicular stomatitis virus (VSV) by PKR-dependent and -independent mechanisms, respectively. Our data also demonstrate that the dsRNA-binding ability of ARV protein sigmaA plays a key role in the resistance of ARV toward IFN by preventing PKR activation. Our findings will contribute to improve the current understanding of the interaction of viruses with the host's innate immune system. Finally, it would be of interest to uncover the mechanisms that allow avian reovirus transcripts to be efficiently translated under conditions (moderate eIF2 phosphorylation) that block the synthesis of cellular proteins.

Phosphorylation of eIF2α on Threonine 169 is not required for Trypanosoma brucei cell cycle arrest during differentiation

The trypanosome life cycle consists of a series of developmental forms each adapted to an environment in the relevant insect and/or mammalian host. The differentiation process from the mammalian bloodstream form to the insect-midgut procyclic form in Trypanosoma brucei occurs in two steps in vivo. First proliferating ‘slender' bloodstream forms differentiate to non-dividing ‘stumpy' forms arrested in G1. Second, in response to environmental cues, stumpy bloodstream forms re-enter the cell cycle and start to proliferate as procyclic forms after a lag during which both cell morphology and gene expression are modified. Nearly all arrested cells have lower rates of protein synthesis when compared to the proliferating equivalent. In eukaryotes, one mechanism used to regulate the overall rate of protein synthesis involves phosphorylation of the alpha subunit of initiation factor eIF2 (eIF2α). The effect of eIF2α phosphorylation is to prevent the action of eIF2B, the guanine nucleotide exchange factor that activates eIF2 for the next rounds of initiation. To investigate the role of the phosphorylation of eIF2α in the life cycle of T. brucei, a cell line was made with a single eIF2α gene that contained the phosphorylation site, threonine 169, mutated to alanine. These cells were capable of differentiating from proliferating bloodstream form cells into arrested stumpy forms in mice and into procyclic forms in vitro and in tsetse flies. These results indicate that translation attenuation mediated by the phosphorylation of eIF2α on threonine 169 is not necessary for the cell cycle arrest associated with these differentiation processes.

Translation initiation factor modifications and the regulation of protein synthesis in apoptotic cells.

The rate of protein synthesis is rapidly down-regulated in mammalian cells following the induction of apoptosis. Inhibition occurs at the level of polypeptide chain initiation and is accompanied by the phosphorylation of the alpha subunit of initiation factor eIF2 and the caspase-dependent cleavage of initiation factors eIF4G, eIF4B, eIF2alpha and the p35 subunit of eIF3. Proteolytic cleavage of these proteins yields characteristic products which may exert regulatory effects on the translational machinery. Inhibition of caspase activity protects protein synthesis from long-term inhibition in cells treated with some, but not all, inducers of apoptosis. This review describes the initiation factor modifications and the possible signalling pathways by which translation may be regulated during apoptosis. We discuss the significance of the initiation factor cleavages and other changes for protein synthesis, and the implications of these events for our understanding of the cellular changes associated with apoptosis.

Specific Interaction of Eukaryotic Translation Initiation Factor 5 (eIF5) with the β-Subunit of eIF2

Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40 S initiation complex (40 S.mRNA. eIF3.Met-tRNAf.eIF2.GTP) and mediates hydrolysis of the bound GTP. To characterize the molecular interactions involved in eIF5 function, we have used 32P-labeled recombinant rat eIF5 as a probe in filter overlay assay to identify eIF5-interacting proteins in crude initiation factor preparations. We observed that eIF5 specifically interacted with the beta subunit of initiation factor eIF2. No other initiation factors including the gamma subunit of eIF2 tested positive in this assay. Furthermore, both yeast and mammalian eIF5 bind to the beta subunit of either mammalian or yeast eIF2. Binding analysis with human eIF2beta deletion mutants expressed in Escherichia coli identified a 22-amino acid domain, between amino acids 68 and 89, as the primary eIF5-binding region of eIF2beta. These results along with our earlier observations that (a) eIF5 neither binds nor hydrolyzes free GTP or GTP bound as Met-tRNAf.eIF2.GTP ternary complex, and (b) eIF5 forms a specific complex with eIF2 suggests that the specific interaction between eIF5 and the beta subunit of eIF2 may be critical for the hydrolysis of GTP during translation initiation.

Localization of the Human Interferon-Induced, ds-RNA Activated p68 Kinase Gene (PRKR) to Chromosome 2p21-p22

The interferon-induced dsRNA-activated protein kinase (PRKR) belongs to a subclass of serine/threonine kinases, involved in the regulation of protein synthesis by phosphorylation of the α subunit of initiation factor eIF2. Somatic cell hybrids segregating human chromosomes were used to assign this kinase to human chromosome 2. Fluorescence in situ hybridization confirmed this assignment and further localized the gene (PRKR) to the boundary region of hands p21 and 22.

Regulation of protein synthesis in rabbit reticulocyte lysates: Thiophosphorylation of initiation factor eIF-2 by heme-regulated protein kinase

The heme-regulated protein kinase, which specifically phosphorylates the 38-kDa subunit of initiation factor eiF-2, can utilize adenosine 5′- O-(3-thiotriphosphate) (ATP[γS]) as a substrate. The rate of thiophosphorylation is 5-6-times slower than that observed with ATP. It is of special interest that thiophosphorylated derivatives of eIF-2 are resistant to dephosphorylation catalyzed by eIF-2 phosphoprotein phosphatase. The thiophosphorylated eIF-2 is less effective in promoting protein synthesis in hemin-deficient lysates under physiological conditions. In addition, ATP[γS] could also be utilized by the self-phosphorylation activity intrinsically associated with HRI.

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).

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).

Mechanism of interferon action: Production and characterization of monoclonal and polyclonal antibodies to the interferon-induced phosphoprotein P 1

Monoclonal and polyclonal antibodies to the interferon-induced phosphoprotein P 1 were prepared using protein P 1 purified from human amnion U cells as the immunogen. Rabbit antiserum to protein P 1 recognized with comparable efficiency P 1 both from human U cells and from mouse L929 cells. Immunoprecipitates that contained protein P 1 also possessed a protein kinase activity that catalyzed the phosphorylation of protein P 1 and the α subunit of initiation factor eIF-2. Three BALB C mouse monoclonal antibodies efficiently recognized human protein P 1, but either did not recognize or recognized very poorly P 1 from mouse cells. A fourth monoclonal antibody against human P 1 recognized mouse P 1 with nearly equal efficiency. Immunoprecipitation of human P 1 with different sequential combinations of the monoclonal antibodies suggest that two antigenic classes of protein P 1 may exist.

Inhibition of viral mRNA translation in interferon-treated L cells infected with reovirus.

Murine L cells were treated with interferon (IFN) concentrations which reduced by 75 to 80% the synthesis of viral mRNA after infection with reovirus. Protein synthesis was not inhibited in these cells up to 6 h after infection, but a large fraction of the viral mRNA was not associated with polyribosomes and sedimented at about 50S. In contrast, most of the reovirus mRNA was associated with polyribosomes in control infected cells. This mRNA was of similar size to non-polyribosomal mRNA from IFN-treated cells when analyzed by Northern blot hybridization with a cloned cDNA for the s2 reovirus mRNA, indicating that the non-polyribosomal mRNA was not appreciably degraded. Viral mRNA was labeled with [3H]uridine and the non-polyribosomal mRNA was isolated from IFN-treated cells. This mRNA could quantitatively bind to 80S initiation complexes when incubated in a rabbit reticulocyte cell-free system. These findings indicated that the non-polyribosomal RNA was translatable, but that its binding to functional initiation complexes was inhibited in IFN-treated cells by a discriminatory mechanism, which did not affect translation of cellular mRNA. Previous experiments showed that mRNA is blocked in 48S complexes when the alpha subunit of initiation factor eIF-2 is phosphorylated by the double-stranded RNA-dependent protein kinase induced by IFN. A localized activation of this kinase could explain the block of viral mRNA in 48S complexes. By labeling the phosphoproteins of IFN-treated cells with 32P, eIF-2 (alpha P) was shown to cosediment with non-polyribosomal mRNA, presumably in 48S complexes.

Inhibition of mRNA binding to ribosomes by localized activation of dsRNA-dependent protein kinase

The initiation of protein synthesis can be regulated in mammalian cells by protein kinases which phosphorylate the alpha subunit of initiation factor eIF-2. This phosphorylation results in a block in the recycling of eIF-2 and in the inhibition of messenger RNA binding to 80S initiation complexes. After eIF-2 alpha is phosphorylated, the mRNA becomes associated with 48S complexes consisting of a 40S ribosomal subunit, eIF-2 (alpha P), GDP and Met-tRNAf. One of the eIF-2 alpha kinases is activated by low concentrations of double-stranded RNA (dsRNA). This kinase (PKds) is present at a basal level in all mammalian cells investigated and its synthesis is induced in cells treated with interferon. The PKds may be involved in the inhibition of translation of viral mRNA in interferon-treated cells infected with RNA viruses, as it is activated by viral replicative complexes. It is not known, however, if the activated PKds preferentially inhibits the translation of viral mRNA when cellular protein synthesis proceeds at a normal rate in infected cells. We now report that mRNA covalently linked to dsRNA is preferentially inhibited from binding to 80S complexes by a localized activation of PKds. This suggests that in interferon-treated cells the binding of some nascent viral mRNAs to functional initiation complexes may be preferentially inhibited by a similar mechanism.

Regulation of protein synthesis in rabbit reticulocyte lysates: effect of of Ca2+, Mg2+, and Mn2+ on self-phosphorylation and heterophosphorylation catalyzed by the heme-regulated and double-stranded-RNA-activated protein kinases.

The influence of divalent cations Mg2+, Mn2+, and Ca2+ on the cyclic-AMP-independent protein kinases of the heme-regulated and double-stranded-RNA-activated translational inhibitory protein kinases on self-phosphorylation and heterophosphorylation of the substrate (the 38 000-dalton subunit of initiation factor eIF-2) has been examined. Results show that Mg2+, Mn2+, and Ca2+ affect the activities of these enzymes in the following fashion. Mg2+ supports both self-phosphorylation and heterophosphorylation efficiently. Mn2+ on the other hand supports self-phosphorylation but to a lesser degree the heterophosphorylation. Ca2+ promotes neither self-phosphorylation nor heterophosphorylation.

Regulation of eukaryotic protein synthesis by protein kinases that phosphorylate initiation factor eIF-2: Further evidence for a common mechanism of inhibition of protein synthesis

The role of reversing factor (RF) in the regulation of protein synthesis by inhibitory protein kinases that phosphorylate the 38,000-dalton subunit of initiation factor eIF-2 has been examined. Results show that as with the heme-regulated protein kinase (HRI), RF restores protein synthesis in reticulocyte lysates inhibited by translational inhibitors from rat liver, wheat germ, Krebs ascites cell, by oxidized glutathione, the protein kinase activated by double stranded RNA (dRI), and the interferon-induced double stranded RNA activated protein kinase from Ehrlich ascites and Hela cells. These findings suggest that RF plays an important role in eukaryotic protein chain initiation cycle.

Regulation of protein synthesis in rabbit reticulocyte lysates: Inhibition of eIF-2 phosphoprotein phosphatase by NaF, pyrophosphate and cations

The nature and the role of eIF-2 phosphoprotein phosphatase in rabbit reticulocyte lysates have been examined. The eIF-2 phosphoprotein phosphatase is inhibited by a variety of divalent metal ions (Cd ++>Ag ++> Cu ++>Pb ++>Zn ++>Co ++>Sr ++>Mo ++) in lysates in situ . In addition, PPi, EDTA and NaF inhibit this enzyme. The eIF-2 phosphoprotein phosphatase is also inhibited by NaHSO 3 and Na 2S 2O 5. Na 2S 2O 5 is, however, more effective. Na 2S 2O 5 has been found to be a potent inhibitor of protein synthesis in lysates. This inhibition is associated with the phosphorylation of the 38,000-dalton subunit of initiation factor eIF-2. eIF-2 overcomes this inhibition. These findings suggest that under optimum conditions of protein synthesis the phosphorylation and dephosphorylation of eIF-2 are in a dynamic state of equilibrium in which dephosphorylation is favored. The inhibition of eIF-2 phosphoprotein phosphatase by Na 2S 2O 5 shifts this equilibrium in favor of eIF-2 phosphorylation, consequently, protein synthesis is inhibited. The sulfhydryl nature of eIF-2 phosphoprotein phosphatase has been established.

Structural requirements of polynucleotides for the activation of (2' - 5')An polymerase and protein kinase.

Two enzymatic pathways are involved in the inhibitory effects of double-stranded (ds)RNA on protein synthesis in cell extracts derived from interferon-treated human fibroblasts or HeLa cells, an oligonucleotide polymerase that synthesizes (2'-5')An from ATP and a protein kinase that phosphorylates the alpha subunit of initiation factor eIF-2 as well as a polypeptide of Mr = 72,000. We have now evaluated the activation of both the (2'-5')An polymerase and protein kinase by a large variety of polynucleotides, triple-stranded and synthetic dsRNAs, homopolymers, alternating copolymers, triple-stranded polymers, purine-purine duplexes and purine-pyrimidine duplexes with modifications at either the pyrimidine or ribose moieties. All these polynucleotides have been the subject of previous interferon induction studies. Some polynucleotides, i.e. (I)n.(C)n and mycophage dsRNA, which have been recognized as excellent interferon inducers, were also potent activators of both (2'-5')An polymerase and protein kinase, whereas non-inducers such as (A)n. (X)n and (A)n. (br5U)n did not activate either the kinase or the polymerase. However, some polymers like (I)n.(br5C)n, (difl)n(C)n and (dIcl)n (C)n, while potent interferon inducers and kinase activators, behaved poorly as activators of the (2'-5')An polymerase. Other polymers, i.e. (dAfl)n (U)n and (A)n.(U)nl (I)n, that do not induce interferon, activated the kinase but not the polymerase. Finally, (I)n (s2c)n, a relatively potent interferon inducer, did not activate either kinase or polymerase. These findings indicate that there is no simple relationship between the interferon-inducing ability of dsRNAs and their stimulating effects on (2'-5')An polymerase and protein kinase activity.

Cross-linking of Met-tRNAf to eIF-2 beta and to the ribosomal proteins S3a and S6 within the eukaryotic inhibition complex, eIF-2 .GMPPCP.Met-tRNAf.small ribosomal subunit.

In the quaternary initiation complex, eIF-2.GMPPCP.Met-tRNAf.40S ribosomal subunit, the Met-tRNAf can be cross-linked to the beta subunit of initiation factor eIF-2 as well as to ribosomal proteins S3a and S6 by treatment with the bifunctional reagent, diepoxybutane. Using 40S subunits, modified in advance with the heterobifunctional reagent, methyl-rho-azido-benzoylaminoacetimidate, Met-tRNAf is covalently bound to the same ribosomal proteins (S3a and S6) upon irradiation of the complex with ultraviolet light. Under both conditions proteins S3a and S6, together with a limited number of other ribosomal proteins, are covalently bound to 18S ribosomal RNA.

Activation of 2',5'-oligo(A) polymerase and protein kinase of interferon-treated HeLa cells by 2'-O-methylated poly (inosinic acid) . poly(cytidylic acid), Correlations with interferon-inducing activity.

An oligonucleotide polymerase and a protein kinase which require double-stranded RNA (dsRNA) for activation are induced in HeLa cells by human fibroblast interferon. The polymerase synthesizes a series of oligonucleotides from ATP, whereas the kinase phosphorylates a polypeptide of Mr = 72,000 and the alpha subunit of initiation factor eIF-2. Partially or fully 2'-O-methylated derivatives of poly(inosinic acid) . poly(cytidylic acid) (rIn . rCn) were used to determine the structural requirements of dsRNA in the activation of these two enzymes. While fully methylated polymers failed to activate either enzyme, partially methylated polymers activated the enzymes in specific manners. The activation of the kinase by the rIn . rCn analogues was affected more severely by the level of methylation than was the activation of the polymerase. Moreover, fully methylated analogues blocked the activation of the kinase by rIn . rCn but not the activation of the polymerase. These observations are consistent with a biphasic model for enzyme activation similar to that proposed for interferon induction, which required the recognition of a relatively small region of rIn . rCn as the last step. Differences in the activation of the polymerase and kinase are explicable on the basis of the polymerase requirement for a smaller recognition region of the rIn . rCn duplex than the kinase. Dependence of polymerase activation on the level of methylation shows striking similarities with the interferon inducing activities of these analogues, suggesting a possible relationship between polymerase activation and interferon induction.

Differential Effects of Two Interferon‐Induced Translational Inhibitors on Initiation of Protein Synthesis

At least two different mechanisms for the inhibition of mRNA translation operate in extracts of interferon-treated L cells. One is mediated by an interferon-induced protein kinase which, when activated by double-stranded RNA and ATP, phosphorylates the small subunit of initiation factor eIF-2. Addition of the purified interferon-induced protein kinase to L cell extracts, strongly reduces the amount of methionyl-tRNA bound to 40-S ribosomal subunits. The second translational inhibition is due to the synthesis of (2'-5')oligo(adenylate) by interferon-induced enzyme E. The oligonucleotide in turn activates a ribonuclease F constitutively present in L cells. Addition of the purified nuclease with its oligonucleotide activator to L cell extracts produces a strong decrease in polyribosome formation and an accumulation of initiation complex. These experiments differentiate the effects of the two interferon-induced inhibitors on mRNA translation.