The E3L and K3L vaccinia virus gene products stimulate translation through inhibition of the double-stranded RNA-dependent protein kinase by different mechanisms.

Vaccinia virus has evolved multiple mechanisms to counteract the interferon-induced antiviral host cell response. Recently, two vaccinia virus gene products were shown to interfere with the activity of the double-stranded RNA-dependent protein kinase (PKR): the K3L gene product and the E3L gene product. We have evaluated the efficiency by which these gene products inhibit PKR and whether they act in a synergistic manner. The effects of the two vaccinia virus gene products were compared in an in vivo system in which translation of a reporter gene (dihydrofolate reductase or eukaryotic translation initiation factor 2 alpha [eIF-2 alpha]) was inhibited because of the localized activation of PKR. In this system, the E3L gene product, and to a lesser extent the K3L gene product, potentiated translation of the reporter gene and inhibited eIF-2 alpha phosphorylation. Analysis in vitro demonstrated that the E3L gene product inhibited PKR approximately 50- to 100-fold more efficiently than the K3L gene product. However, further studies demonstrated that the mechanism of action of these two inhibitors was different. Whereas the E3L inhibitor interfered with the binding of the kinase to double-stranded RNA, the K3L inhibitor did not. We propose that the K3L inhibitor acts through its homology to eIF-2 alpha to interfere with the interaction of eIF-2 alpha with PKR. The two inhibitors did not display a synergistic effect on translation or eIF-2 alpha phosphorylation. In addition, neither K3L nor E3L expression detectably altered cellular protein synthesis.     

The phosphorylation state of eucaryotic initiation factor 2 alters translational efficiency of specific mRNAs.

Phosphorylation of the alpha subunit of the eucaryotic translation initiation factor (eIF-2 alpha) by the double-stranded RNA-activated inhibitor (DAI) kinase correlates with inhibition of translation initiation. The importance of eIF-2 alpha phosphorylation in regulating translation was studied by expression of specific mutants of eIF-2 alpha in COS-1 cells. DNA transfection of certain plasmids could activate DAI kinase and result in poor translation of plasmid-derived mRNAs. In these cases, translation of the plasmid-derived mRNAs was improved by the presence of DAI kinase inhibitors or by the presence of a nonphosphorylatable mutant (serine to alanine) of eIF-2 alpha. The improved translation mediated by expression of the nonphosphorylatable eIF-2 alpha mutant was specific to plasmid-derived mRNA and did not affect global mRNA translation. Expression of a serine-to-aspartic acid mutant eIF-2 alpha, created to mimic the phosphorylated serine, inhibited translation of the mRNAs derived from the transfected plasmid. These results substantiate the hypothesis that DAI kinase activation reduces translation initiation through phosphorylation of eIF-2 alpha and reinforce the importance of phosphorylation of eIF-2 alpha as a way to control initiation of translation in intact cells.     

Translational stimulation by reovirus polypeptide sigma 3: substitution for VAI RNA and inhibition of phosphorylation of the alpha subunit of eukaryotic initiation factor 2.

COS cells transfected with plasmids that activate DAI depend on expression of virus-associated I (VAI) RNA to prevent the inhibitory effects of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) kinase (DAI) and restore the translation of vector-derived dihydrofolate reductase mRNA. This VAI RNA requirement could be completely replaced by reovirus polypeptide sigma 3, consistent with its double-stranded RNA (dsRNA)-binding activity. S4 gene transfection of 293 cells also partially restored adenovirus protein synthesis after infection with the VAI-negative dl331 mutant. In dl331-infected 293 cells, eIF-2 alpha was present mainly in the acidic, phosphorylated form, and trans complementation with polypeptide sigma 3 or VAI RNA decreased the proportion of eIF-2 alpha (P) from approximately 85 to approximately 30%. Activation of DAI by addition of dsRNA to extracts of S4 DNA-transfected COS cells required 10-fold-higher levels of dsRNA than extracts made from cells that were not producing polypeptide sigma 3. In extracts of reovirus-infected mouse L cells, the concentration of dsRNA needed to activate DAI was dependent on the viral serotype used for the infection. Although the proportion of eIF-2 alpha (P) was greater than that in uninfected cells, most of the factor remained in the unphosphorylated form, even at 16 h after infection, consistent with the partial inhibition of host protein synthesis observed with all three viral serotypes. The results indicate that reovirus polypeptide sigma 3 participates in the regulation of protein synthesis by modulating DAI and eIF-2 alpha phosphorylation.     

PKR-dependent mechanisms of gene expression from a subgenomic hepatitis C virus clone

Studies on hepatitis C virus (HCV) replication have been greatly advanced by the development of cell culture models for HCV known as replicon systems. The prototype replicon consists of a subgenomic HCV RNA in which the HCV structural region is replaced by the neomycin phosphotransferase II (NPTII) gene, and translation of the HCV proteins NS3 to NS5 is directed by the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). The interferon (IFN)-inducible protein kinase PKR plays an important role in cell defense against virus infection by impairing protein synthesis as a result of eIF-2alpha phosphorylation. Here, we show that expression of the viral nonstructural (NS) and PKR proteins and eIF-2alpha phosphorylation are all variably regulated in proliferating replicon Huh7 cells. In proliferating cells, induction of PKR protein by IFN-alpha is inversely proportional to viral RNA replication and NS protein expression, whereas eIF-2alpha phosphorylation is induced by IFN-alpha in proliferating but not in serum-starved replicon cells. The role of PKR and eIF-2alpha phosphorylation was further addressed in transient-expression assays in Huh7 cells. These experiments demonstrated that activation of PKR results in the inhibition of EMCV IRES-driven NS protein synthesis from the subgenomic viral clone through mechanisms that are independent of eIF-2alpha phosphorylation. Unlike NS proteins, HCV IRES-driven NPTII protein synthesis from the subgenomic clone was resistant to PKR activation. Interestingly, activation of PKR could induce HCV IRES-dependent mRNA translation from dicistronic constructs, but this stimulatory effect was mitigated by the presence of the viral 3' untranslated region. Thus, PKR may assume multiple roles in modulating HCV replication and protein synthesis, and tight control of PKR activity may play an important role in maintaining virus replication and allowing infection to evade the host's IFN system.     

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.     

Structural requirements for double-stranded RNA binding, dimerization, and activation of the human eIF-2 alpha kinase DAI in Saccharomyces cerevisiae.

The protein kinase DAI is activated upon viral infection of mammalian cells and inhibits protein synthesis by phosphorylation of the alpha subunit of translation initiation factor 2 (eIF-2 alpha). DAI is activated in vitro by double-stranded RNAs (dsRNAs), and binding of dsRNA is dependent on two copies of a conserved sequence motif located N terminal to the kinase domain in DAI. High-level expression of DAI in Saccharomyces cerevisiae cells is lethal because of hyperphosphorylation of eIF-2 alpha; at lower levels, DAI can functionally replace the protein kinase GCN2 and stimulate translation of GCN4 mRNA. These two phenotypes were used to characterize structural requirements for DAI function in vivo, by examining the effects of amino acid substitutions at matching positions in the two dsRNA-binding motifs and of replacing one copy of the motif with the other. We found that both copies of the dsRNA-binding motif are required for high-level kinase function and that the N-terminal copy is more important than the C-terminal copy for activation of DAI in S. cerevisiae. On the basis of these findings, we conclude that the requirements for dsRNA binding in vitro and for activation of DAI kinase function in vivo closely coincide. Two mutant alleles containing deletions of the first or second binding motif functionally complemented when coexpressed in yeast cells, strongly suggesting that the active form of DAI is a dimer. In accord with this conclusion, overexpression of four catalytically inactive alleles containing different deletions in the protein kinase domain interfered with wild-type DAI produced in the same cells. Interestingly, three inactivating point mutations in the kinase domain were all recessive, suggesting that dominant interference involves the formation of defective heterodimers rather than sequestration of dsRNA activators by mutant enzymes. We suggest that large structural alterations in the kinase domain impair an interaction between the two protomers in a DAI dimer that is necessary for activation by dsRNA or for catalysis of eIF-2 alpha phosphorylation.     

Evasion of cellular antiviral responses by human cytomegalovirus TRS1 and IRS1

During infection with human cytomegalovirus (HCMV), cellular protein synthesis continues even as viral proteins are being synthesized in abundance. Thus, HCMV may have a mechanism for counteracting host cell antiviral pathways that act by shutting off translation. Consistent with this view, HCMV infection of human fibroblasts rescues the replication of a vaccinia virus mutant lacking the double-stranded RNA-binding protein gene E3L (VVdeltaE3L). HCMV also prevents the phosphorylation of the eukaryotic translation initiation factor eIF-2alpha, the activation of RNase L, and the shutoff of viral and cellular protein synthesis that otherwise result from VVdeltaE3L infection. To identify the HCMV gene(s) responsible for these effects, we prepared a library of VVdeltaE3L recombinants containing HCMV genomic fragments. By infecting nonpermissive cells with this library and screening for VV gene expression and replication, we isolated a virus containing a 2.8-kb HCMV fragment that rescues replication of VVdeltaE3L. The fragment comprises the 3' end of the J1S open reading frame through the entire TRS1 gene. Analyses of additional VVdeltaE3L recombinants revealed that the protein encoded by TRS1, pTRS1, as well as the closely related IRS1 gene, rescues VVdeltaE3L replication and prevent the shutoff of protein synthesis, the phosphorylation of eIF-2alpha, and activation of RNase L. These results demonstrate that TRS1 and IRS1 are able to counteract critical host cell antiviral response pathways.     

Mechanism of interferon action. Purification and substrate specificities of the double-stranded RNA-dependent protein kinase from untreated and interferon-treated mouse fibroblasts

The double-stranded RNA (dsRNA)-dependent protein kinase which catalyzes the phosphorylation of ribosome-associated protein P1 and the alpha subunit of eukaryotic protein synthesis initiation factor 2 (eIF-2) was purified and characterized from mouse fibroblast L929 cells treated with either natural or recombinant interferon and from untreated cells. The dsRNA-dependent P1/eIF-2 alpha kinase was purified at least 1,500-fold from interferon-treated cells; the kinase activity that catalyzed the phosphorylation of eIF-2 alpha copurified with protein P1. The yield of P1/eIF-2 alpha protein kinase activity obtained following purification from cells treated with interferon was about 5-10 times greater than the yield from an equivalent number of untreated cells. The purified protein kinase remained dsRNA dependent. When P1 kinase was activated by dsRNA, a major phosphopeptide designated Xds was phosphorylated; Xds was not phosphorylated from P1 which had not been activated by dsRNA. The apparent native molecular weight of the purified mouse L929 dsRNA-dependent kinase as determined by sedimentation analysis was about 62,000, comparable to the molecular weight of 67,000 determined for denatured L929 phosphoprotein P1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein kinase was highly selective for the alpha subunit of protein synthesis initiation factor eIF-2 and endogenous protein P1. Kinase activity was dependent upon Mg2+, and the Km for ATP was determined to be 5 X 10(-6) M. Histones (H1, H2A-B, H3, and H4) and protein synthesis initiation factors other than eIF-2 (eIF-3, eIF-4A, eIF-4B, and eIF-5) were not substrates or were very poor substrates for the purified dsRNA-dependent protein kinase. N-Ethylmaleimide, ethylenediaminetetraacetic acid, AMP, pyrophosphate, spermine, spermidine, and high concentrations of potassium inhibited both P1 and eIF-2 alpha phosphorylation by the purified kinase, whereas ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and phenanthroline did not significantly affect the phosphorylation of either protein P1 or eIF-2 alpha.     

Phosphorylation of initiation factor-2 alpha is required for activation of internal translation initiation during cell differentiation.

The long uORF-burdened 5'UTRs of many genes encoding regulatory proteins involved in cell growth and differentiation contain internal ribosomal entry site (IRES) elements. In a previous study we showed that utilization of the weak IRES of platelet-derived growth factor (PDGF2) is activated during megakaryocytic differentiation. The establishment of permissive conditions for IRES-mediated translation during differentiation has been confirmed by our demonstration of the enhanced activity of vascular endothelial growth factor, c-Myc and encephalomyocarditis virus IRES elements under these conditions, although their mRNAs are not naturally expressed in differentiated K562 cells. In contrast with the enhancement of IRES-mediated protein synthesis during differentiation, global protein synthesis is reduced, as judged by polysomal profiles and radiolabelled amino acid incorporation rate. The reduction in protein synthesis rate correlates with increased phosphorylation of the translation initiation factor eIF2 alpha. Furthermore, IRES use is decreased by over-expression of the dominant-negative form of the eIF2 alpha kinase, PKR, the vaccinia virus K3L gene, or the eIF2 alpha-S51A variant which result in decreased eIF2 alpha phosphorylation. These data demonstrate a connection between eIF2 alpha phosphorylation and activation of cellular IRES elements. It suggests that phosphorylation of eIF2 alpha, known to be important for cap-dependent translational control, serves to fine-tune the translation efficiency of different mRNA subsets during the course of differentiation and has the potential to regulate expression of IRES-containing mRNAs under a range of physiological circumstances.     

Regulation of the protein kinase PKR by the vaccinia virus pseudosubstrate inhibitor K3L is dependent on residues conserved between the K3L protein and the PKR substrate eIF2alpha.

The mammalian double-stranded RNA-activated protein kinase PKR is a component of the cellular antiviral defense mechanism and phosphorylates Ser-51 on the alpha subunit of the translation factor eIF2 to inhibit protein synthesis. To identify the molecular determinants that specify substrate recognition by PKR, we performed a mutational analysis on the vaccinia virus K3L protein, a pseudosubstrate inhibitor of PKR. High-level expression of PKR is lethal in the yeast Saccharomyces cerevisiae because PKR phosphorylates eIF2alpha and inhibits protein synthesis. We show that coexpression of vaccinia virus K3L can suppress the growth-inhibitory effects of PKR in yeast, and using this system, we identified both loss-of-function and hyperactivating mutations in K3L. Truncation of, or point mutations within, the C-terminal portion of the K3L protein, homologous to residues 79 to 83 in eIF2alpha, abolished PKR inhibitory activity, whereas the hyperactivating mutation, K3L-H47R, increased the homology between the K3L protein and eIF2alpha adjacent to the phosphorylation site at Ser-51. Biochemical and yeast two-hybrid analyses revealed that the suppressor phenotype of the K3L mutations correlated with the affinity of the K3L protein for PKR and was inversely related to the level of eIF2alpha phosphorylation in the cell. These results support the idea that residues conserved between the pseudosubstrate K3L protein and the authentic substrate eIF2alpha play an important role in substrate recognition, and they suggest that PKR utilizes sequences both near and over 30 residues from the site of phosphorylation for substrate recognition. Finally, by reconstituting part of the mammalian antiviral defense mechanism in yeast, we have established a genetically useful system to study viral regulators of PKR.     

Mechanism of interferon action. Effect of double-stranded RNA and the 5'-O-monophosphate form of 2',5'-oligoadenylate on the inhibition of reovirus mRNA translation in vitro

The effect of reovirus double-stranded RNA (dsRNA) and 5'-O-monophosphate form of 2',5'-oligoadenylate (pA(2'p5'A)2) on the translation and degradation of reovirus messenger RNA and on protein phosphorylation was examined in extracts prepared from interferon-treated mouse L fibroblasts. The following results were obtained. 1) The enhanced degradation of reovirus [3H]mRNA observed in the presence of either dsRNA or the 5'-O-triphosphate form of 2',5'-oligoadenylate (pppA(2'p5'A)3) was completely blocked by pA(2'p5'A)2. 2) The dsRNA-dependent phosphorylation of protein P1 and the alpha subunit of eukaryotic initiation factor (eIF-2) depended in a similar manner upon the concentration of dsRNA and was optimal at low dsRNA concentrations (0.1 to 1 microgram/ml). However, high concentrations of dsRNA (greater than 100 micrograms/ml) drastically reduced the phosphorylation of both P1 and eIF-2 alpha. Neither P1 nor eIF-2 alpha phosphorylation was affected by either pA(2'p5'A)2 or pppA(2'p5'A)3. 3) The translation of reovirus mRNA in vitro was inhibited by the addition of either low concentrations of dsRNA or pppA(2'p5'A)3. Whereas pA(2'p5'A)2 completely reversed the pppA(2'p5'A)3-mediated inhibition of translation, the inhibition mediated by low concentrations of dsRNA was only partially reversed by pA(2'p5'A)2. Under conditions where the pppA-(2'p5'A)3mediated degradation of reovirus mRNA was blocked, the translation of reovirus mRNA was still inhibited by low but not by high concentrations of dsRNA in a manner that correlated with the activation of P1 and eIF-2 alpha phosphorylation. These results suggest that the pppA(2'p5'A)n-dependent ribonuclease is not required and that protein phosphorylation may indeed be sufficient for the dsRNA-dependent inhibition of reovirus mRNA translation in cell-free systems derived from interferon-treated mouse fibroblasts.     

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).     

Activation of the double-stranded RNA-dependent eIF-2 alpha kinase by cellular RNA from 3T3-F442A cells.

The interferon induced double-stranded-RNA-dependent eIF-2 alpha kinase has an established role in mediating part of interferons anti-viral effects. Several studies have suggested that it may have additional functions in cells not infected with virus. The mechanism of activation of the kinase and the consequences of its activity in uninfected cells remain to be determined. Our previous results have indicated that the activation (phosphorylation) of this kinase may be an important regulatory signal to the arrest of growth of mouse 3T3-F442A fibroblasts and their subsequent differentiation to adipocytes. We have found that the phosphorylation of the kinase occurred in vivo in the absence of viral infection and in vitro without the addition of dsRNA. We demonstrate here that total cytoplasmic RNA from 3T3-F442A cells contains a regulatory RNA(s) capable of activating dsRNA-dependent eIF-2 alpha kinase. Fractionation of the cytoplasmic RNA by oligo(dT)-cellulose indicated that the regulatory RNA eluted with the poly(A)-rich RNA fraction. It bound tightly to the dsRNA-dependent eIF-2 alpha kinase and was immune-precipitated with its antibodies as a complex of regulatory RNA and dsRNA-dependent eIF-2 alpha kinase. The regulatory RNA activity was further purified by phenol extraction of immune precipitates containing this complex. These findings indicated that the regulatory RNA forms a specific complex with the dsRNA-dependent eIF-2 alpha kinase. The activity of the regulatory RNA was sensitive to the dsRNA-specific RNase VI but not to proteinase K, DNase I or ssRNA-specific RNase T1. The activation of the dsRNA-dependent eIF-2 alpha kinase by regulatory RNA was prevented by addition of a high concentration of poly(I).poly(C). The regulatory RNA was also shown to activate partially purified dsRNA-dependent eIF-2 alpha kinase prepared from rabbit reticulocyte lysates and to inhibit protein synthesis in reticulocyte lysates. Our findings, that cellular RNAs can specifically activate the dsRNA-dependent eIF-2 alpha kinase, are consistent with a physiological role for the dsRNA-dependent eIF-2 alpha kinase and interferon during cell growth and differentiation. The relationship of the regulatory RNA activity to growth and differentiation of 3T3-F442A cells is discussed.     

The phosphorylation of eukaryotic initiation factor 2: a principle of translational control in mammalian cells

In eukaryotic cells, protein biosynthesis is controlled at the level of polypeptide chain initiation. During the initiation process, eukaryotic initiation factor 2 (eIF-2) catalyzes the binding of Met-tRNAf and GTP to the 40S ribosomal subunit. In a later step, eIF-2 is released from the ribosomal initiation complex, most likely as an eIF-2.GDP complex, and another initiation factor termed eIF-2B is necessary to recycle eIF-2 by displacing GDP by GTP. In rabbit reticulocytes, inhibition of protein synthesis is accompanied by the phosphorylation of the alpha-subunit of eIF-2, a process that does not render eIF-2 inactive, but prevents it from being recycled by eIF-2B. First described in rabbit reticulocytes as inhibitors of translation, two distinct eIF-2 alpha kinases are known: the haemin-controlled kinase (termed HCI) and the double-stranded RNA-activated kinase (termed DAI). eIF-2 alpha phosphorylation appears to be a reversible control mechanism since corresponding phosphatases have been described. Recent reports indicate a correlation between eIF-2 alpha phosphorylation and the inhibition of protein synthesis in several mammalian cell types under a range of physiological conditions. In this review, the physical and functional features of the known eIF-2 alpha kinases are described with respect to their role in mammalian cells and the mode of activation by cellular signals. Furthermore, the possible impact of the eIF-2/eIF-2B ratio and of the subcellular compartmentation of these factors (and the eIF-2 alpha kinases) on mammalian protein synthesis is discussed.     

Abrogation of translation initiation factor eIF-2 phosphorylation causes malignant transformation of NIH 3T3 cells.

The interferon induced double-stranded RNA-activated kinase, PKR, has been suggested to act as a tumor suppressor since expression of a dominant negative mutant of PKR causes malignant transformation. However, the mechanism of transformation has not been elucidated. PKR phosphorylates translation initiation factor eIF-2 alpha on Ser51, resulting in inhibition of protein synthesis and cell growth arrest. Consequently, it is possible that cell transformation by dominant negative PKR mutants is caused by inhibition of eIF-2 alpha phosphorylation. Here, we demonstrate that in NIH 3T3 cells transformed by the dominant negative PKR mutant (PKR delta 6), eIF-2 alpha phosphorylation is dramatically reduced. Furthermore, expression of a mutant form of eIF-2 alpha, which cannot be phosphorylated on Ser51 also caused malignant transformation of NIH 3T3 cells. These results are consistent with a critical role of phosphorylation of eIF-2 alpha in control of cell proliferation, and indicate that dominant negative PKR mutants transform cells by inhibition of eIF-2 alpha phosphorylation.     

Mutations in the GCD7 subunit of yeast guanine nucleotide exchange factor eIF-2B overcome the inhibitory effects of phosphorylated eIF-2 on translation initiation.

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha) impairs translation initiation by inhibiting the guanine nucleotide exchange factor for eIF-2, known as eIF-2B. In Saccharomyces cerevisiae, phosphorylation of eIF-2 alpha by the protein kinase GCN2 specifically stimulates translation of GCN4 mRNA in addition to reducing general protein synthesis. We isolated mutations in several unlinked genes that suppress the growth-inhibitory effect of eIF-2 alpha phosphorylation catalyzed by mutationally activated forms of GCN2. These suppressor mutations, affecting eIF-2 alpha and the essential subunits of eIF-2B encoded by GCD7 and GCD2, do not reduce the level of eIF-2 alpha phosphorylation in cells expressing the activated GCN2c kinase. Four GCD7 suppressors were shown to reduce the derepression of GCN4 translation in cells containing wild-type GCN2 under starvation conditions or in GCN2c strains. A fifth GCD7 allele, constructed in vitro by combining two of the GCD7 suppressors mutations, completely impaired the derepression of GCN4 translation, a phenotype characteristic of deletions in GCN1, GCN2, or GCN3. This double GCD7 mutation also completely suppressed the lethal effect of expressing the mammalian eIF-2 alpha kinase dsRNA-PK in yeast cells, showing that the translational machinery had been rendered completely insensitive to phosphorylated eIF-2. None of the GCD7 mutations had any detrimental effect on cell growth under nonstarvation conditions, suggesting that recycling of eIF-2 occurs efficiently in the suppressor strains. We propose that GCD7 and GCD2 play important roles in the regulatory interaction between eIF-2 and eIF-2B and that the suppressor mutations we isolated in these genes decrease the susceptibility of eIF-2B to the inhibitory effects of phosphorylated eIF-2 without impairing the essential catalytic function of eIF-2B in translation initiation.     

Second-site mutation outside of the U(S)10-12 domain of Deltagamma(1)34.5 herpes simplex virus 1 recombinant blocks the shutoff of protein synthesis induced by activated protein kinase R and partially restores neurovirulence.

Earlier studies have shown that herpes simplex virus type 1 (HSV-1) activated protein kinase R (PKR) but that the product of the product of the gamma(1)34.5 gene binds and redirects the host phosphatase 1 to dephosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha). In consequence, the gamma(1)34.5 gene product averts the threatened shutoff of protein synthesis caused by activated PKR. Serial passages of Deltagamma(1)34.5 mutants in human cells led to isolation of two classes of second-site, compensatory mutants. The first, reported earlier, resulted from the juxtaposition of the alpha promoter of the U(S)12 gene to the coding sequence of the U(S)11 gene. The mutant blocks the phosphorylation of eIF-2alpha but does not restore the virulence phenotype of the wild-type virus. We report another class of second-site, compensatory mutants that do not map to the U(S)10-12 domain of the HSV-1 genome. All mutants in this series exhibit sustained late protein synthesis, higher yields in human cells, and reduced phosphorylation of PKR that appears to be phosphatase dependent. Specific dephosphorylation of eIF-2alpha was not demonstrable. At least one mutant in this series exhibited a partial restoration of the virulence phenotype characteristic of the wild-type virus phenotype. The results suggest that the second-site mutations reflect activation of fossilized functions designed to block the interferon response pathways in cells infected with the progenitor of present HSV.