GCN2-like eIF2α kinase manages the amino acid starvation response in Toxoplasma gondii

The apicomplexan protozoan Toxoplasma gondii is a significant human and veterinary pathogen. As an obligate intracellular parasite, Toxoplasma depends on nutrients provided by the host cell and needs to adapt to limitations in available resources. In mammalian cells, translational regulation via GCN2 phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) is a key mechanism for adapting to nutrient stress. Toxoplasma encodes two GCN2-like protein kinases, TgIF2K-C and TgIF2K-D. We previously showed that TgIF2K-D phosphorylates T. gondii eIF2α (TgIF2α) upon egress from the host cell, which enables the parasite to overcome exposure to the extracellular environment. However, the function of TgIF2K-C remained unresolved. To determine the functions of TgIF2K-C in the parasite, we cloned the cDNA encoding TgIF2K-C and generated knockout parasites of this TgIF2α kinase to study its function during the lytic cycle. The TgIF2K-C knockout did not exhibit a fitness defect compared with parental parasites. However, upon infection of human fibroblasts that were subsequently cultured in glutamine-free medium, the intracellular TgIF2K-C knockout parasites were impeded for induced phosphorylation of TgIF2α and showed a 50% reduction in the number of plaques formed compared with parental parasites. Furthermore, we found that this growth defect in glutamine-free media was phenocopied in parasites expressing only a non-phosphorylatable TgIF2α (TgIF2α-S71A), but not in a TgIF2K-D knockout. These studies suggest that Toxoplasma GCN2-like kinases TgIF2K-C and TgIF2K-D evolved to have distinct roles in adapting to changes in the parasite’s environment.     

Translation regulation by eukaryotic initiation factor-2 kinases in the development of latent cysts in Toxoplasma gondii

A key problem in the treatment of numerous pathogenic eukaryotes centers on their development into latent forms during stress. For example, the opportunistic protist Toxoplasma gondii converts to latent cysts (bradyzoites) responsible for recrudescence of disease. We report that Toxoplasma eukaryotic initiation factor-2alpha (TgIF2alpha) is phosphorylated during stress and establish that protozoan parasites utilize translation control to modulate gene expression during development. Importantly, TgIF2alpha remains phosphorylated in bradyzoites, explaining how these cells maintain their quiescent state. Furthermore, we have characterized novel eIF2 kinases; one in the endoplasmic reticulum and a likely regulator of the unfolded protein response (TgIF2K-A) and another that is a probable responder to cytoplasmic stresses (TgIF2K-B). Significantly, our data suggest that 1) the regulation of protein translation through eIF2 kinases is associated with development, 2) eIF2alpha phosphorylation is employed by cells to maintain a latent state, and 3) endoplasmic reticulum and cytoplasmic stress responses evolved in eukaryotic cells before the early diverging Apicomplexa. Given its importance to pathogenesis, eIF2 kinase-mediated stress responses may provide opportunities for novel therapeutics.     

Autophagy is a cell death mechanism in Toxoplasma gondii

Nutrient sensing and the capacity to respond to starvation is tightly regulated as a means of cell survival. Among the features of the starvation response are induction of both translational repression and autophagy. Despite the fact that intracellular parasite like Toxoplasma gondii within a host cell predicted to be nutrient rich, they encode genes involved in both translational repression and autophagy. We therefore examined the consequence of starvation, a classic trigger of autophagy, on intracellular parasites. As expected, starvation results in the activation of the translational repression system as evidenced by elevation of phosphorylated TgIF2α (TgIF2α-P). Surprisingly, we also observe a rapid and selective fragmentation of the single parasite mitochondrion that leads irreversibly to parasite death. This profound effect was dependent primarily on the limitation of amino acids and involved signalling by the parasite TOR homologue. Notably, the effective blockade of mitochondrial fragmentation by the autophagy inhibitor 3-methyl adenine (3-MA) suggests an autophagic mechanism. In the absence of a documented apoptotic cascade in T. gondii, the data suggest that autophagy is the primary mechanism of programmed cell death in T. gondii and potentially other related parasites.     

Large G3BP-induced granules trigger eIF2α phosphorylation

Stress granules are large messenger ribonucleoprotein (mRNP) aggregates composed of translation initiation factors and mRNAs that appear when the cell encounters various stressors. Current dogma indicates that stress granules function as inert storage depots for translationally silenced mRNPs until the cell signals for renewed translation and stress granule disassembly. We used RasGAP SH3-binding protein (G3BP) overexpression to induce stress granules and study their assembly process and signaling to the translation apparatus. We found that assembly of large G3BP-induced stress granules, but not small granules, precedes phosphorylation of eIF2α. Using mouse embryonic fibroblasts depleted for individual eukaryotic initiation factor 2α (eIF2α) kinases, we identified protein kinase R as the principal kinase that mediates eIF2α phosphorylation by large G3BP-induced granules. These data indicate that increasing stress granule size is associated with a threshold or switch that must be triggered in order for eIF2α phosphorylation and subsequent translational repression to occur. Furthermore, these data suggest that stress granules are active in signaling to the translational machinery and may be important regulators of the innate immune response.     

A critical role of eEF-2K in mediating autophagy in response to multiple cellular stresses

The phosphorylation of the subunit α of eukaryotic translation initiation factor 2 (eIF2α), a critical regulatory event in controlling protein translation, has recently been found to mediate the induction of autophagy. However, the mediators of autophagy downstream of eIF2α remain unknown. Here, we provide evidence that eIF2α phosphorylation is required for phosphorylation of eukaryotic elongation factor 2 (eEF-2) during nutrient starvation. In addition, we show that eukaryotic elongation factor 2 kinase (eEF-2K) is also required for autophagy signaling during ER stress, suggesting that phosphorylation

of eEF-2 may serve as an integrator of various cell stresses for autophagy signaling. On the other hand, although the activation of eEF-2K in response to starvation requires the phosphorylation of eIF2α, additional pathways relying partly on Ca²⁺ flux may control eEF-2K activity during ER stress, as eIF2α phosphorylation is dispensable for both eEF-2 phosphorylation and autophagy in this context.     

Poliovirus switches to an eIF2-independent mode of translation during infection

Inhibition of translation is an integral component of the innate antiviral response and is largely accomplished via interferon-activated phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). To successfully infect a host, a virus must overcome this blockage by either controlling eIF2α phosphorylation or by utilizing a noncanonical mode of translation initiation. Here we show that enterovirus RNA is sensitive to translation inhibition resulting from eIF2α phosphorylation, but it becomes resistant as infection progresses. Further, we show that the cleavage of initiation factor eIF5B during enteroviral infection, along with the viral internal ribosome entry site, plays a role in mediating viral translation under conditions that are nonpermissive for host cell translation. Together, these results provide a mechanism by which enteroviruses evade the antiviral response and provide insight into a noncanonical mechanism of translation initiation.     

Blocking Double-Stranded RNA-Activated Protein Kinase PKR by Japanese Encephalitis Virus Nonstructural Protein 2A

Japanese encephalitis virus (JEV) is an enveloped flavivirus with a single-stranded, positive-sense RNA genome encoding three structural and seven nonstructural proteins. To date, the role of JEV nonstructural protein 2A (NS2A) in the viral life cycle is largely unknown. The interferon (IFN)-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) phosphorylates the eukaryotic translation initiation factor 2α subunit (eIF2α) after sensing viral RNA and results in global translation arrest as an important host antiviral defense response. In this study, we found that JEV NS2A could antagonize PKR-mediated growth inhibition in a galactose-inducible PKR-expressing yeast system. In human cells, PKR activation, eIF2α phosphorylation, and the subsequent translational inhibition and cell death triggered by dsRNA and IFN-α were also repressed by JEV NS2A. Moreover, among the four eIF2α kinases, NS2A specifically blocked the eIF2α phosphorylation mediated by PKR and attenuated the PKR-promoted cell death induced by the chemotherapeutic drug doxorubicin. A single point mutation of NS2A residue 33 from Thr to Ile (T33I) abolished the anti-PKR potential of JEV NS2A. The recombinant JEV mutant carrying the NS2A-T33I mutation showed reduced in vitro growth and in vivo virulence phenotypes. Thus, JEV NS2A has a novel function in blocking the host antiviral response of PKR during JEV infection.     

Development of transgenic mice expressing a conditionally active form of the eIF2α kinase PKR

Phosphorylation of the alpha (α) subunit of the eukaryotic initiation factor 2 (eIF2) at serine 51 is an important mechanism of translational control in response to various forms of environmental stress. In metazoans, eIF2α phosphorylation is mediated by four kinases each of which becomes activated by distinct stimuli. Previous work established that expression of a chimera protein comprising of the bacteria Gyrase B N-terminal (GyrB) domain fused to the kinase domain (KD) of the eIF2α kinase PKR is capable of inducing eIF2α phosphorylation in cultured cells after treatment with the antibiotic coumermycin. Herein, we report the development of transgenic mice expressing the fusion protein GyrB.PKR ubiquitously. Treatment of mice with coumermycin induces eIF2α phosphorylation in vivo as demonstrated by immunoblotting and immunoshistochemistry of mouse tissues. The GyrB.PKR transgene represents a useful model system to investigate the biological effects of the conditional induction of eIF2α phosphorylation in vivo in the absence of parallel signaling pathways that are elicited in response to stress.     

eIF2α kinases control chalone production in Dictyostelium discoideum

Growing Dictyostelium cells secrete CfaD and AprA, two proteins that have been characterized as chalones. They exist within a high-molecular-weight complex that reversibly inhibits cell proliferation, but not growth, via cell surface receptors and a signaling pathway that includes G proteins. How the production of these two proteins is regulated is unknown. Dictyostelium cells possess three GCN2-type eukaryotic initiation factor 2 α subunit (eIF2α) kinases, proteins that phosphorylate the translational initiation factor eIF2α and possess a tRNA binding domain involved in their regulation. The Dictyostelium kinases have been shown to function during development in regulating several processes. We show here that expression of an unregulated, activated kinase domain greatly inhibits cell proliferation. The inhibitory effect on proliferation is not due to a general inhibition of translation. Instead, it is due to enhanced production of a secreted factor(s). Indeed, extracellular CfaD and AprA proteins, but not their mRNAs, are overproduced in cells expressing the activated kinase domain. The inhibition of proliferation is not seen when the activated kinase domain is expressed in cells lacking CfaD or AprA or in cells that contain a nonphosphorylatable eIF2α. We conclude that production of the chalones CfaD and AprA is translationally regulated by eIF2α phosphorylation. Both proteins are upregulated at the culmination of development, and this enhanced production is lacking in a strain that possesses a nonphosphorylatable eIF2α.     

Protein synthesis attenuation by phosphorylation of eIF2α is required for the differentiation of Trypanosoma cruzi into infective forms

Chagas' disease is a potentially life-threatening illness caused by the unicellular protozoan parasite Trypanosoma cruzi. It is transmitted to humans by triatomine bugs where T. cruzi multiplies and differentiates in the digestive tract. The differentiation of proliferative and non-infective epimastigotes into infective metacyclic trypomastigotes (metacyclogenesis) can be correlated to nutrient exhaustion in the gut of the insect vector. In vitro, metacyclic-trypomastigotes can be obtained when epimastigotes are submitted to nutritional stress suggesting that metacyclogenesis is triggered by nutrient starvation. The molecular mechanism underlying such event is not understood. Here, we investigated the role of one of the key signaling responses elicited by nutritional stress in all other eukaryotes, the inhibition of translation initiation by the phosphorylation of the eukaryotic initiation factor 2α (eIF2α), during the in vitro differentiation of T. cruzi. Monospecific antibodies that recognize the phosphorylated Tc-eIF2α form were generated and used to demonstrate that parasites subjected to nutritional stress show increased levels of Tc-eIF2α phosphorylation. This was accompanied by a drastic inhibition of global translation initiation, as determined by polysomal profiles. A strain of T. cruzi overexpressing a mutant Tc-eIF2α, incapable of being phosphorylated, showed a block on translation initiation, indicating that such a nutritional stress in trypanosomatids induces the conserved translation inhibition response. In addition, Tc-eIF2α phosphorylation is critical for parasite differentiation since the overexpression of the mutant eIF2α in epimastigotes abolished metacyclogenesis. This work defines the role of eIF2α phosphorylation as a key step in T. cruzi differentiation.     

eIF2A mediates translation of hepatitis C viral mRNA under stress conditions

Translation of most mRNAs is suppressed under stress conditions. Phosphorylation of the α-subunit of eukaryotic translation initiation factor 2 (eIF2), which delivers initiator tRNA (Met-tRNA(i)) to the P site of the 40S ribosomal subunit, is responsible for such translational suppression. However, translation of hepatitis C viral (HCV) mRNA is refractory to the inhibitory effects of eIF2α phosphorylation, which prevents translation by disrupting formation of the eIF2-GTP-Met-tRNA(i) ternary complex. Here, we report that eIF2A, an alternative initiator tRNA-binding protein, has a key role in the translation of HCV mRNA during HCV infection, in turn promoting eIF2α phosphorylation by activating the eIF2α kinase PKR. Direct interaction of eIF2A with the IIId domain of the HCV internal ribosome entry site (IRES) is required for eIF2A-dependent translation. These data indicate that stress-independent translation of HCV mRNA occurs by recruitment of eIF2A to the HCV IRES via direct interaction with the IIId domain and subsequent loading of Met-tRNA(i) to the P site of the 40S ribosomal subunit.     

The alpha subunit of eukaryotic initiation factor 2B (eIF2B) is required for eIF2-mediated translational suppression of vesicular stomatitis virus

Eukaryotic translation initiation factor 2B (eIF2B) is a heteropentameric guanine nucleotide exchange factor that converts protein synthesis initiation factor 2 (eIF2) from a GDP-bound form to the active eIF2-GTP complex. Cellular stress can repress translation initiation by activating kinases capable of phosphorylating the alpha subunit of eIF2 (eIF2α), which sequesters eIF2B to prevent exchange activity. Previously, we demonstrated that tumor cells are sensitive to viral replication, possibly due to the occurrence of defects in eIF2B that overcome the inhibitory effects of eIF2α phosphorylation. To extend this analysis, we have investigated the importance of eIF2Bα function and report that this subunit can functionally substitute for its counterpart, GCN3, in yeast. In addition, a variant of mammalian eIF2Bα harboring a point mutation (T41A) was able overcome translational inhibition invoked by amino acid depravation, which activates Saccharomyces cerevisiae GCN2 to phosphorylate the yeast eIF2α homolog SUI2. Significantly, we also demonstrate that the loss of eIF2Bα, or the expression of the T41A variant in mammalian cells, is sufficient to neutralize the consequences of eIF2α phosphorylation and render normal cells susceptible to virus infection. Our data emphasize the importance of eIF2Bα in mediating the eIF2 kinase translation-inhibitory activity and may provide insight into the complex nature of viral oncolysis.     

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.     

Innate immune evasion mediated by the Ambystoma tigrinum virus eukaryotic translation initiation factor 2alpha homologue

Ranaviruses (family Iridoviridae, genus Ranavirus) are large, double-stranded DNA (dsDNA) viruses whose replication is restricted to ectothermic vertebrates. Many highly pathogenic members of the genus Ranavirus encode a homologue of the eukaryotic translation initiation factor 2α (eIF2α). Data in a heterologous vaccinia virus system suggest that the Ambystoma tigrinum virus (ATV) eIF2α homologue (vIF2αH; open reading frame [ORF] 57R) is involved in evading the host innate immune response by degrading the interferon-inducible, dsRNA-activated protein kinase, PKR. To test this hypothesis directly, the ATV vIF2αH gene (ORF 57R) was deleted by homologous recombination, and a selectable marker was inserted in its place. The ATVΔ57R virus has a small plaque phenotype and is 8-fold more sensitive to interferon than wild-type ATV (wtATV). Infection of fish cells with the ATVΔ57R virus leads to eIF2α phosphorylation, in contrast to infection with wtATV, which actively inhibits eIF2α phosphorylation. The inability of ATVΔ57R to prevent phosphorylation of eIF2α correlates with degradation of fish PKZ, an interferon-inducible enzyme that is closely related to mammalian PKR. In addition, salamanders infected with ATVΔ57R displayed an increased time to death compared to that of wtATV-infected salamanders. Therefore, in a biologically relevant system, the ATV vIF2αH gene acts as an innate immune evasion factor, thereby enhancing virus pathogenesis.     

Interactome analysis of herpes simplex virus 1 envelope glycoprotein H

Herpes simplex virus 1 (HSV‐1) envelope glycoprotein H (gH) is important for viral entry into cells and nuclear egress of nucleocapsids. To clarify additional novel roles of gH during HSV‐1 replication, host cell proteins that interact with gH were screened for by tandem affinity purification coupled with mass spectrometry‐based proteomics in 293T cells transiently expressing gH. This screen identified 123 host cell proteins as potential gH interactors. Of these proteins, general control nonderepressive‐1 (GCN1), a trans‐acting positive effector of GCN2 kinase that regulates phosphorylation of the α subunit of translation initiation factor 2 (eIF2α), was subsequently confirmed to interact with gH in HSV‐1‐infected cells. eIF2α phosphorylation is known to downregulate protein synthesis, and various viruses have evolved mechanisms to prevent the accumulation of phosphorylated eIF2α in infected cells. Here, it was shown that GCN1 knockdown reduces phosphorylation of eIF2α in HSV‐1‐infected cells and that the gH‐null mutation increases eIF2α in HSV‐1‐infected cells, whereas gH overexpression in the absence of other HSV‐1 proteins reduces eIF2α phosphorylation. These findings suggest that GCN1 can regulate eIF2α phosphorylation in HSV‐1‐infected cells and that the GCN1‐binding viral partner gH is necessary and sufficient to prevent the accumulation of phosphorylated eIF2α. Our database of 123 host cell proteins potentially interacting with gH will be useful for future studies aimed at unveiling further novel functions of gH and the roles of cellular proteins in HSV‐1‐infected cells.     

The small molecule ‘1-(4-biphenylylcarbonyl)-4-(5-bromo-2-methoxybenzyl) piperazine oxalate’ and its derivatives regulate global protein synthesis by inactivating eukaryotic translation initiation factor 2-alpha

By environmental stresses, cells can initiate a signaling pathway in which eukaryotic translation initiation factor 2-alpha (eIF2-α) is involved to regulate the response. Phosphorylation of eIF2-α results in the reduction of overall protein neogenesis, which allows cells to conserve resources and to reprogram energy usage for effective stress control. To investigate the role of eIF2-α in cell stress responses, we conducted a viability-based compound screen under endoplasmic reticulum (ER) stress condition, and identified 1-(4-biphenylylcarbonyl)-4-(5-bromo-2-methoxybenzyl) piperazine oxalate (AMC-01) and its derivatives as eIF2-α-inactivating chemical. Molecular characterization of this signaling pathway revealed that AMC-01 induced inactivation of eIF2-α by phosphorylating serine residue 51 in a dose- and time-dependent manner, while the negative control compounds did not affect eIF2-α phosphorylation. In contrast with ER stress induction by thapsigargin, phosphorylation of eIF2-α persisted for the duration of incubation with AMC-01. By pathway analysis, AMC-01 clearly induced the activation of protein kinase RNA-activated (PKR) kinase and nuclear factor-κB (NF-κB), whereas it did not modulate the activity of PERK or heme-regulated inhibitor (HRI). Finally, we could detect a lower protein translation rate in cells incubated with AMC-01, establishing AMC-01 as a potent chemical probe that can regulate eIF2-α activity. We suggest from these data that AMC-01 and its derivative compounds can be used as chemical probes in future studies of the role of eIF2-α in protein synthesis-related cell physiology.