Translational Control of Alphavirus-Host Interactions: Implications in Viral Evolution, Tropism and Antiviral Response.

Alphaviruses can replicate in arthropods and in many vertebrate species including humankind, but only in vertebrate cells do infections with these viruses result in a strong inhibition of host translation and transcription. Translation shutoff by alphaviruses is a multifactorial process that involves both host- and virus-induced mechanisms, and some of them are not completely understood. Alphavirus genomes contain cis-acting elements (RNA structures and dinucleotide composition) and encode protein activities that promote the translational and transcriptional resistance to type I IFN-induced antiviral effectors. Among them, IFIT1, ZAP and PKR have played a relevant role in alphavirus evolution, since they have promoted the emergence of multiple viral evasion mechanisms at the translational level. In this review, we will discuss how the adaptations of alphaviruses to vertebrate hosts likely involved the acquisition of new features in viral mRNAs and proteins to overcome the effect of type I IFN.

Translational control of gene expression by eIF2 modulates proteostasis and extends lifespan.

Although the stress response in eukaryotes depends on early events triggered in cells by environmental insults, long-term processes such as aging are also affected. The loss of cellular proteostasis greatly impacts aging, which is regulated by the balancing of protein synthesis and degradation systems. As translation is the input event in proteostasis, we decided to study the role of translational activity on cell lifespan. Our hypothesis was that a reduction on translational activity or specific changes in translation may increase cellular longevity. Using mutant strains of Schizosaccharomyces pombe and various stress conditions, we showed that translational reduction caused by phosphorylation of eukaryotic translation initiation factor 2 (eIF2) during the exponential growth phase enhances chronological lifespan (CLS). Furthermore, through next-generation sequence analysis, we found eIF2α phosphorylation-dependent translational activation of some specific genes, especially those involved in autophagy. This fact, together with the observed regulation of autophagy, points to a conserved mechanism involving general and specific control of translation and autophagy as mediators of the role of eIF2α phosphorylation in aging.

Naturally Occurring and Engineered Alphaviruses Sensitive to Double-Stranded-RNA-Activated Protein Kinase Show Restricted Translation in Mammalian Cells, Increased Sensitivity to Interferon, and Marked Oncotropism.

Alphaviruses are insect-borne viruses that alternate between replication in mosquitoes and vertebrate species. Adaptation of some alphaviruses to vertebrate hosts has involved the acquisition of an RNA structure (downstream loop [DLP]) in viral subgenomic mRNAs that confers translational resistance to protein kinase (PKR)-mediated eIF2α phosphorylation. Here, we found that, in addition to promoting eIF2-independent translation of viral subgenomic mRNAs, presence of the DLP structure also increased the resistance of alphavirus to type I interferon (IFN). Aura virus (AURAV), an ecologically isolated relative of Sindbis virus (SV) that is poorly adapted to replication in vertebrate cells, displayed a nonfunctional DLP structure and dramatic sensitivity to type I IFN. Our data suggest that an increased resistance to IFN emerged during translational adaptation of alphavirus mRNA to vertebrate hosts, reinforcing the role that double-stranded RNA (dsRNA)-activated protein kinase (PKR) plays as both a constitutive and IFN-induced antiviral effector. Interestingly, a mutant SV lacking the DLP structure (SV-ΔDLP) and AURAV both showed a marked oncotropism for certain tumor cell lines that have defects in PKR expression and/or activation. AURAV selectively replicated in and killed some cell lines derived from human hepatocarcinoma (HCC) that lacked PKR response to infection or poly(I·C) transfection. The oncolytic activities of SV-ΔDLP and AURAV were also confirmed using tumor xenografts in mice, showing tumor regression activities comparable to wild-type SV. Our data show that translation of alphavirus subgenomic mRNAs plays a central role in IFN susceptibility and cell tropism, suggesting an unanticipated oncolytic potential that some naive arboviruses may have in virotherapy.IMPORTANCE Interferons (IFNs) induce the expression of a number of antiviral genes that protect the cells of vertebrates against viruses and other microbes. The susceptibility of cells to viruses greatly depends on the level and activity of these antiviral effectors but also on the ability of viruses to counteract this antiviral response. Here, we found that the level of one of the main IFN effectors in the cell, the dsRNA-activated protein kinase (PKR), greatly determines the permissiveness of cells to alphaviruses that lack mechanisms to counteract its activation. These naive viruses also showed a hypersensitivity to IFN, suggesting that acquisition of IFN resistance (even partial) has probably been involved in expanding the host range of alphaviruses in the past. Interestingly, some of these naive viruses showed a marked oncotropism for some tumor cell lines derived from human hepatocarcinoma (HCC), opening the possibility of their use in oncolytic therapy to treat human tumors.

Generation of endoplasmic reticulum stress and inhibition of autophagy by plitidepsin induces proteotoxic apoptosis in cancer cells.

Plitidepsin (PLD, Aplidin®), a cyclic depsipeptide originally isolated from the marine tunicate Aplidium albicans, has been recently approved by Australian regulatory authorities for the treatment of multiple myeloma patients. Plitidepsin binds to eEF1A2 and induces oxidative stress, Rac1 activation and JNK1 phosphorylation, triggering a rapid apoptotic program in tumor cells. Since oxidative stress is one of the known sources of endoplasmic reticulum stress, we investigated whether PLD was inducing a bona fide ER stress in HeLa cells and whether this process was essential in the mechanism of action of the compound. Indeed, PLD activated an ER stress-induced unfolded protein response (UPR), including the alternative splicing of XBP1, the proteolytic processing of ATF6 and the phosphorylation of eIF2α and JNK. Interestingly, though PLD induced a strong phosphorylation of eIF2α in all the analyzed cell lines, it did not elicit an increased expression of ATF4 and CHOP, a transcription factor involved in launching UPR-mediated apoptosis. On the contrary, a clear reduction of CHOP protein levels was observed after PLD treatment, most probably due to both the lack of transactivation by ATF4 and its rapid degradation by the ubiquitin/proteasome machinery. Using fibroblasts devoid of each one of the four possible kinases involved in eIF2α phosphorylation, we observed that only PKR was involved in the response to PLD treatment and, accordingly, PKR-/- fibroblasts are shown to be resistant to the apoptogenic activity of the compound. Furthermore, eIF2α phosphorylation itself was shown to be irrelevant for the induction of cell death by PLD. Instead, we reveal that PLD induces an increase in the levels of misfolded proteins while simultaneously inhibiting the autophagic flux. These two effects combined prevent PLD-treated cells from reducing proteotoxic stress and lead to apoptosis. Other anti-myeloma drugs like bortezomib, which target the proteasome, also inhibit the degradation of misfolded proteins through alternate pathways and a synergistic anticancer effect of the PLD plus bortezomib combination has been previously disclosed. The present results extend this synergy to in vivo experiments and provide a mechanistic rationale for this synergy.

An mRNA-binding channel in the ES6S region of the translation 48S-PIC promotes RNA unwinding and scanning.

Loading of mRNA onto the ribosomal 43S pre-initiation complex (PIC) and its subsequent scanning require the removal of the secondary structure of the by RNA helicases such as eIF4A. However, the topology and mechanics of the scanning complex bound to mRNA (48S-PIC) and the influence of its solvent-side composition on the scanning process are poorly known. Here, we found that the ES6S region of the 48S-PIC constitutes an extended binding channel for eIF4A-mediated unwinding of mRNA and scanning. Blocking ES6S inhibited the cap-dependent translation of mRNAs that have structured 5' UTRs (including G-quadruplexes), many of which are involved in signal transduction and growth, but it did not affect IRES-driven translation. Genome-wide analysis of mRNA translation revealed a great diversity in ES6S-mediated scanning dependency. Our data suggest that mRNA threading into the ES6S region makes scanning by 48S PIC slower but more processive. Hence, we propose a topological and functional model of the scanning 48S-PIC.

New roles of the fission yeast eIF2α kinases Hri1 and Gcn2 in response to nutritional stress.

In fission yeast, three distinct eukaryotic initiation factor 2α (eIF2α) kinases (Hri1, Hri2 and Gcn2), regulate protein synthesis in response to various environmental stresses. Thus, Gcn2 is activated early after exposure to hydrogen peroxide (H2O2) and methyl methanesulfonate (MMS), whereas Hri2 is the primary activated eIF2α kinase in response to heat shock. The function of Hri1 is still not completely understood. It is also known that the mitogen-activated protein kinase Sty1 negatively regulates Gcn2 and Hri2 activities under oxidative stress. In this study, we demonstrate that Hri1 is mainly activated, and its expression upregulated, during transition from exponential growth to the stationary phase in response to nutritional limitation. Accordingly, both Hri1 and Gcn2, but not Hri2, are activated upon nitrogen source deprivation. In contrast, Hri2 is stimulated early during glucose starvation. We also found that Gcn2 is implicated in nitrogen starvation-induced growth arrest in the cell cycle G1 phase as well as in the non-selective protein degradation process caused upon this particular cellular stress. Moreover, Gcn2, but not Hri1 or Hri2, is essential for survival of cells growing in minimal medium, upon oxidative stress or glucose limitation. We further show that eIF2α phosphorylation at serine 52 by the eIF2α kinases is necessary for efficient cell cycle arrest in the G1 phase, for the consequent protein degradation and for sexual differentiation, under nitrogen starvation. Therefore, the eIF2α kinase signalling pathway modulates G1 phase cell cycle arrest, cell survival and mating under nutritional stress in the fission yeast Schizosaccharomyces pombe.

Phosphorylation of initiation factor eIF2 in response to stress conditions is mediated by acidic ribosomal P1/P2 proteins in Saccharomyces cerevisiae.

Eukaryotic cells contain an unusually large cytoplasmic pool of P1/P2 phosphoproteins, which form the highly flexible 60S subunit stalk that is required to interact with and activate soluble translation factors. In cells, cytoplasmic P1/P2 proteins are exchanged for ribosome-bound proteins in a process that can modulate ribosome function and translation. Here, we analysed different S. cerevisiae stalk mutants grown under stress conditions that result in eIF2α phosphorylation. These mutants either lack a cytoplasmic pool of stalk proteins or contain free but not ribosome-bound proteins. Only cells that contain free P1/P2 proteins induce eIF2 phosphorylation in vivo in response to glucose starvation or osmotic stress. Moreover, we show that free S. cerevisiae P1/P2 proteins can induce in vitro phosphorylation of the initiation factor eIF2 by stimulating the autophosphorylation and activation of GCN2 kinase. Indeed, these ribosomal proteins do not stimulate other eIF2α kinases, such as PKR and HRI. P1/P2 and the known GCN2 activator deacylated tRNA compete for stimulating the eIF2α kinase activity of GCN2, although the P1/P2 proteins are considerably more active. These findings reveal a capacity of free cytoplasmic ribosomal stalk components to stimulate eIF2α phosphorylation, which in turn would modulate translation in response to specific forms of stress that may be linked with the previously described regulatory function of the ribosomal stalk.

GCN2 has inhibitory effect on human immunodeficiency virus-1 protein synthesis and is cleaved upon viral infection.

The reversible phosphorylation of the alpha-subunit of eukaryotic translation initiation factor 2 (eIF2alpha) is a well-characterized mechanism of translational control in response to a wide variety of cellular stresses, including viral infection. Beside PKR, the eIF2alpha kinase GCN2 participates in the cellular response against viral infection by RNA viruses with central nervous system tropism. PKR has also been involved in the antiviral response against HIV-1, although this antiviral effect is very limited due to the distinct mechanisms evolved by the virus to counteract PKR action. Here we report that infection of human cells with HIV-1 conveys the proteolytic cleavage of GCN2 and that purified HIV-1 and HIV-2 proteases produce direct proteolysis of GCN2 in vitro, abrogating the activation of GCN2 by HIV-1 RNA. Transfection of distinct cell lines with a plasmid encoding an HIV-1 cDNA clone competent for a single round of replication resulted in the activation of GCN2 and the subsequent eIF2alpha phosphorylation. Moreover, transfection of GCN2 knockout cells or cells with low levels of phosphorylated eIF2alpha with the same HIV-1 cDNA clone resulted in a marked increase of HIV-1 protein synthesis. Also, the over-expression of GCN2 in cells led to a diminished viral protein synthesis. These findings suggest that viral RNA produced during HIV-1 infection activates GCN2 leading to inhibition of viral RNA translation, and that HIV-1 protease cleaves GCN2 to overcome its antiviral effect.

Role of mitogen-activated protein kinase Sty1 in regulation of eukaryotic initiation factor 2alpha kinases in response to environmental stress in Schizosaccharomyces pombe.

The mitogen-activated protein kinase (MAPK) Sty1 is essential for the regulation of transcriptional responses that promote cell survival in response to different types of environmental stimuli in Schizosaccharomyces pombe. In fission yeast, three distinct eukaryotic initiation factor 2alpha (eIF2alpha) kinases, two mammalian HRI-related protein kinases (Hri1 and Hri2) and the Gcn2 ortholog, regulate protein synthesis in response to cellular stress conditions. In this study, we demonstrate that both Hri1 and Hri2 exhibited an autokinase activity, specifically phosphorylated eIF2alpha, and functionally replaced the endogenous Saccharomyces cerevisiae Gcn2. We further show that Gcn2, but not Hri1 or Hri2, is activated early after exposure to hydrogen peroxide and methyl methanesulfonate (MMS). Cells lacking Gcn2 exhibit a later activation of Hri2. The activated MAPK Sty1 negatively regulates Gcn2 and Hri2 activities under oxidative stress but not in response to MMS. In contrast, Hri2 is the primary activated eIF2alpha kinase in response to heat shock. In this case, the activation of Sty1 appears to be transitory and does not contribute to the modulation of the eIF2alpha kinase stress pathway. In strains lacking Hri2, a type 2A protein phosphatase is activated soon after heat shock to reduce eIF2alpha phosphorylation. Finally, the MAPK Sty1, but not the eIF2alpha kinases, is essential for survival upon oxidative stress or heat shock, but not upon MMS treatment. These findings point to a regulatory coordination between the Sty1 MAPK and eIF2alpha kinase pathways for a particular range of stress responses.

Dual mechanism for the translation of subgenomic mRNA from Sindbis virus in infected and uninfected cells.

Infection of BHK cells by Sindbis virus (SV) gives rise to a profound inhibition of cellular protein synthesis, whereas translation of viral subgenomic mRNA that encodes viral structural proteins, continues for hours. To gain further knowledge on the mechanism by which this subgenomic mRNA is translated, the requirements for some initiation factors (eIFs) and for the presence of the initiator AUG were examined both in infected and in uninfected cells. To this end, BHK cells were transfected with different SV replicons or with in vitro made SV subgenomic mRNAs after inactivation of some eIFs. Specifically, eIF4G was cleaved by expression of the poliovirus 2A protease (2A(pro)) and the alpha subunit of eIF2 was inactivated by phosphorylation induced by arsenite treatment. Moreover, cellular location of these and other translation components was analyzed in BHK infected cells by confocal microscopy. Cleavage of eIF4G by poliovirus 2A(pro) does not hamper translation of subgenomic mRNA in SV infected cells, but bisection of this factor blocks subgenomic mRNA translation in uninfected cells or in cell-free systems. SV infection induces phosphorylation of eIF2alpha, a process that is increased by arsenite treatment. Under these conditions, translation of subgenomic mRNA occurs to almost the same extent as controls in the infected cells but is drastically inhibited in uninfected cells. Notably, the correct initiation site on the subgenomic mRNA is still partially recognized when the initiation codon AUG is modified to other codons only in infected cells. Finally, immunolocalization of different eIFs reveals that eIF2 alpha and eIF4G are excluded from the foci, where viral RNA replication occurs, while eIF3, eEF2 and ribosomes concentrate in these regions. These findings support the notion that canonical initiation takes place when the subgenomic mRNA is translated out of the infection context, while initiation can occur without some eIFs and even at non-AUG codons in infected cells.

Poly(A)-binding protein-interacting protein 1 binds to eukaryotic translation initiation factor 3 to stimulate translation.

Poly(A)-binding protein (PABP) stimulates translation initiation by binding simultaneously to the mRNA poly(A) tail and eukaryotic translation initiation factor 4G (eIF4G). PABP activity is regulated by PABP-interacting (Paip) proteins. Paip1 binds PABP and stimulates translation by an unknown mechanism. Here, we describe the interaction between Paip1 and eIF3, which is direct, RNA independent, and mediated via the eIF3g (p44) subunit. Stimulation of translation by Paip1 in vivo was decreased upon deletion of the N-terminal sequence containing the eIF3-binding domain and upon silencing of PABP or several eIF3 subunits. We also show the formation of ternary complexes composed of Paip1-PABP-eIF4G and Paip1-eIF3-eIF4G. Taken together, these data demonstrate that the eIF3-Paip1 interaction promotes translation. We propose that eIF3-Paip1 stabilizes the interaction between PABP and eIF4G, which brings about the circularization of the mRNA.

Regulation of poly(A) binding protein function in translation: Characterization of the Paip2 homolog, Paip2B.

The 5' cap and 3' poly(A) tail of eukaryotic mRNAs act synergistically to enhance translation. This synergy is mediated via interactions between eIF4G (a component of the eIF4F cap binding complex) and poly(A) binding protein (PABP). Paip2 (PABP-interacting protein 2) binds PABP and inhibits translation both in vitro and in vivo by decreasing the affinity of PABP for polyadenylated RNA. Here, we describe the functional characteristics of Paip2B, a Paip2 homolog. A full-length brain cDNA of Paip2B encodes a protein that shares 59% identity and 80% similarity with Paip2 (Paip2A), with the highest conservation in the two PABP binding domains. Paip2B acts in a manner similar to Paip2A to inhibit translation of capped and polyadenylated mRNAs both in vitro and in vivo by displacing PABP from the poly(A) tail. Also, similar to Paip2A, Paip2B does not affect the translation mediated by the internal ribosome entry site (IRES) of hepatitis C virus (HCV). However, Paip2A and Paip2B differ with respect to both mRNA and protein distribution in different tissues and cell lines. Paip2A is more highly ubiquitinated than is Paip2B and is degraded more rapidly by the proteasome. Paip2 protein degradation may constitute a primary mechanism by which cells regulate PABP activity in translation.

Poly(A) binding protein (PABP) homeostasis is mediated by the stability of its inhibitor, Paip2.

The poly(A)-binding protein (PABP) is a unique translation initiation factor in that it binds to the mRNA 3' poly(A) tail and stimulates recruitment of the ribosome to the mRNA at the 5' end. PABP activity is tightly controlled by the PABP-interacting protein 2 (Paip2), which inhibits translation by displacing PABP from the mRNA. Here, we describe a close interplay between PABP and Paip2 protein levels in the cell. We demonstrate a mechanism for this co-regulation that involves an E3 ubiquitin ligase, EDD, which targets Paip2 for degradation. PABP depletion by RNA interference (RNAi) causes co-depletion of Paip2 protein without affecting Paip2 mRNA levels. Upon PABP knockdown, Paip2 interacts with EDD, which leads to Paip2 ubiquitination. Supporting a critical role for EDD in Paip2 degradation, knockdown of EDD expression by siRNA leads to an increase in Paip2 protein stability. Thus, we demonstrate that the turnover of Paip2 in the cell is mediated by EDD and is regulated by PABP. This mechanism serves as a homeostatic feedback to control the activity of PABP in cells.

Translational resistance of late alphavirus mRNA to eIF2alpha phosphorylation: a strategy to overcome the antiviral effect of protein kinase PKR.

The double-stranded RNA-dependent protein kinase (PKR) is one of the four mammalian kinases that phosphorylates the translation initiation factor 2alpha in response to virus infection. This kinase is induced by interferon and activated by double-stranded RNA (dsRNA). Phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) blocks translation initiation of both cellular and viral mRNA, inhibiting virus replication. To counteract this effect, most viruses express inhibitors that prevent PKR activation in infected cells. Here we report that PKR is highly activated following infection with alphaviruses Sindbis (SV) and Semliki Forest virus (SFV), leading to the almost complete phosphorylation of eIF2alpha. Notably, subgenomic SV 26S mRNA is translated efficiently in the presence of phosphorylated eIF2alpha. This modification of eIF2 does not restrict viral replication; SV 26S mRNA initiates translation with canonical methionine in the presence of high levels of phosphorylated eIF2alpha. Genetic and biochemical data showed a highly stable RNA hairpin loop located downstream of the AUG initiator codon that is necessary to provide translational resistance to eIF2alpha phosphorylation. This structure can stall the ribosomes on the correct site to initiate translation of SV 26S mRNA, thus bypassing the requirement for a functional eIF2. Our findings show the existence of an alternative way to locate the ribosomes on the initiation codon of mRNA that is exploited by a family of viruses to counteract the antiviral effect of PKR.

Mixed chimerism is frequent after allogeneic peripheral blood stem cell transplantation with positive CD34 selection, and is not reverted by low doses of donor T-cells add-back.

UNLABELLED: The incidence of full donor chimerism (full DC) after CD34+ -selected peripheral blood stem cell transplantation (CD34+ -PBSCT) is controversial. Whereas the initial reports suggested a high incidence of full DC (hypothetically because of the high number of CD34+ cells infused) more recent works describe a high incidence of mixed lymphoid chimerism. There are no data concerning the ability of low-dose donor T-lymphocyte add-back on conversion to full DC. METHODS: We prospectively monitored the chimerism status of 25 patients undergoing CD34+ -PBSCT and the effect on chimerism of delayed low doses of donor T-cell add-back (TCAB). One, two or three doses of TCAB were administered on days +28 (2 x 10(5) CD3+/kg), +60 (2 x 10(5) CD3+/kg) and +90 (2 x 10(6) CD3+/kg), respectively, when on cyclosporine A prophylaxis. RESULTS: Incidence of full DC on day +20 was 56%. However, all but two patients progressed to MC. Fifteen patients were scheduled to TCAB. Six patients with initial MC did not convert to full DC after TCAB. Moreover, seven patients with full DC status progressed to mixed chimerism. CONCLUSIONS: Our results indicate that low doses of TCAB administered under cyclosporine A prophylaxis have no effect on the eradication of the recipient cells. We believe that a high dose of CD34+ cells in the grafts of CD34+ -PBSCT is not enough to achieve stable full DC unless a minimum number of CD3+ cells are infused, or more intensified transplant conditioning regimens are employed.

Activation of GCN2 in UV-irradiated cells inhibits translation.

BACKGROUND: Mammalian cells subjected to ultraviolet (UV) irradiation actively repress DNA replication, transcription, and mRNA translation. While the effects of UV irradiation on DNA replication and transcription have been extensively studied, the mechanism(s) responsible for translational repression are poorly understood. RESULTS: Here, we demonstrate that UV irradiation elicits phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) by activating the kinase GCN2 in a manner that does not require SAPK/JNK or p38 MAP kinase. GCN2-/- cells, and cells expressing nonphosphorylatable eIF2alpha as their only source of eIF2alpha protein, fail to repress translation in response to UV irradiation. CONCLUSIONS: These results provide a mechanism for translation inhibition by UV irradiation and identify a hitherto unrecognized role for mammalian GCN2 as a mediator of the cellular response to UV stress.