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1.  Influenza A Virus Host Shutoff Disables Antiviral Stress-Induced Translation Arrest 
PLoS Pathogens  2014;10(7):e1004217.
Influenza A virus (IAV) polymerase complexes function in the nucleus of infected cells, generating mRNAs that bear 5′ caps and poly(A) tails, and which are exported to the cytoplasm and translated by host machinery. Host antiviral defences include mechanisms that detect the stress of virus infection and arrest cap-dependent mRNA translation, which normally results in the formation of cytoplasmic aggregates of translationally stalled mRNA-protein complexes known as stress granules (SGs). It remains unclear how IAV ensures preferential translation of viral gene products while evading stress-induced translation arrest. Here, we demonstrate that at early stages of infection both viral and host mRNAs are sensitive to drug-induced translation arrest and SG formation. By contrast, at later stages of infection, IAV becomes partially resistant to stress-induced translation arrest, thereby maintaining ongoing translation of viral gene products. To this end, the virus deploys multiple proteins that block stress-induced SG formation: 1) non-structural protein 1 (NS1) inactivates the antiviral double-stranded RNA (dsRNA)-activated kinase PKR, thereby preventing eIF2α phosphorylation and SG formation; 2) nucleoprotein (NP) inhibits SG formation without affecting eIF2α phosphorylation; 3) host-shutoff protein polymerase-acidic protein-X (PA-X) strongly inhibits SG formation concomitant with dramatic depletion of cytoplasmic poly(A) RNA and nuclear accumulation of poly(A)-binding protein. Recombinant viruses with disrupted PA-X host shutoff function fail to effectively inhibit stress-induced SG formation. The existence of three distinct mechanisms of IAV-mediated SG blockade reveals the magnitude of the threat of stress-induced translation arrest during viral replication.
Author Summary
Like all viruses, Influenza A virus (IAV) is absolutely dependent on host-cell protein synthesis machinery. This dependence makes the virus vulnerable to the innate ability of cells to inhibit protein synthesis in response to various types of stress. This inhibition, termed translation arrest, helps cells survive adverse conditions by re-dedicating their energy to stress responses. When cells arrest translation, they form stress granules: depots of untranslated mRNAs and associated proteins. Translation arrest and formation of stress granules can be induced pharmacologically, and in this work we sought to determine whether stress granule induction would be effective in blocking IAV replication. Here we demonstrate that treatment of cells with inducers of stress granules at early times after infection resulted in blockade of viral protein synthesis and stopped viral replication. At later times post-infection, by contrast, IAV proteins prevented pharmacological induction of stress granules. We identified three viral proteins – more than in any virus to date – that work in concert to prevent stress granule formation. Taken together, our studies reveal a multipronged approach for viral suppression of translation arrest, and identify a window of opportunity early in infection when pharmacological induction of stress granules has a strong antiviral effect.
PMCID: PMC4092144  PMID: 25010204
2.  Universal Features of Post-Transcriptional Gene Regulation Are Critical for Plasmodium Zygote Development 
PLoS Pathogens  2010;6(2):e1000767.
A universal feature of metazoan sexual development is the generation of oocyte P granules that withhold certain mRNA species from translation to provide coding potential for proteins during early post-fertilization development. Stabilisation of translationally quiescent mRNA pools in female Plasmodium gametocytes depends on the RNA helicase DOZI, but the molecular machinery involved in the silencing of transcripts in these protozoans is unknown. Using affinity purification coupled with mass-spectrometric analysis we identify a messenger ribonucleoprotein (mRNP) from Plasmodium berghei gametocytes defined by DOZI and the Sm-like factor CITH (homolog of worm CAR-I and fly Trailer Hitch). This mRNP includes 16 major factors, including proteins with homologies to components of metazoan P granules and archaeal proteins. Containing translationally silent transcripts, this mRNP integrates eIF4E and poly(A)-binding protein but excludes P body RNA degradation factors and translation-initiation promoting eIF4G. Gene deletion mutants of 2 core components of this mRNP (DOZI and CITH) are fertilization-competent, but zygotes fail to develop into ookinetes in a female gametocyte-mutant fashion. Through RNA-immunoprecipitation and global expression profiling of CITH-KO mutants we highlight CITH as a crucial repressor of maternally supplied mRNAs. Our data define Plasmodium P granules as an ancient mRNP whose protein core has remained evolutionarily conserved from single-cell organisms to germ cells of multi-cellular animals and stores translationally silent mRNAs that are critical for early post-fertilization development during the initial stages of mosquito infection. Therefore, translational repression may offer avenues as a target for the generation of transmission blocking strategies and contribute to limiting the spread of malaria.
Author Summary
Transmission of malaria relies on ingestion of male and female sexual precursor cells (gametocytes) from the human host by the mosquito vector. Fertilization results in the formation of a diploid zygote that transforms into the ookinete, the motile form of the parasite that is capable of escaping the hostile mosquito midgut environment and truly infecting the mosquito vector. The developmental program of the Plasmodium zygote depends on the availability of mRNA pools transcribed and stored, but not translated, in the female gametocyte. Here we identify the core protein factors that co-operate in the assembly of mRNAs into a translationally silent ribonucleoprotein complex. In the absence of either DOZI or CITH—two key molecules within this complex—gametocytes suffer large scale mRNA de-stabilization that does not affect fertilization but culminates in the abortion of ookinete development soon after zygote formation. We characterize large scale, evolutionarily ancient translational silencing as a principal regulatory element during Plasmodium sexual development.
PMCID: PMC2820534  PMID: 20169188
3.  N-myc Downstream Regulated 1 (NDRG1) Is Regulated by Eukaryotic Initiation Factor 3a (eIF3a) during Cellular Stress Caused by Iron Depletion 
PLoS ONE  2013;8(2):e57273.
Iron is critical for cellular proliferation and its depletion leads to a suppression of both DNA synthesis and global translation. These observations suggest that iron depletion may trigger a cellular “stress response”. A canonical response of cells to stress is the formation of stress granules, which are dynamic cytoplasmic aggregates containing stalled pre-initiation complexes that function as mRNA triage centers. By differentially prioritizing mRNA translation, stress granules allow for the continued and selective translation of stress response proteins. Although the multi-subunit eukaryotic initiation factor 3 (eIF3) is required for translation initiation, its largest subunit, eIF3a, may not be essential for this activity. Instead, eIF3a is a vital constituent of stress granules and appears to act, in part, by differentially regulating specific mRNAs during iron depletion. Considering this, we investigated eIF3a’s role in modulating iron-regulated genes/proteins that are critically involved in proliferation and metastasis. In this study, eIF3a was down-regulated and recruited into stress granules by iron depletion as well as by the classical stress-inducers, hypoxia and tunicamycin. Iron depletion also increased expression of the metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), and a known downstream repressed target of eIF3a, namely the cyclin-dependent kinase inhibitor, p27kip1. To determine if eIF3a regulates NDRG1 expression, eIF3a was inducibly over-expressed or ablated. Importantly, eIF3a positively regulated NDRG1 expression and negatively regulated p27kip1 expression during iron depletion. This activity of eIF3a could be due to its recruitment to stress granules and/or its ability to differentially regulate mRNA translation during cellular stress. Additionally, eIF3a positively regulated proliferation, but negatively regulated cell motility and invasion, which may be due to the eIF3a-dependent changes in expression of NDRG1 and p27kip1 observed under these conditions.
PMCID: PMC3578820  PMID: 23437357
4.  Formation of Antiviral Cytoplasmic Granules during Orthopoxvirus Infection ▿ †  
Journal of Virology  2010;85(4):1581-1593.
Vaccinia virus (VV) mutants lacking the double-stranded RNA (dsRNA)-binding E3L protein (ΔE3L mutant VV) show restricted replication in most cell types, as dsRNA produced by VV activates protein kinase R (PKR), leading to eIF2α phosphorylation and impaired translation initiation. Here we show that cells infected with ΔE3L mutant VV assemble cytoplasmic granular structures which surround the VV replication factories at an early stage of the nonproductive infection. These structures contain the stress granule-associated proteins G3BP, TIA-1, and USP10, as well as poly(A)-containing RNA. These structures lack large ribosomal subunit proteins, suggesting that they are translationally inactive. Formation of these punctate structures correlates with restricted replication, as they occur in >80% of cells infected with ΔE3L mutant VV but in only 10% of cells infected with wild-type VV. We therefore refer to these structures as antiviral granules (AVGs). Formation of AVGs requires PKR and phosphorylated eIF2α, as mouse embryonic fibroblasts (MEFs) lacking PKR displayed reduced granule formation and MEFs lacking phosphorylatable eIF2α showed no granule formation. In both cases, these decreased levels of AVG formation correlated with increased ΔE3L mutant VV replication. Surprisingly, MEFs lacking the AVG component protein TIA-1 supported increased replication of ΔE3L mutant VV, despite increased eIF2α phosphorylation and the assembly of AVGs that lacked TIA-1. These data indicate that the effective PKR-mediated restriction of ΔE3L mutant VV replication requires AVG formation subsequent to eIF2α phosphorylation. This is a novel finding that supports the hypothesis that the formation of subcellular protein aggregates is an important component of the successful cellular antiviral response.
PMCID: PMC3028896  PMID: 21147913
5.  Translation suppression promotes stress granule formation and cell survival in response to cold shock 
Molecular Biology of the Cell  2012;23(19):3786-3800.
Cells respond to stress by inhibition of protein synthesis and subsequent assembly of stress granules (SGs). Cold shock is identified as a novel trigger of SG assembly in yeast and mammals. Cells actively suppress protein synthesis by parallel pathways to induce SG formation and ensure cellular survival at low temperatures.
Cells respond to different types of stress by inhibition of protein synthesis and subsequent assembly of stress granules (SGs), cytoplasmic aggregates that contain stalled translation preinitiation complexes. Global translation is regulated through the translation initiation factor eukaryotic initiation factor 2α (eIF2α) and the mTOR pathway. Here we identify cold shock as a novel trigger of SG assembly in yeast and mammals. Whereas cold shock–induced SGs take hours to form, they dissolve within minutes when cells are returned to optimal growth temperatures. Cold shock causes eIF2α phosphorylation through the kinase PERK in mammalian cells, yet this pathway is not alone responsible for translation arrest and SG formation. In addition, cold shock leads to reduced mitochondrial function, energy depletion, concomitant activation of AMP-activated protein kinase (AMPK), and inhibition of mTOR signaling. Compound C, a pharmacological inhibitor of AMPK, prevents the formation of SGs and strongly reduces cellular survival in a translation-dependent manner. Our results demonstrate that cells actively suppress protein synthesis by parallel pathways, which induce SG formation and ensure cellular survival during hypothermia.
PMCID: PMC3459856  PMID: 22875991
6.  Uncoupling Stress Granule Assembly and Translation Initiation Inhibition 
Molecular Biology of the Cell  2009;20(11):2673-2683.
Cytoplasmic stress granules (SGs) are specialized regulatory sites of mRNA translation that form under different stress conditions known to inhibit translation initiation. The formation of SG occurs via two pathways; the eukaryotic initiation factor (eIF) 2α phosphorylation-dependent pathway mediated by stress and the eIF2α phosphorylation-independent pathway mediated by inactivation of the translation initiation factors eIF4A and eIF4G. In this study, we investigated the effects of targeting different translation initiation factors and steps in SG formation in HeLa cells. By depleting eIF2α, we demonstrate that reduced levels of the eIF2.GTP.Met-tRNAiMet ternary translation initiation complexes is sufficient to induce SGs. Likewise, reduced levels of eIF4B, eIF4H, or polyA-binding protein, also trigger SG formation. In contrast, depletion of the cap-binding protein eIF4E or preventing its assembly into eIF4F results in modest SG formation. Intriguingly, interfering with the last step of translation initiation by blocking the recruitment of 60S ribosome either with 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamideis or through depletion of the large ribosomal subunits protein L28 does not induce SG assembly. Our study identifies translation initiation steps and factors involved in SG formation as well as those that can be targeted without induction of SGs.
PMCID: PMC2688547  PMID: 19369421
7.  The DEAD-box protein Ded1 modulates translation by the formation and resolution of an eIF4F-mRNA complex 
Molecular Cell  2011;43(6):962-972.
The translation, localization, and degradation of cytoplasmic mRNAs are controlled by the formation and rearrangement of their mRNPs. The conserved Ded1/DDX3 DEAD-box protein functions in an unknown manner to affect both translation initiation and repression. We demonstrate that Ded1 first functions by directly interacting with eIF4G to assemble a Ded1-mRNA-eIF4F complex, which accumulates in stress granules. Following ATP hydrolysis by Ded1, the mRNP exits stress granules and completes translation initiation. Thus, Ded1 functions both as a repressor of translation, by assembling an mRNP stalled in translation initiation, and as an ATP-dependent activator of translation, by resolving the stalled mRNP. These results identify Ded1 as a translation initiation factor that assembles and remodels an intermediate complex in translation initiation.
PMCID: PMC3268518  PMID: 21925384
8.  Mammalian Orthoreovirus Escape from Host Translational Shutoff Correlates with Stress Granule Disruption and Is Independent of eIF2α Phosphorylation and PKR▿  
Journal of Virology  2011;85(17):8798-8810.
In response to mammalian orthoreovirus (MRV) infection, cells initiate a stress response that includes eIF2α phosphorylation and protein synthesis inhibition. We have previously shown that early in infection, MRV activation of eIF2α phosphorylation results in the formation of cellular stress granules (SGs). In this work, we show that as infection proceeds, MRV disrupts SGs despite sustained levels of phosphorylated eIF2α and, further, interferes with the induction of SGs by other stress inducers. MRV interference with SG formation occurs downstream of eIF2α phosphorylation, suggesting the virus uncouples the cellular stress signaling machinery from SG formation. We additionally examined mRNA translation in the presence of SGs induced by eIF2α phosphorylation-dependent and -independent mechanisms. We found that irrespective of eIF2α phosphorylation status, the presence of SGs in cells correlated with inhibition of viral and cellular translation. In contrast, MRV disruption of SGs correlated with the release of viral mRNAs from translational inhibition, even in the presence of phosphorylated eIF2α. Viral mRNAs were also translated in the presence of phosphorylated eIF2α in PKR−/− cells. These results suggest that MRV escape from host cell translational shutoff correlates with virus-induced SG disruption and occurs in the presence of phosphorylated eIF2α in a PKR-independent manner.
PMCID: PMC3165827  PMID: 21715487
9.  Upf1 Phosphorylation Triggers Translational Repression during Nonsense-Mediated mRNA Decay 
Cell  2008;133(2):314-327.
In mammalian cells, nonsense-mediated mRNA decay (NMD) generally requires that translation terminates sufficiently upstream of a post-splicing exon junction complex (EJC) during a pioneer round of translation. The subsequent binding of Upf1 to the EJC triggers Upf1 phosphorylation. We provide evidence that phospho-Upf1 functions after nonsense codon recognition during steps that involve the translation initiation factor eIF3 and mRNA decay factors. Phospho-Upf1 interacts directly with eIF3 and inhibits the eIF3-dependent conversion of 40S/Met-tRNAiMet/mRNA to translationally competent 80S/Met-tRNAiMet/mRNA initiation complexes to repress continued translation initiation. Consistent with phospho-Upf1 impairing eIF3 function, NMD fails to detectably target nonsense-containing transcripts that initiate translation independently of eIF3 from the CrPV IRES. There is growing evidence that translational repression is a key transition that precedes mRNA delivery to the degradation machinery. Our results uncover a critical step during NMD that converts a pioneer translation initiation complex to a translationally compromised mRNP.
PMCID: PMC4193665  PMID: 18423202
10.  Biomass Conversion Inhibitors Furfural and 5-Hydroxymethylfurfural Induce Formation of Messenger RNP Granules and Attenuate Translation Activity in Saccharomyces cerevisiae 
Various forms of stress can cause an attenuation of bulk translation activity and the accumulation of nontranslating mRNAs into cytoplasmic messenger RNP (mRNP) granules termed processing bodies (P-bodies) and stress granules (SGs) in eukaryotic cells. Furfural and 5-hydroxymethylfurfural (HMF), derived from lignocellulosic biomass, inhibit yeast growth and fermentation as stressors. Since there is no report regarding their effects on the formation of cytoplasmic mRNP granules, here we investigated whether furfural and HMF cause the assembly of yeast P-bodies and SGs accompanied by translational repression. We found that furfural and HMF cause the attenuation of bulk translation activity and the assembly of cytoplasmic mRNP granules in Saccharomyces cerevisiae. Notably, a combination of furfural and HMF induced the remarkable repression of translation initiation and SG formation. These findings provide new information about the physiological effects of furfural and HMF on yeast cells, and also suggest the potential usefulness of cytoplasmic mRNP granules as a warning sign or index of the deterioration of cellular physiological status in the fermentation of lignocellulosic hydrolysates.
PMCID: PMC3591974  PMID: 23275506
11.  OGFOD1, a Novel Modulator of Eukaryotic Translation Initiation Factor 2α Phosphorylation and the Cellular Response to Stress ▿ †  
Molecular and Cellular Biology  2010;30(8):2006-2016.
Cells possess mechanisms that permit survival and recovery from stress, several of which regulate the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). We identified the human OGFOD1 protein as a novel stress granule component that regulates the phosphorylation of eIF2α and the resumption of translation in cells recovering from arsenite-induced stress. Coimmunoprecipitation studies revealed that OGFOD1 associates with a small subset of stress granule proteins (G3BP1, USP10, Caprin1, and YB-1) and the ribosome in both unstressed and stressed cells. Overexpression of OGFOD1 led to increased abundance of phosphorylated eIF2α, both in unstressed cells and in cells exposed to arsenite-induced stress, and to accelerated apoptosis during stress. Conversely, knockdown of OGFOD1 resulted in smaller amounts of phosphorylated eIF2α and a faster accumulation of polyribosomes in cells recovering from stress. Finally, OGFOD1 interacted with both eIF2α and the eIF2α kinase heme-regulated inhibitor (HRI), which was identified as a novel stress granule resident. These findings argue that OGFOD1 plays important proapoptotic roles in the regulation of translation and HRI-mediated phosphorylation of eIF2α in cells subjected to arsenite-induced stress.
PMCID: PMC2849474  PMID: 20154146
12.  P-Bodies and Stress Granules: Possible Roles in the Control of Translation and mRNA Degradation 
The control of translation and mRNA degradation is important in the regulation of eukaryotic gene expression. In general, translation and steps in the major pathway of mRNA decay are in competition with each other. mRNAs that are not engaged in translation can aggregate into cytoplasmic mRNP granules referred to as processing bodies (P-bodies) and stress granules, which are related to mRNP particles that control translation in early development and neurons. Analyses of P-bodies and stress granules suggest a dynamic process, referred to as the mRNA Cycle, wherein mRNPs can move between polysomes, P-bodies and stress granules although the functional roles of mRNP assembly into higher order structures remain poorly understood. In this article, we review what is known about the coupling of translation and mRNA degradation, the properties of P-bodies and stress granules, and how assembly of mRNPs into larger structures might influence cellular function.
In eukaryotic cells, translation and mRNA decay are coordinated. Nontranslating mRNAs can accumulate in P-bodies (with mRNA decay machinery) and stress granules (with translation initiation components).
PMCID: PMC3428773  PMID: 22763747
13.  Evidence That Ternary Complex (eIF2-GTP-tRNAiMet)–Deficient Preinitiation Complexes Are Core Constituents of Mammalian Stress Granules 
Molecular Biology of the Cell  2002;13(1):195-210.
Environmental stress-induced phosphorylation of eIF2α inhibits protein translation by reducing the availability of eIF2-GTP-tRNAiMet, the ternary complex that joins initiator tRNAMet to the 43S preinitiation complex. The resulting untranslated mRNA is dynamically routed to discrete cytoplasmic foci known as stress granules (SGs), a process requiring the related RNA-binding proteins TIA-1 and TIAR. SGs appear to be in equilibrium with polysomes, but the nature of this relationship is obscure. We now show that most components of the 48S preinitiation complex (i.e., small, but not large, ribosomal subunits, eIF3, eIF4E, eIF4G) are coordinately recruited to SGs in arsenite-stressed cells. In contrast, eIF2 is not a component of newly assembled SGs. Cells expressing a phosphomimetic mutant (S51D) of eIF2α assemble SGs of similar composition, confirming that the recruitment of these factors is a direct consequence of blocked translational initiation and not due to other effects of arsenite. Surprisingly, phospho-eIF2α is recruited to SGs that are disassembling in cells recovering from arsenite-induced stress. We discuss these results in the context of a translational checkpoint model wherein TIA and eIF2 are functional antagonists of translational initiation, and in which lack of ternary complex drives SG assembly.
PMCID: PMC65082  PMID: 11809833
14.  PKCα Binds G3BP2 and Regulates Stress Granule Formation Following Cellular Stress 
PLoS ONE  2012;7(4):e35820.
Protein kinase C (PKC) isoforms regulate a number of processes crucial for the fate of a cell. In this study we identify previously unrecognized interaction partners of PKCα and a novel role for PKCα in the regulation of stress granule formation during cellular stress. Three RNA-binding proteins, cytoplasmic poly(A)+ binding protein (PABPC1), IGF-II mRNA binding protein 3 (IGF2BP3), and RasGAP binding protein 2 (G3BP2) all co-precipitate with PKCα. RNase treatment abolished the association with IGF2BP3 and PABPC1 whereas the PKCα-G3BP2 interaction was largely resistant to this. Furthermore, interactions between recombinant PKCα and G3BP2 indicated that the interaction is direct and PKCα can phosphorylate G3BP2 in vitro. The binding is mediated via the regulatory domain of PKCα and the C-terminal RNA-binding domain of G3BP2. Both proteins relocate to and co-localize in stress granules, but not to P-bodies, when cells are subjected to stress. Heat shock-induced stress granule assembly and phosphorylation of eIF2α are suppressed following downregulation of PKCα by siRNA. In conclusion this study identifies novel interaction partners of PKCα and a novel role for PKCα in regulation of stress granules.
PMCID: PMC3335008  PMID: 22536444
15.  Dynamic Oscillation of Translation and Stress Granule Formation Mark the Cellular Response to Virus Infection 
Cell host & microbe  2012;12(1):10.1016/j.chom.2012.05.013.
Virus infection-induced global protein synthesis suppression is linked to assembly of stress granules (SGs), cytosolic aggregates of stalled translation preinitiation complexes. To study long-term stress responses, we developed an imaging approach for extended observation and analysis of SG dynamics during persistent hepatitis C virus (HCV) infection. In combination with type 1 interferon, HCV infection induces highly dynamic assembly/disassembly of cytoplasmic SGs, concomitant with phases of active and stalled translation, delayed cell division, and prolonged cell survival. Double-stranded RNA (dsRNA), independent of viral replication, is sufficient to trigger these oscillations. Translation initiation factor eIF2α phosphorylation by protein kinase R mediates SG formation and translation arrest. This is antagonized by the upregulation of GADD34, the regulatory subunit of protein phosphatase 1 dephosphorylating eIF2α. Stress response oscillation is a general mechanism to prevent long-lasting translation repression and a conserved host cell reaction to multiple RNA viruses, which HCV may exploit to establish persistence.
PMCID: PMC3873964  PMID: 22817989
16.  Importance of eIF2α Phosphorylation and Stress Granule Assembly in Alphavirus Translation Regulation 
Molecular Biology of the Cell  2005;16(8):3753-3763.
Alphavirus infection results in the shutoff of host protein synthesis in favor of viral translation. Here, we show that during Semliki Forest virus (SFV) infection, the translation inhibition is largely due to the activation of the cellular stress response via phosphorylation of eukaryotic translation initiation factor 2α subunit (eIF2α). Infection of mouse embryo fibroblasts (MEFs) expressing a nonphosphorylatable mutant of eIF2α does not result in efficient shutoff, despite efficient viral protein production. Furthermore, we show that the SFV translation enhancer element counteracts the translation inhibition imposed by eIF2α phosphorylation. In wild-type MEFs, viral infection induces the transient formation of stress granules (SGs) containing the cellular TIA-1/R proteins. These SGs are disassembled in the vicinity of viral RNA replication, synchronously with the switch from cellular to viral gene expression. We propose that phosphorylation of eIF2α and the consequent SG assembly is important for shutoff to occur and that the localized SG disassembly and the presence of the enhancer aid the SFV mRNAs to elude general translational arrest.
PMCID: PMC1182313  PMID: 15930128
17.  Poliovirus Unlinks TIA1 Aggregation and mRNA Stress Granule Formation▿ 
Journal of Virology  2011;85(23):12442-12454.
In response to environmental stress and viral infection, mammalian cells form foci containing translationally silenced mRNPs termed stress granules (SGs). As aggregates of stalled initiation complexes, SGs are defined by the presence of translation initiation machinery in addition to mRNA binding proteins. Here, we report that cells infected with poliovirus (PV) can form SGs early that contain T-cell-restricted intracellular antigen 1 (TIA1), translation initiation factors, RNA binding proteins, and mRNA. However, this response is blocked as infection progresses, and a type of pseudo-stress granule remains at late times postinfection and contains TIA but lacks translation initiation factors, mRNA binding proteins, and most polyadenylated mRNA. This result was observed using multiple stressors, including viral infection, oxidative stress, heat shock, and endoplasmic reticulum stress. Multiple proteins required for efficient viral internal ribosome entry site-dependent translation are localized to SGs under stress conditions, providing a potential rationale for the evolution and maintenance of the SG inhibition phenotype. Further, the expression of a noncleavable form of the RasGAP-SH3 domain binding protein in PV-infected cells enables SGs whose constituents are consistent with the presence of stalled 48S translation preinitiation complexes to persist throughout infection. These results indicate that in poliovirus-infected cells, the functions of TIA self-aggregation and aggregation of stalled translation initiation complexes into stress granules are severed, leading to novel foci that contain TIA1 but lack other stress granule-defining components.
PMCID: PMC3209409  PMID: 21957303
18.  Hepatitis C Virus (HCV) Induces Formation of Stress Granules Whose Proteins Regulate HCV RNA Replication and Virus Assembly and Egress 
Journal of Virology  2012;86(20):11043-11056.
Stress granules (SGs) are cytoplasmic structures that are induced in response to environmental stress, including viral infections. Here we report that hepatitis C virus (HCV) triggers the appearance of SGs in a PKR- and interferon (IFN)-dependent manner. Moreover, we show an inverse correlation between the presence of stress granules and the induction of IFN-stimulated proteins, i.e., MxA and USP18, in HCV-infected cells despite high-level expression of the corresponding MxA and USP18 mRNAs, suggesting that interferon-stimulated gene translation is inhibited in stress granule-containing HCV-infected cells. Finally, in short hairpin RNA (shRNA) knockdown experiments, we found that the stress granule proteins T-cell-restricted intracellular antigen 1 (TIA-1), TIA1-related protein (TIAR), and RasGAP-SH3 domain binding protein 1 (G3BP1) are required for efficient HCV RNA and protein accumulation at early time points in the infection and that G3BP1 and TIA-1 are required for intracellular and extracellular infectious virus production late in the infection, suggesting that they are required for virus assembly. In contrast, TIAR downregulation decreases extracellular infectious virus titers with little effect on intracellular RNA content or infectivity late in the infection, suggesting that it is required for infectious particle release. Collectively, these results illustrate that HCV exploits the stress granule machinery at least two ways: by inducing the formation of SGs by triggering PKR phosphorylation, thereby downregulating the translation of antiviral interferon-stimulated genes, and by co-opting SG proteins for its replication, assembly, and egress.
PMCID: PMC3457181  PMID: 22855484
19.  Screening of small molecules affecting mammalian P-body assembly uncovers links with diverse intracellular processes and organelle physiology 
RNA Biology  2013;10(11):1661-1669.
Processing bodies (P-bodies) are cytoplasmatic mRNP granules containing non-translating mRNAs and proteins from the mRNA decay and silencing machineries. The mechanism of P-body assembly has been typically addressed by depleting P-body components. Here we apply a complementary approach and establish an automated cell-based assay platform to screen for molecules affecting P-body assembly. From a unique library of compounds derived from myxobacteria, 30 specifically inhibited P-body assembly. Gephyronic acid A (GA), a eukaryotic protein synthesis inhibitor, showed the strongest effect. GA also inhibited, under stress conditions, phosphorylation of eIF2α and stress granule formation. Other hits uncovered interesting novel links between P-body assembly, lipid metabolism, and internal organelle physiology. The obtained results provide a chemical toolbox to manipulate P-body assembly and function.
PMCID: PMC3907476  PMID: 24418890
P-body assembly; eIF2α; gephyronic acid A; inhibitors; myxobacterial metabolites; processing bodies; stress granules
20.  Host Factors That Interact with the Pestivirus N-Terminal Protease, Npro, Are Components of the Ribonucleoprotein Complex 
Journal of Virology  2014;88(18):10340-10353.
The viral N-terminal protease Npro of pestiviruses counteracts cellular antiviral defenses through inhibition of IRF3. Here we used mass spectrometry to identify a new role for Npro through its interaction with over 55 associated proteins, mainly ribosomal proteins and ribonucleoproteins, including RNA helicase A (DHX9), Y-box binding protein (YBX1), DDX3, DDX5, eIF3, IGF2BP1, multiple myeloma tumor protein 2, interleukin enhancer binding factor 3 (IEBP3), guanine nucleotide binding protein 3, and polyadenylate-binding protein 1 (PABP-1). These are components of the translation machinery, ribonucleoprotein particles (RNPs), and stress granules. Significantly, we found that stress granule formation was inhibited in MDBK cells infected with a noncytopathic bovine viral diarrhea virus (BVDV) strain, Kyle. However, ribonucleoproteins binding to Npro did not inhibit these proteins from aggregating into stress granules. Npro interacted with YBX1 though its TRASH domain, since the mutant C112R protein with an inactive TRASH domain no longer redistributed to stress granules. Interestingly, RNA helicase A and La autoantigen relocated from a nuclear location to form cytoplasmic granules with Npro. To address a proviral role for Npro in RNP granules, we investigated whether Npro affected RNA interference (RNAi), since interacting proteins are involved in RISC function during RNA silencing. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) silencing with small interfering RNAs (siRNAs) followed by Northern blotting of GAPDH, expression of Npro had no effect on RNAi silencing activity, contrasting with other viral suppressors of interferon. We propose that Npro is involved with virus RNA translation in the cytoplasm for virus particle production, and when translation is inhibited following stress, it redistributes to the replication complex.
IMPORTANCE Although the pestivirus N-terminal protease, Npro, has been shown to have an important role in degrading IRF3 to prevent apoptosis and interferon production during infection, the function of this unique viral protease in the pestivirus life cycle remains to be elucidated. We used proteomic mass spectrometry to identify novel interacting proteins and have shown that Npro is present in ribosomal and ribonucleoprotein particles (RNPs), indicating a translational role in virus particle production. The virus itself can prevent stress granule assembly from these complexes, but this inhibition is not due to Npro. A proviral role to subvert RNA silencing through binding of these host RNP proteins was not identified for this viral suppressor of interferon.
PMCID: PMC4178888  PMID: 24965446
21.  Direct interaction between STAT3 and EIF2AK2 controls fatty acid-induced autophagy 
Autophagy  2013;9(3):415-417.
A chemical screen designed to identify novel inducers of autophagy led to the discovery that signal transducer and activator of transcription 3 (STAT3) inhibitors can potently stimulate the autophagic flux. Although STAT3 is best known as a pro-inflammatory and oncogenic transcription factor, mechanistic analyses revealed that autophagy is regulated by the cytoplasmic, not nuclear, pool of STAT3. Cytoplasmic STAT3 normally interacts with the eukaryotic translation initiation factor 2, subunit 1α, 35kDa (EIF2S1/eIF2α) kinase 2/protein kinase, RNA-activated (EIF2AK2/PKR), a sensor of double-stranded RNA. This interaction, which could be recapitulated using recombinant proteins in pull-down experiments, involves the catalytic domain of EIF2AK2 as well as the SH2 domain of STAT3, which can adopt a fold similar to that of EIF2S1. Thus, STAT3 may act as a competitive inhibitor of EIF2AK2. Indeed, pharmacological or genetic inhibition of STAT3 stimulates EIF2AK2-dependent EIF2S1 phosphorylation and autophagy. Conversely, the overexpression of wild-type STAT3 as well as of STAT3 mutants that cannot be phosphorylated by JAK2 or are excluded from the nucleus inhibits autophagy. However, STAT3 mutants that fail to interact with EIF2AK2 are unable to suppress autophagy. Both STAT3-targeting agents (i.e., Stattic, JSI-124 and WP1066) and EIF2AK2 activators (such as the double-strand RNA mimetic polyinosinic:polycytidylic acid) are capable of disrupting the inhibitory interaction between STAT3 and EIF2AK2 in cellula, yet only the latter does so in cell-free systems in vitro. A further screen designed to identify EIF2AK2-dependent autophagy inducers revealed that several fatty acids including palmitate trigger autophagy via a pathway that involves the disruption of the STAT3-EIF2AK2 complex as well as the phosphorylation of mitogen-activated protein kinase 8/c-Jun N-terminal kinase 1 (MAPK8/JNK1) and EIF2S1. These results reveal an unsuspected crosstalk between cellular metabolism (fatty acids), pro-inflammatory signaling (STAT3), innate immunity (EIF2AK2), and translational control (EIF2S1) that regulates autophagy.
PMCID: PMC3590262  PMID: 23221979
EIF2S1S51A; endoplasmic reticulum; IRS1; palmitate; polyI:C; STAT3Y705F
22.  Angiogenin-induced tRNA fragments inhibit translation initiation 
Molecular cell  2011;43(4):613-623.
Angiogenin is a stress-activated ribonuclease that cleaves tRNA within anticodon loops to produce tRNA-derived stress-induced fragments (tiRNAs). Transfection of natural or synthetic tiRNAs inhibits protein synthesis and triggers the phospho-eIF2α independent assembly of stress granules (SGs), essential components of the stress response program. We show that selected tiRNAs inhibit protein synthesis by displacing eIF4G/eIF4A from uncapped>capped RNAs. tiRNAs also displace eIF4F, but not eIF4E:4EBP1, from isolated m7G cap. We identify a terminal oligoguanine motif that is required to displace the eIF4F complex, inhibit translation, and induce SG assembly. We show that the tiRNA-associated translational silencer YB-1 contributes to angiogenin-, tiRNA-, and oxidative stress-induced translational repression. Our data reveal some of the mechanisms by which stress-induced tRNA cleavage inhibits protein synthesis and activates a cytoprotective stress response program.
PMCID: PMC3160621  PMID: 21855800
23.  壽Visibly stressed: the role of eIF2, TIA-1, and stress granules in protein translation 
Cell Stress & Chaperones  2002;7(2):213-221.
Eukaryotic cells express a family of eukaryotic translation initiation factor 2 alpha (eIF2α) kinases (eg, PKR, PERK-PEK, GCN2, HRI) that are individually activated in response to distinct types of environmental stress. Phosphorylation of eIF2α by one or more of these kinases reduces the concentration of eIF2–guanosine triphosphate (GTP)–transfer ribonucleic acid for methionine (tRNAMet), the ternary complex that loads tRNAMet onto the small ribosomal subunit to initiate protein translation. When ternary complex levels are reduced, the related RNA-binding proteins TIA-1 and TIAR promote the assembly of a noncanonical preinitiation complex that lacks eIF2-GTP-tRNAMet. The TIA proteins dynamically sort these translationally incompetent preinitiation complexes into discrete cytoplasmic domains known as stress granules (SGs). RNA-binding proteins that stabilize or destabilize messenger RNA (mRNA) are also recruited to SGs during stress. Thus, TIA-1 and TIAR act downstream of eIF2α phosphorylation to promote SG assembly and facilitate mRNA triage during stress. The role of the SG in the integration of translational efficiency, mRNA stability, and the stress response is discussed.
PMCID: PMC514820  PMID: 12380690
24.  Reovirus Induces and Benefits from an Integrated Cellular Stress Response 
Journal of Virology  2006;80(4):2019-2033.
Following infection with most reovirus strains, viral protein synthesis is robust, even when cellular translation is inhibited. To gain further insight into pathways that regulate translation in reovirus-infected cells, we performed a comparative microarray analysis of cellular gene expression following infection with two strains of reovirus that inhibit host translation (clone 8 and clone 87) and one strain that does not (Dearing). Infection with clone 8 and clone 87 significantly increased the expression of cellular genes characteristic of stress responses, including the integrated stress response. Infection with these same strains decreased transcript and protein levels of P58IPK, the cellular inhibitor of the eukaryotic initiation factor 2α (eIF2α) kinases PKR and PERK. Since infection with host shutoff-inducing strains of reovirus impacted cellular pathways that control eIF2α phosphorylation and unphosphorylated eIF2α is required for translation initiation, we examined reovirus replication in a variety of cell lines with mutations that impact eIF2α phosphorylation. Our results revealed that reovirus replication is more efficient in the presence of eIF2α kinases and phosphorylatable eIF2α. When eIF2α is phosphorylated, it promotes the synthesis of ATF4, a transcription factor that controls cellular recovery from stress. We found that the presence of this transcription factor increased reovirus yields 10- to 100-fold. eIF2α phosphorylation also led to the formation of stress granules in reovirus-infected cells. Based on these results, we hypothesize that eIF2α phosphorylation facilitates reovirus replication in two ways—first, by inducing ATF4 synthesis, and second, by creating an environment that places abundant reovirus transcripts at a competitive advantage for limited translational components.
PMCID: PMC1367166  PMID: 16439558
25.  Poliovirus infection induces the co-localization of cellular protein SRp20 with TIA-1, a cytoplasmic stress granule protein 
Virus research  2013;176(0):223-231.
Different types of environmental stress cause mammalian cells to form cytoplasmic foci, termed stress granules, which contain mRNPs that are translationally silenced. These foci are transient and dynamic, and contain components of the cellular translation machinery as well as certain mRNAs and RNA binding proteins. Stress granules are known to be induced by conditions such as hypoxia, nutrient deprivation, and oxidative stress, and a number of cellular factors have been identified that are commonly associated with these foci. More recently it was discovered that poliovirus infection also induces the formation of stress granules, although these cytoplasmic foci appear to be somewhat compositionally unique. Work described here examined the punctate pattern of SRp20 (a host cell mRNA splicing protein) localization in the cytoplasm of poliovirus-infected cells, demonstrating the partial co-localization of SRp20 with the stress granule marker protein TIA-1. We determined that SRp20 does not co-localize with TIA-1, however, under conditions of oxidative stress, indicating that the close association of these two proteins during poliovirus infection is not representative of a general response to cellular stress. We confirmed that the expression of a dominant negative version of TIA-1 (TIA-1-PRD) results in the dissociation of stress granules. Finally, we demonstrated that expression of wild type TIA-1 or dominant negative TIA-1-PRD in cells during poliovirus infection does not dramatically affect viral translation. Taken together, these studies provide a new example of the unique cytoplasmic foci that form during poliovirus infection.
PMCID: PMC3742715  PMID: 23830997
picornavirus; poliovirus; stress granules; SRp20; TIA-1; protein co-localization

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