YB-1 regulates stress granule formation and tumor progression by translationally activating G3BP1

Under cell stress, global protein synthesis is inhibited to preserve energy. One mechanism is to sequester and silence mRNAs in ribonucleoprotein complexes known as stress granules (SGs), which contain translationally silent mRNAs, preinitiation factors, and RNA-binding proteins. Y-box binding protein 1 (YB-1) localizes to SGs, but its role in SG biology is unknown. We now report that YB-1 directly binds to and translationally activates the 5′ untranslated region (UTR) of G3BP1 mRNAs, thereby controlling the availability of the G3BP1 SG nucleator for SG assembly. YB-1 inactivation in human sarcoma cells dramatically reduces G3BP1 and SG formation in vitro. YB-1 and G3BP1 expression are highly correlated in human sarcomas, and elevated G3BP1 expression correlates with poor survival. Finally, G3BP1 down-regulation in sarcoma xenografts prevents in vivo SG formation and tumor invasion, and completely blocks lung metastasis in mouse models. Together, these findings demonstrate a critical role for YB-1 in SG formation through translational activation of G3BP1, and highlight novel functions for SGs in tumor progression.     

Analysis of the DNA binding site of Escherichia coli RecA protein

To investigate the DNA binding site of RecA protein, we constructed 15 recA mutants having alterations in the regions homologous to the other ssDNA binding proteins. The in vivo analyses showed that the mutational change at Arg²⁴³, Lys²⁴⁸, Tyr²⁶⁴, or simultaneously at Lys⁶ and Lys¹⁹, or Lys⁶ and Lys²³ caused severe defects in the recA functions, while other mutational changes did not. Purified RecA-K6A-K23A (Lys⁶ and Lys²³ changed to Ala and Ala, respectively) protein was indistinguishable from the wild-type RecA protein in its binding to DNA. However, the RecA-R243A (Arg²⁴³ changed to Ala) and RecA-Y264A (Tyr²⁶⁴ changed to Ala) proteins were defective in binding to both ss- and ds-DNA. In self-oligomerization property, RecA-R243A was proficient but RecA-Y264A was deficient, suggesting that the RecA-R243A protein had a defect in DNA binding site and the RecA-Y264A protein was defective in its interaction with the adjacent RecA molecule. The region of residues 243–257 including the Arg²⁴³ is highly homologous to the DNA binding motif in the ssDNA binding proteins, while the eukaryotic RecA homologues have a similar structure at the amino-terminal side proximal to the nucleotide binding core. The region of residues 243–257 would be a part of the DNA binding site. The other parts of this site would be the Tyr¹⁰³ and the region of residues 178–183, which were cross-linked to ssDNA. These three regions lie in a line in the crystal structure.     

Sequestration of G3BP coupled with efficient translation inhibits stress granules in Semliki Forest virus infection

Dynamic, mRNA-containing stress granules (SGs) form in the cytoplasm of cells under environmental stresses, including viral infection. Many viruses appear to employ mechanisms to disrupt the formation of SGs on their mRNAs, suggesting that they represent a cellular defense against infection. Here, we report that early in Semliki Forest virus infection, the C-terminal domain of the viral nonstructural protein 3 (nsP3) forms a complex with Ras-GAP SH3-domain–binding protein (G3BP) and sequesters it into viral RNA replication complexes in a manner that inhibits the formation of SGs on viral mRNAs. A viral mutant carrying a C-terminal truncation of nsP3 induces more persistent SGs and is attenuated for propagation in cell culture. Of importance, we also show that the efficient translation of viral mRNAs containing a translation enhancer sequence also contributes to the disassembly of SGs in infected cells. Furthermore, we show that the nsP3/G3BP interaction also blocks SGs induced by other stresses than virus infection. This is one of few described viral mechanisms for SG disruption and underlines the role of SGs in antiviral defense.     

Production of a Dominant-Negative Fragment Due to G3BP1 Cleavage Contributes to the Disruption of Mitochondria-Associated Protective Stress Granules during CVB3 Infection

Stress granules (SGs) are dynamic cytosolic aggregates containing messenger ribonucleoproteins and target poly-adenylated (A)-mRNA. A key component of SGs is Ras-GAP SH3 domain binding protein-1 (G3BP1), which in part mediates protein-protein and protein-RNA interactions. SGs are modulated during infection by several viruses, however, the function and significance of this process remains poorly understood. In this study, we investigated the interplay between SGs and Coxsackievirus type B3 (CVB3), a member of the Picornaviridae family. Our studies demonstrated that SGs were formed early during CVB3 infection; however, G3BP1-positive SGs were actively disassembled at 5 hrs post-infection, while poly(A)-positive RNA granules persisted. Furthermore, we confirmed G3BP1 cleavage by 3C^pro at Q325. We also demonstrated that overexpression of G3BP1-SGs negatively impacted viral replication at the RNA, protein, and viral progeny levels. Using electron microscopy techniques, we showed that G3BP1-positive SGs localized near mitochondrial surfaces. Finally, we provided evidence that the C-terminal cleavage product of G3BP1 inhibited SG formation and promoted CVB3 replication. Taken together, we conclude that CVB3 infection selectively targets G3BP1-SGs by cleaving G3BP1 to produce a dominant-negative fragment that further inhibits G3BP1-SG formation and facilitates viral replication.     

Spare interactions of highly potent [Arg14,Lys15]nociceptin for cooperative induction of ORL1 receptor activation

[Arg¹⁴,Lys¹⁵]Nociceptin is a very potent for ORL1 receptor, showing a few times stronger binding activity and much more enhanced biological activity than endogenous nociceptin. This synergistic outcome has been suggested to be due to the interaction with the receptor aromatic and/or acidic amino acid residues crucial to receptor activation. In order to identify such receptor residues in the second ORL1 extracellular loop, we prepared a series of recombinant mutant receptors. The mutant receptor Gln205Ala was found to be as active as wild-type ORL1 for both nociceptin and [Arg¹⁴,Lys¹⁵]nociceptin. In contrast, Asp206Ala and Tyr207Ala exhibited considerably reduced activity for [Arg¹⁴,Lys¹⁵]nociceptin, exhibiting no synergistic activity enhancement. These results suggest that Asp206 and Tyr207 are directly involved in the interaction with nociceptin-[Arg¹⁴,Lys¹⁵]. Trp208Ala was found to bind strongly both nociceptin and [Arg¹⁴,Lys¹⁵]nociceptin, although it elicited no biological activity. All these results indicate that the consecutive amino acid residues Asp206, Tyr207, and Trp208 are critical to the activation of the ORL1 receptor, but not to nociceptin-binding.     

Cytoplasmic hGle1A regulates stress granules by modulation of translation

When eukaryotic cells respond to stress, gene expression pathways change to selectively export and translate subsets of mRNAs. Translationally repressed mRNAs accumulate in cytoplasmic foci known as stress granules (SGs). SGs are in dynamic equilibrium with the translational machinery, but mechanisms controlling this are unclear. Gle1 is required for DEAD-box protein function during mRNA export and translation. We document that human Gle1 (hGle1) is a critical regulator of translation during stress. hGle1 is recruited to SGs, and hGLE1 small interfering RNA–mediated knockdown perturbs SG assembly, resulting in increased numbers of smaller SGs. The rate of SG disassembly is also delayed. Furthermore, SG hGle1-depletion defects correlate with translation perturbations, and the hGle1 role in SGs is independent of mRNA export. Interestingly, we observe isoform-specific roles for hGle1 in which SG function requires hGle1A, whereas mRNA export requires hGle1B. We find that the SG defects in hGle1-depleted cells are rescued by puromycin or DDX3 expression. Together with recent links of hGLE1 mutations in amyotrophic lateral sclerosis patients, these results uncover a paradigm for hGle1A modulating the balance between translation and SGs during stress and disease.     

Encephalomyocarditis Virus Disrupts Stress Granules,the Critical Platform for Triggering Antiviral Innate Immune Responses

In response to stress, cells induce ribonucleoprotein aggregates, termed stress granules (SGs). SGs are transient loci containing translation-stalled mRNA, which is eventually degraded or recycled for translation. Infection of some viruses, including influenza A virus with a deletion of nonstructural protein 1 (IAVΔNS1), induces SG-like protein aggregates. Previously, we showed that IAVΔNS1-induced SGs are required for efficient induction of type I interferon (IFN). Here, we investigated SG formation by different viruses using green fluorescent protein (GFP)-tagged Ras-Gap SH3 domain binding protein 1 (GFP-G3BP1) as an SG probe. HeLa cells stably expressing GFP-G3BP1 were infected with different viruses, and GFP fluorescence was monitored live with time-lapse microscopy. SG formations by different viruses was classified into 4 different patterns: no SG formation, stable SG formation, transient SG formation, and alternate SG formation. We focused on encephalomyocarditis virus (EMCV) infection, which exhibited transient SG formation. We found that EMCV disrupts SGs by cleavage of G3BP1 at late stages of infection (>8 h) through a mechanism similar to that used by poliovirus. Expression of a G3BP1 mutant that is resistant to the cleavage conferred persistent formation of SGs as well as an enhanced induction of IFN and other cytokines at late stages of infection. Additionally, knockdown of endogenous G3BP1 blocked SG formation with an attenuated induction of IFN and potentiated viral replication. Taken together, our findings suggest a critical role of SGs as an antiviral platform and shed light on one of the mechanisms by which a virus interferes with host stress and subsequent antiviral responses.     

The binding of Arg- and Lys-peptides to single standed polyribonucleotides and its effect on the polymer conformation

The interactions between basic oligopeptides (Lys₂, Lys₃, Arg₂, and Arg₃) and single stranded polynucleotides (poly(A), poly(C), poly(I) and poly(U) were investigated at low ion concentration by UV spectroscopy, circular dichroism and field jump relaxation. Various domains of binding were detected: 1) High concentrations (up to 1 mM) of some peptides induce opalescencs followed by coacervation- Arg₃ causes coacervation in all polynucleotides used, yet Lys₃ only in poly(I). In the case of poly(I) the threshold concentration for coacervation is much lower for Arg₃ (150 μM) than for Lys₃ (500 μM). 2) Medium concentrations (?10 μM) of Arg₃ and Lys₃ induce helix formation in poly(U). In the case of poly(I) cooperative helix formation is only induced by Lys₃, but not by Arg₃. 3) The onset of peptide association is observed at very low peptide concentrations (?1 μM) already by using the field jump method. The association is reflected by a relaxation process, that can be described by a single exponential within experimental accuracy. Measurements of relaxation time constants as a function of the peptide concentration provide information on the association constants K, the number of nucleotide residues per binding place n and the rate constants k_R and k_D. Using a simple model with independent and “separate” binding sites, K for Arg₃ and Lys₃ is found to be in the range of 10⁶ to 10⁷ M^?1. In the case of Arg₂ and K is lower by a factor of about 10. For various polynucleotides K_Arg3 is slightly higher than K_Lys3. except in the case of poly(I), where K_Arg3/K_Lys3 ≈ 5. Similar data are obtained by application of a “sphere model” (see below). These results provide quantitative evidence for specific hydrogen bonding between the guanidino group of Arg and inosine. They also explain the absence of helix formation for poly(I) + Arg₃: Arg blocks the hydrogen bonding sites of inosine. Thus cooperative coupling leads in this case to a considerable amplification of specificity in the peptide-polynucleotide interaction Both field jump and stopped flow data demonstrate a high mobility of the peptide lisands along the polymer, resulting in a redistribution being fast compared with the overall binding step. Based on this result the relaxation data are analysed by a “sphere” model, which considers a) excluded binding under the condition of fast Ugand distribution along the lattice and b) the connection of sites into a polymer sphere. The rate constants obtained by this model are in the range of 4 × 10¹¹ M^?1 s^?1. These high values reflect the large reaction distance for polymers of chain lengths around 1000. A comparison with rate constants obtained previously for oligomer complexes indicates that the recombination rate is approximately a function of the square root of the nucleotide chain length, which is directly related to the mean radius of coiled polymers.     

Mammalian orthoreovirus escape from host translational shutoff correlates with stress granule disruption and is independent of eIF2alpha phosphorylation and PKR

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.     

A conserved basic residue cluster is essential for the protein quality control function of the Arabidopsis calreticulin 3

Calreticulin (CRT) is a highly conserved chaperone-like lectin that regulates Ca²⁺ homeostasis and participates in protein quality control in the endoplasmic reticulum (ER). Most of our CRT knowledge came from mammalian studies, but our understanding of plant CRTs is limited. Many plants contain more than two CRTs that form two distinct groups: CRT1/CRT2 and CRT3. Previous studies on plant CRTs were focused on their Ca²⁺-binding function, but recent studies revealed a crucial role for the Arabidopsis CRT3 in ER retention of a mutant brassinosteroid receptor, brassinosteroid-insensitive 1-9 (bri1-9) and in complete folding of a plant immunity receptor EF-Tu Receptor (EFR). However, little is known about the molecular basis of the functional specification of the CRTs. We have recently shown that the C-terminal domain of CRT3, which is rich in basic residues, is essential for retaining bri1-9 in the ER; however, its role in assisting EFR folding has not been studied. Here, we used an insertional mutant of CRT3, ebs2-8 (EMS mutagenized bri1 suppressor 2-8), in the bri1-9 background as a genetic system to investigate the functional importance of two basic residue clusters in the CRT3′s C-terminal domain. Complementation experiments of ebs2-8 bri1-9 with mutant CRT3^M transgenes showed that a highly conserved basic tetrapeptide Arg³⁹²Arg³⁹³Arg³⁹⁴Lys³⁹⁵ is essential but a less conserved basic tetrapeptide Arg⁴⁰¹Arg⁴⁰²Arg⁴⁰³Arg⁴⁰⁴ is dispensable for the quality control function of CRT3 that retains bri1-9 in the ER and facilitates the complete folding of EFR.     

Importance of eIF2alpha phosphorylation and stress granule assembly in alphavirus translation regulation

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 2alpha subunit (eIF2alpha). Infection of mouse embryo fibroblasts (MEFs) expressing a nonphosphorylatable mutant of eIF2alpha 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 eIF2alpha 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 eIF2alpha 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.     

Direct binding of the Alu binding protein dimer SRP9/14 to 40S ribosomal subunits promotes stress granule formation and is regulated by Alu RNA

Stress granules (SGs) are formed in response to stress, contain mRNAs, 40S ribosomal subunits, initiation factors, RNA-binding and signaling proteins, and promote cell survival. Our study describes a novel function of the protein heterodimer SRP9/14 and Alu RNA in SG formation and disassembly. In human cells, SRP9/14 exists assembled into SRP, bound to Alu RNA and as a free protein. SRP9/14, but not SRP, localizes to SGs following arsenite or hippuristanol treatment. Depletion of the protein decreases SG size and the number of SG-positive cells. Localization and function of SRP9/14 in SGs depend primarily on its ability to bind directly to the 40S subunit. Binding of SRP9/14 to 40S and Alu RNA is mutually exclusive indicating that the protein alone is bound to 40S in SGs and that Alu RNA might competitively regulate 40S binding. Indeed, by changing the effective Alu RNA concentration in the cell or by expressing an Alu RNA binding-defective protein we were able to influence SG formation and disassembly. Our findings suggest a model in which SRP9/14 binding to 40S promotes SG formation whereas the increase in cytoplasmic Alu RNA following stress promotes disassembly of SGs by disengaging SRP9/14 from 40S.     

All-atom models of the membrane-spanning domain of HIV-1 gp41 from metadynamics

The 27-residue membrane-spanning domain (MSD) of the HIV-1 glycoprotein gp41 bears conserved sequence elements crucial to the biological function of the virus, in particular a conserved GXXXG motif and a midspan arginine. However, structure-based explanations for the roles of these and other MSD features remain unclear. Using molecular dynamics and metadynamics calculations of an all-atom, explicit solvent, and membrane-anchored model, we study the conformational variability of the HIV-1 gp41 MSD. We find that the MSD peptide assumes a stable tilted α-helical conformation in the membrane. However, when the side chain of the midspan Arg ⁶⁹⁴ “snorkels” to the outer leaflet of the viral membrane, the MSD assumes a metastable conformation where the highly-conserved N-terminal core (between Lys⁶⁸¹ and Arg⁶⁹⁴ and containing the GXXXG motif) unfolds. In contrast, when the Arg⁶⁹⁴ side chain snorkels to the inner leaflet, the MSD peptide assumes a metastable conformation consistent with experimental observations where the peptide kinks at Phe⁶⁹⁷ to facilitate Arg⁶⁹⁴ snorkeling. Both of these models suggest specific ways that gp41 may destabilize viral membrane, priming the virus for fusion with a target cell.     

The Molecular Mechanism Underlying Recruitment and Insertion of Lipid-Anchored LC3 Protein into Membranes

Lipid modification of cytoplasmic proteins initiates membrane engagement that triggers diverse cellular processes. Despite the abundance of lipidated proteins in the human proteome, the key determinants underlying membrane recognition and insertion are poorly understood. Here, we define the course of spontaneous membrane insertion of LC3 protein modified with phosphatidylethanolamine using multiple coarse-grain simulations. The partitioning of the lipid anchor chains proceeds through a concerted process, with its two acyl chains inserting one after the other. Concurrently, a conformational rearrangement involving the α-helix III of LC3, especially in the three basic residues Lys⁶⁵, Arg⁶⁸, and Arg⁶⁹, ensures stable insertion of the phosphatidylethanolamine anchor into membranes. Mutational studies validate the crucial role of these residues, and further live-cell imaging analysis shows a substantial reduction in the formation of autophagic vesicles for the mutant proteins. Our study captures the process of water-favored LC3 protein recruitment to the membrane and thus opens, to our knowledge, new avenues to explore the cellular dynamics underlying vesicular trafficking.     

Expression of sarco/endoplasmic reticulum Ca ATPase (SERCA) 3 proteins in two major conformational states in native human cell membranes

The SERCA family includes 3 genes (SERCA1-3), each of which giving rise to various isoforms. To date, detailed structural data is only available for the SERCA1a isoform. Here, limited trypsinolysis of either human platelet membranes or recombinant SERCA3a in HEK-293 cells followed by Western blotting using antibodies covering different regions of the SERCA3(a) protein revealed two, kinetically distinct, Early (ETF) and Late (LTF) Tryptic Fragmentations. The ETF uses many tryptic sites while the LTF uses a unique tryptic site. Using site-directed mutagenesis: i) Arg³³⁴, Arg³⁹⁶ and Arg⁶³⁸ were directly assigned to the ETF and ii) Arg¹⁹⁸ was assigned as the only tryptic site to the LTF. Arg⁶⁷¹, Lys⁷¹²/Lys⁷¹³ and Lys⁷²⁸ were also found to modulate the ETF. SERCA inhibitors Tg and tBHQ induced modest inhibition of the ETF. In contrast, the addition of CaCl₂, EGTA or AlF₄⁻ strikingly modified the ETF without any effect on the LTF. Trypsinolysis of the other recombinant SERCA3b-3f isoforms revealed: i) same ETF and LTF as SERCA3a, with variations of the length of the C-terminal fragments; ii) Arg¹⁰⁰² as an additional tryptic site in SERCA3b-3e isoforms. Taken together, the two distinct SERCA3 fragmentation profiles sign the co-expression of SERCA3 proteins in two conformational states in cell membranes.     

Uncoupling Stress Granule Assembly and Translation Initiation Inhibition

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-tRNAi^Met 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.     

Biophysical Investigations of Complement Receptor 2 (CD21 and CR2)-Ligand Interactions Reveal Amino Acid Contacts Unique to Each Receptor-Ligand Pair

Human complement receptor type 2 (CR2 and CD21) is a cell membrane receptor, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B lymphocyte responses and bridges innate and acquired immunity. The most distally located SCRs, SCR1–2, mediate the interaction of CR2 with its four known ligands (C3d, EBV gp350, IFNα, and CD23). To ascertain specific interacting residues on CR2, we utilized NMR studies wherein gp350 and IFNα were titrated into ¹⁵N-labeled SCR1–2, and chemical shift changes indicative of specific inter-molecular interactions were identified. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1–2. With regard to gp350, the binding region of CR2 is primarily focused on SCR1 and the inter-SCR linker, specifically residues Asn¹¹, Arg¹³, Ala²², Arg²⁸, Ser³², Arg³⁶, Lys⁴¹, Lys⁵⁷, Tyr⁶⁴, Lys⁶⁷, Tyr⁶⁸, Arg⁸³, Gly⁸⁴, and Arg⁸⁹. With regard to IFNα, the binding is similar to the CR2-C3d interaction with specific residues being Arg¹³, Tyr¹⁶, Arg²⁸, Ser⁴², Lys⁴⁸, Lys⁵⁰, Tyr⁶⁸, Arg⁸³, Gly⁸⁴, and Arg⁸⁹. We also report thermodynamic properties of each ligand-receptor pair determined using isothermal titration calorimetry. The CR2-C3d interaction was characterized as a two-mode binding interaction with K_d values of 0.13 and 160 μm, whereas the CR2-gp350 and CR2-IFNα interactions were characterized as single site binding events with affinities of 0.014 and 0.035 μm, respectively. The compilation of chemical binding maps suggests specific residues on CR2 that are uniquely important in each of these three binding interactions.     

Critical roles of RNA helicase DDX3 and its interactions with eIF4E/PABP1 in stress granule assembly and stress response

Upon environmental insults, SGs (stress granules) aid cell survival by serving as sites of translational silencing. RNA helicase DDX3 was reported to associate with SGs. However, its role in SG physiology remains undefined. We have demonstrated previously that DDX3 acts as an eIF4E (eukaryotic initiation factor 4E)-inhibitory protein to suppress translation. In the present study, we indentified the SG marker PABP1 [poly(A)-binding protein 1] as another direct interaction partner of DDX3. We established various stimuli as novel stressors that direct DDX3 with eIF4E and PABP1 into SGs, but not to processing bodies. Interestingly, down-regulation of DDX3 interfered with SG assembly, led to nuclear accumulation of PABP1 and reduced cell viability following stress. Conversely, supplementation with a shRNA (short hairpin RNA)-resistant DDX3 restored SG formation, the translocation of PABP1 into SGs and cell survival. Notably, the SG-inducing capacity of DDX3 is independent of its ATPase and helicase activities, but mapped to the eIF4E-binding region. Moreover, the eIF4E-binding-defective mutant DDX3 was impaired in its SG-inducing ability and protective effect on cell survival under adverse conditions. All together, the present study has characterized DDX3 as a pivotal SG-nucleating factor and illustrates co-ordinative roles for DDX3, eIF4E and PABP1 in integrating environmental stress with translational regulation.