Clues to the function of bacterial microcompartments from ancillary genes.

Bacterial microcompartments (BMCs) are prokaryotic organelles. Their bounding membrane is a selectively permeable protein shell, encapsulating enzymes of specialized metabolic pathways. While the function of a BMC is dictated by the encapsulated enzymes which vary with the type of the BMC, the shell is formed by conserved protein building blocks. The genes necessary to form a BMC are typically organized in a locus; they encode the shell proteins, encapsulated enzymes as well as ancillary proteins that integrate the BMC function into the cell's metabolism. Among these are transcriptional regulators which usually found at the beginning or end of a locus, and transmembrane proteins that presumably function to conduct the BMC substrate into the cell. Here, we describe the types of transcriptional regulators and permeases found in association with BMC loci, using a recently collected data set of more than 7000 BMC loci distributed over 45 bacterial phyla, including newly discovered BMC loci. We summarize the known BMC regulation mechanisms, and highlight how much remains to be uncovered. We also show how analysis of these ancillary proteins can inform hypotheses about BMC function; by examining the ligand-binding domain of the regulator and the transporter, we propose that nucleotides are the likely substrate for an enigmatic uncharacterized BMC of unknown function.

Hyper-truncated Asn355- and Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase.

Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remains elusive. To this end, we have characterised the structure-biosynthesis-activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants and a previously unreported low-abundance monoprotomer. Longitudinal profiling of maturing, mature, granule-separated and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, molecular modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity.

Extracellular adenosine enhances the ability of PMNs to kill Streptococcus pneumoniae by inhibiting IL-10 production.

Polymorphonuclear leukocytes (PMNs) are crucial for initial control of Streptococcus pneumoniae (pneumococcus) lung infection; however, as the infection progresses their persistence in the lungs becomes detrimental. Here we explored why the antimicrobial efficacy of PMNs declines over the course of infection. We found that the progressive inability of PMNs to control infection correlated with phenotypic differences characterized by a decrease in CD73 expression, an enzyme required for production of extracellular adenosine (EAD). EAD production by CD73 was crucial for the ability of both murine and human PMNs to kill S. pneumoniae. In exploring the mechanisms by which CD73 controlled PMN function, we found that CD73 mediated its antimicrobial activity by inhibiting IL-10 production. PMNs from wild-type mice did not increase IL-10 production in response to S. pneumoniae; however, CD73-/- PMNs up-regulated IL-10 production upon pneumococcal infection in vitro and during lung challenge. IL-10 inhibited the ability of WT PMNs to kill pneumococci. Conversely, blocking IL-10 boosted the bactericidal activity of CD73-/- PMNs as well as host resistance of CD73-/- mice to pneumococcal pneumonia. CD73/IL-10 did not affect apoptosis, bacterial uptake, and intracellular killing or production of antimicrobial neutrophil elastase and myeloperoxidase. Rather, inhibition of IL-10 production by CD73 was important for optimal reactive oxygen species (ROS) production by PMNs. ROS contributed to PMN antimicrobial function as their removal or detoxification impaired the ability of PMNs to efficiently kill S. pneumoniae. This study demonstrates that CD73 controls PMN antimicrobial phenotype during S. pneumoniae infection.

Localization of BCR-ABL to Stress Granules Contributes to Its Oncogenic Function.

The oncogenic tyrosine kinase BCR-ABL activates a variety of signaling pathways and plays a causative role in the pathogenesis of chronic myelogenous leukemia (CML); however, the subcellular distribution of this chimeric protein remains controversial. Here, we report that BCR-ABL is localized to stress granules and that its granular localization contributes to BCR-ABL-dependent leukemogenesis. BCR-ABL-positive granules were not colocalized with any markers for membrane-bound organelles but were colocalized with HSP90a, a component of RNA granules. The number of such granules increased with thapsigargin treatment, confirming that the granules were stress granules. Given that treatment with the ABL kinase inhibitor imatinib and elimination of the N-terminal region of BCR-ABL abolished granule formation, kinase activity and the coiled-coil domain are required for granule formation. Whereas wild-type BCR-ABL rescued the growth defect in IL-3-depleted Ba/F3 cells, mutant BCR-ABL lacking the N-terminal region failed to do so. Moreover, forced tetramerization of the N-terminus-deleted mutant could not restore the growth defect, indicating that granule formation, but not tetramerization, through its N-terminus is critical for BCR-ABL-dependent oncogenicity. Our findings together provide new insights into the pathogenesis of CML by BCR-ABL and open a window for developing novel therapeutic strategies for this disease.Key words: BCR-ABL, subcellular localization, stress granule.

Enterovirus 71 inhibits cytoplasmic stress granule formation during the late stage of infection.

Stress granules (SGs) are host translationally silent ribonucleo-proteins formed in cells in response to multiple types of environmental stress, including viral infection. We previously showed that the nuclear protein, 68-kDa Src-associated in mitosis protein (Sam68), is recruited to cytoplasm and form the Sam68-positive SGs at 6 hpi, but the Sam68-positive SGs disassembled beyond 12 hpi, suggesting that the SGs might be inhibited during the late stage of Enterovirus 71 (EV71) infection. However, the mechanism and function of this process remains poorly understood. Thus in this study, we demonstrated that EV71 initially induced SGs formation at the early stage of EV71 infection, and confirmed that 2Apro of EV71 was the key viral component that triggered SG formation. In contrast, SGs were diminished as EV71 infection proceeding. At the same time, arsenite-induced SGs were also blocked at the late stage of EV71 infection. This disruption of SGs was caused by viral protease 3Cpro-mediated G3BP1 cleavage. Furthermore, we demonstrated that over-expression of G3BP1-SGs negatively impacted viral replication at the cytopathic effect (CPE), protein, RNA, and viral titer levels. Our novel finding may not only help us to better understand the mechanism how EV71 interacts with the SG response, but also provide mechanistic linkage between cellular stress responses and innate immune activation during EV71 infection.

ZFAND1 Recruits p97 and the 26S Proteasome to Promote the Clearance of Arsenite-Induced Stress Granules.

Stress granules (SGs) are cytoplasmic assemblies of mRNPs stalled in translation initiation. They are induced by various stress conditions, including exposure to the environmental toxin and carcinogen arsenic. While perturbed SG turnover is linked to the pathogenesis of neurodegenerative diseases, the molecular mechanisms underlying SG formation and turnover are still poorly understood. Here, we show that ZFAND1 is an evolutionarily conserved regulator of SG clearance. ZFAND1 interacts with two key factors of protein degradation, the 26S proteasome and the ubiquitin-selective segregase p97, and recruits them to arsenite-induced SGs. In the absence of ZFAND1, SGs lack the 26S proteasome and p97, accumulate defective ribosomal products, and persist after arsenite removal, indicating their transformation into aberrant, disease-linked SGs. Accordingly, ZFAND1 depletion is epistatic to the expression of pathogenic mutant p97 with respect to SG clearance, suggesting that ZFAND1 function is relevant to the multisystem degenerative disorder IBMPFD/ALS.

Insights into the role of endonuclease V in RNA metabolism in Trypanosoma brucei.

Inosine may arise in DNA as a result of oxidative deamination of adenine or misincorporation of deoxyinosine triphosphate during replication. On the other hand, the occurrence of inosine in RNA is considered a normal and essential modification induced by specific adenosine deaminases acting on mRNA and tRNA. In prokaryotes, endonuclease V (EndoV) can recognize and cleave inosine-containing DNA. In contrast, mammalian EndoVs preferentially cleave inosine-containing RNA, suggesting a role in RNA metabolism for the eukaryotic members of this protein family. We have performed a biochemical characterization of EndoV from the protozoan parasite Trypanosoma brucei. In vitro, TbEndoV efficiently processes single-stranded RNA oligonucleotides with inosine, including A to I-edited tRNA-like substrates but exhibits weak activity over DNA, except when a ribonucleotide is placed 3' to the inosine. Immunolocalization studies performed in procyclic forms indicate that TbEndoV is mainly cytosolic yet upon nutritional stress it redistributes and accumulates in stress granules colocalizing with the DEAD-box helicase TbDhh1. RNAi-mediated depletion of TbEndoV results in moderate growth defects in procyclic cells while the two EndoV alleles could be readily knocked out in bloodstream forms. Taken together, these observations suggest an important role of TbEndoV in RNA metabolism in procyclic forms of the parasite.

Role of granule proteases in the life and death of neutrophils.

Neutrophils constitute a crucial component of the innate immune defenses against microbes. Produced in the bone marrow and patrolling in blood vessels, neutrophils are recruited to injured tissues and are immediately active to contain pathogen invasion. Neutrophils undergo programmed cell death by multiple, context-specific pathways, which have consequences on immunopathology and disease outcome. Studies in the last decade indicate additional functions for neutrophils - or a subset of neutrophils - in modulating adaptive responses and tumor progression. Neutrophil granules contain abundant amounts of various proteases, which are directly implicated in protective and pathogenic functions of neutrophils. It now emerges that neutral serine proteases such as cathepsin G and proteinase-3 also contribute to the neutrophil life cycle, but do so via different pathways than that of the aspartate protease cathepsin D and that of mutants of the serine protease elastase. The aim of this review is to appraise the present knowledge of the function of neutrophil granule proteases and their inhibitors in neutrophil cell death, and to integrate these findings in the current understandings of neutrophil life cycle and programmed cell death pathways.

Regulation of Human Endonuclease V Activity and Relocalization to Cytoplasmic Stress Granules.

Endonuclease V (EndoV) is an enzyme with specificity for inosines in nucleic acids. Whereas the bacterial homologs are active on both DNA and RNA, the mammalian variants only cleave RNA, at least when assayed with recombinant proteins. Here we show that ectopically expressed, as well as endogenously expressed human (h)EndoV, share the same enzymatic properties as the recombinant protein and cleaves RNA with inosine but not DNA. In search for proteins interacting with hEndoV, polyadenylate-binding protein C1 (PABPC1) was identified. The association between PABPC1 and hEndoV is RNA dependent and furthermore, PABPC1 stimulates hEndoV activity and affinity for inosine-containing RNA. Upon cellular stress, PABPC1 relocates to cytoplasmic stress granules that are multimolecular aggregates of stalled translation initiation complexes formed to aid cell recovery. Arsenite, as well as other agents, triggered relocalization also of hEndoV to cytoplasmic stress granules. As inosines in RNA are highly abundant, hEndoV activity is likely regulated in cells to avoid aberrant cleavage of inosine-containing transcripts. Indeed, we find that hEndoV cleavage is inhibited by normal intracellular ATP concentrations. The ATP stores inside a cell do not overlay stress granules and we suggest that hEndoV is redistributed to stress granules as a strategy to create a local environment low in ATP to permit hEndoV activity.

G3BP-Caprin1-USP10 complexes mediate stress granule condensation and associate with 40S subunits.

Mammalian stress granules (SGs) contain stalled translation preinitiation complexes that are assembled into discrete granules by specific RNA-binding proteins such as G3BP. We now show that cells lacking both G3BP1 and G3BP2 cannot form SGs in response to eukaryotic initiation factor 2α phosphorylation or eIF4A inhibition, but are still SG-competent when challenged with severe heat or osmotic stress. Rescue experiments using G3BP1 mutants show that phosphomimetic G3BP1-S149E fails to rescue SG formation, whereas G3BP1-F33W, a mutant unable to bind G3BP partner proteins Caprin1 or USP10, rescues SG formation. Caprin1/USP10 binding to G3BP is mutually exclusive: Caprin binding promotes, but USP10 binding inhibits, SG formation. G3BP interacts with 40S ribosomal subunits through its RGG motif, which is also required for G3BP-mediated SG formation. We propose that G3BP mediates the condensation of SGs by shifting between two different states that are controlled by the phosphorylation of S149 and by binding to Caprin1 or USP10.

Granule Associated Serine Proteases of Hematopoietic Cells - An Analysis of Their Appearance and Diversification during Vertebrate Evolution.

Serine proteases are among the most abundant granule constituents of several hematopoietic cell lineages including mast cells, neutrophils, cytotoxic T cells and NK cells. These proteases are stored in their active form in the cytoplasmic granules and in mammals are encoded from four different chromosomal loci: the chymase locus, the met-ase locus, the T cell tryptase and the mast cell tryptase locus. In order to study their appearance during vertebrate evolution we have performed a bioinformatic analysis of related genes and gene loci from a large panel of metazoan animals from sea urchins to placental mammals for three of these loci: the chymase, met-ase and granzyme A/K loci. Genes related to mammalian granzymes A and K were the most well conserved and could be traced as far back to cartilaginous fish. Here, the granzyme A and K genes were found in essentially the same chromosomal location from sharks to humans. However in sharks, no genes clearly identifiable as members of the chymase or met-ase loci were found. A selection of these genes seemed to appear with bony fish, but sometimes in other loci. Genes related to mammalian met-ase locus genes were found in bony fish. Here, the most well conserved member was complement factor D. However, genes distantly related to the neutrophil proteases were also identified in this locus in several bony fish species, indicating that this locus is also old and appeared at the base of bony fish. In fish, a few of the chymase locus-related genes were found in a locus with bordering genes other than the mammalian chymase locus and some were found in the fish met-ase locus. This indicates that a convergent evolution rather than divergent evolution has resulted in chymase locus-related genes in bony fish.

Syk Is Recruited to Stress Granules and Promotes Their Clearance through Autophagy.

Syk is a cytoplasmic kinase that serves multiple functions within the immune system to couple receptors for antigens and antigen-antibody complexes to adaptive and innate immune responses. Recent studies have identified additional roles for the kinase in cancer cells, where its expression can either promote or suppress tumor cell growth, depending on the context. Proteomic analyses of Syk-binding proteins identified several interacting partners also found to be recruited to stress granules. We show here that the treatment of cells with inducers of stress granule formation leads to the recruitment of Syk to these protein-RNA complexes. This recruitment requires the phosphorylation of Syk on tyrosine and results in the phosphorylation of proteins at or near the stress granule. Grb7 is identified as a Syk-binding protein involved in the recruitment of Syk to the stress granule. This recruitment promotes the formation of autophagosomes and the clearance of stress granules from the cell once the stress is relieved, enhancing the ability of cells to survive the stress stimulus.

Impact of the serum- and glucocorticoid-inducible kinase 1 on platelet dense granule biogenesis and secretion.

BACKGROUND: Platelet secretion is critical to development of acute thrombotic occlusion. Platelet dense granules contain a variety of important hemostatically active substances. Nevertheless, biogenesis of platelet granules is poorly understood. OBJECTIVES: Serum- and glucocorticoid-inducible kinase 1 (SGK1) has been shown to be highly expressed in platelets and megakaryocytes, but its role in the regulation of platelet granule biogenesis and its impact on thrombosis has not been investigated so far. METHODS AND RESULTS: Electron microscopy analysis of the platelet ultrastructure revealed a significant reduction in the number and packing of dense granules in platelets lacking SGK1 (sgk1(-/-) ). In sgk1(-/-) platelets serotonin content was significantly reduced and activation-dependent secretion of ATP, serotonin and CD63 significantly impaired. In vivo adhesion after carotis ligation was significantly decreased in platelets lacking SGK1 and occlusive thrombus formation after FeCl3 -induced vascular injury was significantly diminished in sgk1(-/-) mice. Transcript levels and protein abundance of dense granule biogenesis regulating GTPase Rab27b were significantly reduced in sgk1(-/-) platelets without affecting Rab27b mRNA stability. In MEG-01 cells transfection with constitutively active (S422) (D) SGK1 but not with inactive (K127) (N) SGK1 significantly enhanced Rab27b mRNA levels. Sgk1(-/-) megakaryocytes show significantly reduced expression of Rab27b and serotonin/CD63 levels compared with sgk1(+/+) megakaryocytes. Proteome analysis identified nine further vesicular transport proteins regulated by SGK1, which may have an impact on impaired platelet granule biogenesis in sgk1(-/-) platelets independent of Rab27b. CONCLUSIONS: The present observations identify SGK1 as a novel powerful regulator of platelet dense granule biogenesis, platelet secretion and thrombus formation. SGK1 is at least partially effective because it regulates transcription of Rab27b in megakaryocytes.

The histone deacetylase sirtuin 2 is a new player in the regulation of platelet function.

BACKGROUND: Histone deacetylases (HDACs) play a key role in signaling in many cell types. However, little is known about the participation of HDACs, particularly sirtuins (SIRTs), in platelet reactivity. OBJECTIVE: To investigate the role of HDACs in platelets, we examined the effects of SIRT inhibition on platelet function and protein acetylation in human platelets. METHODS: We used washed platelets obtained from healthy subjects. Cambinol (SIRT1 and SIRT2 inhibitor), AGK2 (specific SIRT2 inhibitor) and EX527 (specific SIRT1 inhibitor) were used as SIRT inhibitors. Platelets were stimulated with collagen, thrombin, or U46619, and platelet responses were determined according to optical aggregometry findings, dense granule release, and cytosolic calcium levels (Fura-2AM fluorescence). Protein acetylation and phosphorylation were assessed by immunoblotting. RESULTS: SIRT inhibition remarkably reduced platelet responses (aggregation, granule release, and cytosolic calcium level; P < 0.05). SIRT2 was present in platelets at the level of mRNA and protein, and its specific inhibition reduced platelet responses. The acetylated protein pattern observed in resting platelets changed during platelet aggregation. Inhibition of SIRT2 increased the acetylation of Akt kinase, which in turn blocked agonist-induced Akt phosphorylation and glycogen synthase kinase-3ß phosphorylation, which are markers of Akt activity. Finally, collagen-induced aggregation provoked Akt acetylation. CONCLUSIONS: Regulation of protein acetylation by SIRT2 plays a central role in platelet function. The effects of SIRT2 are mediated in part by the acetylation and inhibition of Akt. These results open a new avenue for research into the control of platelet function, and may help to identify new therapeutic targets.

Tryptase, a novel angiogenic factor stored in mast cell granules.

Human mast cells (MCs) are a rich reservoir of neutral proteases, packed in large amounts in their granules and comprising a high fraction of all cellular proteins. Among these proteases, tryptase is involved in angiogenesis after its release from activated MC granules, as it has been demonstrated in different in vitro and in vivo assays. Moreover, tryptase-positive MCs increase in number and vascularization increases in a linear fashion in different solid and hematological tumors. This complex interplay between MCs and tumor angiogenesis have led to consider the therapeutic use of angiogenesis inhibitors, which specifically target the angiogenic activity of tryptase, such as gabexate mesilate and nafamostat mesilate, two inhibitors of trypsin-like serine proteases.

Loss of PIKfyve in platelets causes a lysosomal disease leading to inflammation and thrombosis in mice.

PIKfyve is essential for the synthesis of phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] and for the regulation of endolysosomal membrane dynamics in mammals. PtdIns(3,5)P2 deficiency causes neurodegeneration in mice and humans, but the role of PtdIns(3,5)P2 in non-neural tissues is poorly understood. Here we show that platelet-specific ablation of PIKfyve in mice leads to accelerated arterial thrombosis, and, unexpectedly, also to inappropriate inflammatory responses characterized by macrophage accumulation in multiple tissues. These multiorgan defects are attenuated by platelet depletion in vivo, confirming that they reflect a platelet-specific process. PIKfyve ablation in platelets induces defective maturation and excessive storage of lysosomal enzymes that are released upon platelet activation. Impairing lysosome secretion from PIKfyve-null platelets in vivo markedly attenuates the multiorgan defects, suggesting that platelet lysosome secretion contributes to pathogenesis. Our findings identify PIKfyve as an essential regulator for platelet lysosome homeostasis, and demonstrate the contributions of platelet lysosomes to inflammation, arterial thrombosis and macrophage biology.

Granule proteases of hematopoietic cells, a family of versatile inflammatory mediators - an update on their cleavage specificity, in vivo substrates, and evolution.

Cells from several of the hematopoietic cell lineages including mast cells, basophils, neutrophils, cytotoxic T cells, and natural killer (NK) cells store proteases at very high levels within their cytoplasmic granules. In mast cells, these proteases can account for up to 35% of the total cellular protein, and the absolute majority of these belong to the chymotrypsin-related serine protease family. A number of very diverse functions have been identified for these proteases, including apoptosis induction, blood pressure regulation, inactivation of insect and snake toxins, intestinal parasite expulsion, killing of bacteria and fungi, induction, mobilization, or degradation of cytokines, and the degradation of connective tissue components. A very broad spectrum of primary cleavage specificities has also been observed, including chymase, tryptase, asp-ase, elastase, and met-ase specificities, which highlights the large flexibility in the active site of these proteases. Mast cells primarily express chymases and tryptases with chymotryptic or tryptic primary cleavage specificities, respectively. Neutrophils have several enzymes with chymase, elastase, and tryptase specificities. T cells and NK cells express between 5 and 14 different granzymes, depending on the species, and these enzymes have tryptase, asp-ase, chymase, and met-ase specificities. This review focuses on the appearance of these proteases during vertebrate evolution, their primary and extended cleavage specificities, and their potential in vivo substrates. The in vivo substrates and functions are a particular challenging issue because several of these enzymes have a relatively broad specificity and may therefore cleave a wide range of different substrates.

Proteins that contain a functional Z-DNA-binding domain localize to cytoplasmic stress granules.

Long double-stranded RNA may undergo hyper-editing by adenosine deaminases that act on RNA (ADARs), where up to 50% of adenosine residues may be converted to inosine. However, although numerous RNAs may undergo hyper-editing, the role for inosine-containing hyper-edited double-stranded RNA in cells is poorly understood. Nevertheless, editing plays a critical role in mammalian cells, as highlighted by the analysis of ADAR-null mutants. In particular, the long form of ADAR1 (ADAR1(p150)) is essential for viability. Moreover, a number of studies have implicated ADAR1(p150) in various stress pathways. We have previously shown that ADAR1(p150) localized to cytoplasmic stress granules in HeLa cells following either oxidative or interferon-induced stress. Here, we show that the Z-DNA-binding domain (Zα(ADAR1)) exclusively found in ADAR1(p150) is required for its localization to stress granules. Moreover, we show that fusion of Zα(ADAR1) to either green fluorescent protein (GFP) or polypyrimidine binding protein 4 (PTB4) also results in their localization to stress granules. We additionally show that the Zα domain from other Z-DNA-binding proteins (ZBP1, E3L) is likewise sufficient for localization to stress granules. Finally, we show that Z-RNA or Z-DNA binding is important for stress granule localization. We have thus identified a novel role for Z-DNA-binding domains in mammalian cells.

MDA5 localizes to stress granules, but this localization is not required for the induction of type I interferon.

Virus infection can initiate a type I interferon (IFN-α/ß) response via activation of the cytosolic RNA sensors retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). Furthermore, it can activate kinases that phosphorylate eukaryotic translation initiation factor 2α (eIF2α), which leads to inhibition of (viral) protein translation and formation of stress granules (SG). Most viruses have evolved mechanisms to suppress these cellular responses. Here, we show that a mutant mengovirus expressing an inactive leader (L) protein, which we have previously shown to be unable to suppress IFN-α/ß, triggered SG formation in a protein kinase R (PKR)-dependent manner. Furthermore, we show that infection of cells that are defective in SG formation yielded higher viral RNA levels, suggesting that SG formation acts as an antiviral defense mechanism. Since the induction of both IFN-α/ß and SG is suppressed by mengovirus L, we set out to investigate a potential link between these pathways. We observed that MDA5, the intracellular RNA sensor that recognizes picornaviruses, localized to SG. However, activation of the MDA5 signaling pathway did not trigger and was not required for SG formation. Moreover, cells that were unable to form SG-by protein kinase R (PKR) depletion, using cells expressing a nonphosphorylatable eIF2α protein, or by drug treatment that inhibits SG formation-displayed a normal IFN-α/ß response. Thus, although MDA5 localizes to SG, this localization seems to be dispensable for induction of the IFN-α/ß pathway.