Tau protein plays an important role in the biology of stress granules and in the stress response of neurons, but the nature of these biochemical interactions is not known. Here we show that the interaction of tau with RNA and the RNA binding protein TIA1 is sufficient to drive phase separation of tau at physiological concentrations, without the requirement for artificial crowding agents such as polyethylene glycol (PEG). We further show that phase separation of tau in the presence of RNA and TIA1 generates abundant tau oligomers. Prior studies indicate that recombinant tau readily forms oligomers and fibrils in vitro in the presence of polyanionic agents, including RNA, but the resulting tau aggregates are not particularly toxic. We discover that tau oligomers generated during copartitioning with TIA1 are significantly more toxic than tau aggregates generated by incubation with RNA alone or phase-separated tau complexes generated by incubation with artificial crowding agents. This pathway identifies a potentially important source for generation of toxic tau oligomers in tau-related neurodegenerative diseases. Our results also reveal a general principle that phase-separated RBP droplets provide a vehicle for coassortment of selected proteins. Tau selectively copartitions with TIA1 under physiological conditions, emphasizing the importance of TIA1 for tau biology. Other RBPs, such as G3BP1, are able to copartition with tau, but this happens only in the presence of crowding agents. This type of selective mixing might provide a basis through which membraneless organelles bring together functionally relevant proteins to promote particular biological activities.

Keywords: Alzheimer's disease; RNA binding proteins; fibrillar tau; liquid–liquid phase separation (LLPS); oligomeric tau.

G3BPs are members of an RNA-binding protein family and their aberrant expression is common in various cancers and there is growing evidence that G3BPs possess antiviral activities and are targeted by various viruses. G3BPs have also been implicated in both stabilization and degradation of specific mRNAs as well as translational control of mRNA targets. G3BPs have been shown to control translation of interferon-stimulated genes (ISGs), implying that G3BPs are involved in the regulation of the interferon system in response to viral infections and/or cellular stress. The interferon induced transmembrane (IFITM1, IFITM2 and IFITM3) proteins are antiviral proteins, and are also involved in cancer progression and metastasis. Therefore, these genes were selected in the studies reported here as potential transcript targets of G3BPs. Furthermore, G3BPs are involved in the regulation of the MEK pathway which also impacts on the translation of ISGs. Therefore, the role of this pathway was also analysed in regulation of IFITM1-3 proteins. Overall, this research study suggests that G3BPs are essential for the accumulation of IFITM1-3 proteins and intersect twice in the regulation of IFITM1-3 expression, first through MEK pathway and then through an interaction with the 3'-UTRs of its target transcripts. However, it is still to be determined whether the two apparent functions are part of a single control mechanism or the two functions are mutually exclusive.

RNA-protein interactions play a pivotal role in the function of picornavirus internal ribosome entry site (IRES) elements. Here we analysed the impact of Ras GTPase SH3 domain binding protein 1 (G3BP1) in the IRES activity of foot-and-mouth disease virus (FMDV). We found that G3BP1 interacts directly with three distinct sequences of the IRES element using RNA electrophoretic mobility-shift assays. Analysis of the interaction with domain 5 indicated that the G3BP1 binding-site is placed at the single-stranded region although it allows large sequence heterogeneity and the hairpin located upstream of this region enhances retarded complex formation. In addition, G3BP1 interacts directly with the polypyrimidine tract-binding protein and the translation initiation factor 4B (eIF4B) through the C-terminal region. Moreover, G3BP1 is cleaved during FMDV infection yielding two fragments, Ct-G3BP1 and Nt-G3BP1. Both fragments inhibit cap- and IRES-dependent translation, but the Ct-G3BP1 fragment shows a stronger effect on IRES-dependent translation. Assembly of complexes with G3BP1 results in a significantly reduced local flexibility of the IRES element, consistent with the negative effect of this protein. Our results highlight the IRES-binding capacity of G3BP1 and illustrate its function as a translation inhibitor.

Foot-and-mouth disease virus (FMDV) is one of the most notorious pathogens in the global livestock industry. To establish an infection, FMDV needs to counteract host antiviral responses. Several studies have shown how FMDV suppresses the type I interferon (IFN) response; however, whether FMDV modulates the integrated autophagy and innate immunity remains largely unknown. Here, the porcine Ras-GAP SH3-binding protein 1 (G3BP1) was shown to promote the RIG-I-like helicases (RLH) signaling by upregulating expression of RIG-I and MDA5. The FMDV 3A interacted with G3BP1 to inhibit G3BP1 expression and G3BP1-mediated RLH signaling by upregulating the expression of autophagy-related protein LRRC25. In addition, 3A proteins of other picornaviruses, including SVV 3A, EV71 3A, and EMCV 3A, also showed similar actions. Taken together, we elucidated for the first time, a novel mechanism by which FMDV has evolved to inhibit IFN signaling and counteract host innate antiviral responses by autophagy.Importance We show that FMDV 3A inhibits RIG-I-like helicases signaling by degrading G3BP1 protein. Furthermore, FMDV 3A reduces G3BP1 by upregulating expression of autophagy-related protein LRRC25. Additionally, other picornavirus 3A, such as SVV 3A, EV71 3A, and EMCV 3A, also degrade G3BP1 by upregulating LRRC25 expression. This study will help to improve the design of current vaccines and aid the development of novel control strategies to combat FMD.

Foot-and-mouth disease (FMD) is a highly contagious, severe viral illness notifiable to the World Organization for Animal Health. The causative agent, FMD virus (FMDV), replicates rapidly and efficiently inhibits host translation and the innate immune response for it has developed multiple tactics to evade host defenses and takes over gene expression machinery in the host cell. Here, we report a systemic analysis of the proteome and phosphoproteome of FMDV-infected cells. Bioinformatics analysis suggested that FMDV infection shuts off host cap-dependent translation, but leaves intact internal ribosome entry site (IRES)-mediated translation for viral proteins. Interestingly, several FMDV IRES-transacting factors, including G3BP stress granule assembly factor 1 (G3BP1), were dephosphorylated during FMDV infection. Ectopic expression of G3BP1 inhibited FMDV IRES activity, promoted assembly of stress granules, and activated innate immune responses, collectively suppressing FMDV replication. To counteract these host protective responses, FMDV-induced dephosphorylation of G3BP1, compromising its inhibitory effect on viral IRES. In addition, FMDV also proteolytically cleaved G3BP1 by its 3C protease (3Cpro). G3BP1 was cleaved at glutamic acid-284 (E284) by FMDV 3Cpro, and this cleavage completely lost the abilities of G3BP1 to activate innate immunity and to inhibit FMDV replication. Together, these data provide new insights into the post-translational mechanisms by which FMDV limits host stress and antiviral responses and indicate that G3BP1 dephosphorylation and its proteolysis by viral protease are important factors in the failure of host defense against FMDV infection.

Tourrière et al. (2013. J. Cell Biol. https://doi.org/10.1083/jcb.200212128) reported that G3BP1-S149 dephosphorylation promotes stress granule formation. We show that constructs used to establish this conclusion contain additional mutations causing these phenotypes, and that S149 phosphorylation status does not change upon stress.

Stress granules (SGs) are dynamic cytoplasmic aggregates containing mRNA, RNA-binding proteins, and translation factors that form in response to cellular stress. SGs have been shown to contribute to the pathogenesis of several human diseases, but their role in vascular diseases is unknown. This study shows that SGs accumulate in vascular smooth muscle cells (VSMCs) and macrophages during atherosclerosis. Approach and Results: Immunohistochemical analysis of atherosclerotic plaques from LDLR^-/- mice revealed an increase in the stress granule-specific markers Ras-G3BP1 (GTPase-activating protein SH3 domain-binding protein) and PABP (poly-A-binding protein) in intimal macrophages and smooth muscle cells that correlated with disease progression. In vitro, PABP+ and G3BP1+ SGs were rapidly induced in VSMC and bone marrow-derived macrophages in response to atherosclerotic stimuli, including oxidized low-density lipoprotein and mediators of mitochondrial or oxidative stress. We observed an increase in eIF2α (eukaryotic translation initiation factor 2-alpha) phosphorylation, a requisite for stress granule formation, in cells exposed to these stimuli. Interestingly, SG formation, PABP expression, and eIF2α phosphorylation in VSMCs is reversed by treatment with the anti-inflammatory cytokine interleukin-19. Microtubule inhibitors reduced stress granule accumulation in VSMC, suggesting cytoskeletal regulation of stress granule formation. SG formation in VSMCs was also observed in other vascular disease pathologies, including vascular restenosis. Reduction of SG component G3BP1 by siRNA significantly altered expression profiles of inflammatory, apoptotic, and proliferative genes.

These results indicate that SG formation is a common feature of the vascular response to injury and disease, and that modification of inflammation reduces stress granule formation in VSMC.

Motile ciliopathies are characterized by specific defects in cilia beating that result in chronic airway disease, subfertility, ectopic pregnancy, and hydrocephalus. While many patients harbor mutations in the dynein motors that drive cilia beating, the disease also results from mutations in so-called dynein axonemal assembly factors (DNAAFs) that act in the cytoplasm. The mechanisms of DNAAF action remain poorly defined. Here, we show that DNAAFs concentrate together with axonemal dyneins and chaperones into organelles that form specifically in multiciliated cells, which we term DynAPs, for dynein axonemal particles. These organelles display hallmarks of biomolecular condensates, and remarkably, DynAPs are enriched for the stress granule protein G3bp1, but not for other stress granule proteins or P-body proteins. Finally, we show that both the formation and the liquid-like behaviors of DynAPs are disrupted in a model of motile ciliopathy. These findings provide a unifying cell biological framework for a poorly understood class of human disease genes and add motile ciliopathy to the growing roster of human diseases associated with disrupted biological phase separation.

The adhesion blocking antibody 3S3 was used to probe the regulation of alpha5beta1 integrin mediated adhesion in K562 cells. This antibody prevented cellular adherence but it did not interfere with ligand binding by cells or purified integrin. Interaction with 3S3 induced change in the cytoskeletal organization resulting in extensive filopodia formation. The antibody also prevented ligand and anti-integrin antibody induced phosphorylation of FAK in a trans acting fashion. MS based analysis of 3S3 induced integrin containing complexes identified rasGAP SH3 binding protein 1, G3BP1, as a component of these structures. The G3BP1 binding molecule, rasGap120, was also identified in the complexes. Microscopic examination confirmed the recruitment of a component of cellular G3BP1 and rasGap120 pools to sites of integrin cross-linking. G3BP1 was also observed in the 3S3 induced filopodia. In untreated cells, G3BP1 was shown to associate with submembranous regions involved in cellular polarization. Collectively, these results suggest that G3BP1 and rasGap120 can be recruited to sites of integrin ligation where they may play a role in cytoskeletal reorganization. Such changes may result in reduced adhesive potential and account for the 3S3 effects on cellular adhesion. It should be emphasized that these results do not necessarily indicate a direct interaction of integrin with G3BP1 and rasGap120.

It is well established that promyelocytic leukaemia nuclear bodies (PML NBs) play important roles in DNA damage responses (DDR). After irradiation, PML NBs dynamically recruit or release important proteins involved in cell-cycle regulation, DNA repair and apoptosis. As PML protein is the key molecule of PML NBs' dynamic assembling, we aimed to characterize the PML-interacting proteins in (60)Co-irradiated MCF-7 cells. A proteomic approach using CoIP, mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of 124 proteins that may associate with PML after irradiation. Bioinformatic analysis of the identified proteins showed that most of them were related to characterized PML functions, such as transcriptional regulation, cell-cycle regulation, cell-death regulation and response to stress. Four proteins, B23, MVP, G3BP1 and DHX9, were verified to co-localize with PML differentially before and after ionizing radiation (IR) treatment. The proteins identified in this study will significantly improve our understanding of the dynamic organization and multiple functions of PML NBs in DDR.

The RIG-I-like receptor (RLR) pathway is essential for detecting cytosolic viral RNA to trigger the production of type I interferons (IFNα/β) that initiate an innate antiviral response. Through systematic assessment of a wide variety of genomics data, we discovered 10 molecular signatures of known RLR pathway components that collectively predict novel members. We demonstrate that RLR pathway genes, among others, tend to evolve rapidly, interact with viral proteins, contain a limited set of protein domains, are regulated by specific transcription factors, and form a tightly connected interaction network. Using a Bayesian approach to integrate these signatures, we propose likely novel RLR regulators. RNAi knockdown experiments revealed a high prediction accuracy, identifying 94 genes among 187 candidates tested (~50%) that affected viral RNA-induced production of IFNβ. The discovered antiviral regulators may participate in a wide range of processes that highlight the complexity of antiviral defense (e.g. MAP3K11, CDK11B, PSMA3, TRIM14, HSPA9B, CDC37, NUP98, G3BP1), and include uncharacterized factors (DDX17, C6orf58, C16orf57, PKN2, SNW1). Our validated RLR pathway list (http://rlr.cmbi.umcn.nl/), obtained using a combination of integrative genomics and experiments, is a new resource for innate antiviral immunity research.

BACKGROUND

Current therapies to treat cancer, although successful for some patients, have significant side-effects and a high number of patients have disease that is either non-responsive or which develops resistance. Our goal was to design a small peptide that possesses similar functions to an antibody drug conjugate with regard to targeting and killing cancer cells, but that overcomes size restrictions.

METHODS

We designed a novel cancer-specific killer peptide created by fusion of the toxic peptide (KLAKLAK)2 with the cancer recognition peptide LTVSPWY.

RESULTS

This bi-functional peptide showed toxicity to breast cancer, prostate cancer, and neuroblastoma cell lines. Only low toxicity to non-cancer cells, colon cancer, lung cancer, and lymphoma cell lines was observed. In vivo injections of the bi-functional peptide caused tumor growth retardation compared to mice treated with control peptides. The bi-functional peptide caused retardation of MDA-MB-435S tumors in vivo and increased survival to 80% at day 100 after tumor implantation, whereas all control animals died at day 70. Previous reports showed that the recognition moiety LTVSPWY targets the tumor-associated antigen HER2. Here we found that our new peptide TP-Tox also excerts toxic effects on HER2-negative cell lines. Therefore, we searched for the molecular target of the bi-specific peptide using immunoprecipitation and mass spectrometry. Our data suggest a possible interaction with RAS GTPase-activating protein binding protein 1 (G3BP1).

CONCLUSIONS

We designed a bi-functional peptide of 23 amino acids and demonstrated its ability to bind and kill several cancer cell lines in vitro and to strongly increase survival in breast cancer bearing mice in vivo. This novel toxin could be used in future cancer therapies and warrants further pre-clinical and clinical exploration.

Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.

Keywords: G3BP1; G3BP2; TSC complex; cancer; lysosome; mTORC1; metabolism; neuronal function; stress granule.

Cyclic GMP-AMP synthase (cGAS) is a major sensor of cytosolic DNA from invading pathogens and damaged cellular organelles. Activation of cGAS promotes liquid-like phase separation and formation of membraneless cytoplasmic structures. Here, we found that cGAS bound G3BP1, a double-stranded nucleic acid helicase involved in the formation of stress granules. Loss of G3BP1 blocked subcellular cGAS condensation and suppressed the interferon response to intracellular DNA and DNA virus particles in cells. Furthermore, an RNA-dependent association with PKR promoted G3BP1 foci formation and cGAS-dependent interferon responses. Together, these results indicate that PKR promotes the formation of G3BP1-dependent, membraneless cytoplasmic structures necessary for the DNA-sensing function of cGAS in human cells. These data suggest that there is a previously unappreciated link between nucleic acid sensing pathways, which requires the formation of specialized subcellular structures.

Virus infection leads to activation of the interferon-induced endoribonuclease, RNase L, which results in degradation of viral and cellular RNAs. Both cellular and viral RNA cleavage products of RNase L bind pattern recognition receptors (PRR) like Retinoic acid-inducible I (Rig-I) and or melanoma differentiation-associated protein 5 (MDA5) to further amplify interferon (IFN) production and antiviral response. Although much is known about the mechanics of ligand binding and PRR activation, how the cells coordinate RNA sensing to signaling response and interferon production remains unclear. We show that RNA cleavage products of RNase L activity induce formation of antiviral stress granule (avSG) by regulating activation of double-stranded RNA (dsRNA)-dependent protein kinase R (PKR), and recruit antiviral proteins Rig-I, PKR, OAS and RNase L to avSG. Biochemical analysis of purified avSG showed interaction of key stress granule protein, G3BP1, with only PKR and Rig-I and not with OAS or RNase L. AvSG assembly during RNase L activation is required for IRF3-mediated IFN production and not IFN signaling or proinflammatory cytokine induction. Consequently, cells lacking avSG formation or RNase L signaling produced less IFN and showed higher susceptibility during Sendai virus infection demonstrating the importance of avSG in RNase L-mediated host defense. During viral infection, we propose a role for RNase L-cleaved RNAs in inducing avSG containing antiviral proteins to provide a platform for efficient interaction of RNA ligands with pattern recognition receptors to enhance IFN production to effectively mount antiviral response.IMPORTANCE Double-stranded RNAs produced during viral infections serve as pathogen associated molecular patterns (PAMPs) and bind pattern recognition receptors to stimulate IFN production. RNase L is an IFN-regulated endoribonuclease that is activated in virus-infected cells and cleaves single-stranded viral and cellular RNAs. The RNase L-cleaved dsRNAs signal to Rig-like helicases to amplify IFN production. This study identifies a novel role of antiviral stress granules induced by RNase L as an antiviral signaling hub to coordinate the RNA ligands with cognate receptors to mount effective host response during viral infections.

LINE-1 (L1) retrotransposons are autonomous transposable elements that can affect gene expression and genome integrity. Potential consequences of exogenous viral infections for L1 activity have not been studied to date. Here, we report that hepatitis C virus (HCV) infection causes a significant increase of endogenous L1-encoded ORF1 protein (L1ORF1p) levels and translocation of L1ORF1p to HCV assembly sites at lipid droplets. HCV replication interferes with retrotransposition of engineered L1 reporter elements, which correlates with HCV RNA-induced formation of stress granules and can be partially rescued by knockdown of the stress granule protein G3BP1. Upon HCV infection, L1ORF1p localizes to stress granules, associates with HCV core in an RNA-dependent manner and translocates to lipid droplets. While HCV infection has a negative effect on L1 mobilization, L1ORF1p neither restricts nor promotes HCV infection. In summary, our data demonstrate that HCV infection causes an increase of endogenous L1 protein levels and that the observed restriction of retrotransposition of engineered L1 reporter elements is caused by sequestration of L1ORF1p in HCV-induced stress granules.

Aging affects numerous aspects of cell biology, but the senescence-associated changes in the stress response are only beginning to emerge. To obtain mechanistic insights into these events, we examined the formation of canonical and non-canonical stress granules (SGs) in the cytoplasm. SG generation is a key event after exposure to physiological or environmental stressors. It requires the SG-nucleating proteins G3BP1 and TIA-1/TIAR and stress-related signaling events. To analyze SG formation, we used two independent models of somatic cell aging. In both model systems, cellular senescence impaired the assembly of two SG classes: (i) it compromised the formation of canonical SGs, and (ii) skewed the production of non-canonical SGs. We dissected the mechanisms underlying these senescence-dependent changes in granule biogenesis and identified several specific targets that were modulated by aging. Thus, we demonstrate a depletion of G3BP1 and TIA-1/TIAR in senescent cells and show that the loss of G3BP1 contributed to impaired SG formation. We further reveal that aging reduced Sp1 levels; this transcription factor regulated G3BP1 and TIA-1/TIAR abundance. The assembly of canonical SGs relies on the phosphorylation of translation initiation factor eIF2α. We show that senescence can cause eIF2α hyperphosphorylation. CReP is a subunit of protein phosphatase 1 and critical to reverse the stress-dependent phosphorylation of eIF2α. We demonstrate that the loss of CReP correlated with the aging-related hyperphosphorylation of eIF2α. Together, we have identified significant changes in the stress response of aging cells and provide mechanistic insights. Based on our work, we propose that the decline in SG formation can provide a new biomarker to evaluate cellular aging.

Tailoring stress responsesWhen faced with environmental stress, cells respond by shutting down cellular processes such as translation and nucleocytoplasmic transport. At the same time, cells preserve cytoplasmic messenger RNAs in structures known as stress granules, and many cellular proteins are modified by the covalent addition of ubiquitin, which has long been presumed to reflect degradation of stress-damaged proteins (see the Perspective by Dormann). Maxwell et al. show that cells generate distinct patterns of ubiquitination in response to different stressors. Rather than reflecting the degradation of stress-damaged proteins, this ubiquitination primes cells to dismantle stress granules and reinitiate normal cellular activities once the stress is removed. Gwon et al. show that persistent stress granules are degraded by autophagy, whereas short-lived granules undergo a process of disassembly that is autophagy independent. The mechanism of this disassembly depends on the initiating stress.Science, abc3593 and abf6548, this issue p. eabc3593 and p. eabf6548; see also abj2400, p. 1393.

Stress granule formation is important for stress response in normal cells and could lead to chemotherapy resistance in cancer cells. Aberrant stress granule dynamics are also known to disrupt proteostasis, affect RNA metabolism, and contribute to neuronal cell death. Meanwhile, circadian abnormality is an aging-related risk factor for cancer and neurodegeneration. Whether stress granule dynamics are circadian regulated is entirely unknown. Here we show that the formation of stress granules varied by zeitgeber time in mouse liver. Moreover, altering circadian regulation by silencing the core circadian gene Bmal1 in a cell line expressing an endogenous GFP-tagged G3BP1 significantly increased stress granule dynamics, while the overexpression of Bmal1 decreased them. Surprisingly, increased stress granule dynamics and formation by transient decrease of BMAL1 coincided with increased resistance to stress-induced cell death. The circadian regulation of stress granules was mediated by oscillating eIF2α expression. At zeitgeber time when BMAL1 and eIF2α were at nadir, reduction of unphosphorylated eIF2α could significantly alter the ratio of phosphorylated/total eIF2α and quickly lead to increased formation of stress granules. Therefore, diurnal oscillating eIF2α connects the circadian cue to a cellular stress response mechanism that is vital for both neurodegeneration and cancer.

Stress granules (SGs) are formed in the cytoplasm under environmental stress, including viral infection. Human enterovirus D68 (EV-D68) is a highly pathogenic virus, which can cause serious respiratory and neurological diseases. At present, there is no effective drug or vaccine against EV-D68 infection, and the relationship between EV-D68 infection and SGs is poorly understood. This study revealed the biological function of SGs in EV-D68 infection. Our results suggest that EV-D68 infection induced the accumulation of SG markers proteins, G3BP1, TIA1, and HUR, in the cytoplasm of infected host cells during early infection but inhibited their accumulation during the late stage. Simultaneously, we revealed that EV-D68 infection induces HUR, TIA1, and G3BP1 co-localization, which marks the formation of typical SGs dependent on PKR and eIF2α phosphorylation. In addition, we found that TIA1, HUR and G3BP1 were capable of targeting the 3'-untranslated regions (UTR) of EV-D68 RNA to inhibit viral replication. However, the formation of SGs in response to arsenite (Ars) gradually decreased as the infection progressed, and G3BP1 was cleaved in the late stage as a strategy to antagonize SGs. Our findings have important implications in understanding the mechanism of interaction between EV-D68 and the host, while providing a potential target for the development of antiviral drugs.IMPORTANCE EV-D68 is a serious threat to human health, and there are currently no effective treatments or vaccines. SGs play an important role in cellular innate immunity as a target with antiviral effects. This manuscript describes the formation of SGs induced by EV-D68 early infection, but inhibited during the late stage of infection. Moreover, TIA1, HUR, and G3BP1 can chelate a specific site of the 3'-UTR of EV-D68 to inhibit viral replication, and this interaction is sequence- and complex-dependent. However, this inhibition can be antagonized by overexpression of the minireplicon. These findings increase our understanding of EV-D68 infection and may help identify new antiviral targets that can inhibit viral replication and limit the pathogenesis of EV-D68.

Cells reprogram ongoing translation in response to viral infection, resulting in formation of stress granules (SGs), while viruses have evolved a variety of strategies to antagonize the host SG response. Previous literature reported that in BHK-1 cells, infection with dengue virus (DENV) interfered with the SG formation. In the current study, we further investigated SG formation in human epithelial A549 cells by detecting subcellular localization of two SG hallmarks, TIA-1 and G3BP1. In response to DENV type 2 (DENV2) and type 3 (DENV3) infection, G3BP1, but not TIA-1, was recruited into cytoplasmic granules in some cells, and viral protein synthesis was significantly impaired in the G3BP1-granule-containing cells. Knockdown of G3BP1 significantly rescued the dsRNA-mediated suppression of DENV2 replication. Furthermore, our data showed that the phosphorylation of protein kinase regulated by dsRNA (PKR) and eIF2α, as well as accumulation of dsRNA, mainly occurred at the late stage of viral infection. This work revealed that in DENV-infected A549 cells, G3BP1 granules were assembled independently of TIA-1 and had a negative impact on viral replication. This extends our understanding of the antagonistic relationship between the SG response and dengue virus infection.

Cellular senescence is a known driver of carcinogenesis and age-related diseases, yet senescence is required for various physiological processes. However, the mechanisms and factors that control the negative effects of senescence while retaining its benefits are still elusive. Here, we show that the rasGAP SH3-binding protein 1 (G3BP1) is required for the activation of the senescent-associated secretory phenotype (SASP). During senescence, G3BP1 achieves this effect by promoting the association of the cyclic GMP-AMP synthase (cGAS) with cytosolic chromatin fragments. In turn, G3BP1, through cGAS, activates the NF-κB and STAT3 pathways, promoting SASP expression and secretion. G3BP1 depletion or pharmacological inhibition impairs the cGAS-pathway preventing the expression of SASP factors without affecting cell commitment to senescence. These SASPless senescent cells impair senescence-mediated growth of cancer cells in vitro and tumor growth in vivo. Our data reveal that G3BP1 is required for SASP expression and that SASP secretion is a primary mediator of senescence-associated tumor growth.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with no cure. The reports showed the role of nearby astrocytes around the motor neurons as one among the causes of the disease. However, the exact mechanistic insights are not explored so far. Thus, in the present investigations, we employed the induced pluripotent stem cells (iPSCs) of Cu/Zn-SOD1^L39R linked ALS patient to convert them into the motor neurons (MNs) and astrocytes. We report that the higher expression of stress granule (SG) marker protein G3BP1, and its co-localization with the mutated Cu/Zn-SOD1^L39R protein in patient's MNs and astrocytes are linked with AIF1-mediated upregulation of caspase 3/7 and hyper activated autophagy. We also observe the astrocyte-mediated non-cell autonomous neurotoxicity on MNs in ALS. The secretome of the patient's iPSC-derived astrocytes exerts significant oxidative stress in MNs. The findings suggest the hyperactive status of autophagy in MNs, as witnessed by the co-distribution of LAMP1, P62 and LC3 I/II with the autolysosomes. Conversely, the secretome of normal astrocytes has shown neuroprotection in patient's iPSC-derived MNs. The whole-cell patch-clamp assay confirms our findings at a physiological functional level in MNs. Perhaps for the first time, we are reporting that the MN degeneration in ALS triggered by the hyper-activation of autophagy and induced apoptosis in both cell-autonomous and non-cell autonomous conditions.

Keywords: Amyotrophic lateral sclerosis; Astrocytes; Autophagy; Conditioned medium; Motor neuron disease; Non-cell autonomous neurotoxicity; SOD1.