Stress granules (SGs) are sites of mRNA storage that are formed in response to various conditions of stress, including viral infections. Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has been devastating the swine industry worldwide since the late 1980s. In this study, we found that infection of PRRSV strain WUH3 (genotype 2 PRRSV) induced stable formation of robust SGs in MARC-145 cells, as demonstrated by the recruitment of marker proteins of SGs, including TIA1, G3BP1, and eIF3η. Treatment with specific inhibitors or siRNAs against the stress kinases that are involved in SG formation revealed that PRRSV induced SG formation through a PERK (protein kinase R-like endoplasmic reticulum kinase)-dependent mechanism. Impairment of SG assembly by concomitant knockdown of the SG marker proteins (TIA1, G3BP1, and TIAR) did not affect PRRSV growth, while significantly enhanced PRRSV-induced NF-κB subunit p65 phosphorylation and inflammatory cytokine production. Taken together, our results demonstrate that PRRSV induces SG formation via a PERK-dependent pathway and that SGs are involved in the signaling pathway of the PRRSV-induced inflammatory response in MARC-145 cells.

The co-chaperone HspBP1 interacts with members of the hsp70 family, but also provides chaperone-independent functions. We report here novel biological properties of HspBP1 that are relevant to the formation of cytoplasmic stress granules (SGs). SG assembly is a conserved reaction to environmental or pathological insults and part of the cellular stress response. Our study reveals that HspBP1 (1) is an integral SG constituent, and (2) a regulator of SG assembly. Oxidative stress relocates HspBP1 to SGs, where it co-localizes with granule marker proteins and polyA-RNA. Mass spectrometry and co-immunoprecipitation identified novel HspBP1-binding partners that are critical for SG biology. Specifically, HspBP1 associates with the SG proteins G3BP1, HuR and TIA-1/TIAR. HspBP1 also interacts with polyA-RNA in vivo and binds directly RNA homopolymers in vitro. Multiple lines of evidence and single-granule analyses demonstrate that HspBP1 is crucial for SG biogenesis. Thus, HspBP1 knockdown interferes with stress-induced SG assembly. By contrast, HspBP1 overexpression promotes SG formation in the absence of stress. Notably, the hsp70-binding domains of HspBP1 regulate SG production in unstressed cells. Taken together, we identified novel HspBP1 activities that control SG formation. These features expand HspBP1's role in the cellular stress response and provide new mechanistic insights into SG biogenesis.

While biomolecular condensates have emerged as an important biological phenomenon, mechanisms regulating their composition and the ways that viruses hijack these mechanisms remain unclear. The mosquito-borne alphaviruses cause a range of diseases from rashes and arthritis to encephalitis, and no licensed drugs are available for treatment or vaccines for prevention. The alphavirus virulence factor nonstructural protein 3 (nsP3) suppresses the formation of stress granules (SGs)-a class of cytoplasmic condensates enriched with translation initiation factors and formed during the early stage of infection. nsP3 has a conserved N-terminal macrodomain that hydrolyzes ADP-ribose from ADP-ribosylated proteins and a C-terminal hypervariable domain that binds the essential SG component G3BP1. Here, we show that macrodomain hydrolase activity reduces the ADP-ribosylation of G3BP1, disassembles virus-induced SGs, and suppresses SG formation. Expression of nsP3 results in the formation of a distinct class of condensates that lack translation initiation factors but contain G3BP1 and other SG-associated RNA-binding proteins. Expression of ADP-ribosylhydrolase-deficient nsP3 results in condensates that retain translation initiation factors as well as RNA-binding proteins, similar to SGs. Therefore, our data reveal that ADP-ribosylation controls the composition of biomolecular condensates, specifically the localization of translation initiation factors, during alphavirus infection.

Keywords: ADP-ribosylation; alphavirus; biomolecular condensates; macrodomain; stress granules.

Background: Metastasis is the key cause of death in ovarian cancer patients. To figure out the biological nature of cancer metastasis is essential for developing effective targeted therapy. Here we investigate how long non-coding RNA (lncRNA) SPOCD1-AS from ovarian cancer extracellular vesicles (EVs) remodel mesothelial cells through a mesothelial-to-mesenchymal transition (MMT) manner and facilitate peritoneal metastasis.

Methods: EVs purified from ovarian cancer cells and ascites of patients were applied to mesothelial cells. The MMT process of mesothelial cells was assessed by morphology observation, western blot analysis, migration assay and adhesion assay. Altered lncRNAs of EV-treated mesothelial cells were screened by RNA sequencing and identified by qRT-PCR. SPOCD1-AS was overexpressed or silenced by overexpression lentivirus or shRNA, respectively. RNA pull-down and RNA immunoprecipitation assays were conducted to reveal the mechanism by which SPOCD1-AS remodeled mesothelial cells. Interfering peptides were synthesized and applied. Ovarian cancer orthotopic implantation mouse model was established in vivo.

Results: We found that ovarian cancer-secreted EVs could be taken into recipient mesothelial cells, induce the MMT phenotype and enhance cancer cell adhesion to mesothelial cells. Furthermore, SPOCD1-AS embedded in ovarian cancer-secreted EVs was transmitted to mesothelial cells to induce the MMT process and facilitate peritoneal colonization in vitro and in vivo. SPOCD1-AS induced the MMT process of mesothelial cells via interacting with G3BP1 protein. Additionally, G3BP1 interfering peptide based on the F380/F382 residues was able to block SPOCD1-AS/G3BP1 interaction, inhibit the MMT phenotype of mesothelial cells, and diminish peritoneal metastasis in vivo.

Conclusions: Our findings elucidate the mechanism associated with EVs and their cargos in ovarian cancer peritoneal metastasis and may provide a potential approach for metastatic ovarian cancer therapeutics.

Keywords: Extracellular vesicles; G3BP1; Interfering peptides; Mesothelial-to-mesenchymal transition; Ovarian cancer; Peritoneal metastasis; SPOCD1-AS.

The newly emerging coronavirus SARS-CoV-2 causes severe lung disease and substantial mortality. How the virus evades host defense for efficient replication is not fully understood. In this report, we found that the SARS-CoV-2 nucleocapsid protein (NP) impaired stress granule (SG) formation induced by viral RNA. SARS-CoV-2 NP associated with the protein kinase PKR after dsRNA stimulation. SARS-CoV-2 NP did not affect dsRNA-induced PKR oligomerization, but impaired dsRNA-induced PKR phosphorylation (a hallmark of its activation) as well as SG formation. SARS-CoV-2 NP also targeted the SG-nucleating protein G3BP1 and impaired G3BP1-mediated SG formation. Deficiency of PKR or G3BP1 impaired dsRNA-triggered SG formation and increased SARS-CoV-2 replication. The NP of SARS-CoV also targeted both PKR and G3BP1 to impair dsRNA-induced SG formation, whereas the NP of MERS-CoV targeted PKR, but not G3BP1 for the impairment. Our findings suggest that SARS-CoV-2 NP promotes viral replication by impairing formation of antiviral SGs, and reveal a conserved mechanism on evasion of host antiviral responses by highly pathogenic human betacoronaviruses.

Invasion and multiplication of the facultative, cytosolic, enteropathogen Shigella flexneri within the colonic epithelial lining leads to an acute inflammatory response, fever and diarrhea. During the inflammatory process, infected cells are subjected to numerous stresses including heat, oxidative stress and genotoxic stress. The evolutionarily conserved pathway of cellular stress management is the formation of stress granules that store translationally inactive cellular mRNAs and interfere with cellular signalling pathways by sequestering signalling components. In this study, we investigated the ability of S. flexneri-infected cells to form stress granules in response to exogenous stresses. We found that S. flexneri infection inhibits movement of the stress granule markers eIF3 and eIF4B into stress granules and prevents the aggregation of G3BP1 and eIF4G-containing stress granules. This inhibition occurred only with invasive, but not with non-invasive bacteria and occurred in response to stresses that induce translational arrest through the phosphorylation of eIF2α and by treating cells with pateamine A, a drug that induces stress granules by inhibiting the eIF4A helicase. The S. flexneri-mediated stress granule inhibition could be largely phenocopied by the microtubule-destabilizing drug nocodazole and while S. flexneri infection did not lead to microtubule depolymerization, infection greatly enhanced acetylation of alpha-tubulin. Our data suggest that qualitative differences in the microtubule network or subversion of the microtubule-transport machinery by S. flexneri may be involved in preventing the full execution of this cellular stress response.

The nuclear transport factor 2-like (NTF2-like) domain of human G3BP1 was subcloned, overexpressed in Escherichia coli and purified. Crystals were obtained using the hanging-drop vapour-diffusion method. Diffraction data were collected to 3.6 Å resolution using synchrotron radiation. The crystals belonged to the hexagonal space group P6(3)22, with unit-cell parameters a=b=89.84, c=70.02 Å. The crystals contained one molecule per asymmetric unit, with an estimated solvent content of 56%. Initial phases were obtained by molecular replacement.

Although the V-gene segments coding for the TCR alpha and delta chains are mixed together in the alpha delta locus and are recombined by the same processes, some gene segments (TRAV) are rearranged only with TCR Jalpha gene segments, some (TRDV) only with TCR Ddelta gene segments and some (TRADV) with both. To date, no molecular signal is known that can characterize these three different types of gene segments. Studying the recombination signal sequences (RSS) of all mouse TCR V-gene segments we observed that 80% of the TRAV contain a palindrome sequence (CTGCAG) or its related variant CTGTAG in their 23-bp spacer. Using gel-shift assays we show that these sequences are specifically recognized by some nuclear proteins that are expressed by fresh thymocytes, fresh lymphocytes and tumor cells. Recombination assays on plasmid substrates in a pre-B cell line showed that RSS containing the CTGCAG sequence can impair recombination. From the protein fractions containing the CTGCAG-binding activity, three proteins were identified: G3BP1 (a nucleic-acid-binding protein with a proposed helicase activity) and two proteins from the high-mobility group (HMG) family--HMGB2 and HMGB3. We hypothesize that these proteins can affect recombination at the TCR alpha delta locus.

Ribosome-associated quality control (RQC) purges aberrant mRNAs and nascent polypeptides in a multi-step molecular process initiated by the E3 ligase ZNF598 through sensing of ribosomes collided at aberrant mRNAs and monoubiquitination of distinct small ribosomal subunit proteins. We show that G3BP1-family-USP10 complexes are required for deubiquitination of RPS2, RPS3, and RPS10 to rescue modified 40S subunits from programmed degradation. Knockout of USP10 or G3BP1 family proteins increased lysosomal ribosomal degradation and perturbed ribosomal subunit stoichiometry, both of which were rescued by a single K214R substitution of RPS3. While the majority of RPS2 and RPS3 monoubiquitination resulted from ZNF598-dependent sensing of ribosome collisions initiating RQC, another minor pathway contributed to their monoubiquitination. G3BP1 family proteins have long been considered RNA-binding proteins, however, our results identified 40S subunits and associated mRNAs as their predominant targets, a feature shared by stress granules to which G3BP1 family proteins localize under stress.

Stress granules are membraneless organelles composed of numerous components including ribonucleoproteins. The stress granules are characterized by a dynamic complex assembly in response to various environmental stressors, which has been implicated in the coordinated regulation of diverse biological pathways, to exert a protective role against stress-induced cell death. Here, we show that stress granule formation is induced by morusin, a novel phytochemical displaying antitumor capacity through barely known mechanisms. Morusin-mediated induction of stress granules requires activation of protein kinase R (PKR) and subsequent eIF2α phosphorylation. Notably, genetic inactivation of stress granule formation mediated by G3BP1 knockout sensitized cancer cells to morusin treatment. This protective function against morusin-mediated cell death can be attributed at least in part to the sequestration of receptors for activated C kinase-1 (RACK1) within the stress granules, which reduces caspase-3 activation. Collectively, our study provides biochemical evidence for the role of stress granules in suppressing the antitumor capacity of morusin, proposing that morusin treatment, together with pharmacological inhibition of stress granules, could be an efficient strategy for targeting cancer.

Keywords: PKR; RACK1; cell death; eIF2α; morusin; stress granule.

Ras-GTPase activating SH3 domain-binding protein 1 (G3BP1) is a multifunctional binding protein involved in the development of a variety of human cancers. However, the role of G3BP1 in breast cancer progression remains largely unknown. In this study, we report that G3BP1 is upregulated and correlated with poor prognosis in breast cancer. Overexpression of G3BP1 promotes breast cancer cell proliferation by stimulating β-catenin signaling, which upregulates a number of proliferation-related genes. We further show that G3BP1 improves the stability of β-catenin by inhibiting its ubiquitin-proteasome degradation rather than affecting the transcription of β-catenin. Mechanistically, elevated G3BP1 interacts with and inactivates GSK-3β to suppress β-catenin phosphorylation and degradation. Disturbing the G3BP1-GSK-3β interaction accelerates the degradation of β-catenin, impairing the proliferative capacity of breast cancer cells. Our study demonstrates that the regulatory mechanism of the G3BP1/GSK-3β/β-catenin axis may be a potential therapeutic target for breast cancer.

Keywords: G3BP1; GSK-3β phosphorylation; Wnt/β-catenin signaling pathway; breast cancer; peptide antagonist; protein stability.

In response to virus-induced shutoff host protein synthesis, dynamic aggregates containing mRNA, RNA-binding proteins and translation factors termed stress granules (SGs) often accumulate within the cytoplasm. SGs typically form following phosphorylation and inactivation of the eukaryotic translation initiation factor 2α (eIF2α), a substrate of the double-stranded RNA (dsRNA)-activated kinase protein kinase R (PKR). The detection of innate immune sensors and effectors like PKR at SGs suggests a role in pathogen nucleic acid sensing. However, the functional importance of SGs in host innate responses is unclear and has primarily been examined in response to infection with select RNA viruses. During infection with the DNA virus herpes simplex virus 1 (HSV-1), the virus-encoded virion host shutoff (VHS) endoribonuclease is required to restrict interferon production, PKR activation, and SG formation, although the relationship between these activities remains incompletely understood. Here, we show that in cells infected with a VHS-deficient HSV-1 (ΔVHS) dsRNA accumulated and localized to SGs. Surprisingly, formation of dsRNA and its concentration at SGs was not required for beta interferon mRNA induction, indicating that suppression of type I interferon induction by VHS does not stem from its control of dsRNA accumulation. Instead, STING signaling downstream of cGMP-AMP synthase (cGAS)-dependent DNA sensing is required for beta interferon induction. In contrast, significantly less PKR activation is observed when SG assembly is disrupted by ISRIB, an inhibitor of phosphorylated eIF2α-mediated translation repression, or depleting SG scaffolding proteins G3BP1 or TIA1. This demonstrates that PKR activation is intimately linked to SG formation and that SGs form important hubs to potentiate PKR activation during infection.IMPORTANCE Formation of cytoplasmic stress granules that are enriched for innate immune sensors and effectors is suppressed during many viral infections. It is unclear, however, to what extent this is a side effect of viral efforts to maintain protein synthesis or intentional disruption of a hub for innate immune sensing. In this study, we utilize a herpes simplex virus 1 mutant lacking the RNA nuclease VHS which upon infection induces SGs, PKR activation, and beta interferon to address this question. We show that dsRNA is localized to SGs and that SGs can function to promote PKR activation in the context of a DNA virus infection, but we find no evidence to support their importance for interferon induction during HSV-1 infection.

Flightless I (FliI) is a calcium-dependent, actin severing and capping protein that localizes to cell matrix adhesions, contributes to the generation of cell extensions, and colocalizes with Ras. Currently, the mechanism by which FliI interacts with Ras to enable assembly of actin-based cell protrusions is not defined. R-Ras, but not K-ras, H-ras, or N-ras, associated with the leucine-rich region (LRR) of FliI. Mutations of the proline-rich region of R-ras (P202A, P203A) prevented this association. Knockdown of Ras GTPase-activating SH3 domain-binding protein (G3BP1) or Rasgap120 by small interfering RNA inhibited the formation of cell extensions and prevented interaction of R-ras and G3BP1 in FliI wild-type (WT) cells. Pull-down assays using G3BP1 fusion proteins showed a strong association of R-ras with the C-terminus of G3BP1 (amino acids 236-466), which also required the LRR of FliI. In cells that expressed the truncated N-terminus or C-terminus of G3BP1, the formation of cell extensions was blocked. Endogenous Rasgap120 interacted with the N-terminus of G3BP1 (amino acids 1-230). We conclude that in cells plated on collagen FliI-LRR interacts with R-ras to promote cell extension formation and that FliI is required for the interaction of Rasgap120 with G3BP1 to regulate R-ras activity and growth of cell extensions.

The elaborate control of biogenesis and turnover is essential for circular RNAs (circRNAs) to exert their functions properly in eukaryotic cells, whereas how circRNAs are degraded remains unclear. A recent study by Fischer et al. reveals a novel structure-mediated circRNA decay that selectively degrades highly structured RNAs by UPF1 and G3BP1.

Streptococcus agalactiae is a common pathogen in aquatic animals, especially tilapia, that hinders aquaculture development and leads to serious economic losses. Previously, a S. agalactiae strain named HN016 was identified from infected tilapia, and the attenuated strain YM001 was subsequently obtained by continuous passaging in Tryptic Soy Broth (TSB) medium. YM001 has been demonstrated as a safe vaccine for S. agalactiae infection in tilapia. To understand the molecular bases of the virulence of these two strains, we performed proteomic and transcriptomic analysis to reveal the protein and gene expression changes in the liver and intestine during the infection process. HN016 significantly decreased the contents of white blood cells (WBCs), neutrophils (NEUs), red blood cells (RBCs) and hematocrit (HCT) and increased the levels of total protein (TP), albumin (ALB) and globulin (GLO), while no such significant differences were observed when comparing the control with YM001. During the infection process, pathogenic peptidoglycan hydrolase, CSPA and membrane proteins were significantly differentially expressed between YM001 and HN016. Furthermore, both proteome and transcriptome data showed that the complement and coagulation cascades pathway and the antigen processing and presentation pathway were stimulated in the liver and intestine, respectively, by YM001 infection compared to HN016 infection. The interaction network analysis of key virulence genes from pathogens suggested that CSPA, as a key node, affects the expression of DOLPP1, MIPEP, PA2G4, OCIAD1, G3BP1 and CLIC5 with a positive correlation. The present evidence suggests that during the infection process, CSPA was the key genes contributing to low virulence in YM001.

Mammalian Ras-GTPase-activating protein SH3-domain-binding proteins (G3BPs) are a highly conserved family of RNA-binding proteins that link kinase receptor-mediated signaling to RNA metabolism. Mammalian G3BP1 is a multifunctional protein that functions in viral immunity. Here, we show that the Arabidopsis thaliana homolog of human G3BP1 negatively regulates plant immunity. Arabidopsis g3bp1 mutants showed enhanced resistance to the virulent bacterial pathogen Pseudomonas syringae pv. tomato. Pathogen resistance was mediated in Atg3bp1 mutants by altered stomatal and apoplastic immunity. Atg3bp1 mutants restricted pathogen entry into stomates showing insensitivity to bacterial coronatine-mediated stomatal reopening. AtG3BP1 was identified as a negative regulator of defense responses, which correlated with moderate up-regulation of salicylic acid biosynthesis and signaling without growth penalty.

We report that several viruses from the human enterovirus group B cause massive vimentin rearrangements during lytic infection. Comprehensive studies suggested that viral protein synthesis was triggering the vimentin rearrangements. Blocking the host cell vimentin dynamics with IDPN did not significantly affect the production of progeny viruses and only moderately lowered the synthesis of structural proteins such as VP1. In contrast, the synthesis of the non-structural proteins 2A, 3C, and 3D was drastically lowered. This led to attenuation of the cleavage of the host cell substrates PABP and G3BP1 and reduced caspase activation, thus leading to prolonged cell survival. Furthermore, the localization of the proteins differed in the infected cells. Capsid protein VP1 was found diffusely around the cytoplasm, whereas 2A and 3D followed vimentin distribution. Based on protein blotting, lower amounts of non-structural proteins did not result from proteasomal degradation, but from lower synthesis without intact vimentin cage structure. In contrast, inhibition of Hsp90 chaperone activity, which regulates P1 maturation, lowered the amount of VP1, but had less effect on 2A. The results suggest that, the vimentin dynamics regulate viral non-structural protein synthesis while having no effect on structural protein synthesis or overall infection efficiency. The results presented here shed new light on differential fate of structural and non-structural proteins of enteroviruses, having consequences on host cell survival.Importance A virus needs the host cell in order to replicate and produce new progeny viruses. For this, the virus takes over the host cell and modifies it to become a factory for viral proteins. Irrespective of the specific virus family, these proteins can be divided into structural and non-structural proteins. Structural proteins are the building blocks for the new progeny virions, whereas the non-structural proteins orchestrate the take-over of the host cell and its functions. Here we have shown a mechanism that viruses exploit in order to regulate the host cell. We show that viral protein synthesis induces vimentin cages, which promote production of specific viral proteins that eventually control apoptosis and the host cell death. This study specifies vimentin as the key regulator of these events and indicates that viral proteins have different fates in the cells depending on their association with vimentin cages.

Liquid-liquid phase separation enables compartmentalization of biomolecules in cells, notably RNA and associated proteins in the nucleus. Besides having critical functions in RNA processing, there is a major interest in deciphering the molecular mechanisms of compartmentalization orchestrated by RNA-binding proteins such as TDP-43 (also known as TARDBP) and FUS because of their link to neuron diseases. However, tools for probing compartmentalization in cells are lacking. Here, we developed a method to analyze the mixing and demixing of two different phases in a cellular context. The principle is the following: RNA-binding proteins are confined on microtubules and quantitative parameters defining their spatial segregation are measured along the microtubule network. Through this approach, we found that four mRNA-binding proteins, HuR (also known as ELAVL1), G3BP1, TDP-43 and FUS form mRNA-rich liquid-like compartments on microtubules. TDP-43 is partly miscible with FUS but immiscible with either HuR or G3BP1. We also demonstrate that mRNA is essential to capture the mixing and demixing behavior of mRNA-binding proteins in cells. Taken together, we show that microtubules can be used as platforms to understand the mechanisms underlying liquid-liquid phase separation and their deregulation in human diseases.

COVID-19 is currently a highly pressing health threat and therapeutic strategies to mitigate the infection impact are urgently needed. Characterization of the SARS-CoV-2 interactome in infected cells may represent a powerful tool to identify cellular proteins hijacked by viruses for their life cycle and develop host-oriented antiviral therapeutics. Here we report the proteomic characterization of host proteins interacting with SARS-CoV-2 Nucleoprotein in infected Vero E6 cells. We identified 24 high-confidence proteins mainly playing a role in RNA metabolism and translation, including RNA helicases and scaffold proteins involved in the formation of stress granules, cytoplasmic aggregates of messenger ribonucleoproteins that accumulate as a result of stress-induced translation arrest. Analysis of stress granules upon SARS-CoV-2 infection showed that these structures are not induced in infected cells, neither eIF2α phosphorylation, an upstream event leading to stress-induced translation inhibition. Notably, we found that G3BP1, a stress granule component that associates with the Nucleoprotein, is required for efficient SARS-CoV-2 replication. Moreover, we showed that the Nucleoprotein-interacting RNA helicase DDX3X colocalizes with viral RNA foci and its inhibition by small molecules or small interfering RNAs significantly reduces viral replication. Altogether, these results indicate that SARS-CoV-2 subverts the stress granule machinery and exploits G3BP1 and DDX3X for its replication cycle, offering groundwork for future development of host-directed therapies.

Keywords: COVID-19; DEAD RNA helicase; G3BP1; antiviral drugs; proteomics; stress granules.

Ras‑GTPase‑activating protein SH3 domain‑binding protein 1 (G3BP1) has been reported to be of importance in the occurrence and development of colon cancer. However, the underlying mechanisms remain largely unknown. Therefore, the aim of the present study was to investigate the role of Wnt/β‑catenin signaling in G3BP1‑mediated colon cancer progression. The expression of G3BP1 in colon tissues and cells was detected via reverse transcription‑quantitative PCR, western blotting and immunohistochemistry. Gain‑of‑function assays were performed in colon cancer RKO cells, which have a relatively low expression of G3BP1, while loss‑of‑function assays were performed in SW620 colon cancer cells, which have a relatively high expression of G3BP1. Cell proliferation, apoptosis and tumorigenesis were assessed using Cell Counting Kit‑8, flow cytometry and tumor‑bearing mice assays, respectively. The results demonstrated that G3BP1 expression was significantly upregulated in colon cancer tissues and cells compared with healthy colon tissues and cells. It was found that high expression of G3BP1 was closely associated with the poor prognosis and advanced clinical process in patients with colon cancer. Overexpression of G3BP1 in RKO cells enhanced their proliferative ability and decreased their apoptosis tendency, while knockdown of G3BP1 inhibited SW620 cell proliferation and induced apoptosis. In addition, G3BP1 interacted with β‑catenin and upregulated its expression and nuclear accumulation. It was identified that β‑catenin knockdown abolished the effects of G3BP1 on the enhancement of cell proliferation in vitro and tumor formation in vivo, as well as the inhibition of cell apoptosis. In conclusion, the present study demonstrated that G3BP1 promoted the progression of colon cancer by activating β‑catenin signaling, which provided novel evidence for the role of G3BP1 in colon cancer.

Porcine epidemic diarrhoea virus (PEDV) is a single-stranded, positive-sense RNA virus that belongs to the Coronaviridae. PEDV causes severe diarrhoea and dehydration in nursing piglets, which leads to significant economic losses to the swine industry worldwide. Stress granules (SGs) are sites of mRNA storage that are formed under various stress conditions including viral infections. Increasing evidence suggests that SGs function in antiviral innate immunity of host cells to limit virus replication. Ras-GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) is a key stress granule-resident protein that nucleates stress granule assembly. Depletion of G3BP1 inhibits SGs formation and overexpression of G3BP1 nucleates SGs assembly. We observed that knockdown of G3BP1 by silencing RNA significantly increased PEDV replication. Overexpression of exogenous G3BP1, on the other hand, lowered virus replication by 100-fold compared to vector control. An increase in the levels of mRNAs of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) was also observed in PEDV-infected G3BP1 depleted cells compared to PEDV-infected control cells. Taken together, our results suggest that G3BP1 plays an antiviral role and impairs PEDV replication.

Stress granules (SGs) are dynamic sites of cytosolic mRNA storage that are formed in response to stress conditions, including viral infection. SGs have been implicated in regulating several aspects of the host immune response to various pathogens. Porcine reproductive and respiratory syndrome virus (PRRSV), an economically-important global swine pathogen, reportedly induced SGs during replication, although the underlying mechanisms are poorly defined. In this study, we delineated the molecular mechanisms regulating the SG response to PRRSV infection. Using confocal microscopy, we first demonstrated that infection with PRRSV strain VR2385 induces an accumulation of the SG markers G3BP1, G3BP2, TIAR, eIF3b, and USP10 as well as mRNAs into punctate structures in the cytoplasm of infected host cells. Subsequently, we demonstrated that the PRRSV-induced SGs were in close proximity to viral replication complexes (VRCs) and processing bodies (P-bodies), and that SG formation was coordinated with inhibition of host cellular translation. Treatment of infected cells with cycloheximide disrupted the PRRSV-induced SGs. Furthermore, impairment of SG assembly by the shRNA-mediated knockdown of G3BP1, G3BP2 and USP10 did not affect viral replication. Collectively, these results demonstrate that PRRSV infection induces formation of SGs associated with VRCs, which is coordinated with the suppression of host cell protein synthesis. This is the first study to extensively characterize the formation and underlying mechanism of bona fide SGs during PRRSV infection. Our findings have important implications in understanding the mechanism of PRRSV-host interactions.

In aortic vascular smooth muscle (VSM), the canonical Wnt receptor LRP6 inhibits protein arginine (Arg) methylation, a new component of noncanonical Wnt signaling that stimulates nuclear factor of activated T cells (viz NFATc4). To better understand how methylation mediates these actions, MS was performed on VSM cell extracts from control and LRP6-deficient mice. LRP6-dependent Arg methylation was regulated on >500 proteins; only 21 exhibited increased monomethylation (MMA) with concomitant reductions in dimethylation. G3BP1, a known regulator of arteriosclerosis, exhibited a >30-fold increase in MMA in its C-terminal domain. Co-transfection studies confirm that G3BP1 (G3BP is Ras-GAP SH3 domain-binding protein) methylation is inhibited by LRP6 and that G3BP1 stimulates NFATc4 transcription. NFATc4 association with VSM osteopontin (OPN) and alkaline phosphatase (TNAP) chromatin was increased with LRP6 deficiency and reduced with G3BP1 deficiency. G3BP1 activation of NFATc4 mapped to G3BP1 domains supporting interactions with RIG-I (retinoic acid inducible gene I), a stimulus for mitochondrial antiviral signaling (MAVS) that drives cardiovascular calcification in humans when mutated in Singleton-Merten syndrome (SGMRT2). Gain-of-function SGMRT2/RIG-I mutants increased G3BP1 methylation and synergized with osteogenic transcription factors (Runx2 and NFATc4). A chemical antagonist of G3BP, C108 (C108 is 2-hydroxybenzoic acid, 2-[1-(2-hydroxyphenyl)ethylidene]hydrazide CAS 15533-09-2), down-regulated RIG-I-stimulated G3BP1 methylation, Wnt/NFAT signaling, VSM TNAP activity, and calcification. G3BP1 deficiency reduced RIG-I protein levels and VSM osteogenic programs. Like G3BP1 and RIG-I deficiency, MAVS deficiency reduced VSM osteogenic signals, including TNAP activity and Wnt5-dependent nuclear NFATc4 levels. Aortic calcium accumulation is decreased in MAVS-deficient LDLR-/- mice fed arteriosclerotic diets. The G3BP1/RIG-I/MAVS relay is a component of Wnt signaling. Targeting this relay may help mitigate arteriosclerosis.

Stress granules (SGs) are membrane-less ribonucleoprotein condensates that form in response to various stress stimuli via phase separation. SGs act as a protective mechanism to cope with acute stress, but persistent SGs have cytotoxic effects that are associated with several age-related diseases. Here, we demonstrate that the testis-specific protein, MAGE-B2, increases cellular stress tolerance by suppressing SG formation through translational inhibition of the key SG nucleator G3BP. MAGE-B2 reduces G3BP protein levels below the critical concentration for phase separation and suppresses SG initiation. Knockout of the MAGE-B2 mouse ortholog or overexpression of G3BP1 confers hypersensitivity of the male germline to heat stress in vivo. Thus, MAGE-B2 provides cytoprotection to maintain mammalian spermatogenesis, a highly thermosensitive process that must be preserved throughout reproductive life. These results demonstrate a mechanism that allows for tissue-specific resistance against stress and could aid in the development of male fertility therapies.

Keywords: DDX5; G3BP; MAGE-B2; RNA binding protein; cancer; cancer-testis antigen; spermatogonial stem cells; stress granule; testis; translation.