TRIM25 predominately associates with anti-viral stress granules.

Stress granules (SGs) are induced by various environmental stressors, resulting in their compositional and functional heterogeneity. SGs play a crucial role in the antiviral process, owing to their potent translational repressive effects and ability to trigger signal transduction; however, it is poorly understood how these antiviral SGs differ from SGs induced by other environmental stressors. Here we identify that TRIM25, a known driver of the ubiquitination-dependent antiviral innate immune response, is a potent and critical marker of the antiviral SGs. TRIM25 undergoes liquid-liquid phase separation (LLPS) and co-condenses with the SG core protein G3BP1 in a dsRNA-dependent manner. The co-condensation of TRIM25 and G3BP1 results in a significant enhancement of TRIM25's ubiquitination activity towards multiple antiviral proteins, which are mainly located in SGs. This co-condensation is critical in activating the RIG-I signaling pathway, thus restraining RNA virus infection. Our studies provide a conceptual framework for better understanding the heterogeneity of stress granule components and their response to distinct environmental stressors.

Critical role of G3BP1 in bovine parainfluenza virus type 3 (BPIV3)-inhibition of stress granules formation and viral replication.

Background: It remains unclear whether BPIV3 infection leads to stress granules formation and whether G3BP1 plays a role in this process and in viral replication. This study aims to clarify the association between BPIV3 and stress granules, explore the effect of G3BP1 on BPIV3 replication, and provide significant insights into the mechanisms by which BPIV3 evades the host's antiviral immunity to support its own survival. Methods: Here, we use Immunofluorescence staining to observe the effect of BPIV3 infection on the assembly of stress granules. Meanwhile, the expression changes of eIF2α and G3BP1 were determined. Overexpression or siRNA silencing of intracellular G3BP1 levels was examined for its regulatory control of BPIV3 replication. Results: We identify that the BPIV3 infection elicited phosphorylation of the eIF2α protein. However, it did not induce the assembly of stress granules; rather, it inhibited the formation of stress granules and downregulated the expression of G3BP1. G3BP1 overexpression facilitated the formation of stress granules within cells and hindered viral replication, while G3BP1 knockdown enhanced BPIV3 expression. Conclusion: This study suggest that G3BP1 plays a crucial role in BPIV3 suppressing stress granule formation and viral replication.

Phosphorylation of G3BP1 is involved in the regulation of PDCoV-induced inflammatory response.

Stress granules (SGs), the main component is GTPase-activating protein-binding protein 1 (G3BP1), which are assembled during viral infection and function to sequester host and viral mRNAs and proteins, are part of the antiviral responses. In this study, we found that porcine deltacoronavirus (PDCoV) infection induced stable formation of robust SGs in cells through a PERK (protein kinase R-like endoplasmic reticulum kinase)-dependent mechanism. Overexpression of SGs marker proteins G3BP1 significantly reduced PDCoV replication in vitro, while inhibition of endogenous G3BP1 enhanced PDCoV replication. Moreover, PDCoV infected LLC-PK1 cells raise the phosphorylation level of G3BP1. By overexpression of the G3BP1 phosphorylated protein or the G3BP1 dephosphorylated protein, we found that phosphorylation of G3BP1 is involved in the regulation of PDCoV-induced inflammatory response. Taken together, our study presents a vital aspect of the host innate response to invading pathogens and reveals attractive host targets for antiviral target.

Translational arrest and mRNA decay are independent activities of alphaherpesvirus virion host shutoff proteins.

The herpes simplex virus 1 (HSV1) virion host shutoff (vhs) protein is an endoribonuclease that regulates the translational environment of the infected cell, by inducing the degradation of host mRNA via cellular exonuclease activity. To further understand the relationship between translational shutoff and mRNA decay, we have used ectopic expression to compare HSV1 vhs (vhsH) to its homologues from four other alphaherpesviruses - varicella zoster virus (vhsV), bovine herpesvirus 1 (vhsB), equine herpesvirus 1 (vhsE) and Marek's disease virus (vhsM). Only vhsH, vhsB and vhsE induced degradation of a reporter luciferase mRNA, with poly(A)+ in situ hybridization indicating a global depletion of cytoplasmic poly(A)+ RNA and a concomitant increase in nuclear poly(A)+ RNA and the polyA tail binding protein PABPC1 in cells expressing these variants. By contrast, vhsV and vhsM failed to induce reporter mRNA decay and poly(A)+ depletion, but rather, induced cytoplasmic G3BP1 and poly(A)+ mRNA- containing granules and phosphorylation of the stress response proteins eIF2α and protein kinase R. Intriguingly, regardless of their apparent endoribonuclease activity, all vhs homologues induced an equivalent general blockade to translation as measured by single-cell puromycin incorporation. Taken together, these data suggest that the activities of translational arrest and mRNA decay induced by vhs are separable and we propose that they represent sequential steps of the vhs host interaction pathway.

Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication and pathogenicity.

G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibits stress granule assembly and interacts with G3BP1/2 via an ITFG motif, including residue F17, in the N protein. Prior studies examining the impact of the G3PB1-N interaction on SARS-CoV-2 replication have produced inconsistent findings, and the role of this interaction in pathogenesis is unknown. Here, we use structural and biochemical analyses to define the residues required for G3BP1-N interaction and structure-guided mutagenesis to selectively disrupt this interaction. We find that N-F17A mutation causes highly specific loss of interaction with G3BP1/2. SARS-CoV-2 N-F17A fails to inhibit stress granule assembly in cells, has decreased viral replication, and causes decreased pathology in vivo. Further mechanistic studies indicate that the N-F17-mediated G3BP1-N interaction promotes infection by limiting sequestration of viral genomic RNA (gRNA) into stress granules.

Escherichia coli-Induced cGLIS3-Mediated Stress Granules Activate the NF-κB Pathway to Promote Intrahepatic Cholangiocarcinoma Progression.

Patients with concurrent intrahepatic cholangiocarcinoma (ICC) and hepatolithiasis generally have poor prognoses. Hepatolithiasis is once considered the primary cause of ICC, although recent insights indicate that bacteria in the occurrence of hepatolithiasis can promote the progression of ICC. By constructing in vitro and in vivo ICC models and patient-derived organoids (PDOs), it is shown that Escherichia coli induces the production of a novel RNA, circGLIS3 (cGLIS3), which promotes tumor growth. cGLIS3 binds to hnRNPA1 and G3BP1, resulting in the assembly of stress granules (SGs) and suppression of hnRNPA1 and G3BP1 ubiquitination. Consequently, the IKKα mRNA is blocked in SGs, decreasing the production of IKKα and activating the NF-κB pathway, which finally results in chemoresistance and produces metastatic phenotypes of ICC. This study shows that a combination of Icaritin (ICA) and gemcitabine plus cisplatin (GP) chemotherapy can be a promising treatment strategy for ICC.

PRKAA2, MTOR, and TFEB in the regulation of lysosomal damage response and autophagy.

Lysosomes function as critical signaling hubs that govern essential enzyme complexes. LGALS proteins (LGALS3, LGALS8, and LGALS9) are integral to the endomembrane damage response. If ESCRT fails to rectify damage, LGALS-mediated ubiquitination occurs, recruiting autophagy receptors (CALCOCO2, TRIM16, and SQSTM1) and VCP/p97 complex containing UBXN6, PLAA, and YOD1, initiating selective autophagy. Lysosome replenishment through biogenesis is regulated by TFEB. LGALS3 interacts with TFRC and TRIM16, aiding ESCRT-mediated repair and autophagy-mediated removal of damaged lysosomes. LGALS8 inhibits MTOR and activates TFEB for ATG and lysosomal gene transcription. LGALS9 inhibits USP9X, activates PRKAA2, MAP3K7, ubiquitination, and autophagy. Conjugation of ATG8 to single membranes (CASM) initiates damage repair mediated by ATP6V1A, ATG16L1, ATG12, ATG5, ATG3, and TECPR1. ATG8ylation or CASM activates the MERIT system (ESCRT-mediated repair, autophagy-mediated clearance, MCOLN1 activation, Ca2+ release, RRAG-GTPase regulation, MTOR modulation, TFEB activation, and activation of GTPase IRGM). Annexins ANAX1 and ANAX2 aid damage repair. Stress granules stabilize damaged membranes, recruiting FLCN-FNIP1/2, G3BP1, and NUFIP1 to inhibit MTOR and activate TFEB. Lysosomes coordinate the synergistic response to endomembrane damage and are vital for innate and adaptive immunity. Future research should unveil the collaborative actions of ATG proteins, LGALSs, TRIMs, autophagy receptors, and lysosomal proteins in lysosomal damage response.

G3BP1 Interact with JAK2 mRNA to Promote the Malignant Progression of Nasopharyngeal Carcinoma via Activating JAK2/STAT3 Signaling Pathway.

Ras-GTPase-activating protein (GAP)-binding protein 1 (G3BP1) is an RNA-binding protein implicated in various malignancies. However, its role in nasopharyngeal carcinoma (NPC) remains elusive. This study elucidates the potential regulation mechanisms of G3BP1 and its significance in NPC advancement. Through knockdown and overexpression approaches, we validate G3BP1's oncogenic role by promoting proliferation, migration, and invasion in vitro and in vivo. Moreover, G3BP1 emerges as a key regulator of the JAK2/STAT3 signaling pathway, augmenting JAK2 expression via mRNA binding. Notably, epigallocatechin gallate (EGCG), a green tea-derived antioxidant, counteracts G3BP1-mediated pathway activation. Clinical analysis reveals heightened G3BP1, JAK2, and p-STAT3 as powerful prognostic markers, with G3BP1's expression standing as an independent indicator of poorer outcomes for NPC patients. In conclusion, the study unveils the oncogenic prowess of G3BP1, its orchestration of the JAK2/STAT3 signaling pathway, and its pivotal role in NPC progression.

Profiling of RNA-binding protein binding sites by in situ reverse transcription-based sequencing.

RNA-binding proteins (RBPs) regulate diverse cellular processes by dynamically interacting with RNA targets. However, effective methods to capture both stable and transient interactions between RBPs and their RNA targets are still lacking, especially when the interaction is dynamic or samples are limited. Here we present an assay of reverse transcription-based RBP binding site sequencing (ARTR-seq), which relies on in situ reverse transcription of RBP-bound RNAs guided by antibodies to identify RBP binding sites. ARTR-seq avoids ultraviolet crosslinking and immunoprecipitation, allowing for efficient and specific identification of RBP binding sites from as few as 20 cells or a tissue section. Taking advantage of rapid formaldehyde fixation, ARTR-seq enables capturing the dynamic RNA binding by RBPs over a short period of time, as demonstrated by the profiling of dynamic RNA binding of G3BP1 during stress granule assembly on a timescale as short as 10 minutes.

Targeting the NTF2-like domain of G3BP1: Novel modulators of intracellular granule dynamics.

Stress granule (SG) is a temporary cellular structure that plays a crucial role in the regulation of mRNA and protein sequestration during various cellular stress conditions. SG enables cells to cope with stress more effectively, conserving vital energy and resources. Focusing on the NTF2-like domain of G3BP1, a key protein in SG dynamics, we explore to identify and characterize novel small molecules involved in SG modulation without external stressors. Through in silico molecular docking approach to simulate the interaction between various compounds and the NTF2-like domain of G3BP1, we identified three compounds as potential candidates that could bind to the NTF2-like domain of G3BP1. Subsequent immunofluorescence experiments demonstrated that these compounds induce the formation of SG-like, G3BP1-positive granules. Importantly, the granule formation by these compounds occurs independent from the phosphorylation of eIF2α, a common mechanism in SG formation, suggesting that it might offer a new strategy for influencing SG dynamics implicated in various diseases.

Proteomic analysis of SARS-CoV-2 particles unveils a key role of G3BP proteins in viral assembly.

Considerable progress has been made in understanding the molecular host-virus battlefield during SARS-CoV-2 infection. Nevertheless, the assembly and egress of newly formed virions are less understood. To identify host proteins involved in viral morphogenesis, we characterize the proteome of SARS-CoV-2 virions produced from A549-ACE2 and Calu-3 cells, isolated via ultracentrifugation on sucrose cushion or by ACE-2 affinity capture. Bioinformatic analysis unveils 92 SARS-CoV-2 virion-associated host factors, providing a valuable resource to better understand the molecular environment of virion production. We reveal that G3BP1 and G3BP2 (G3BP1/2), two major stress granule nucleators, are embedded within virions and unexpectedly favor virion production. Furthermore, we show that G3BP1/2 participate in the formation of cytoplasmic membrane vesicles, that are likely virion assembly sites, consistent with a proviral role of G3BP1/2 in SARS-CoV-2 dissemination. Altogether, these findings provide new insights into host factors required for SARS-CoV-2 assembly with potential implications for future therapeutic targeting.

CircNUP50 is a novel therapeutic target that promotes cisplatin resistance in ovarian cancer by modulating p53 ubiquitination.

BACKGROUND: Most patients with ovarian cancer (OC) treated with platinum-based chemotherapy have a dismal prognosis owing to drug resistance. However, the regulatory mechanisms of circular RNA (circRNA) and p53 ubiquitination are unknown in platinum-resistant OC. We aimed to identify circRNAs associated with platinum-resistant OC to develop a novel treatment strategy. METHODS: Platinum-resistant circRNAs were screened through circRNA sequencing and validated using quantitative reverse-transcription PCR in OC cells and tissues. The characteristics of circNUP50 were analysed using Sanger sequencing, oligo (dT) primers, ribonuclease R and fluorescence in situ hybridisation assays. Functional experimental studies were performed in vitro and in vivo. The mechanism underlying circNUP50-mediated P53 ubiquitination was investigated through circRNA pull-down analysis and mass spectrometry, luciferase reporters, RNA binding protein immunoprecipitation, immunofluorescence assays, cycloheximide chase assays, and ubiquitination experiments. Finally, a platinum and si-circNUP50 co-delivery nanosystem (Psc@DPP) was constructed to treat platinum-resistant OC in an orthotopic animal model. RESULTS: We found that circNUP50 contributes to platinum-resistant conditions in OC by promoting cell proliferation, affecting the cell cycle, and reducing apoptosis. The si-circNUP50 mRNA sequencing and circRNA pull-down analysis showed that circNUP50 mediates platinum resistance in OC by binding p53 and UBE2T, accelerating p53 ubiquitination. By contrast, miRNA sequencing and circRNA pull-down experiments indicated that circNUP50 could serve as a sponge for miR-197-3p, thereby upregulating G3BP1 to mediate p53 ubiquitination, promoting OC platinum resistance. Psc@DPP effectively overcame platinum resistance in an OC tumour model and provided a novel idea for treating platinum-resistant OC using si-circNUP50. CONCLUSIONS: This study reveals a novel molecular mechanism by which circNUP50 mediates platinum resistance in OC by modulating p53 ubiquitination and provides new insights for developing effective therapeutic strategies for platinum resistance in OC.

G3BP1 and SLU7 Jointly Promote Immune Evasion by Downregulating MHC-I via PI3K/Akt Activation in Bladder Cancer.

Immune checkpoint inhibitors (ICIs) show promise as second-line treatment for advanced bladder cancer (BLCA); however, their responsiveness is limited by the immune evasion mechanisms in tumor cells. This study conduct a Cox regression analysis to screen mRNA-binding proteins and reveals an association between Ras GTPase-activating protein-binding protein 1 (G3BP1) and diminished effectiveness of ICI therapy in patients with advanced BLCA. Subsequent investigation demonstrates that G3BP1 enhances immune evasion in BLCA cells by downregulating major histocompatibility complex class I (MHC-I) through phosphoinositide 3-kinase (PI3K)/Akt signaling activation. Mechanistically, G3BP1 interacts with splicing factor synergistic lethal with U5 snRNA 7 (SLU7) to form a complex with poly(A)-binding protein cytoplasmic 1 and eukaryotic translation initiation factor 4 gamma 1. This complex stabilizes the closed-loop structure of the mRNAs of class IA PI3Ks and consequently facilitates their translation and stabilization, thereby activating PI3K/Akt signaling to downregulate MHC-I. Consistently, targeting G3BP1 with epigallocatechin gallate (EGCG) impedes immune evasion and sensitizes BLCA cells to anti-programmed cell death (PD)-1 antibodies in mice. Thus, G3BP1 and SLU7 collaboratively contribute to immune evasion in BLCA, indicating that EGCG is a precision therapeutic agent to enhance the effectiveness of anti-PD-1 therapy.

A novel G3BP1-GFP reporter human lung cell system enabling real-time monitoring of stress granule dynamics for in vitro lung toxicity assessment.

Under various cellular stress conditions, including exposure to toxic chemicals, RNA-binding proteins (RBPs), including Ras GTPase-activating protein-binding protein 1 (G3BP1), aggregate and form stress granule complexes, which serve as hallmarks of cellular stress. The existing methods for analyzing stress granule assembly have limitations in the rapid detection of dynamic cellular stress and ignore the effects of constitutively overexpressed RBP on cellular stress and stress-related processes. Therefore, to overcome these limitations, we established a G3BP1-GFP reporter in a human lung epithelial cell line using CRISPR/Cas9-based knock-in as an alternative system for stress granule analysis. We showed that the G3BP1-GFP reporter system responds to stress conditions and forms a stress granule complex similar to that of native G3BP1. Furthermore, we validated the stress granule response of an established cell line under exposure to various household chemicals. Overall, this novel G3BP1-GFP reporter human lung cell system is capable of monitoring stress granule dynamics in real time and can be used for assessing the lung toxicity of various substances in vitro.

Inhibition of cGAS-STING pathway by stress granules after activation of M2 macrophages by human mesenchymal stem cells against drug induced liver injury.

OBJECTIVE: Cells can produce stress granules (SGs) to protect itself from damage under stress. The cGAS-STING pathway is one of the important pattern recognition pathways in the natural immune system. This study was investigated whether human mesenchymal stem cells (hMSCs) could protect the liver by inducing M2 macrophages to produce SGs during acute drug induced liver injury (DILI) induced by acetaminophen (APAP). METHODS: After intragastric administration of APAP in vivo to induce DILI mice model, hMSCs were injected into the tail vein. The co-culture system of hMSCs and M2 macrophages was established in vitro. It was further use SGs inhibitor anisomicin to intervene M2 macrophages. The liver histopathology, liver function, reactive oxygen species (ROS) level, apoptosis pathway, endoplasmic reticulum stress (ERS) level, SGs markers (G3BP1/TIA-1), cGAS-STING pathway, TNF-α, IL-6, IL-1ß mRNA levels in liver tissue and M2 macrophages were observed. RESULTS: In vivo experiments, it showed that hMSCs could alleviate liver injury, inhibite the level of ROS, apoptosis and ERS, protect liver function in DILI mice. The mount of M2 was increased in the liver. hMSCs could also induce the production of SGs, inhibit the cGAS-STING pathway and reduce TNF-α, IL-6, IL-1ß mRNA expression. The results in vitro showed that hMSCs could induce the production of SGs in macrophages, inhibit the cGAS-STING pathway, promote the secretion of IL-4 and IL-13, and reduce TNF-α, IL-6, IL-1ß mRNA level in cells. In the process of IL-4 inducing M2 macrophage activation, anisomycin could inhibit the production of SGs, activate the cGAS-STING pathway, and promote the inflammatory factor TNF-α, IL-6, IL-1ß mRNA expression in cells. CONCLUSIONS: HMSCs had a protective effect on acute DILI in mice induced by APAP. Its mechanism might involve in activating M2 type macrophages, promoting the production of SGs, inhibiting the cGAS-STING pathway, and reducing the expression of pro-inflammatory factors in macrophages, to reduce hepatocytes damage.

A bivalent inhibitor against TDRD3 to suppress phase separation of methylated G3BP1.

The formation of membrane-less organelles is driven by multivalent weak interactions while mediation of such interactions by small molecules remains an unparalleled challenge. Here, we uncovered a bivalent inhibitor that blocked the recruitment of TDRD3 by the two methylated arginines of G3BP1. Relative to the monovalent inhibitor, this bivalent inhibitor demonstrated an enhanced binding affinity to TDRD3 and capability to suppress the phase separation of methylated G3BP1, TDRD3, and RNAs, and in turn inhibit the stress granule growth in cells. Our result paves a new path to mediate multivalent interactions involved in SG assembly for potential combinational chemotherapy by bivalent inhibitors.

Convergent gene pair dSH3 and irr regulate Pi and Fe homeostasis in Bradyrhizobium diazoefficiens USDA110 and symbiotic nitrogen fixation efficiency.

The nitrogen-fixing bacteroids inhabit inside legume root nodules must manage finely the utilization of P and Fe, the two most critical elements, due to their antagonistic interactions. While the balance mechanism for them remains unclear. A double SH3 domain-containing protein (dSH3) in the Bradyrhizobium diazoefficiens USDA110 was found to inhibit the alkaline phosphatase activity, thereby reducing P supply from organophosphates. The dSH3 gene is adjacent to the irr gene, which encodes the iron response repressor and regulates Fe homeostasis under Fe-limited conditions. Their transcription directions converge to a common intergenic sequence (IGS) region, forming a convergent transcription. Extending the IGS region through Tn5 transposon or pVO155 plasmid insertion significantly down-regulated expression of this gene pair, leading to a remarkable accumulation of P and an inability to grow under Fe-limited conditions. Inoculation of soybean with either of the insertion mutants resulted in N2-fixing failure. However, the IGS-deleted mutant showed no visible changes in N2-fixing efficiency on soybean compared to that inoculated with wild type. These findings reveal a novel regulative strategy in the IGS region and its flanking convergent gene pair for antagonistic utilization of P and Fe in rhizobia and coordination of N2-fixing efficiency.

DHX38 enhances proliferation, metastasis, and EMT progression in NSCLC through the G3BP1-mediated MAPK pathway.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a prevalent and aggressive malignancy with limited therapeutic options. Despite advances in treatment, NSCLC remains a major cause of cancer-related death worldwide. Tumor heterogeneity and therapy resistance present challenges in achieving remission. Research is needed to provide molecular insights, identify new targets, and develop personalized therapies to improve outcomes. METHODS: The protein expression level and prognostic value of DHX38 in NSCLC were explored in public databases and NSCLC tissue microarrays. DHX38 knockdown and overexpression cell lines were established to evaluate the role of DHX38 in NSCLC. In vitro and in vivo functional experiments were conducted to assess proliferation and metastasis. To determine the underlying molecular mechanism of DHX38 in human NSCLC, proteins that interact with DHX38 were isolated by IP and identified by LC-MS. KEGG analysis of DHX38-interacting proteins revealed the molecular pathway of DHX38 in human NSCLC. Abnormal pathway activation was verified by Western blot analysis and immunohistochemical (IHC) staining. A molecule-specific inhibitor was further used to explore potential therapeutic targets for NSCLC. The pathway-related target that interacted with DHX38 was verified by co-immunoprecipitation(co-IP) experiments. In cell lines with stable DHX38 overexpression, the target protein was knocked down to explore its complementary effect on DHX38 overexpression-induced tumor promotion. RESULTS: The protein expression of DHX38 was increased in NSCLC, and patients with high DHX38 expression levels had a poor prognosis. In vitro and in vivo experiments showed that DHX38 promoted the proliferation, migration and invasion of human NSCLC cells. DHX38 overexpression caused abnormal activation of the MAPK pathway and promoted epithelial-mesenchymal transition (EMT) in tumours. SCH772984, a novel specific ERK1/2 inhibitor, significantly reduced the increases in cell proliferation, migration and invasion caused by DHX38 overexpression. The co-IP experiments confirmed that DHX38 interacted with the Ras GTPase-activating protein-binding protein G3BP1. DHX38 regulated the expression of G3BP1. Knocking down G3BP1 in cells with stable DHX38 overexpression prevented DHX38-induced tumor cell proliferation, migration and invasion. Silencing G3BP1 reversed the MAPK pathway activation and EMT induced by DHX38 overexpression. CONCLUSION: In NSCLC, DHX38 functions as a tumor promoter. DHX38 modulates G3BP1 expression, leading to the activation of the MAPK signaling pathway, thus promoting tumor cell proliferation, metastasis, and the progression of epithelial-mesenchymal transition (EMT) in non-small cell lung cancer.

SIRT2 negatively regulates the cGAS-STING pathway by deacetylating G3BP1.

SIRT2, a cytoplasmic member of the Sirtuin family, has important roles in immunity and inflammation. However, its function in regulating the response to DNA virus infection remains elusive. Here, we find that SIRT2 is a unique regulator among the Sirtuin family that negatively modulates the cGAS-STING-signaling pathway. SIRT2 is down-regulated after Herpes simplex virus-1 (HSV-1) infection, and SIRT2 deficiency markedly elevates the expression levels of type I interferon (IFN). SIRT2 inhibits the DNA binding ability and droplet formation of cGAS by interacting with and deacetylating G3BP1 at K257, K276, and K376, leading to the disassembly of the cGAS-G3BP1 complex, which is critical for cGAS activation. Administration of AGK2, a selective SIRT2 inhibitor, protects mice from HSV-1 infection and increases the expression of IFN and IFN-stimulated genes. Our study shows that SIRT2 negatively regulates cGAS activation through G3BP1 deacetylation, suggesting a potential antiviral strategy by modulating SIRT2 activity.

Alphavirus-induced transcriptional and translational shutoffs play major roles in blocking the formation of stress granules.

IMPORTANCE: Our study highlights the mechanisms behind the cell's resistance to stress granule (SG) formation after infection with Old World alphaviruses. Shortly after infection, the replication of these viruses hinders the cell's ability to form SGs, even when exposed to chemical inducers such as sodium arsenite. This resistance is primarily attributed to virus-induced transcriptional and translational shutoffs, rather than interactions between the viral nsP3 and the key components of SGs, G3BP1/2, or the ADP-ribosylhydrolase activity of nsP3 macro domain. While interactions between G3BPs and nsP3 are essential for the formation of viral replication complexes, their role in regulating SG development appears to be small, if any. Cells harboring replicating viruses or replicons with lower abilities to inhibit transcription and/or translation, but expressing wild-type nsP3, retain the ability for SG development. Understanding these mechanisms of regulation of SG formation contributes to our knowledge of viral replication and the intricate relationships between alphaviruses and host cells.