Natural evidence of coronaviral 2′-O-methyltransferase activity affecting viral pathogenesis via improved substrate RNA binding

Previous studies through targeted mutagenesis of K-D-K-E motif have demonstrated that 2′-O-MTase activity is essential for efficient viral replication and immune evasion.However,the K-D-K-E catalytic motif of 2′-O-MTase is highly conserved across numerous viruses,including flaviviruses,vaccinia viruses,coronaviruses,and extends even to mammals.Here,we observed a stronger 2′-O-MTase activity in SARS-CoV-2 compared to SARS-CoV,despite the presence of a consistently active catalytic center.We further identified critical residues(Leu-36,Asn-138 and Ile-153)which served as determinants of discrepancy in 2′-O-MTase activity between SARS-CoV-2 and SARS-CoV.These residues significantly enhanced the RNA binding affinity of 2′-O-MTase and boosted its versatility toward RNA substrates.Of interest,a triple substitution(Leu^(36)→Ile^(36),Asn^(138)→His^(138),Ile^(153)→Leu^(153),from SARS-CoV-2 to SARS-CoV)within nsp16 resulted in a proportional reduction in viral 2′-O-methylation and impaired viral replication.Furthermore,it led to a significant upregulation of type I interferon(IFN-I)and proinflammatory cytokines both in vitro and vivo,relying on the cooperative sensing of melanoma differentiation-associated protein 5(MDA5)and laboratory of genetics and physiology 2(LGP2).In conclusion,our findings demonstrated that alterations in residues other than K-D-K-E of 2′-O-MTase may affect viral replication and subsequently influence pathogenesis.Monitoring changes in nsp16 residues is crucial as it may aid in identifying and assessing future alteration in viral pathogenicity resulting from natural mutations occurring in nsp16.

C-reactive protein to albumin ratio predict responses to programmed cell death-1 inhibitors in hepatocellular carcinoma patients

BACKGROUND Over the years,programmed cell death-1(PD-1)inhibitors have been routinely used for hepatocellular carcinoma(HCC)treatment and yielded improved survival outcomes.Nonetheless,significant heterogeneity surrounds the outcomes of most studies.Therefore,it is critical to search for biomarkers that predict the efficacy of PD-1 inhibitors in patients with HCC.AIM To investigate the role of the C-reactive protein to albumin ratio(CAR)in evaluating the efficacy of PD-1 inhibitors for HCC.METHODS The clinical data of 160 patients with HCC treated with PD-1 inhibitors from January 2018 to November 2022 at the First Affiliated Hospital of Guangxi Medical University were retrospectively analyzed.RESULTS The optimal cut-off value for CAR based on progression-free survival(PFS)was determined to be 1.20 using x-tile software.Cox proportional risk model was used to determine the factors affecting prognosis.Eastern Cooperative Oncology Group performance status[hazard ratio(HR)=1.754,95%confidence interval(95%CI)=1.045-2.944,P=0.033],CAR(HR=2.118,95%CI=1.057-4.243,P=0.034)and tumor number(HR=2.932,95%CI=1.246-6.897,P=0.014)were independent prognostic factors for overall survival.CAR(HR=2.730,95%CI=1.502-4.961,P=0.001),tumor number(HR=1.584,95%CI=1.003-2.500,P=0.048)and neutrophil to lymphocyte ratio(HR=1.120,95%CI=1.022-1.228,P=0.015)were independent prognostic factors for PFS.Two nomograms were constructed based on independent prognostic factors.The C-index index and calibration plots confirmed that the nomogram is a reliable risk prediction tool.The ROC curve and decision curve analysis confirmed that the nomogram has a good predictive effect as well as a net clinical benefit.CONCLUSION Overall,we reveal that the CAR is a potential predictor of short-and long-term prognosis in patients with HCC treated with PD-1 inhibitors.If further verified,CAR-based nomogram may increase the number of markers that predict individualized prognosis.

Radiomics and nomogram of magnetic resonance imaging for preoperative prediction of microvascular invasion in small hepatocellular carcinoma

BACKGROUND Microvascular invasion(MVI)of small hepatocellular carcinoma(sHCC)(≤3.0 cm)is an independent prognostic factor for poor progression-free and overall survival.Radiomics can help extract imaging information associated with tumor pathophysiology.AIM To develop and validate radiomics scores and a nomogram of gadolinium ethoxybenzyl-diethylenetriamine pentaacetic acid(Gd-EOB-DTPA)-enhanced magnetic resonance imaging(MRI)for preoperative prediction of MVI in sHCC.METHODS In total,415 patients were diagnosed with sHCC by postoperative pathology.A total of 221 patients were retrospectively included from our hospital.In addition,we recruited 94 and 100 participants as independent external validation sets from two other hospitals.Radiomics models of Gd-EOB-DTPA-enhanced MRI and diffusion-weighted imaging(DWI)were constructed and validated using machine learning.As presented in the radiomics nomogram,a prediction model was developed using multivariable logistic regression analysis,which included radiomics scores,radiologic features,and clinical features,such as the alpha-fetoprotein(AFP)level.The calibration,decision-making curve,and clinical usefulness of the radiomics nomogram were analyzed.The radiomic nomogram was validated using independent external cohort data.The areas under the receiver operating curve(AUC)were used to assess the predictive capability.RESULTS Pathological examination confirmed MVI in 64(28.9%),22(23.4%),and 16(16.0%)of the 221,94,and 100 patients,respectively.AFP,tumor size,non-smooth tumor margin,incomplete capsule,and peritumoral hypointensity in hepatobiliary phase(HBP)images had poor diagnostic value for MVI of sHCC.Quantitative radiomic features(1409)of MRI scans)were extracted.The classifier of logistic regression(LR)was the best machine learning method,and the radiomics scores of HBP and DWI had great diagnostic efficiency for the prediction of MVI in both the testing set(hospital A)and validation set(hospital B,C).The AUC of HBP was 0.979,0.970,and 0.803,respectively,and the AUC of DWI was 0.971,0.816,and 0.801(P<0.05),respectively.Good calibration and discrimination of the radiomics and clinical combined nomogram model were exhibited in the testing and two external validation cohorts(C-index of HBP and DWI were 0.971,0.912,0.808,and 0.970,0.843,0.869,respectively).The clinical usefulness of the nomogram was further confirmed using decision curve analysis.CONCLUSION AFP and conventional Gd-EOB-DTPA-enhanced MRI features have poor diagnostic accuracies for MVI in patients with sHCC.Machine learning with an LR classifier yielded the best radiomics score for HBP and DWI.The radiomics nomogram developed as a noninvasive preoperative prediction method showed favorable predictive accuracy for evaluating MVI in sHCC.

Interferon-γ induces immunosuppression in salivary adenoid cystic carcinoma by regulating programmed death ligand 1 secretion

Interferon-γ(IFN-γ), a key effector molecule in anti-tumor immune response, has been well documented to correlate with the intratumoral infiltration of immune cells. Of interest, however, a high level of IFN-γ has been reported in salivary adenoid cystic carcinoma(SACC), which is actually a type of immunologically cold cancer with few infiltrated immune cells. Investigating the functional significance of IFN-γ in SACC would help to explain such a paradoxical phenomenon. In the present study, we revealed that,compared to oral squamous cell carcinoma cells(a type of immunologically hot cancer), SACC cells were less sensitive to the growthinhibition effect of IFN-γ. Moreover, the migration and invasion abilities of SACC cells were obviously enhanced upon IFN-γ treatment.In addition, our results revealed that exposure to IFN-γ significantly up-regulated the level of programmed death ligand 1(PD-L1) on SACC cell-derived small extracellular vesicles(s EVs), which subsequently induced the apoptosis of CD8+T cells through antagonizing PD-1. Importantly, it was also found that SACC patients with higher levels of plasma IFN-γ also had higher levels of circulating s EVs that carried PD-L1 on their surface. Our study unveils a mechanism that IFN-γ induces immunosuppression in SACC via s EV PD-L1,which would account for the scarce immune cell infiltration and insensitivity to immunotherapy.

Di-and tri-methylation of histone H3K36 play distinct roles in DNA double-strand break repair

Histone H3 Lys36(H3K36)methylation and its associated modifiers are crucial for DNA double-strand break(DSB)repair,but the mechanism governing whether and how different H3K36 methylation forms impact repair pathways is unclear.Here,we unveil the distinct roles of H3K36 dimethylation(H3K36me2)and H3K36 trimethylation(H3K36me3)in DSB repair via non-homologous end joining(NHEJ)or homologous recombination(HR).Yeast cells lacking H3K36me2 or H3K36me3 exhibit reduced NHEJ or HR efficiency.y Ku70 and Rfa1 bind H3K36me2-or H3K36me3-modified peptides and chromatin,respectively.Disrupting these interactions impairs y Ku70 and Rfa1 recruitment to damaged H3K36me2-or H3K36me3-rich loci,increasing DNA damage sensitivity and decreasing repair efficiency.Conversely,H3K36me2-enriched intergenic regions and H3K36me3-enriched gene bodies independently recruit y Ku70 or Rfa1 under DSB stress.Importantly,human KU70 and RPA1,the homologs of y Ku70 and Rfa1,exclusively associate with H3K36me2 and H3K36me3 in a conserved manner.These findings provide valuable insights into how H3K36me2 and H3K36me3 regulate distinct DSB repair pathways,highlighting H3K36 methylation as a critical element in the choice of DSB repair pathway.

Disulfiram ameliorates STING/MITA-dependent inflammation and autoimmunity by targeting RNF115

STING(also known as MITA)is an adaptor protein that mediates cytoplasmic DNA-triggered signaling,and aberrant activation of STING/MITA by cytosolic self-DNA or gain-of-function mutations causes severe inflammation.Here,we show that STING-mediated inflammation and autoimmunity are promoted by RNF115 and alleviated by the RNF115 inhibitor disulfiram(DSF).Knockout of RNF115 or treatment with DSF significantly inhibit systemic inflammation and autoimmune lethality and restore immune cell development in Trex1^(–/–)mice and STING^(N153S/WT) bone marrow chimeric mice.In addition,knockdown or pharmacological inhibition of RNF115 substantially downregulate the expression of IFN-α,IFN-γand proinflammatory cytokines in PBMCs from patients with systemic lupus erythematosus(SLE)who exhibit high concentrations of dsDNA in peripheral blood.Mechanistically,knockout or inhibition of RNF115 impair the oligomerization and Golgi localization of STING in various types of cells transfected with cGAMP and in organs and cells from Trex1^(–/–)mice.Interestingly,knockout of RNF115 inhibits the activation and Golgi localization of STINGN153S as well as the expression of proinflammatory cytokines in myeloid cells but not in endothelial cells or fibroblasts.Taken together,these findings highlight the RNF115-mediated cell type-specific regulation of STING and STINGN153S and provide potential targeted intervention strategies for STING-related autoimmune diseases.

Ptip safeguards the epigenetic control of skeletal stem cell quiescence and potency in skeletogenesis

Stem cells remain in a quiescent state for long-term maintenance and preservation of potency;this process requires fine-tuning regulatory mechanisms.In this study,we identified the epigenetic landscape along the developmental trajectory of skeletal stem cells(SSCs)in skeletogenesis governed by a key regulator,Ptip(also known as Paxip1,Pax interaction with transcription-activation domain protein-1).Our results showed that Ptip is required for maintaining the quiescence and potency of SSCs,and loss of Ptip in type II collagen(Col2)^(+)progenitors causes abnormal activation and differentiation of SSCs,impaired growth plate morphogenesis,and long bone dysplasia.We also found that Ptip suppressed the glycolysis of SSCs through downregulation of phosphoglycerate kinase 1(Pgk1)by repressing histone H3 lysine 27 acetylation(H3K27ac)at the promoter region.Notably,inhibition of glycolysis improved the function of SSCs despite Ptip deficiency.To the best of our knowledge,this is the first study to establish an epigenetic framework based on Ptip,which safeguards skeletal stem cell quiescence and potency through metabolic control.This framework is expected to improve SSC-based treatments of bone developmental disorders.

Dental stem cell-derived extracellular vesicles as promising therapeutic agents in the treatment of diseases

Dental stem cells(DSCs), an important source of mesenchymal stem cells(MSCs), can be easily obtained by minimally invasive procedures and have been used for the treatment of various diseases. Classic paradigm attributed the mechanism of their therapeutic action to direct cell differentiation after targeted migration, while contemporary insights into indirect paracrine effect opened new avenues for the mystery of their actual low engraftment and differentiation ability in vivo. As critical paracrine effectors, DSC-derived extracellular vesicles(DSC-EVs) are being increasingly linked to the positive effects of DSCs by an evolving body of in vivo studies. Carrying bioactive contents and presenting therapeutic potential in certain diseases, DSC-EVs have been introduced as promising treatments. Here, we systematically review the latest in vivo evidence that supports the therapeutic effects of DSC-EVs with mechanistic studies. In addition, current challenges and future directions for the clinical translation of DSC-EVs are also highlighted to call for more attentions to the(Ⅰ) distinguishing features of DSC-EVs compared with other types of MSC-EVs,(Ⅱ)heterogeneity among different subtypes of DSC-derived EVs,(Ⅲ) action modes of DSC-EVs,(Ⅳ) standardization for eligible DSC-EVs and(Ⅴ) safety guarantee for the clinical application of DSC-EVs. The present review would provide valuable insights into the emerging opportunities of DSC-EVs in future clinical applications.

Resolving the lineage relationship between malignant cells and vascular cells in glioblastomas

Glioblastoma multiforme(GBM),a highly malignant and heterogeneous brain tumor,contains various types of tumor and non-tumor cells.Whether GBM cells can trans-differentiate into non-neural cell types,including mural cells or endothelial cells(ECs),to support tumor growth and invasion remains controversial.Here we generated two genetic GBM models de novo in immunocompetent mouse brains,mimicking essential pathological and molecular features of human GBMs.Lineage-tracing and transplantation studies demonstrated that,although blood vessels in GBM brains underwent drastic remodeling,evidence of trans-differentiation of GBM cells into vascular cells was barely detected.Intriguingly,GBM cells could promiscuously express markers for mural cells during gliomagenesis.Furthermore,single-cell RNA sequencing showed that patterns of copy number variations(CNVs)of mural cells and ECs were distinct from those of GBM cells,indicating discrete origins of GBM cells and vascular components.Importantly,single-cell CNV analysis of human GBM specimens also suggested that GBM cells and vascular cells are likely separate lineages.Rather than expansion owing to trans-differentiation,vascular cell expanded by proliferation during tumorigenesis.Therefore,cross-lineage trans-differentiation of GBM cells is very unlikely to occur during gliomagenesis.Our findings advance understanding of cell lineage dynamics during gliomagenesis,and have implications for targeted treatment of GBMs.

Amelioration of ethanol-induced oxidative stress and alcoholic liver disease by in vivo RNAi targeting Cyp2e1

Alcoholic liver disease(ALD)results from continuous and heavy alcohol consumption.The current treatment strategy for ALD is based on alcohol withdrawal coupled with antioxidant drug intervention,which is a long process with poor efficacy and low patient compliance.Alcohol-induced CYP2E1 upregulation has been demonstrated as a key regulator of ALD,but CYP2E1 knockdown in humans was impractical,and pharmacological inhibition of CYP2E1 by a clinically relevant approach for treating ALD was not shown.In this study,we developed a RNAi therapeutics delivered by lipid nanoparticle,and treated mice fed on Lieber-DeCarli ethanol liquid diet weekly for up to 12 weeks.This RNAi-based inhibition of Cyp2e1 expression reduced reactive oxygen species and oxidative stress in mouse livers,and contributed to improved ALD symptoms in mice.The liver fat accumulation,hepatocyte inflammation,and fibrosis were reduced in ALD models.Therefore,this study suggested the feasibility of RNAi targeting to CYP2E1 as a potential therapeutic tool to the development of ALD.

Mitochondrial DNA-triggered innate immune response:mechanisms and diseases

Various cellular stress conditions trigger mitochondrial DNA(mtDNA)release from mitochondria into the cytosol.The released mtDNA is sensed by the cGAS-MITA/STING pathway,resulting in the induced expression of type I interferon and other effector genes.These processes contribute to the innate immune response to viral infection and other stress factors.The deregulation of these processes causes autoimmune diseases,inflammatory metabolic disorders and cancer.Therefore,the cGAS-MITA/STING pathway is a potential target for intervention in infectious,inflammatory and autoimmune diseases as well as cancer.In this review,we focus on the mechanisms underlying the mtDNA-triggered activation of the cGAS-MITA/STING pathway,the effects of the pathway under various physiological and pathological conditions,and advances in the development of drugs that target cGAS and MITA/STING.

Combined targeting of CCL7 and Flt3L to promote the expansion and infiltration of cDC1s in tumors enhances T-cell activation and anti-PD-1 therapy effectiveness in NSCLC

mmune checkpoint inhibitors(ICIs),represented by anti-PD-1/PD-L1 antibodies,have been widely applied in various cancers,and the efficacy of ICIs is closely associated with the tumor immune microenvironment(TIME)[1,2].We previously demonstrated that the alveolar macrophage-derived chemokine CCL7 recruited conventional type 1 dendritic cells(cDC1s)to remodel the TIME,thereby promoting the expansion of T cells to inhibit non-small cell lung cancer(NSCLC)progression in KrasLSL-G12D/+Tp53fl/fl(KP)and KrasLSL-G12D/+Lkb1fl/fl(KL)mouse models[3].Here,we showed that the fusion protein PD-1Ab7,in which CCL7 was fused with the single-chain variable fragment region(scFv)of an anti-PD-1 antibody(PD-1Ab),exhibited antitumor activity superior to that of PD-1Ab in a manner dependent on cDC1s.In addition,Fms-like tyrosine kinase 3 ligand(Flt3L)synergized with PD-1Ab7 to inhibit NSCLC progression in both the KP and the KL mouse models.Mechanistically,Flt3L promoted the generation and proliferation of cDC1s,whereas PD-1Ab7 increased the infiltration and migration of cDC1s in the TIME to potentiate the activation and proliferation of T cells.These findings not only highlight the essential roles of the PD-1Ab-based chemokine fusion strategy in targeting cDC1s and T cells to potentiate the efficacy of ICIs for cancer prevention but also provide therapeutic lead molecules for antitumor therapy.

Efficient non-viral delivery of macromolecules in human primary hematopoietic stem cells and lymphocytes

Dear Editor,Electroporation(EP)is a non-viral method for introducing macromolecules such as DNA,protein,and messenger RNA(mRNA)into mammalian cells for both basic research and ex vivo therapy(Glass et al.,2018).Due to its nature,EP offers great promise as a delivery method for gene therapy without causing risks associated with the viral vector-mediated delivery(Yin et al.,2017;Qiu et al.,2022).

SEL1L preserves CD8^(+) T-cell survival and homeostasis by fine-tuning PERK signaling and the IL-15 receptor-mediated mTORC1 axis

SEL1L-mediated endoplasmic reticulum-associated degradation(ERAD)plays critical roles in controlling protein homeostasis by degrading misfolded or terminal unfolded proteins.However,it remains unclear how SEL1L regulates peripheral T-cell survival and homeostasis.Herein,we found that SEL1L deficiency led to a greatly reduced frequency and number of mature T cells,which was further validated by adoptive transfer experiments or bone marrow chimera experiments,accompanied by the induction of multiple forms of cell death.Furthermore,SEL1L deficiency selectively disrupted naïve CD8+T-cell homeostasis,as indicated by the severe loss of the naïve T-cell subset but an increase in the memory T-cell subset.We also found that SEL1L deficiency fueled mTORC1/c-MYC activation and induced a metabolic shift,which was largely attributable to enhanced expression of the IL-15 receptorαandβchains.Mechanistically,single-cell transcriptomic profiling and biochemical analyses further revealed that Sel1l−/−CD8+T cells harbored excessive ER stress,particularly aberrant activation of the PERK-ATF4-CHOP-Bim pathway,which was alleviated by supplementing IL-7 or IL-15.Importantly,PERK inhibition greatly resolved the survival defects of Sel1l−/−CD8+T cells.In addition,IRE1αdeficiency decreased mTORC1 signaling in Sel1l−/−naïve CD8+T cells by downregulating the IL-15 receptorαchain.Altogether,these observations suggest that the ERAD adaptor molecule SEL1L acts as an important checkpoint for preserving the survival and homeostasis of peripheral T cells by regulating the PERK signaling cascade and IL-15 receptor-mediated mTORC1 axis.

A Non-canonical Excitatory PV RGC–PV SC Visual Pathway for Mediating the Looming-evoked Innate Defensive Response

Parvalbumin-positive retinal ganglion cells(PV+RGCs)are an essential subset of RGCs found in various species.However,their role in transmitting visual information remains unclear.Here,we characterized PV+RGCs in the retina and explored the functions of the PV+RGC-mediated visual pathway.By applying multiple viral tracing strategies,we investigated the downstream of PV+RGCs across the whole brain.Interestingly,we found that the PV+RGCs provided direct monosynaptic input to PV+excitatory neurons in the superficial layers of the superior colliculus(SC).Ablation or suppression of SC-projecting PV+RGCs abolished or severely impaired the flight response to looming visual stimuli in mice without affecting visual acuity.Furthermore,using transcriptome expression profiling of individual cells and immunofluorescence colocalization for RGCs,we found that PV+RGCs are predominant glutamatergic neurons.Thus,our findings indicate the critical role of PV+RGCs in an innate defensive response and suggest a non-canonical subcortical visual pathway from excitatory PV+RGCs to PV+SC neurons that regulates looming visual stimuli.These results provide a potential target for intervening and treating diseases related to this circuit,such as schizophrenia and autism.

Mettl3依赖的m^(6)A甲基化调控CD8^(+)T细胞效应分化和记忆形成

Efficient immune responses rely on the proper differentiation of CD8^(+)T cells into effector and memory cells.Here,we show a critical requirement of N^6-Methyladenosine(m^(6)A)methyltransferase Mettl3 during CD8^(+)T cell responses upon acute viral infection.Conditional deletion of Mettl3 in CD8^(+)T cells impairs effector expansion and terminal differentiation in an m^(6)A-dependent manner,subsequently affecting memory formation and the secondary response of CD8^(+)T cells.Our combined RNA-seq and m^(6)AmiCLIP-seq analyses reveal that Mettl3 deficiency broadly impacts the expression of cell cycle and transcriptional regulators.Remarkably,Mettl3 binds to the Tbx21 transcript and stabilizes it,promoting effector differentiation of CD8^(+)T cells.Moreover,ectopic expression of T-bet partially restores the defects in CD8^(+)T cell differentiation in the absence of Mettl3.Thus,our study highlights the role of Mettl3 in regulating multiple target genes in an m^(6)A-dependent manner and underscores the importance of m^(6)A modification during CD8^(+)T cell response.

C1QTNF5 is a novel attachment factor that facilitates the entry of influenza A virus

Influenza A virus(IAV)binds sialic acid receptors on the cell surface to enter the host cells,which is the key step in initiating infection,transmission and pathogenesis.Understanding the factors that contribute to the highly efficient entry of IAV into human cells will help elucidate the mechanism of viral entry and pathogenicity,and provide new targets for intervention.In the present study,we reported a novel membrane protein,C1QTNF5,which binds to the hemagglutinin protein of IAV and promotes IAV infection in vitro and in vivo.We found that the HA1 region of IAV hemagglutinin is critical for the interaction with C1QTNF5 protein,and C1QTNF5 interacts with hemagglutinin mainly through its N-terminus(1–103 aa).In addition,we further demonstrated that overexpression of C1QTNF5 promotes IAV entry,while blocking the interaction between C1QTNF5 and IAV hemagglutinin greatly inhibits viral entry.However,C1QTNF5 does not function as a receptor to mediate IAV infection in sialic acid-deficient CHO-Lec2 cells,but promotes IAV to attach to these cells,suggesting that C1QTNF5 is an important attachment factor for IAV.This work reveals C1QTNF5 as a novel IAV attachment factor and provides a new perspective for antiviral strategies.

Flux Balance Analysis Reveals Potential Anti-HIV-1 Metabolic Targets

Background:Human immunodeficiency virus type 1(HIV-1)remains a persistent global health challenge.Therefore,a continuous exploration of novel therapeutic strategies is essential.A comprehensive understanding of how HIV-1 utilizes the cellular metabolism machinery for replication can provide insights into new therapeutic approaches.Methods:In this study,we performed a flux balance analysis using a genome-scale metabolic model(GEM)integrated with an HIV-1 viral biomass objective function to identify potential targets for anti–HIV-1 interventions.We generated a GEM by integrating an HIV-1 production reaction into CD4+T cells and optimized for both host and virus optimal states as objective functions to depict metabolic profiles of cells in the status for optimal host biomass maintenance or for optimal HIV-1 virion production.Differential analysis was used to predict biochemical reactions altered optimal for HIV-1 production.In addition,we conducted in silico simulations involving gene and reaction knock-outs to identify potential anti–HIV-1 targets,which were subsequently validated by human phytohemagglutinin(PHA)blasts infected with HIV-1.Results:Differential analysis identified several altered biochemical reactions,including increased lysine uptake and oxidative phosphorylation(OXPHOS)activities in the virus optima compared with the host optima.In silico gene and reaction knock-out simulations revealed de novo pyrimidine synthesis,and OXPHOS could serve as potential anti–HIV-1 metabolic targets.In vitro assay confirmed that targeting OXPHOS using metformin could suppress the replication of HIV-1 by 56.6%(385.4±67.5 pg/mL in the metformintreated group vs.888.4±32.3 pg/mL in the control group,P<0.001).Conclusion:Our integrated host-virus genome-scale metabolic study provides insights on potential targets(OXPHOS)for anti-HIV therapies.

Dephosphorylation of cGAS by PPP6C impairs its substrate binding activity and innate antiviral response

The cyclic GMP-AMP (cGAMP) synthase (cGAS) plays a critical role in host defense by sensing cytosollc DNA derived from microbial pathogens or mls-located cellular DNA.Upon DNA binding,cGAS utilizes GTP and ATP as substrates to synthesize cGAMP,leading to MITA-mediated innate immune response.In this study,we identified the phosphatase PPP6C as a negative regulator of cGASmediated innate immune response.PPP6C is constitutively associated with cGAS in un-stimulated cells.DNA virus infection causes rapid disassociation of PPP6C from cGAS,resulting in phosphorylation of human cGAS S435 or mouse cGAS S420 in its catalytic pocket.Mutation of this eerine residue of cGAS impairs its ability to synthesize cGAMP upon DNA virus infection.In vitro experiments indicate that S420-phosphorylated mcGAS has higher affinity to GTP and enzymatic activity.PPP6Cdeficiency promotes innate immune response to DNA virus in various cells.Our findings suggest that PPP6Cmediated dephosphorylation of a catalytic pocket serine residue of cGAS impairs its substrate binding activity and innate immune response,which provides a mechanism for keeping the DNA sensor cGAS inactive in the absence of infection to avoid autoimmune response.

The membrane-associated E3 ubiquitin ligase MARCH3 downregulates the IL-6 receptor and suppresses colitis-associated carcinogenesis

The IL-6-STAT3 axis is critically involved in inflammation-associated carcinogenesis(IAC).How this axis is regulated to modulate IAC remains unknown.Here,we show that the plasma membrane-associated E3 ubiquitin ligase MARCH3 negatively regulates STAT3 activation triggered by IL-6,as well as another IL-6 subfamily member,Oncostatin M(OSM).MARCH3 is associated with the IL-6 receptorα-chain(IL-6Rα)and its coreceptor gp130.Biochemical experiments indicated that MARCH3 mediates the polyubiquitination of IL-6Rαat K401 and gp130 at K849 following IL-6 stimulation,leading to their translocation to and degradation in lysosomes.MARCH3 deficiency increases IL-6-and OSM-triggered activation of STAT3 and induction of downstream effector genes in various cell types.MARCH3 deficiency enhances dextran sulfate sodium(DSS)-induced STAT3 activation,increases the expression of inflammatory cytokines,and exacerbates colitis,as well as azoxymethane(AOM)/DSS-induced colitis-associated cancer in mice.In addition,MARCH3 is downregulated in human colorectal cancer tissues and associated with poor survival across different cancer types.Our findings suggest that MARCH3 is a pivotal negative regulator of IL-6-induced STAT3 activation,inflammation,and inflammation-associated carcinogenesis.