Primate hemorrhagic fever-causing arteriviruses are poised for spillover to humans.

Simian arteriviruses are endemic in some African primates and can cause fatal hemorrhagic fevers when they cross into primate hosts of new species. We find that CD163 acts as an intracellular receptor for simian hemorrhagic fever virus (SHFV; a simian arterivirus), a rare mode of virus entry that is shared with other hemorrhagic fever-causing viruses (e.g., Ebola and Lassa viruses). Further, SHFV enters and replicates in human monocytes, indicating full functionality of all of the human cellular proteins required for viral replication. Thus, simian arteriviruses in nature may not require major adaptations to the human host. Given that at least three distinct simian arteriviruses have caused fatal infections in captive macaques after host-switching, and that humans are immunologically naive to this family of viruses, development of serology tests for human surveillance should be a priority.

iRhom2 is essential for innate immunity to DNA viruses by mediating trafficking and stability of the adaptor STING.

STING is a central adaptor in the innate immune response to DNA viruses. However, the manner in which STING activity is regulated remains unclear. We identified iRhom2 ('inactive rhomboid protein 2') as a positive regulator of DNA-virus-triggered induction of type I interferons. iRhom2 deficiency markedly impaired DNA-virus- and intracellular-DNA-induced signaling in cells, and iRhom2-deficient mice were more susceptible to lethal herpes simplex virus type 1 (HSV-1) infection. iRhom2 was constitutively associated with STING and acted in two distinct processes to regulate STING activity. iRhom2 recruited the translocon-associated protein TRAPß to the STING complex to facilitate trafficking of STING from the endoplasmic reticulum to perinuclear microsomes. iRhom2 also recruited the deubiquitination enzyme EIF3S5 to maintain the stability of STING through removal of its K48-linked polyubiquitin chains. These results suggest that iRhom2 is essential for STING activity, as it regulates TRAPß-mediated translocation and EIF3S5-mediated deubiquitination of STING.

Structural basis for directional chitin biosynthesis.

Chitin, the most abundant aminopolysaccharide in nature, is an extracellular polymer consisting of N-acetylglucosamine (GlcNAc) units1. The key reactions of chitin biosynthesis are catalysed by chitin synthase2-4, a membrane-integrated glycosyltransferase that transfers GlcNAc from UDP-GlcNAc to a growing chitin chain. However, the precise mechanism of this process has yet to be elucidated. Here we report five cryo-electron microscopy structures of a chitin synthase from the devastating soybean root rot pathogenic oomycete Phytophthora sojae (PsChs1). They represent the apo, GlcNAc-bound, nascent chitin oligomer-bound, UDP-bound (post-synthesis) and chitin synthase inhibitor nikkomycin Z-bound states of the enzyme, providing detailed views into the multiple steps of chitin biosynthesis and its competitive inhibition. The structures reveal the chitin synthesis reaction chamber that has the substrate-binding site, the catalytic centre and the entrance to the polymer-translocating channel that allows the product polymer to be discharged. This arrangement reflects consecutive key events in chitin biosynthesis from UDP-GlcNAc binding and polymer elongation to the release of the product. We identified a swinging loop within the chitin-translocating channel, which acts as a 'gate lock' that prevents the substrate from leaving while directing the product polymer into the translocating channel for discharge to the extracellular side of the cell membrane. This work reveals the directional multistep mechanism of chitin biosynthesis and provides a structural basis for inhibition of chitin synthesis.

The Zinc-Finger Protein ZCCHC3 Binds RNA and Facilitates Viral RNA Sensing and Activation of the RIG-I-like Receptors.

Recognition of viral RNA by the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) initiates innate antiviral immune response. How the binding of viral RNA to and activation of the RLRs are regulated remains enigmatic. In this study, we identified ZCCHC3 as a positive regulator of the RLRs including RIG-I and MDA5. ZCCHC3 deficiency markedly inhibited RNA virus-triggered induction of downstream antiviral genes, and ZCCHC3-deficient mice were more susceptible to RNA virus infection. ZCCHC3 was associated with RIG-I and MDA5 and functions in two distinct processes for regulation of RIG-I and MDA5 activities. ZCCHC3 bound to dsRNA and enhanced the binding of RIG-I and MDA5 to dsRNA. ZCCHC3 also recruited the E3 ubiquitin ligase TRIM25 to the RIG-I and MDA5 complexes to facilitate its K63-linked polyubiquitination and activation. Thus, ZCCHC3 is a co-receptor for RIG-I and MDA5, which is critical for RLR-mediated innate immune response to RNA virus.

Genome enrichment of rare and unknown species from complicated microbiomes by nanopore selective sequencing.

Rare species are vital members of a microbial community, but retrieving their genomes is difficult because of their low abundance. The ReadUntil (RU) approach allows nanopore devices to sequence specific DNA molecules selectively in real time, which provides an opportunity for enriching rare species. Despite the robustness of enriching rare species by reducing the sequencing depth of known host sequences, such as the human genome, there is still a gap in RU-based enriching of rare species in environmental samples whose community composition is unclear, and many rare species have poor or incomplete reference genomes in public databases. Therefore, here we present metaRUpore to overcome this challenge. When we applied metaRUpore to a thermophilic anaerobic digester (TAD) community and human gut microbial community, it reduced coverage of the high-abundance populations and modestly increased (∼2×) the genome coverage of the rare taxa, facilitating successful recovery of near-finished metagenome-assembled genomes (nf-MAGs) of rare species. The simplicity and robustness of the approach make it accessible for laboratories with moderate computational resources, and hold the potential to become the standard practice in future metagenomic sequencing of complicated microbiomes.

Lysozyme 1 Inflamed CCR2+ Macrophages Promote Obesity-Induced Cardiac Dysfunction.

BACKGROUND: Macrophages are key players in obesity-associated cardiovascular diseases, which are marked by inflammatory and immune alterations. However, the pathophysiological mechanisms underlying macrophage's role in obesity-induced cardiac inflammation are incompletely understood. Our study aimed to identify the key macrophage population involved in obesity-induced cardiac dysfunction and investigate the molecular mechanism that contributes to the inflammatory response. METHODS AND RESULTS: Though analyzing in-depth cardiac macrophage clusters identified by single macrophage RNA-sequencing, we find that the Ccr2 cluster undergoes a functional transition from homeostatic maintenance to proinflammation. Our data highlight specific changes in macrophage behavior during cardiac dysfunction under metabolic challenge. Consistently, inducible ablation of CCR2+CX3CR1+ macrophages by a dual recombinase-based lineage-tracing approach or selective deletion of macrophage C-C chemokine receptor 2 (CCR2) prevents obesity-induced cardiac dysfunction. At the mechanistic level, we demonstrate that the obesity-induced functional shift of CCR2-expressing macrophages is mediated by the CCR2/ATF3/lysozyme 1/NF-κB (nuclear factor kappa B) signaling. Finally, we uncover a noncanonical role for lysozyme 1 as a transcription activator, binding to the RelA promoter, driving NF-κB signaling, and strongly promoting inflammation and cardiac dysfunction in obesity. CONCLUSIONS: Our findings suggest that lysozyme 1 may represent a potential target for the diagnosis of obesity-induced inflammation and the treatment of obesity-induced heart disease.

Protein Kinase A Is a Master Regulator of Physiological and Pathological Cardiac Hypertrophy.

BACKGROUND: The sympathoadrenergic system and its major effector PKA (protein kinase A) are activated to maintain cardiac output coping with physiological or pathological stressors. If and how PKA plays a role in physiological cardiac hypertrophy (PhCH) and pathological CH (PaCH) are not clear. METHODS: Transgenic mouse models expressing the PKA inhibition domain (PKAi) of PKA inhibition peptide alpha (PKIalpha)-green fluorescence protein (GFP) fusion protein (PKAi-GFP) in a cardiac-specific and inducible manner (cPKAi) were used to determine the roles of PKA in physiological CH during postnatal growth or induced by swimming, and in PaCH induced by transaortic constriction (TAC) or augmented Ca2+ influx. Kinase profiling was used to determine cPKAi specificity. Echocardiography was used to determine cardiac morphology and function. Western blotting and immunostaining were used to measure protein abundance and phosphorylation. Protein synthesis was assessed by puromycin incorporation and protein degradation by measuring protein ubiquitination and proteasome activity. Neonatal rat cardiomyocytes (NRCMs) infected with AdGFP (GFP adenovirus) or AdPKAi-GFP (PKAi-GFP adenovirus) were used to determine the effects and mechanisms of cPKAi on myocyte hypertrophy. rAAV9.PKAi-GFP was used to treat TAC mice. RESULTS: (1) cPKAi delayed postnatal cardiac growth and blunted exercise-induced PhCH; (2) PKA was activated in hearts after TAC due to activated sympathoadrenergic system, the loss of endogenous PKIα (PKA inhibition peptide α), and the stimulation by noncanonical PKA activators; (3) cPKAi ameliorated PaCH induced by TAC and increased Ca2+ influxes and blunted neonatal rat cardiomyocyte hypertrophy by isoproterenol and phenylephrine; (4) cPKAi prevented TAC-induced protein synthesis by inhibiting mTOR (mammalian target of rapamycin) signaling through reducing Akt (protein kinase B) activity, but enhancing inhibitory GSK-3α (glycogen synthase kinase-3α) and GSK-3ß signals; (5) cPKAi reduced protein degradation by the ubiquitin-proteasome system via decreasing RPN6 phosphorylation; (6) cPKAi increased the expression of antihypertrophic atrial natriuretic peptide (ANP); (7) cPKAi ameliorated established PaCH and improved animal survival. CONCLUSIONS: Cardiomyocyte PKA is a master regulator of PhCH and PaCH through regulating protein synthesis and degradation. cPKAi can be a novel approach to treat PaCH.

A genomic and epigenomic atlas of prostate cancer in Asian populations.

Prostate cancer is the second most common cancer in men worldwide1. Over the past decade, large-scale integrative genomics efforts have enhanced our understanding of this disease by characterizing its genetic and epigenetic landscape in thousands of patients2,3. However, most tumours profiled in these studies were obtained from patients from Western populations. Here we produced and analysed whole-genome, whole-transcriptome and DNA methylation data for 208 pairs of tumour tissue samples and matched healthy control tissue from Chinese patients with primary prostate cancer. Systematic comparison with published data from 2,554 prostate tumours revealed that the genomic alteration signatures in Chinese patients were markedly distinct from those of Western cohorts: specifically, 41% of tumours contained mutations in FOXA1 and 18% each had deletions in ZNF292 and CHD1. Alterations of the genome and epigenome were correlated and were predictive of disease phenotype and progression. Coding and noncoding mutations, as well as epimutations, converged on pathways that are important for prostate cancer, providing insights into this devastating disease. These discoveries underscore the importance of including population context in constructing comprehensive genomic maps for disease.

SoyMD: a platform combining multi-omics data with various tools for soybean research and breeding.

Advanced multi-omics technologies offer much information that can uncover the regulatory mechanisms from genotype to phenotype. In soybean, numerous multi-omics databases have been published. Although they cover multiple omics, there are still limitations when it comes to the types and scales of omics datasets and analysis methods utilized. This study aims to address these limitations by collecting and integrating a comprehensive set of multi-omics datasets. This includes 38 genomes, transcriptomes from 435 tissue samples, 125 phenotypes from 6686 accessions, epigenome data involving histone modification, transcription factor binding, chromosomal accessibility and chromosomal interaction, as well as genetic variation data from 24 501 soybean accessions. Then, common analysis pipelines and statistical methods were applied to mine information from these multi-omics datasets, resulting in the successful establishment of a user-friendly multi-omics database called SoyMD (https://yanglab.hzau.edu.cn/SoyMD/#/). SoyMD provides researchers with efficient query options and analysis tools, allowing them to swiftly access relevant omics information and conduct comprehensive multi-omics data analyses. Another notable feature of SoyMD is its capability to facilitate the analysis of candidate genes, as demonstrated in the case study on seed oil content. This highlights the immense potential of SoyMD in soybean genetic breeding and functional genomics research.

AnimalMetaOmics: a multi-omics data resources for exploring animal microbial genomes and microbiomes.

The Animal Meta-omics landscape database (AnimalMetaOmics, https://yanglab.hzau.edu.cn/animalmetaomics#/) is a comprehensive and freely available resource that includes metagenomic, metatranscriptomic, and metaproteomic data from various non-human animal species and provides abundant information on animal microbiomes, including cluster analysis of microbial cognate genes, functional gene annotations, active microbiota composition, gene expression abundance, and microbial protein identification. In this work, 55 898 microbial genomes were annotated from 581 animal species, including 42 924 bacterial genomes, 12 336 virus genomes, 496 archaea genomes and 142 fungi genomes. Moreover, 321 metatranscriptomic datasets were analyzed from 31 animal species and 326 metaproteomic datasets from four animal species, as well as the pan-genomic dynamics and compositional characteristics of 679 bacterial species and 13 archaea species from animal hosts. Researchers can efficiently access and acquire the information of cross-host microbiota through a user-friendly interface, such as species, genomes, activity levels, expressed protein sequences and functions, and pan-genome composition. These valuable resources provide an important reference for better exploring the classification, functional diversity, biological process diversity and functional genes of animal microbiota.

FAM91A1-TBC1D23 complex structure reveals human genetic variations susceptible for PCH.

Pontocerebellar hypoplasia (PCH) is a group of rare neurodevelopmental disorders with limited diagnostic and therapeutic options. Mutations in WDR11, a subunit of the FAM91A1 complex, have been found in patients with PCH-like symptoms; however, definitive evidence that the mutations are causal is still lacking. Here, we show that depletion of FAM91A1 results in developmental defects in zebrafish similar to that of TBC1D23, an established PCH gene. FAM91A1 and TBC1D23 directly interact with each other and cooperate to regulate endosome-to-Golgi trafficking of KIAA0319L, a protein known to regulate axonal growth. Crystal structure of the FAM91A1-TBC1D23 complex reveals that TBC1D23 binds to a conserved surface on FAM91A1 by assuming a Z-shaped conformation. More importantly, the interaction between FAM91A1 and TBC1D23 can be used to predict the risk of certain TBC1D23-associated mutations to PCH. Collectively, our study provides a molecular basis for the interaction between TBC1D23 and FAM91A1 and suggests that disrupted endosomal trafficking underlies multiple PCH subtypes.

Functional role of carbohydrate-binding modules in multi-modular chitinase OfChtII.

The primary distinction between insect and bacterial chitin degradation systems lies in the presence of a multi-modular endo-acting chitinase ChtII, in contrast to a processive exo-acting chitinase. Although the essential role of ChtII during insect development and its synergistic action with processive chitinase during chitin degradation have been established, the mechanistic understanding of how it deconstructs chitin remains largely elusive. Here OfChtII from the insect Ostrinia furnacalis was investigated employing comprehensive approaches encompassing biochemical and microscopic analyses. The results demonstrated that OfChtII truncations with more carbohydrate-binding modules (CBMs) exhibited enhanced hydrolysis activity, effectively yielding a greater proportion of fibrillary fractions from the compacted chitin substrate. At the single-molecule level, the CBMs in these OfChtII truncations have been shown to primarily facilitate chitin substrate association rather than dissociation. Furthermore, a greater number of CBMs was demonstrated to be essential for the enzyme to effectively bind to chitin substrates with high crystallinity. Through real-time imaging by high-speed atomic force microscopy, the OfChtII-B4C1 truncation with three CBMs was observed to shear chitin fibers, thereby generating fibrillary fragments and deconstructing the compacted chitin structure. This work pioneers in revealing the nanoscale mechanism of endo-acting multi-modular chitinase involved in chitin degradation, which provides an important reference for the rational design of chitinases or other glycoside hydrolases.

Conditional splicing system for tight control of viral overlapping genes.

Viral genomes frequently harbor overlapping genes, complicating the development of virus-vectored vaccines and gene therapies. This study introduces a novel conditional splicing system to precisely control the expression of such overlapping genes through recombinase-mediated conditional splicing. We refined site-specific recombinase (SSR) conditional splicing systems and explored their mechanisms. The systems demonstrated exceptional inducibility (116,700-fold increase) with negligible background expression, facilitating the conditional expression of overlapping genes in adenovirus-associated virus (AAV) and human immunodeficiency virus type 1. Notably, this approach enabled the establishment of stable AAV producer cell lines, encapsulating all necessary packaging genes. Our findings underscore the potential of the SSR-conditional splicing system to significantly advance vector engineering, enhancing the efficacy and scalability of viral-vector-based therapies and vaccines. IMPORTANCE: Regulating overlapping genes is vital for gene therapy and vaccine development using viral vectors. The regulation of overlapping genes presents challenges, including cytotoxicity and impacts on vector capacity and genome stability, which restrict stable packaging cell line development and broad application. To address these challenges, we present a "loxp-splice-loxp"-based conditional splicing system, offering a novel solution for conditional expression of overlapping genes and stable cell line establishment. This system may also regulate other cytotoxic genes, representing a significant advancement in cell engineering and gene therapy as well as biomass production.

Macrophages derived from human induced pluripotent stem cells (iPSCs) serve as a high-fidelity cellular model for investigating HIV-1, dengue, and influenza viruses.

Macrophages are important target cells for diverse viruses and thus represent a valuable system for studying virus biology. Isolation of primary human macrophages is done by culture of dissociated tissues or from differentiated blood monocytes, but these methods are both time consuming and result in low numbers of recovered macrophages. Here, we explore whether macrophages derived from human induced pluripotent stem cells (iPSCs)-which proliferate indefinitely and potentially provide unlimited starting material-could serve as a faithful model system for studying virus biology. Human iPSC-derived monocytes were differentiated into macrophages and then infected with HIV-1, dengue virus, or influenza virus as model human viruses. We show that iPSC-derived macrophages support the replication of these viruses with kinetics and phenotypes similar to human blood monocyte-derived macrophages. These iPSC-derived macrophages were virtually indistinguishable from human blood monocyte-derived macrophages based on surface marker expression (flow cytometry), transcriptomics (RNA sequencing), and chromatin accessibility profiling. iPSC lines were additionally generated from non-human primate (chimpanzee) fibroblasts. When challenged with dengue virus, human and chimpanzee iPSC-derived macrophages show differential susceptibility to infection, thus providing a valuable resource for studying the species-tropism of viruses. We also show that blood- and iPSC-derived macrophages both restrict influenza virus at a late stage of the virus lifecycle. Collectively, our results substantiate iPSC-derived macrophages as an alternative to blood monocyte-derived macrophages for the study of virus biology. IMPORTANCE: Macrophages have complex relationships with viruses: while macrophages aid in the removal of pathogenic viruses from the body, macrophages are also manipulated by some viruses to serve as vessels for viral replication, dissemination, and long-term persistence. Here, we show that iPSC-derived macrophages are an excellent model that can be exploited in virology.

Sumoylation Promotes the Stability of the DNA Sensor cGAS and the Adaptor STING to Regulate the Kinetics of Response to DNA Virus.

During viral infection, sensing of cytosolic DNA by the cyclic GMP-AMP synthase (cGAS) activates the adaptor protein STING and triggers an antiviral response. Little is known about the mechanisms that determine the kinetics of activation and deactivation of the cGAS-STING pathway, ensuring effective but controlled innate antiviral responses. Here we found that the ubiquitin ligase Trim38 targets cGas for sumoylation in uninfected cells and during the early phase of viral infection. Sumoylation of cGas prevented its polyubiquitination and degradation. Trim38 also sumoylated Sting during the early phase of viral infection, promoting both Sting activation and protein stability. In the late phase of infection, cGas and Sting were desumoylated by Senp2 and subsequently degraded via proteasomal and chaperone-mediated autophagy pathways, respectively. Our findings reveal an essential role for Trim38 in the innate immune response to DNA virus and provide insight into the mechanisms that ensure optimal activation and deactivation of the cGAS-STING pathway.

A vitamin-C-derived DNA modification catalysed by an algal TET homologue.

Methylation of cytosine to 5-methylcytosine (5mC) is a prevalent DNA modification found in many organisms. Sequential oxidation of 5mC by ten-eleven translocation (TET) dioxygenases results in a cascade of additional epigenetic marks and promotes demethylation of DNA in mammals1,2. However, the enzymatic activity and function of TET homologues in other eukaryotes remains largely unexplored. Here we show that the green alga Chlamydomonas reinhardtii contains a 5mC-modifying enzyme (CMD1) that is a TET homologue and catalyses the conjugation of a glyceryl moiety to the methyl group of 5mC through a carbon-carbon bond, resulting in two stereoisomeric nucleobase products. The catalytic activity of CMD1 requires Fe(II) and the integrity of its binding motif His-X-Asp, which is conserved in Fe-dependent dioxygenases3. However, unlike previously described TET enzymes, which use 2-oxoglutarate as a co-substrate4, CMD1 uses L-ascorbic acid (vitamin C) as an essential co-substrate. Vitamin C donates the glyceryl moiety to 5mC with concurrent formation of glyoxylic acid and CO2. The vitamin-C-derived DNA modification is present in the genome of wild-type C. reinhardtii but at a substantially lower level in a CMD1 mutant strain. The fitness of CMD1 mutant cells during exposure to high light levels is reduced. LHCSR3, a gene that is critical for the protection of C. reinhardtii from photo-oxidative damage under high light conditions, is hypermethylated and downregulated in CMD1 mutant cells compared to wild-type cells, causing a reduced capacity for photoprotective non-photochemical quenching. Our study thus identifies a eukaryotic DNA base modification that is catalysed by a divergent TET homologue and unexpectedly derived from vitamin C, and describes its role as a potential epigenetic mark that may counteract DNA methylation in the regulation of photosynthesis.

CottonMD: a multi-omics database for cotton biological study.

Cotton is an important economic crop, and many loci for important traits have been identified, but it remains challenging and time-consuming to identify candidate or causal genes/variants and clarify their roles in phenotype formation and regulation. Here, we first collected and integrated the multi-omics datasets including 25 genomes, transcriptomes in 76 tissue samples, epigenome data of five species and metabolome data of 768 metabolites from four tissues, and genetic variation, trait and transcriptome datasets from 4180 cotton accessions. Then, a cotton multi-omics database (CottonMD, http://yanglab.hzau.edu.cn/CottonMD/) was constructed. In CottonMD, multiple statistical methods were applied to identify the associations between variations and phenotypes, and many easy-to-use analysis tools were provided to help researchers quickly acquire the related omics information and perform multi-omics data analysis. Two case studies demonstrated the power of CottonMD for identifying and analyzing the candidate genes, as well as the great potential of integrating multi-omics data for cotton genetic breeding and functional genomics research.

CRAMdb: a comprehensive database for composition and roles of microbiome in animals.

CRAMdb (a database for composition and roles of animal microbiome) is a comprehensive resource of curated and consistently annotated metagenomes for non-human animals. It focuses on the composition and roles of the microbiome in various animal species. The main goal of the CRAMdb is to facilitate the reuse of animal metagenomic data, and enable cross-host and cross-phenotype comparisons. To this end, we consistently annotated microbiomes (including 16S, 18S, ITS and metagenomics sequencing data) of 516 animals from 475 projects spanning 43 phenotype pairs to construct the database that is equipped with 9430 bacteria, 278 archaea, 2216 fungi and 458 viruses. CRAMdb provides two main contents: microbiome composition data, illustrating the landscape of the microbiota (bacteria, archaea, fungi, and viruses) in various animal species, and microbiome association data, revealing the relationships between the microbiota and various phenotypes across different animal species. More importantly, users can quickly compare the composition of the microbiota of interest cross-host or body site and the associated taxa that differ between phenotype pairs cross-host or cross-phenotype. CRAMdb is freely available at (http://www.ehbio.com/CRAMdb).

Odd viscosity-induced Hall-like transport of an active chiral fluid.

Broken time-reversal and parity symmetries in active spinner fluids imply a nondissipative "odd viscosity," engendering phenomena unattainable in traditional passive or active fluids. Here we show that the odd viscosity itself can lead to a Hall-like transport when the active chiral fluid flows through a quenched matrix of obstacles, reminiscent of the anomalous Hall effect. The Hall-like velocity depends significantly on the spinner activity and longitudinal flow due to the interplay between odd viscosity and spinner-obstacle collisions. Our findings underscore the importance of odd viscosity in active chiral matter and elucidate its essential role in the anomalous Hall-like effect.

Endocytosis triggers V-ATPase-SYK-mediated priming of cGAS activation and innate immune response.

The current view of nucleic acid-mediated innate immunity is that binding of intracellular sensors to nucleic acids is sufficient for their activation. Here, we report that endocytosis of virus or foreign DNA initiates a priming signal for the DNA sensor cyclic GMP-AMP synthase (cGAS)-mediated innate immune response. Mechanistically, viral infection or foreign DNA transfection triggers recruitment of the spleen tyrosine kinase (SYK) and cGAS to the endosomal vacuolar H+ pump (V-ATPase), where SYK is activated and then phosphorylates human cGASY214/215 (mouse cGasY200/201) to prime its activation. Upon binding to DNA, the primed cGAS initiates robust cGAMP production and mediator of IRF3 activation/stimulator of interferon genes-dependent innate immune response. Consistently, blocking the V-ATPase-SYK axis impairs DNA virus- and transfected DNA-induced cGAMP production and expression of antiviral genes. Our findings reveal that V-ATPase-SYK-mediated tyrosine phosphorylation of cGAS following endocytosis of virus or other cargos serves as a priming signal for cGAS activation and innate immune response.