Cell Discovery

G-quadruplexes (G4s) are critical nucleic acid secondary structures that play pivotal roles in regulating gene expression. In this study, we conducted a proteome-wide in silico analysis across multiple viruses causing hemorrhagic fevers to identify candidate proteins containing a conserved G4-binding motif. Four peptides belonging to Marburg, Ebola, Hantaan and Yellow fever viruses were shown to bind to G4 in vitro. We selected the NS3 protease domain of Yellow Fever virus for further validation. Biochemical assays demonstrated that the NS3 protease domain binds G4 structures with high specificity and affinity, particularly favoring the parallel conformation. Molecular docking and simulations further revealed that the NS3 protease domain interacts with the terminal G-tetrads and loop regions of G4 via key residues, including PHE40, adopting an insertion and stacking composite binding mode. These findings expand our understanding of virus - G4 interactions and offer novel potential targets for G4-based antiviral strategies. Bullet points- We screened viruses causing hemorrhagic fevers for potential G4-binding peptides. - Four peptides belonging to Marburg, Ebola, Hantaan and Yellow fever viruses were shown to bind to G4 in vitro. - Biochemical assays demonstrated that the NS3 protease domain of YFV binds G4 structures with high specificity and affinity.

The inositol monophosphatase (IMPase) orthologue is pivotal for virulence, pathogenesis, and biofilm regulation, and is therefore considered a potential drug target in Pseudomonas aeruginosa and other bacterial pathogens. The mammalian IMPase orthologue is an established drug target for bipolar disorder. The precise catalytic mechanism in this class of enzymes remains obscure despite five to six decades of extensive efforts and detailed studies of substrate, transition-state analogue, and product-bound structures. Here, we have solved the crystal structures of the IMPase orthologue from Pseudomonas aeruginosa (PaIMPase), capturing pre- and post-catalytic snapshots of metal-substrate- and metal-product-mimic-bound states. Moreover, we solved the metal-substrate transition-state-analogue-bound crystal structure of the enzyme. Critical evaluation of these high-resolution crystal structures of PaIMPase complexed with substrate, transition-state analogue, and product mimic (myo-inositol and phosphate) supports three Mg2+-dependent catalytic mechanisms of PaIMPase. The structural snapshots indicate that, at the enzyme active site, a metal (Mg2+)-coordinating water molecule, activated by two bound Mg2+ ions and the active-site-proximal Threonine/Aspartate dyad, attacks the central phosphorus atom of the bound substrate, leading to formation of a trigonal bipyramidal transition state. Following that, the immediate breakdown of the P-O bond results in the formation of inositolate and phosphate ions. The second water molecule, activated by another Mg2+ dyad, facilitates the departure of myo-inositol and phosphate from the active site. The detailed mechanistic insights gained from this work may offer a foundation for the rational design of precise inhibitors against PaIMPase.

Highly pathogenic avian influenza viruses (HPAIVs) continue to cause substantial disease in birds and mammals, with repeated H5N1 spillovers highlighting the need for broadly protective antiviral strategies. Here we develop a programmable RNA-targeting antiviral platform based on RfxCas13d and evaluate its activity in avian cells. Screening of five Cas13 orthologs in chicken DF1 fibroblasts revealed RfxCas13d as the most potent and well tolerated effector. Virus-specific CRISPR RNAs (crRNAs) targeting conserved regions of positive- and negative-sense influenza RNA were tested against A/WSN/033[H1N1] and multiple HPAIV isolates, including a member of clade 2.3.4.4b H5N1. Targeting positive-sense RNA conferred superior influenza inhibitory activity and further enhanced by multiplexed crRNA expression. These findings establish RfxCas13d as a versatile RNA-guided antiviral platform and provide a route for broad-spectrum influenza control through conserved RNA targeting.

Trypanosoma cruzi is the causative agent of Chagas disease, a neglected illness with outdated treatments. Bromodomain factors (BDFs) are essential proteins that recognize acetylated lysines and have strong therapeutic potential. They form part of epigenetic complexes that regulate chromatin accessibility and, therefore, gene expression. However, little is known about their structure in trypanosomatids. Here, we used a combination of experimental and bioinformatic approaches to infer the stoichiometry and structure of T. cruzi bromodomain-containing complexes. By reconstructing the proximity networks of five BDFs using TurboID-directed proximity labeling, we identified highly interconnected components that assemble into the CRKT and NuA4 complexes. Using novel structure prediction strategies that systematically explore the stoichiometric space, we inferred that CRKT assembles into three distinct modules and NuA4 in two, with different degrees of interaction dynamics. The core module of CRKT contains two copies of each component, including BDF3, BDF5, and BDF8, arranged in a subcomplex with central symmetry. The catalytic module of CRKT has three subunits, including the histone acetyltransferase 2 (HAT2), while the BET (bromodomain and extra-terminal) module has one unit of both BDF4 and BDF1. The catalytic module of NuA4 closely resembles the yeast piccolo-NuA4 module and contains HAT1, while the TINTIN module associates with the catalytic module via the C-terminal domain of BDF6. These insights shed light on the structure and composition of epigenetic complexes in trypanosomatids, opening new avenues for rational drug design aimed at disrupting their function.

Hantaviruses are zoonotic negative-sense RNA viruses that cause two severe diseases; haemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) for which no approved antiviral therapies are available. To identify host-directed modulators of hantavirus infection in the available annotated drug space, we performed a drug repurposing screen in A549 cells and HUVECs, using live Puumala virus (PUUV). We identified and validated 70 drugs with antiviral activity across these 2 different cell systems. Functional clustering confirmed the known infection-inhibitory effect of several group of compounds, including inhibitors of heat shock proteins, mTOR pathway or nucleotide synthesis. In addition, we also identified compounds yet unexplored as antivirals against Hantaviruses, such as certain antibiotics. This dataset provides a systematic map of host pathways influencing PUUV infection and highlights candidate compounds and cellular processes that warrant further investigation.

Long noncoding RNAs (LncRNAs) have emerged as a class of important regulatory ncRNAs and are known to fine-tune numerous cellular processes including proliferation, differentiation and development; however, their role in quiescence still remains largely unexplored. A miRNA host gene lncRNA, MIR503HG, has been reported to play important role in cancer development. Here, we demonstrate the role of MIR503HG lncRNA in regulating cellular quiescence. MIR503HG displays elevated levels in human diploid fibroblasts induced to undergo quiescence. Depletion of MIR503HG in HDFs affects the entry of cells into quiescence but has no effect on cell cycle progression, suggesting its role in quiescence attainment and/or maintenance. Additionally, MIR503HG depletion led to a drastic decrease in the levels of miR508 target, PTEN with a concomitant increase in pAkt levels, indicating its role in negative regulation of miR508. Further, we demonstrate that the lncRNA MIR503HG regulates PTEN levels by acting as a ceRNA for miR508 to maintain cellular quiescence. Our studies illustrate that MIR503HG can function synergistically with miR503 to maintain cells under quiescence and both the miRNA-HG and the miRNA encoded by its gene locus synergistically control the same biological process in different ways by regulating different downstream genes.

The molecular mechanisms driving SLFN11 chromatin recruitment remain partially elucidated. Using high-throughput imaging of 162 oncology-focused compounds in U2OS cells with inducible SLFN11 expression, we discovered that deubiquitinase (DUB) inhibitors drive massive SLFN11 recruitment to chromatin, preferentially at promoter regions while concurrently suppressing transcription. DUB inhibitors such as VLX-1570 promote ubiquitin-dependent enrichment of SLFN11 without detectable DNA damage, distinct from the camptothecin-induced RPA-associated SLFN11 foci formed at stressed replication forks. Yet, SLFN11 chromatin recruitment both by DUB inhibitors and DNA damage are suppressed by TAK243 demonstrating their ubiquitylation dependency. RNF168 is required for SLFN11 ubiquitylation and its subsequent chromatin association, and ubiquitylation within SLFN11s middle linker domain (lysines 390, 391, and 429) with K27-linked polyubiquitin chains is essential for the chromatin recruitment of SLFN11. These findings suggest the importance of SLFN11 ubiquitylation by RNF168 for SLFN11 chromatin recruitment and SLFN11 transcriptional regulatory role at promoter regions.

Bovine viral diarrhea virus (BVDV, genus Pestivirus, family Flaviviridae) is a notifiable pathogen of cattle which significantly impacts animal health, welfare, and the economy. Several cellular factors important for BVDV infection, such as Jiv, CD46 and ADAM17, have already been identified providing new targets development of effective defense strategies. However, our knowledge about BVDV host factor requirements remains limited, as no genome-wide studies of BVDV host resistance factors were performed to date, in part due to lack of accessible whole genome libraries. To close this gap, we have designed a novel bovine whole genome knockout library and successfully used it to identify a set of BVDV host resistance factors. The validity of our approach is highlighted by the strong selection of cells with inactivated ADAM17 and TMEM41B, which have both been described to be of pivotal importance for BVDV infection. In addition, guides targeting VMP1, recently identified as an important factor for flavivirus infection, were also significantly enriched in our screen. Furthermore, we found differential selection of several proteins essential for triggering autophagy, providing additional strong evidence of this process underlying key cellular functions involved in resistance to BVDV.

Inflammasomes lead to activation of inflammatory caspases, which induce pyroptosis and an inflammatory immune response to control microbial infections. Inflammasomes are tightly regulated to avoid lethal sepsis and chronic autoimmune conditions. However, posttranslational regulation of inflammatory caspases remains poorly defined. We constructed 375 individual ubiquitin ligase knockout lines by CRISPR-Cas9, performed an unbiased screening, and identified Muscle Excess 3B (MEX3B), an RNA-binding protein and ubiquitin ligase, as a positive regulator of the caspase-4 inflammasome. Genetic depletion of MEX3B inhibited not only the caspase-4 but also NLRP3 and NLRC4 inflammasomes, regarding caspase activation, pyroptosis, and secretion of inflammasome-dependent cytokines, in human cells and murine primary macrophages. This MEX3B function required its RNA-binding, but not ubiquitin ligase activity. These results suggest that MEX3B is a pan-inflammasome regulator and a potential therapeutic target for inflammation.

It remains unclear whether podocyte loss directly causes acute renal tubular cell (RTC) damage and interstitial fibrosis, thereby leading to renal failure. Here, we applied intermedilysin (ILY)-mediated human CD59 (hCD59) cell ablation to generate an acute, specific podocyte-ablation mouse model. Cre-induced hCD59 transgenics (ihCD59) were crossed with Nphs2Cre to generate ihCD59+/-/Nphs2Cre+/- mice. The specific and rapid podocyte-ablation mediated by ILY injection directly caused RTC necrosis, leading to renal failure and even death within 2-3 days in a dose-dependent manner. Treating mice that received an ILY lethal dose with peritoneal dialysis or administering a non-lethal dose, we extended their survival beyond six weeks and found that mice developed interstitial fibrosis and glomerulosclerosis with persistent proteinuria and tubule damage. Podocyte-ablation caused massive disruption of glomerular function at week 1, and then partial recovery by week 2. Genes and pathways of TLRs and apoptosis, and mitochondrial functions were respectively upregulated and downregulated in both ablated-podocyte mouse and biopsied-glomerulonephritis patient kidney samples. Together, this rapid podocyte-ablation causes acute RTC necrosis that progresses to interstitial fibrosis in this mouse model, which is applicable for dissecting mechanisms underlying podocyte injury-mediated tubular damage and glomerular repair, with the potential to reveal novel therapeutic targets for kidney diseases.

The cGAS-STING pathway has been widely recognized as a critical DNA-sensing pathway that plays a broad-spectrum antiviral role. Livestock, especially pigs, represents one of the most important meat sources. In this study, we identified a key lysine 61 (K61) of porcine STING (pSTING) that plays an essential role in its degradation and antiviral signaling in a species-specific manner, with K61 as the major lysine of pSTING for K48-linked ubiquitination. After virus infection, pSTING recruits the E3 ligase, RNF5, which specifically assembles a K48-linked ubiquitin chain at K61, thereby mediating pSTING proteasomal degradation and reducing its antiviral activity. Meanwhile, the deubiquitylation of K61 is mediated mainly by deubiquitinase USP20, which enhances the stability and antiviral activity of pSTING. Together, given the relatively few lysine numbers in livestock STINGs and species-specific K61 regulation of pSTING stability and antiviral function, the K61 and its specific regulatory enzymes of pSTING could serve as potential targets for breeding of antiviral pigs and design of antiviral drugs, respectively.

G protein-coupled receptors (GPCRs) are critical regulators of human physiology and major drug targets. Although structural studies have provided valuable insights, determining GPCR structures remains challenging, especially for inactive state receptors. Recent advances in cryo-electron microscopy (cryo-EM) have enabled structural determination of small GPCRs by using fusion partner proteins and binders to increase molecular weight. However, current methods require extensive experimental screening of fusion constructs. Widely adopted strategies, such as BRIL-Fab complexes, also face limitations due to inherent flexibility. Here, we introduce a streamlined and universal pipeline that integrates an in silico fusion construct screening program, NOAH (NOAH: NOn-experimental, AI-assisted High-throughput construct screening), with a de novo designed fusion protein called ARK1 (ARtificially-designed fiducial marKer). We validate the efficacy of NOAH by determining the structures of the vasopressin V2 receptor (V2R) bound to the clinical antagonist tolvaptan and the partial agonist OPC51803, as well as the bradykinin B2 receptor (B2R) bound to the clinical antagonist icatibant, thereby elucidating their activation and deactivation mechanisms. Furthermore, we demonstrate the capability of NOAH-ARK1 by solving the tolvaptan-bound V2R structure at higher resolution and showcase the methods versatility by determining the structure of lysophosphatidic acid receptor 2 (LPA2) bound to the antagonist Ki16425. This approach eliminates the need for time-consuming and labor-intensive construct optimization, providing a rapid and widely applicable solution for high-resolution GPCR structure determination and drug discovery.

The biosynthetic pathway of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is a universal and essential metabolic process in many organisms, providing the activated sulfate donor required for the synthesis of diverse sulfated metabolites. However, this pathway has undergone substantial evolutionary diversification among species. In Entamoeba histolytica, PAPS biosynthesis occurs within the mitosomes, mitochondrion-related organelles (MROs), representing a distinctive example of lineage-specific evolutionary adaptation. PAPS synthesis proceeds through a conserved two-step, which is sequentially catalyzed by ATP sulfurylase (AS) and adenosine 5'-phosphosulfate (APS) kinase (APSK). In this study, we focused on E. histolytica APSK (EhAPSK). EhAPSK contains an additional AS-like domain (SLD), although its functional role remains unclear. Here, we determined the crystal structure of full-length EhAPSK at 2.60 [A] resolution and the structure of the truncated EhAPSK lacking APS kinase domain (KD) (EhAPSK{Delta}KD) at 2.10 [A] resolution. Structural analyses revealed that the SLD engages in dynamic contacts with the KD. Furthermore, deletion of the domain and mutational analyses indicated that the SLD significantly influences the catalytic activity of the KD. Based on these findings, we propose a new regulatory mechanism in which transient interdomain interactions modulate APS kinase activity, representing an unique evolutionary adaptation of E. histolytica.

African swine fever (ASF) is a highly pathogenic disease caused by the African swine fever virus (ASFV) infection, which can affect pigs of all ages and breeds, posing significant threat to the global pig farming industry. The ASFV p30 protein is an early-expressed viral structural protein; however, its function is not fully understood. In this study, the interaction of viral p30 with host TRIM21 was identified. The ectopic TRIM21 inhibited ASFV replication, while knockdown or knockout of TRIM21 promoted ASFV replication. Further, p30 was found to interact with RIG-I-like receptor (RLR) signaling adaptor MAVS, and during ASFV infection, p30-TRIM21-MAVS interacted with each other. Mechanistically, TRIM21 activated the K27 polyubiquitination of MAVS to induce IRF3 mediated type I interferon (IFN) production, whereas p30 counteracted TRIM21 activated MAVS K27 polyubiquitination to evade RLR signaling mediated antiviral IFN induction. In summary, our study revealed a novel function of ASFV p30, and provided new insights into the immune evasion of ASFV.

Genetic regulation of splicing uniquely contributes to trait-associated genome-wide association studies (GWAS) signals. However, quantitative trait loci (QTL) analysis using short-read sequencing of bulk tissues fails to capture full-length and cell-type-specific isoforms. Here, we present an isoform-level lung cell atlas from 129 never-smoking Korean women using single-cell long-read RNA-sequencing, identifying abundant unannotated and cell-type-specific isoforms. Isoform-level signatures of 37 lung cell types display a larger difference and therefore improve cell-type classification compared to gene-level expression. Notably, isoform-QTLs (isoQTLs) detect unannotated and/or cell-type-specific isoforms with independent genetic regulation from expression-QTL (eQTL), supported by enriched splicing functional elements. IsoQTLs nominate susceptibility isoforms from previously unexplained lung function and cancer GWAS loci, via eQTL-independent signals. We highlight a potentially functional novel variant of PPIL6 in multiciliated cells underlying lung cancer risk through alternative splicing. This isoform-level resource advances our understanding of cell-type-specific isoform regulation and its contribution to lung traits and diseases.

Silencing complexes formed by PIWI-clade Argonaute (Ago) proteins and PIWI-interacting RNAs (piRNAs) are essential guardians of genome integrity, controlling the deleterious activities of transposable elements (TEs) in animal germline. However, our understanding of PIWI-piRNA-directed TE silencing remains incomplete. Here, we systemically characterize the proximity proteome of PIWI members, Piwi, Aubergine (Aub), and Ago3 in the germline of Drosophila ovaries. Functional screening identifies previously uncharacterized factors involved in TE silencing, including H3K4me3 writer and transcriptional coactivator Set1. Transcriptome analysis reveals that Set1 acts as an indispensable repressor for TEs, particularly those forming telomeres. The involvement of Set1 in Piwi pathway is further supported by its critical role in the production of antisense, TE-targeting piRNAs. Notably, catalytic activity of Set1 is dispensable for TE silencing. Genome-wide chromatin binding analysis using CUT&Tag demonstrates that Set1 preferentially associates with TE sequences and localizes to subtelomeric piRNA cluster loci, suggesting a role in promoting piRNA precursor transcription through direct binding. Collectively, these findings uncover a noncanonical function of Set1 in Piwi-mediated TE silencing and telomere control in germline nuclei.

Viral infections are one of the main environmental factors triggering type 1 diabetes (T1D). Pancreatic alpha cells are more resistant than beta cells to diabetogenic viruses, partially explaining their survival in T1D. Similarly, bats have enhanced viral resistance, suggesting putative convergent evolution in antiviral mechanisms. Herein, we compared global gene expression in bat fibroblasts under basal conditions or exposed to double-stranded RNA to human alpha and beta cells and found that alpha cells exhibit greater similarity than beta cells to the antiviral responses of bat cells, as well as stronger intrinsic resistance to viral infection. Interferon-stimulated gene 15 (ISG15), a key regulator of antiviral responses in humans and bats, has higher expression in alpha compared to beta cells in five single-cell RNASeq datasets from human islet cells and in human induced pluripotent stem cell (hiPSC)-derived alpha-like cells. ISG15 knockdown in human insulin-producing EndoC-{beta}H1 cells and human islets increases apoptosis under basal conditions and after IFN exposure, exacerbates IFN responses and increases cell death and viral production after infection with the diabetogenic virus coxsackievirus B1, while its overexpression protects EndoC-{beta}H1 cells from the virus. Collectively, the present results demonstrate that alpha cells but not beta cells have similarities with the virus resistance gene program present in bats and identify ISG15 as an important factor for islet cells to cope with viral and diabetogenic stresses.

Despite of the highly potent antiretroviral therapies, HIV-1 establishes persistent infection and causes chronic inflammation in AIDS patients. Beyond CD4+ T cells, HIV-1 infects myeloid cells, including circulating monocytes and tissue-resident macrophages, and integrates with host genomes to form stable viral reservoirs. To achieve a functional HIV cure, latency-promoting agents (LPAs) have been developed for the "block-and-lock" strategy to reinforce deep HIV-1 latency and permanently silence proviruses. However, most LPAs have been tested mainly in CD4+ T cells, and their efficacy in myeloid cells remains unclear. In this study, we reported that levosimendan (LSM), a drug approved for clinic use to treat heart failures, is able to inhibit HIV lytic infection and reactivation in myeloid cells. LSM blocked viral lytic reactivation in HIV-1 latently infected monocytic cells (TH89GFP, U1) and microglial cells (HC69). LSM also inhibited HIV infection in human induced pluripotent stem cell (iPSC) derived microglia (iMG), primary human resident liver macrophages (Kupffer cells) as well as human monocyte-derived macrophages (MDMs). Furthermore, we demonstrated that overexpression of a predicted drug target of LSM, the conserved serine/threonine kinase RIOK1 (RIO kinase 1), overcomes LSMs anti-HIV effect. Overall, our studies concluded that LSM is a promising LPA to inhibit HIV-1 infection in myeloid cells in the RIOK1-dependent manner.

Obesity, a global health crisis affecting 16% of the world population, is characterized by chronic inflammation that contributes to health complications such as type 2 diabetes and metabolic dysfunction-associated steatotic liver disease (MASLD). Emerging evidence suggests that self-DNA released from dying cells aberrantly activates inflammatory responses during obesity. However, the role of extracellular deoxyribonucleases (DNASEs), which at steady state regulate abundance of extracellular self-DNA, remains poorly understood in this context. Here, we show that individuals with obesity exhibit elevated levels of circulating cell-free DNA (cfDNA) with a distinctive end-motif signature, anti-DNASE1L3 autoantibodies and a reduction in circulating DNASE activity. These cfDNA alterations correlate with the severity of obesity and can be corrected by therapeutic intervention such as bariatric surgery. Similarly, mice fed a high-fat diet (HFD) displayed increased cfDNA levels and decreased DNASE activity. Genetic deficiency of the extracellular nuclease DNASE1L3 in mice worsened HFD-induced metabolic complications, including glucose intolerance, insulin resistance, MASLD, and metabolic tissue inflammation. Conversely, targeted supplementation of DNASE1L3 in the liver using adeno-associated viral vectors protected obese mice from developing MASLD and liver inflammation. These findings uncover a novel role of DNASE1L3 in controlling obesity-associated inflammation and its potential therapeutic use for preventing metabolic disease.

Chronic hepatitis B persists due to the stability of nuclear covalently closed circular DNA (cccDNA), which maintains viral transcription despite prolonged antiviral therapy, highlighting the need for strategies that suppress cccDNA via host-targeted mechanisms. Here, we identify Spiperone, a clinically approved compound, as a repurposed anti-HBV candidate with strong translational potential. Spiperone robustly reduced HBsAg, HBeAg, viral DNA, and pgRNA across HepG2.2.15, HBV-infected HepG2-NTCP-C4 and HepaRG cells, and multiple in vivo models, including HBV transgenic, hydrodynamic injection, and AAV- HBV1.04x models. Notably, intrahepatic cccDNA was significantly diminished. In combination, Spiperone potentiated tenofovir activity, exhibiting synergistic effects, while both intraperitoneal and oral administration reduced antigenemia and viremia. Mechanistically, Spiperone activated the PERK-eIF2-ATF4 arm of the ER stress response, coupled with mitochondrial perturbation and cytosolic release of oxidized mitochondrial DNA, leading to activation of IFI16-STING-IRF3 signaling. This cascade induced type I interferon (IFN-I) and interferon-stimulated genes. ChIP-qPCR further demonstrated reduced enrichment of activating histone marks on cccDNA, consistent with transcriptional repression. Collectively, these findings position Spiperone as a host-directed antiviral that converges ER stress-linked innate immunity and epigenetic repression to suppress cccDNA, supporting its advancement in combination strategies toward a functional cure for chronic HBV infection.