Cell Research

Influenza virus poses a potential risk of triggering the next global pandemic. In-depth investigation into the mechanisms underlying influenza virus replication and pathogenicity will provide robust support for controlling influenza virus infection. Although post-translational modifications are known to regulate viral infection, the role of lactylation in influenza virus replication remains elusive. In this study, influenza virus ribonucleoprotein complex subunits are found to be lactylated. Specifically, ATAT1 promotes viral polymerase acidic protein (PA) lactylation and enhances viral replication. In contrast, SIRT1 mediates de-lactylation of PA and exerts an inhibitory effect on viral replication. Further investigations reveal lactylation of PA at residues K605 and K609 is essential for viral replication and pathogenicity. Mechanistically, PA K605/609 residues are localized at the interaction interface of the ANP32-mediated polymerase asymmetric dimer; mutation at these residues inhibits polymerase asymmetric dimerization, thereby impairing RNA production during viral genome replication. Collectively, this study uncovers a novel mechanism by which influenza virus hijacks host enzymes to mediate PA lactylation, and expands the molecular regulatory network of influenza virus infection.

Brain organoids offer an invaluable model system for studying human brain development and disease. However, the establishment of high-fidelity brain organoids with multiple cell lineages including vasculature and immune cells remains a huge challenge. Here, we present a new strategy to generate human cerebral organoids with vasculature and microglia-like cells using genetic code expansion technology (GCE-T) via site-specific protein engineering. The strategy integrates orthogonal genetic translation machinery in hPSCs via PiggyBac transposon system, enabling temporally control of ETV2 expression and endothelial differentiation in hPSC-derived cerebral organoids. The vascularized human cerebral organoids (vhCOs) exhibit coordinated development of multiple cell lineages and blood-brain barrier (BBB) features. Moreover, vhCOs form perfusable vascular network after transplanted in the immune-deficient mice. Single-nucleus RNA sequencing reveals enhanced neurovascular interactions, multi-brain-regional identities, diverse neuronal subtypes and specialized endothelial subclusters in vhCOs, closely resembling human fetal brain. Strikingly, we identify enriched microglia-like cells comprising three distinct subtypes in vhCOs, which contribute to microglia-vascular interactions and synergistically modulate vascular development. Upon Zika virus (ZIKV) infection, vhCOs show neurovascular dysfunction and impaired microglia development, offering new insights into viral-induced neurodevelopmental disorders. This study offers a unique platform for producing more valuable brain organoids with vasculature and immune components, opening a new avenue to advance organoid research and applications.

Dopamine is a neurotransmitter essential for cognition, and its dysregulation is associated with neurological diseases1,2. Historically, dopamine has been understood to signal exclusively through metabotropic receptors3. Delta-type ionotropic glutamate receptors (GluDs), which have recently been established as ligand-gated ion channels4,5, are fundamental for synaptic maintenance, are implicated in neurological disorders, and co-localize with dopaminergic machinery. Here, we report that dopamine is a direct agonist of GluDs, eliciting ionotropic activity, as visualized by cryo-electron microscopy (cryo-EM), bilayer recordings, mutagenesis, and patch clamp recordings. Dopamine binds to the GluD ligand binding domain, inducing clamshell closure and channel activation through a distinct molecular interface. GluD channel activity is tightly regulated by G-proteins, which act as molecular switches to tune GluD activity: free G{beta}{gamma} inhibits ligand-gating, while G or inactive G-protein heterotrimers enable dopamine-induced GluD currents. This tuning of GluD activity by G-proteins is uncoupled in a point mutation associated with neurodegeneration. These findings expand mechanisms of neuronal dopaminergic signaling, uncover how G-proteins tune GluD channel activity, and provide a framework for targeting GluDs in neurological diseases.

Influenza neuraminidase (NA) is a promising target for universal flu vaccines, yet eliciting potent B-cell responses against its conserved epitopes remains challenging. Here, we developed a membrane-anchored, folding-domain-free NA (mNA) that elicited superior head-specific germinal center B cell and antibody responses compared to soluble tetrameric NA. In non-human primates, mNA immunization induced cross-reactive memory B cell (MBC) responses, expanding clones with the conserved "DR" motif in HCDR3, a hallmark of human broadly reactive NA antibodies. These MBCs conferred cross-inhibitory activity against diverse NA variants and in vivo cross-protection. Cryo-EM analysis revealed that the 554-C2 clone targets the conserved enzymatic pocket via the "DR" motif, while the 554-C1 clone recognizes previously uncharacterized epitopes at the interface between two adjacent N2 monomers, effectively reducing plaque formation by contemporary H3N2 strains. Our findings highlight the immunological advantages of membrane-anchoring, providing a robust strategy for designing next-generation vaccines against influenza and other pathogens.

Chronic inflammation and aging skew hematopoiesis toward myelopoiesis at the expense of lymphoid output. We screened type 2 and anti-inflammatory cytokines to identify extrinsic signals capable of restoring lymphoid lineage commitment in hematopoietic stem and progenitor cells (HSPCs). Interleukin-4 (IL-4) specifically inhibited inflammation-induced myelopoiesis and shifted multipotent progenitor (MPP) differentiation toward the lymphoid lineage. IL-4 activated a STAT6-dependent transcriptional program in MPPs, increasing expression of lymphoid-specific genes. Mechanistically, the receptor tyrosine kinase FLT3, highly expressed in MPPs, interacted with IL-4R to facilitate STAT6 activation. In vivo, IL-4 reversed inflammation-induced hematopoietic imbalance and accelerated lymphoid recovery. In aged mice, IL-4 administration shifted the MPP composition toward lymphoid bias and restored B and T lymphocyte output. Long-term IL-4 treatment of aged mice improved immune, metabolic, physical, and cognitive functions; these rejuvenating effects were recapitulated by transplantation of IL-4-treated HSPCs. Promoting IL-4 signaling on MPPs may enable correcting hematopoietic dysregulation in inflammatory and aging-related conditions.

Human endogenous retroviruses (hERVs) are remnants of ancestral retroviral infections that have shaped the human genome through their capacity to mobilize and retrotranspose. Among these, hERV-K (HML-2) remains the most recently active family and its dysregulation is strongly associated with diverse cancers and neurodegenerative pathologies, yet the structural basis of its integration remains poorly understood. Here, we combine activity assays with high-resolution cryo-electron microscopy to resolve the hERV-K integration machinery in three distinct states: asymmetric target-DNA engagement, strand transfer, and pharmacological inhibition. Our structures reveal a compact architecture defined by a unique organization of the outer integrase domains, which distinguishes hERV-K from other known retroviral intasomes. Biochemical validation confirms the catalytic competence of this compact tetrameric assembly, which relies on specialized polar motifs to optimize synaptic stability while retaining sensitivity to competitive antagonism by strand transfer inhibitors. Notably, beyond canonical restriction by Raltegravir, we discovered that the drug binding stabilizes an unanticipated, "closed" conformation not observed in previously characterized intasomes. Together, these findings elucidate the molecular mechanism of endogenous retroviral integration and provide a structural framework for rational therapeutic targeting of hERV-K-driven diseases.

Rhinoviruses are the leading cause of acute respiratory illnesses and comprise more than 170 types that constantly circulate in humans worldwide. Beyond common colds, rhinoviruses can trigger severe symptoms, particularly in young children, older adults and people with asthma or chronic obstructive pulmonary disease. Despite their clinical and socio-economic impact, no approved vaccine or antiviral treatment exist. Here, we uncovered the interaction of the host AAA+ ATPase RUVBL1/2 with rhinovirus non-structural protein 2C and we demonstrated that RUVBL1/2 is strictly and specifically required for the replication of the viral RNA of the most prevalent and pathogenic rhinovirus species. Pharmacological inhibition of RUVBL1/2 ATPase activity efficiently inhibited rhinovirus replication in a human nasal epithelium model, even post-infection. Moreover, serial viral passaging in the presence of a RUVBL1/2 inhibitor did not lead to the emergence of resistance. These findings reveal an unexpected and strong host dependency with promising potential for antiviral targeting.

Mixed-lineage leukemia translocated to 3 (MLLT3) is vital for maintaining the stemness of hematopoietic stem cells. Loss of MLLT3 in megakaryocyte (MK)-erythrocyte progenitor (MEP) cells leads to its differentiation into MKs. Despite its significance in stemness, the regulatory mechanism of MLLT3 during differentiation remains elusive. In this study, we investigate the regulatory role of histone deacetylase 6 (HDAC6) in modulating MLLT3 levels via heat shock protein 90 (Hsp90) activation during myeloid lineage differentiation into MKs, monocytes, and macrophages. We found that HDAC6 activates Hsp90 through deacetylation, enabling Hsp90 to retain MLLT3 in the cytoplasm where protein kinase C (PKC) phosphorylates MLLT3 at serine residues; leading to loss of MLLT3 during MK and macrophage differentiation but not during monocyte differentiation. This research provides valuable insights into the regulatory mechanisms underlying myeloid lineage commitment and opens new avenues for future investigations into stem cell biology and therapeutic applications.

The Rcs phosphorelay regulates gene expression in response to cell envelope stress and is critical for the virulence of pathogenic bacteria, including Klebsiella pneumoniae, due to its regulation of genes related to extracellular capsule, cell division, and motility. The RcsC histidine kinase, RcsD phosphotransfer protein and RcsB response regulator, which form the core of the Rcs phosphorelay, are negatively regulated by the unique inner membrane protein IgaA via interaction with RcsD. An outer membrane lipoprotein, RcsF, activates signaling by interaction with IgaA, but the precise activation mechanisms remain unclear. In this study, we determined the structures of IgaA and the IgaA/RcsF complex using Cryo-electron microscopy (Cryo-EM). We also determined the structures of RcsC and RcsD, which both form homodimers stabilized by hydrophobic interactions, creating ladder-like structures. Combining the Cryo-EM structures, AlphaFold3 structure predictions of IgaA/RcsD and RcsF/IgaA/RcsD, and genetic studies, we describe a model for how RcsF modifies the IgaA/RcsD interaction, lifting negative regulation and activating the Rcs phosphorelay. Our findings provide a high-resolution depiction of the Rcs stress response system and suggest potential targets for small molecule inhibitors.

The regulation of human hematopoietic stem cell (HSC) function through its native environment is virtually unknown. Cross-species chimeras, particularly humanized mice, are essential tools for investigating human HSC function in vivo. However, conventional models often require toxic conditioning that impairs niche and donor cell function, or simplify niche complexity. We utilize NSGW41 mice, which harbor a KIT receptor mutation, to achieve robust human leukocyte engraftment without prior treatment. We demonstrate that KIT-proficient human HSCs possess a clear advantage, effectively outcompeting endogenous murine stem cells and progenitors to establish stable, multilineage human hematopoiesis. Crucially, the murine niche undergoes significant plastic adaptation in response to humanization. We identify that mesenchymal stromal cells (MSCs) expand and undergo a transcriptional shift, transitioning from a mixed adipo- or osteo-primed state toward predominantly Lepr+ adipo-primed HSC-supporting cells. This adaptation is vital; the depletion of Lepr+ MSCs or the targeted deletion of Stem Cell Factor (SCF) from these cells leads to the mobilization or loss of human HSC engraftment, respectively. These findings provide compelling evidence for functional cross-species niche-HSC communication, identifying Lepr+MSCs as primary regulators of human HSC maintenance in xenotransplantation models. By mapping this molecular dialogue, our work establishes a physiological in vivo platform to study human HSC biology and evaluate niche-targeted therapeutic interventions to improve transplantation outcomes.

Manufacturing red blood cells (RBCs) from human induced pluripotent stem cells (iPSCs) can improve our understanding of embryonic erythropoiesis, foster innovative treatments for RBC-related diseases, and ultimately address clinical blood supply shortages. However, existing systems face low efficiency, enucleation failure, and uncertainty about the develop-mental wave of cultured RBCs. We successfully used self-organized hemanoids to improve iPSC-derived RBC generation. Based on the hypothesis that cellular interactions and 3D organization promote hematopoietic cell fate, we aimed to thoroughly characterize hemanoids. We visualized the spatiotemporal emergence of hematopoiesis by generating a CD43-GFP reporter iPSC line. Imaging and spatial transcriptomics analysis provided de-tailed insight into the hemanoid architecture, identifying stromal cells and hepatoblasts as potential erythropoiesis-supportive elements. The developmental stage mirrors extraembryonic hematopoiesis. Given the difficulties of accessing these early stages in vivo, our system offers a platform not only for further clinical translation but also for exploring hu-man embryonic blood wave dynamics.

Bacteria have evolved multiple immune systems to resist phage invasion, however, only a small part of the defensive mechanisms have been clearly uncovered. In this study, we report a type III Druantia two-component defense system, DruH-DruE, identified from Mycobacterium smegmatis. The DruH-DruE prevents phage DNA cyclization and replication.DruE can be replaced from the defense system by either homolog in M. tuberculosis or M. smegmatis. The physical interaction between this two components is essential for fighting against phage infection. Mutations in the interaction sites led to the loss of phage-defending function of the system. The broad-spectrum antiphage ability of the defense system could be activated by the small tail protein Gp25 of phage A10ZJ24. This study fills a major gap in current knowledge of antiphage mechanism of type III Druantia defense system, expanding our understanding of the immune mechanisms in prokaryotic cells.

Obesity is one of the most prevalent diseases worldwide. Increasing thermogenesis to enhance energy expenditure has emerged as a promising therapeutic strategy. In an effort to identify new regulatory targets in thermogenic adipocytes, we found that SOX8 is correlated with obesity and serves as a novel marker of classical brown adipocytes in both humans and mice, upregulating during acute cold exposure. Functional studies further demonstrated that adipocyte-specific knockdown of SOX8 leads to obesity and metabolic dysfunction in mice. Mechanistically, SOX8 directly interacts with USP7 and stabilizes PGC-1 by reducing its K48-linked polyubiquitination. AAV-Rec2-mediated SOX8 overexpression initially enhanced energy expenditure, improved insulin sensitivity, and alleviated metabolic dysfunction in HFD-fed mice. However, prolonged SOX8 overexpression induced compensatory metabolic maladaptation, characterized by reduced energy expenditure, impaired glucose homeostasis, and mitochondrial structural disruption. These findings reveal a novel SOX8-USP7-PGC-1 regulatory axis in brown adipocytes, and reveal a previously unrecognized time-dependent effect of sustained thermogenic activation, highlighting SOX8 as a promising therapeutic target for obesity and metabolic syndrome.

The anteroposterior collinear expression of Hox genes is a hallmark of animal development that underpins the diversification of body plans1 and life cycles2. However, the origin and drivers of this coordinated expression remain elusive: while vertebrates rely on complex cluster-wide Hox gene regulation3-8, insects define gene-specific, sub-cluster regulatory domains9-11. Here, we discover a new mode of Hox gene regulation in segmented worms (Annelida). By combining chromatin conformation data with histone modifications profiling in Owenia fusiformis, we show that a large distal enhancer forms developmentally regulated, long-range contacts across the Hox cluster, and its activation coincides with the consolidation of a cluster-wide topologically associating domain (TAD), loss of Polycomb-mediated repression, and Hox gene transcription. This chromatin structure also occurs in the annelids Dimorphilus gyrociliatus and Capitella teleta, the latter showing additional subTAD structures that correlate with Hoxs temporal collinearity12. Moreover, related phyla with intact, organised Hox clusters and spatial collinearity, such as nemerteans and chitons, show annelid-like chromatin organisations, whereas phyla with disorganised13 Hox clusters do not. Coordinated Hox gene regulation from a "global control region" is thus ancestral to Lophotrochozoa, indicating that complex regulatory logics based on cluster-wide, long-range chromatin interactions with distal enhancers evolved convergently in vertebrates and spiralians.

Despite effective antivirals, Hepatitis C remains a global health burden, reflecting the difficulty of eliciting broadly neutralizing antibodies (bnAbs). For the Hepatitis C virus, bnAb responses are biased toward VH1-69-encoded Abs targeting a conserved epitope on the E2 glycoprotein, suggesting that effective immunogens must engage the corresponding germline B cell receptors. However, the structural basis of germline recognition remains unclear. Here, we show that germline-reverted VH1-69 bnAbs recognize the E2 core domain in a manner closely resembling mature Abs binding. High-resolution structures reveal a conserved mode of engagement, indicating that key epitope features are accessible prior to affinity maturation. Guided by these insights, we engineered E2 variants to enhance germline Ab interactions and stability. Although these designs increased affinity, they did not improve engagement of naive B cells, highlighting a key constraint in germline-targeting strategies. Together, our findings define principles governing germline recognition and inform the rational design of an HCV vaccine.

Sphingosine-1-phosphate (S1P) - a key bioactive component of high-density lipoproteins (HDL) - is instrumental in mediating HDLs cardiovascular benefits, largely by enhancing endothelial barrier integrity1, 2. Here, we discovered that S1P induces Notch1 activation, and this Notch activation is required to enhance Rac1 activity and adherens junction assembly, which in turn stimulates endothelial barrier integrity. S1P rapidly activates Notch1 by stimulating the G-coupled protein receptor, S1P Receptor 1 (S1PR1) to drive internalization of the Notch ligand Delta-like protein 4 (Dll4). Notably, this internalization of Dll4 and subsequent activation of Notch does not involve traditional G-protein signaling; instead, S1P-bound S1PR1 forms a complex with Dll4 via the scaffolding protein MPDZ, and the undergoes co-endocytosis. Importantly, the loss or inhibition of Notch, Dll4, S1PR1, or MPDZ results in barrier defects. These findings elucidate a novel S1PR1-Dll4-MPDZ-Notch1 signaling axis that coordinates S1P and Notch signaling to regulate of endothelial cell signaling and barrier function.

The intestinal brush border (BB), composed of densely packed microvilli on enterocytes, is essential for nutrient absorption and host defense. Its organization relies on the intermicrovillar adhesion complex (IMAC), mediated by protocadherins CDHR2 and CDHR5. Despite their clinical relevance in inflammatory bowel disease and several carcinomas, structural details of IMAC assemblies have remained elusive. Herein, we report the Cryo-EM structure of the adhesive complex at 3.4 [A] resolution, revealing a heterotetrameric ensemble composed of a dimer of CDHR2 and a dimer of CDHR5. This assembly ensures uniform adhesive strength between neighboring microvilli, and facilitates hexagonal packing of microvilli. Biophysical analyses and molecular dynamics simulations revealed a kinked, Ca{superscript 2}-free linker between domains EC3 and EC4 of CDHR5 conferring the necessary flexibility to withstand the shear stress caused during intestinal peristalsis. Collectively, these findings provide a structural framework for understanding BB organization and suggest strategies for therapeutics targeting IMAC in intestinal disorders.

Despite the success of direct-acting antivirals, preventing hepatitis C virus (HCV) reinfection remains a critical global challenge. To address this, we leveraged deep learning-based de novo protein design to engineer mini-proteins targeting the large extracellular loop (LEL) of the HCV co-receptor CD81. These mini-proteins are predicted to precisely dock into CD81-LEL, occluding the critical binding interface required for the HCV E2 glycoprotein. Through biophysical screening, we identified mini-19, a lead candidate with sub-nanomolar affinity (KD = 0.5 nM) and high thermostability up to 95{degrees}C. Flow cytometry and super-resolution imaging confirmed that mini-19 specifically recognizes the native topology of CD81 on cells and extracellular vesicles. Functionally, mini-19 neutralized HCVcc infection (IC50 = 1.2 nM). Molecular dynamics simulations demonstrated that mini-19 acts as a structural clamp, restricting the fusogenic conformational plasticity of the receptor. By targeting a conserved host factor rather than the rapidly mutating viral envelope, mini-19 provides a high genetic barrier to resistance. Beyond offering a prophylactic strategy against HCV, our findings establish a stable biologic platform for targeting tetraspanin-enriched microdomains and modulating host-pathogen interactions.

Transient receptor potential vanilloid 1 (TRPV1) ion channel mediates thermosensation and pain and is a target for non-addictive analgesics; however, clinical candidates have failed due to thermoregulatory side effects. Limited structural data for human TRPV1 (hTRPV1) bound to clinically relevant antagonists has constrained mechanistic insight. Using chemoinformatics-informed cryo-EM and BRET assays, we define the structural basis of antagonism across diverse chemotypes, including failed clinical compounds. A structure of hTRPV1 bound to 6-iodo-dihydrocapsaicin shows how a single substitution converts an agonist into an antagonist. Additional structures with Asivatrep, Mavatrep, and JNJ-17203212 reveal vanilloid pocket plasticity and divergent interaction networks, including lipid co-binding. Despite this diversity, antagonists converge on a conserved inhibited state, showing high potency is maintained across flexible binding modes. These findings redefine our understanding of hTRPV1 antagonism and illustrate how chemically diverse ligands stabilize an inhibited state in polymodal ion channels, laying groundwork for next-generation analgesics with improved safety.

The SLC6 family is a major target for neuropsychiatric therapeutics. Human B0AT2 (SLC6A15) regulates cerebral amino acid homeostasis and glutamatergic transmission and has been linked to major depressive disorder, yet its transport and inhibition mechanisms remain unclear. Here we report cryo-EM structures of human B0AT2 in the apo state and in complex with three substrates (proline, leucine, and methionine) and two inhibitors (loratadine and tiagabine), capturing outward-open, early substrate-bound intermediate, outward-occluded, and inward-open conformations along the transport cycle. These structures reveal a local conformational tuning at the canonical substrate-binding pocket (S1), in which rearrangement of Phe308 remodels the pocket geometry to tune substrate accommodation and selectivity. Loratadine stabilizes an outward-occluded state via allosteric inhibition at the extracellular S2 pocket, whereas tiagabine stabilizes the inward-open state through cooperative multi-site inhibition involving the S1 site and two previously unrecognized intracellular cavities (S3 and S4). Together with functional assays and mutagenesis, these data define the molecular basis of B0AT2 substrate selectivity and state-dependent inhibition. Notably, the two newly identified intracellular cavities are broadly conserved within the SLC6 family, reflecting a common intracellular vestibular architecture and enabling the rational design of conformation-selective modulators for neuropsychiatric disorders.