Color vision in catarrhine primates relies on red-, green-, and blue-sensitive cone pigments that share an 11-cis-retinal chromophore but differ in absorption maxima. Red and green pigments arose by recent gene duplication and differ at only a few residues. Here, we report cryo-electron microscopy structures of red and green cone pigments from the cynomolgus macaque (Macaca fascicularis) integrated with low-temperature vibrational spectroscopy and quantum mechanical and molecular mechanical modeling. The red-green spectral shift is dominated by threonine 285, the hydroxyl dipole of which modulates chromophore electrostatics, whereas steric effects appear modest. We also identified membrane-facing lateral openings in cone pigments but not in inactive rhodopsin. Comparisons with active-state structures suggest activation-dependent gating, and mutational and spectroscopic analyses support a role for this opening in retinal uptake and rapid pigment regeneration.

Lipid-binding domains, traditionally isolated from natural proteins, are essential tools for probing membrane lipid dynamics and specialized cellular compartments. Despite diverse applications, a general strategy for their engineering remains elusive. Here we present a robust and high-throughput method for monitoring protein-lipid interactions, named the cell surface liposome binding (CLiB) assay. Using the assay, we conducted directed evolution of the PX domain from SnxA, isolating high-affinity variants specific for phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). Combining the CLiB assay with next-generation sequencing enabled parallel analysis of >6,000 clones, comprehensively identifying key residues critical for lipid binding. An engineered variant, PX-SnxAGV, functioned as a lipid biosensor in yeast and mammalian cells, visualizing PI(3,5)P2-enriched membrane subdomains upon hyperosmotic shock and during microautophagy, thereby suggesting localized PI(3,5)P2 synthesis within spatially restricted regions. This study provides a framework for on-demand generation of lipid-binding probes, facilitating the discovery of membrane compartments characterized by unique lipid compositions.

Streptococcus pyogenes (Strep A), a formidable human pathogen, is notorious for causing life-threatening diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome, often complicated by thrombosis and coagulation abnormalities. While macrophage inflammasome activation has been widely studied in Strep A pathogenesis, the contribution of vascular endothelial cells-key regulators of immunity and coagulation-remains largely unexplored. This study aimed to determine whether endothelial NLRP3 inflammasome activation drives immunothrombosis during invasive Strep A infection and to define the roles of streptolysin O (SLO) and streptolysin S (SLS) in this mechanism. Using a murine intramuscular infection model in wild-type and NOD-like receptor family pyrin domain-containing 3 (NLRP3)-deficient mice together with infection of human microvascular endothelial cells with wild-type or toxin-deficient Strep A strains, we found that Strep A robustly activated the endothelial NLRP3 inflammasome, leading to caspase-1 activation, IL-1β secretion, endothelial pyroptosis, and a cascade of immunothrombotic events. Genetic ablation of NLRP3 or pharmacological inhibition of caspase-1 significantly attenuated inflammasome activation, immunothrombosis, and tissue injury. Both SLO and SLS were essential for these pathogenic effects. These findings illuminate a hitherto unrecognized endothelial inflammasome-mediated axis in Strep A infection and pave the way for innovative therapeutic strategies targeting endothelial immune responses to combat invasive streptococcal diseases.

Background Group A Streptococcus (GAS; Streptococcus pyogenes), which causes a broad spectrum of diseases, has been found to invade cells to avoid host immune clearance and antibiotic killing. Our previous findings have shown that the virulence factors of GAS—NAD-glycohydrolase depletes intracellular NAD+ to inhibit xenophagy, and streptolysin O increases the production of intracellular reactive oxygen species (ROS) to promote ineffective LC3-associated phagocytosis (LAP), thereby impairing GAS clearance in endothelial cells. However, how endothelial cells counteract these strategies for GAS clearance has yet to be comprehensively investigated. Methods We therefore speculated that resveratrol (RSV), a potent antioxidant and NAD+-dependent deacetylase—sirtuin activator, could be a potential antibacterial drug upon GAS infection in endothelial cells. To investigate the effect and the underlying mechanism of RSV on GAS infection, RSV was supplemented to human microvascular endothelial cell line-1 (HMEC-1) upon GAS infection, followed by detection of bacterial growth and examination the cellular regulation to LAP and xenophagy pathway. Results RSV significantly inhibited intracellular GAS multiplication in endothelial cells through increasing acidification and double-membrane formation of LC3-positive GAS-containing vacuoles. RSV upregulated the expression of autophagy-related proteins but downregulated the LAP-related proteins in GAS-infected endothelial cells. Knockdown of sirtuin 3 (SIRT3) dismissed the effect of RSV on enhancement of autophagic clearance of GAS and suppression of mtROS, which may participate in regulation of acidification of LC3-positive GAS-containing vacuoles and LAP-related proteins expression. Conclusion RSV promotes intracellular GAS clearance in endothelial cells by shifting the ineffective LAP pathway to functional xenophagy through SIRT3-mediated inhibition of mtROS.

Structures of nucleoprotein (N)-RNA complexes of the Bornaviridae, a virus family in the order Mononegavirales, have not been reported. Here, using cryo-electron microscopy (cryo-EM), we report high-resolution structures of Borna disease virus 1 (BoDV-1) N-RNA complex assemblies, including a dominant hexameric ring-like complex and less populated heptameric and octameric forms, the first RNA-bound N structures reported from this family. These structures reveal key features of N-RNA engagement and a BoDV-1-specific stoichiometry of eight nucleotides per N, providing a framework for comparison with related negative-strand RNA viruses. In addition to these RNA-bound complexes, we identified multiple RNA-free oligomers, indicating substantial conformational flexibility of N. Mutational analyses identified residues essential for nucleocapsid formation and RNA synthesis. Cryo-EM of mutant complexes captured RNA-free assemblies, suggesting that initial N oligomerization precedes RNA binding. These findings clarify the structural organization of the N-RNA complex and suggest how oligomeric plasticity contributes to nucleocapsid assembly.

Large-scale prioritization of protein–ligand interactions requires dynamic information at screening scale. We present a docking-to-dynamics workflow that evaluates poses by ensembles of ultrashort molecular dynamics (MD) trajectories (0.1–5 ns) and an online ligand-RMSD stability metric, MDscore. In a conditional six-target retrospective benchmark of 88 active and 702 inactive compounds, MD-score achieved pose-aware ROC-AUC of 0.868 at top ten poses, while sampling analyses indicated that broad pose and velocity sampling was more useful than extending a small number of trajectories. Runtime steering, which terminates unstable poses and redistributes unfinished tasks, scaled the workflow to 1,234,620 docked poses from 124,245 generated compounds on Fugaku supercomputer while reducing simulated time to 24% of exhaustive evaluation. In a Marburg virus nucleoprotein screen, the workflow prioritized compounds that yielded three initial minigenome-active hits among 76 tested compounds. These results position ultrashort MD as a practical dynamic filtering layer between docking, data-driven prediction and higher-cost validation methods.

Applying molecular dynamics (MD) at discovery scale is limited by the cost of evaluating thousands to millions of candidate poses. We present a docking-to-dynamics workflow that makes MD compatible with screening-scale evaluation by applying ensembles of ultrashort trajectories (0.1–5 ns) to each pose. MD-score, a ligand-RMSD stability metric, serves as both a pose-ranking score and, with a threshold, an online stopping criterion. In a conditional six-target benchmark of 88 actives and 702 inactives, MD-score achieved a pose-aware ROC-AUC of 0.868 for the top ten poses, while broad pose and velocity sampling was more effective than extending fewer trajectories. Runtime steering scaled evaluation to 1,234,620 EGFR poses while reducing simulated time to 24% of exhaustive evaluation. A prospective Marburg virus nucleoprotein screen yielded three initial minigenome-active hits among 76 tested compounds. These results position ultrashort MD as a scalable dynamic filter between docking, data-driven prediction and higher-cost validation.

Secretory immunoglobulin A (sIgA), the first line of defense against mucosal epithelial infections, is a multimer. Although multimeric sIgA shows higher antiviral activity than monomeric IgA, the mechanism underlying this enhancement and the functional differences among IgA subclasses remain unclear. Here, we generated recombinant monomeric and multimeric IgA1 and IgA2m2 antibodies from clone F045-092, which targets the head domain of influenza A virus (IAV) hemagglutinin (HA) and inhibits viral entry, and compared their functional properties. Monomeric IgA1 inhibited viral entry but not viral release. Multimerization of IgA1 enhanced viral entry inhibition and conferred the additional function of inhibiting viral release. In contrast, monomeric IgA2m2 inhibited both viral entry and release and its multimerization enhanced these inhibitory effects. Importantly, treatment of IAV particles with multimeric sIgA or monomeric IgA2m2 induced viral aggregation via IgA-mediated tethering of neighboring virus particles, as visualized via cryo-electron tomography. Analysis of F(ab')2 and Fab fragments from F045-092 IgA2m2 demonstrated that bivalency is essential for antibody-mediated viral aggregation (AVA) by monomeric IgA2m2, further supporting a direct tethering mechanism among virus particles. In conclusion, multimerization of the HA head-targeting IgA tends to enhance the inhibition of viral entry and release through AVA, contributing to its enhanced antiviral activity. Additionally, different IgA subclasses with identical variable regions can exhibit distinct properties in the monomeric form, and subclass switching may confer additional functions. Our findings contribute to the development of virus-aggregating antibodies as a new therapeutic approach that efficiently inhibits viral entry and release at the mucosal epithelium.

Autophagy is induced by nutrient starvation to recycle intracellular constituents; however, its activity must subsequently be attenuated during prolonged nutrient deprivation. The mechanisms underlying this attenuation in mammalian cells remain incompletely understood. Here, using complementary HaloTag-based assays, we show that autophagic activity declines during prolonged starvation in HeLa cells. A genome-wide CRISPR/Cas9 knockout screen designed to identify cells that sustain autophagic activity under these conditions identified protocadherin 17 (PCDH17) as a regulator of autophagy attenuation. PCDH17 depletion maintained autophagic activity during prolonged starvation without detectably altering mTORC1 signaling, ULK1 abundance, or the proximal machinery of autophagosome formation. Instead, PCDH17 depletion increased lysosomal abundance, acidification, and proteolytic activity, whereas PCDH17 overexpression produced reciprocal effects. We further identified a lysosome-associated PCDH17 subpopulation that is supplied predominantly through the biosynthetic ER-Golgi pathway. This pool undergoes proteolytic processing and lysosomal turnover, with starvation preferentially accelerating degradation of the C-terminal fragment while preserving a comparatively stable N-terminal fragment. Together, these findings identify PCDH17 as an unexpected negative regulator of lysosomal function and demonstrate that modulation of lysosomal degradative capacity contributes to autophagy attenuation during prolonged starvation.

The COVID-19 pandemic and recurring outbreaks of infectious diseases underscore the urgent demand for multiplex diagnostics capable of rapid and accurate pathogen identification. Although multiplex nucleic acid amplification tests (NAATs) are widely used for diagnosing diverse infectious diseases, their inherent amplification bias and long turnaround times highlight the demand for faster and reliable alternatives. Here, we present multicolor SATORI (mSATORI), an amplification-free single-molecule genetic test that leverages the complementary activities of CRISPR-Cas13a and Cas13b to achieve simultaneous detection of dual RNA targets. mSATORI identified Influenza A and SARS-CoV-2 RNAs within ∼10 min, with analytical limits of detection (LoD) of 86 aM and 52 aM, respectively. Validation using clinical specimens demonstrated robust diagnostic performance, achieving femtomolar limits of detection (550 aM for Influenza A and 640 aM for SARS-CoV-2), along with sensitivities exceeding 80% and specificities of 100%. Collectively, these results establish mSATORI as a platform for next-generation molecular diagnostics, with broad implications for clinical implementation, outbreak preparedness, and global infectious disease surveillance.

The innate immune response to viral infection needs to be tightly regulated to ensure effective pathogen clearance while avoiding excessive immune activation. During SARS-CoV-2 infection, however, the immune system often fails to elicit appropriate responses, resulting in cytokine-release syndrome in patients with COVID-19. In this study, we show that reduced expression of Regnase-1, an RNase that negatively regulates immune cell activation, confers resistance to infection with the mouse-adapted SARS-CoV-2 MA10 strain. In Regnase-1+/- mice, altered neutrophil function contributed to the amelioration of MA10-induced pneumonia. Single-cell RNA sequencing of lung tissue during MA10 infection revealed four distinct neutrophil subsets, and among these, a subset characterized by an interferon-stimulated gene (ISG) signature was decreased in Regnase-1+/- mice. Furthermore, Regnase-1+/- neutrophils exhibited reduced ISG expression without corresponding changes in proinflammatory gene expression. Regnase-1 was found to repress the expression of Tsc22d3, a gene involved in the negative regulation of interferon responses, through its 3' untranslated region. Collectively, these findings suggest that Regnase-1 attenuates resistance to SARS-CoV-2 MA10 infection by promoting excessive interferon responses in neutrophils.

PTEN-induced kinase 1 (PINK1) is a mitochondrial serine/threonine kinase that plays a central role in Parkin-dependent mitophagy. Mutations in PINK1 are associated with familial Parkinson’s disease. PINK1 is a high-affinity client of the HSP90–CDC37 complex and is stabilized by this chaperone system. However, the molecular mechanism by which HSP90–CDC37 facilitates the folding of PINK1 remains unclear. Here, we present a cryogenic electron microscopy structure of the human PINK1–HSP90–CDC37 complex. The β5 strand of the PINK1 N-lobe is accommodated in the central channel of the HSP90 dimer, which holds the PINK1 kinase domain in a partially unfolded state. The C-lobe and unique C-terminal extension (CTE) of PINK1 is folded. HSP90 covers the CTE of PINK1, which overlaps with interaction sites for TOM5, TOM20, and the PINK1 N-helix. The HPNI motif of CDC37 interacts with the C-lobe of PINK1, mimicking the HPNI motif in the N-lobe. The pathogenic mutation L347P is suggested to disrupt these interactions, while H271Q is located within the HPNI motif in the N-lobe of PINK1. These findings provide structural insights into the folding of PINK1 and its dysfunction in Parkinson’s disease.

Microautophagy is an intracellular degradation process in which degradatory organelles, such as the lysosome, directly take up substrates by invagination and/or protrusion of their membranes. Here, we provide evidence that Rab32-positive, lysosome-related organelles in macrophages incorporate various other organelles, including endosomes and mitochondria. Our data indicates that, upon exposure to a mitochondria-damaging reagent, mitochondria can be directly engulfed by the lysosome-like organelles independently of macroautophagy or ESCRT machinery. Rab32 GTPase, phosphatidylinositol 3,5-bisphosphates, ubiquitination, and p62/SQSTM1 are crucial for this degradation. Furthermore, the degree of M1 polarization of macrophages, which is facilitated by metabolic reprogramming into increased glycolysis via mitochondrial elimination, is significantly reduced in Rab32/38 double-knockout macrophages. Thus, microautophagy plays a role in the physiological regulation of macrophages.

Double-stranded RNA (dsRNA), which induces an innate immune response against viral infections, is rarely detected in influenza A virus (IAV)-infected cells. Nevertheless, we previously reported that the influenza A viral ribonucleoprotein (vRNP) complex generates looped dsRNAs during RNA synthesis in vitro. This finding suggests that IAV possesses a specific mechanism for sequestering dsRNA within infected cells, thereby enabling viral evasion of the innate immune response. Here, we found that dsRNAs were detected in infected cells lacking the expression of viral non-structural protein 1 (NS1) and nuclear export protein (NEP), both encoded by the same RNA segment. Indeed, the looped dsRNA-vRNP complexes were isolated from IAV-infected cells. Interestingly, NS1 molecules masked the entire looped dsRNA generated by vRNP in vitro, implying a potential role for NS1 in segregating viral dsRNA from cytoplasmic dsRNA sensors. Furthermore, dsRNAs were sequestered within the nucleus of wild-type IAV-infected cells, whereas their translocation to the cytoplasm was observed in NS1-deleted mutant virus-infected cells expressing M1 and NEP. This result indicates the possibility that dsRNA is transported to the cytoplasm in association with vRNP. Notably, the cytoplasmic translocation of dsRNA triggered the nuclear translocation of interferon regulatory factor 3, suggesting the capability of dsRNA in inducing the innate immune response. These findings highlight IAV's distinctive strategy for circumventing innate immunity by sequestration of dsRNAs.IMPORTANCEIt is widely recognized that double-stranded RNA (dsRNA) produced during viral infection triggers an innate immune response. However, the influenza A virus (IAV) has been thought to rarely produce dsRNA within infected cells. Here, we detected dsRNA in the nucleus of IAV-infected cells which lacked the expression of viral non-structural protein 1 (NS1) and nuclear export protein (NEP), both encoded by a single RNA segment. High-speed atomic force microscopy demonstrated that NS1 entirely concealed dsRNA produced by the viral ribonucleoprotein complexes, thereby segregating it from cytoplasmic dsRNA sensors that trigger the innate immune response. Interestingly, cytoplasmic translocation of dsRNA was observed in cells infected with an NS1-deleted mutant virus, where M1 and NEP were expressed, resulting in the nuclear translocation of interferon regulatory factor 3. Collectively, our findings suggest that IAV adeptly sequesters dsRNA to evade the innate immune system.

The development of microphysiological systems for preclinical research is often hindered by the limited availability of reliable cell sources, especially when multiple organs or tissues from a single patient are needed for comparative studies of the host innate immune response. In this study, we develop human airway-on-chip and alveolus-on-chip models using lung progenitor cells derived from isogenic induced pluripotent stem cells. Our results using SARS-CoV-2 and influenza reveal distinct initial innate immune responses in the airway- and alveolus-on-chip models. SARS-CoV-2-infected airway chips show a robust early interferon-dependent innate immune response, while alveolus chips show dysregulated and delayed interferon activation alongside a significantly upregulated chemokine pathway. In contrast, influenza infection induces a more pronounced innate immune response and greater cellular damage in both chips compared with SARS-CoV-2. Consequently, airway- and alveolus-on-chip models derived from induced pluripotent stem cells offer a viral pathology platform with screening potential for future therapeutic agents. Airway-on-chip and alveolus-on-chip models using induced-pluripotent-stem-cell-derived lung progenitor cells show innate immune responses to common viruses.

Live-cell imaging enables visualization of the spatiotemporal dynamics of signals in cells. Intracytoplasmic movement of nucleocapsids is crucial during the life cycle of enveloped viruses; however, the molecular mechanisms governing their assembly and transport are not fully understood. Using a Marburg virus (MARV) live-cell imaging system, we identified three nucleocapsid proteins-nucleoprotein (NP), VP35, and VP24-that are necessary and sufficient to form transport-competent nucleocapsid-like structures (NCLSs). These findings are consistent with observations in Ebola virus (EBOV). Interestingly, despite incompatibility among these proteins, VP30 interacts with nucleocapsid proteins from both MARV and EBOV, supporting viral transcription and replication in heterologous systems. Furthermore, we show that the conserved PPxPxY motif at the C-terminus of NP regulates NP-VP30 interactions in both homologous and heterologous contexts and is crucial for VP30 association with NCLSs. Because this motif is conserved across filoviruses, it represents a promising target for antiviral development. Our findings advance the understanding of nucleocapsid formation and offer new avenues for therapeutic intervention against MARV and EBOV.IMPORTANCEThis study provides crucial insights into the molecular mechanisms of nucleocapsid assembly and transport in filoviruses, specifically Marburg virus (MARV) and Ebola virus (EBOV). Using advanced live-cell imaging, we uncovered how the conserved PPxPxY motif in nucleoprotein (NP) mediates its interactions with VP30, thereby regulating nucleocapsid formation and viral replication. Notably, while VP30's role differs between MARV and EBOV, the underlying mechanism of NP-VP30 interaction via this motif appears conserved across filoviruses, making it a promising target for broad-spectrum antiviral strategies. These findings deepen our understanding of nucleocapsid protein compatibility and virus-host interactions, offering new avenues for therapeutic intervention against these deadly pathogens.

Influenza A virus (IAV) has an eight-segmented, single-stranded, negative-sense viral genomic RNA (vRNA). Each vRNA strand associates with nucleoproteins and an RNA-dependent RNA polymerase complex to form a viral ribonucleoprotein (vRNP) complex. IAV vRNPs adopt a flexible double-helical configuration that varies in length. Although the transcription and replication of vRNA take place in the context of vRNPs, the precise structural conformation of vRNPs during RNA synthesis remains partially elucidated. To unravel the intricate ultrastructure of the vRNP, it is necessary to purify it while preserving its native functionality. Herein, we introduce a comprehensive protocol for the purification of IAV vRNPs using glycerol gradient ultracentrifugation. Furthermore, we provide a method for the high-speed atomic force microscopy observation of vRNPs during viral RNA synthesis.

The Ebola virus, a member of the Filoviridae family, causes severe hemorrhagic fever in humans. Filamentous virions contain a helical nucleocapsid responsible for genome transcription, replication, and packaging into progeny virions. The nucleocapsid consists of a helical nucleoprotein (NP)–viral genomic RNA complex forming the core structure, to which VP24 and VP35 bind externally. Two NPs, each paired with a VP24 molecule, constitute a repeating unit. However, the detailed nucleocapsid structure remains unclear. Here, we determine the nucleocapsid-like structure within virus-like particles at 4.6 Å resolution using single-particle cryo-electron microscopy. Mutational analysis identifies specific interactions between the two NPs and two VP24s and demonstrates that each of the two VP24s in different orientations distinctively regulates nucleocapsid assembly, viral RNA synthesis, intracellular transport of the nucleocapsid, and infectious virion production. Our findings highlight the sophisticated mechanisms underlying the assembly and functional regulation of the nucleocapsid and provide insights into antiviral development. Here, the authors use cryo-electron microscopy to structurally characterise the Ebola virus nucleocapsid within virus-like particles, uncovering how specific interactions regulate viral genome synthesis, assembly, and infectious particle production.

Sphingosine-1-phosphate (S1P) is one of the most extensively studied bioactive lipids that transduces signals via the S1P receptor (S1PR) family (S1PR1-5), a class of G-protein-coupled receptors (GPCRs), to regulate immune cell migration, vascular permeability, and pain modulation. However, the mechanism for achieving specificity in downstream signaling remains poorly understood. Here, we present cryogenic electron microscopic structures of the S1PR3-G αq complex bound to endogenous agonists: d18:1 S1P or d16:1 S1P. Both agonists shared the same binding pocket and binding mode despite the different signaling intensities of the S1PR3-G αq signal pathway. By comparing the structures of two agonist-bound complexes, combined with mutagenesis studies, we identified key amino acids, Phe119 3.33 and Arg136 3.50 , that play crucial roles in differential agonist recognition and receptor activation. Furthermore, structural comparisons with previously determined S1PR3-G αi complex or G-protein-free S1PR3 structures, along with mutagenesis analysis, revealed dynamic intracellular loop 2 conformations and specific amino acid interactions that contribute to G-protein selectivity. Notably, we identified amino acids at the 34.50 and 34.53 positions within ICL2 as critical for specific interactions with G proteins. These findings provide better understanding of the mechanism of GPCR activation and unique perspectives that can be applied to other class A GPCRs, leading to the possibility of optimized drug development.

The SARS-CoV-2 pandemic provided important insights into the relationship between infectious diseases and the human genome. A genomic region encoding the 2′-5′-oligoadenylate synthetase (OAS) family proteins that sense viral genomic RNAs and trigger an antiviral response contains single nucleotide polymorphisms (SNPs) associated with SARS-CoV-2 infection susceptibility. A high-risk SNP identified at the splice acceptor site of OAS1 exon 6—a terminal exon—alters the proportion of various splicing isoforms of OAS1 and its activity. However, the actual causality of this SNP or splicing to infection susceptibility remains unknown. In this study, it was found that serine–arginine-rich splicing factor 6 (SRSF6) binds to the splice donor site of the human OAS1 exon 5. SRSF6 determines the selected alternative terminal exon when the risk allele disrupts the splice acceptor site. Subsequently, an inhibitor for CDC-like kinase was rationally selected as a candidate splicing modulator. RNA-Seq and RT-PCR analyses revealed that this inhibitor can induce splice switching of OAS1 mRNAs in the human lung adenocarcinoma cell line Calu-3. Under the inhibitor treatment, the cells exhibited reduced SARS-CoV-2 infection rates. Meanwhile, the colonic epithelial cell line Caco-2 expressed non-risk type OAS1 mRNA isoforms that did not undergo splice-switching or demonstrate altered SARS-CoV-2 sensitivity following treatment with the inhibitor. These results indicate that a high-risk SNP in OAS1 influences cell susceptibility to SARS-CoV-2 infection by inducing splice-switching at its terminal exon. Additionally, chemical splicing modifiers may prove beneficial in overcoming this genomic vulnerability.