Mitochondrial Protein Synthesis Adapts to Influx of Nuclear-Encoded Protein.

Mitochondrial ribosomes translate membrane integral core subunits of the oxidative phosphorylation system encoded by mtDNA. These translation products associate with nuclear-encoded, imported proteins to form enzyme complexes that produce ATP. Here, we show that human mitochondrial ribosomes display translational plasticity to cope with the supply of imported nuclear-encoded subunits. Ribosomes expressing mitochondrial-encoded COX1 mRNA selectively engage with cytochrome c oxidase assembly factors in the inner membrane. Assembly defects of the cytochrome c oxidase arrest mitochondrial translation in a ribosome nascent chain complex with a partially membrane-inserted COX1 translation product. This complex represents a primed state of the translation product that can be retrieved for assembly. These findings establish a mammalian translational plasticity pathway in mitochondria that enables adaptation of mitochondrial protein synthesis to the influx of nuclear-encoded subunits.

Proteome-scale recombinant standards and a robust high-speed search engine to advance cross-linking MS-based interactomics.

Advancing data analysis tools for proteome-wide cross-linking mass spectrometry (XL-MS) requires ground-truth standards that mimic biological complexity. Here we develop well-controlled XL-MS standards comprising hundreds of recombinant proteins that are systematically mixed for cross-linking. We use one standard dataset to guide the development of Scout, a search engine for XL-MS with MS-cleavable cross-linkers. Using other, independent standard datasets and published datasets, we benchmark the performance of Scout and existing XL-MS software. We find that Scout offers an excellent combination of speed, sensitivity and false discovery rate control. The results illustrate how our large recombinant standard can support the development of XL-MS analysis tools and evaluation of XL-MS results.

A leadership-level culture cycle intervention changes teachers' culturally inclusive beliefs and practices.

Despite an abundance of support for culturally inclusive learning environments, there is little consensus regarding how to change educational contexts to effectively and sustainably foster cultural inclusion. To address this gap, we report findings from a research-practice partnership that leveraged the Culture Cycle Framework (CCF) to expand educators' praxis to include both independent and interdependent models of self. Most U.S. schools validate independent cultural models (i.e., those that prioritize individuality, uniqueness, and personal agency) and overlook interdependent models (i.e., those that prioritize connectedness, relationality, and collective well-being), which are more common among students from marginalized racial and socioeconomic backgrounds. Using a quasi-experimental longitudinal design, we trained school leadership to integrate ideas about cultural inclusion (i.e., validating the importance of both independent and interdependent cultural models) into school-wide flagship practices. We assessed downstream indicators of culture change by surveying teachers and students across the district and found that a) leadership-level training enhanced school-wide beliefs about cultural inclusion, b) teachers' endorsement of culturally inclusive beliefs predicted their use of culturally inclusive practices, and c) teachers' use of culturally inclusive practices predicted enhanced psychosocial and academic outcomes among students. This research represents a comprehensive culture change effort using the CCF and illustrates a means of fostering inclusion-focused educational culture change and assessing downstream consequences of culture change initiatives.

Functional importance and divergence of plant apurinic/apyrimidinic endonucleases in somatic and meiotic DNA repair.

Apurinic/apyrimidinic (AP) sites are one of the most abundant DNA lesions and are mainly repaired by AP endonucleases (APEs). While most eukaryotic genomes encode two APEs, plants usually possess three APEs, namely APE1L, APE2, and ARP. To date, the biological relevance and functional divergence of plant APEs are unclear. Here, we show that the three plant APEs have ancient origins, with the APE1L clade being plant-specific. In Arabidopsis thaliana, simultaneously mutating APE1L and APE2, but not ARP alone or in combination with either APE1L or APE2, results in clear developmental defects linked to genotoxic stress. Genetic analyses indicated that the three plant APEs have different substrate preferences in vivo. ARP is mainly responsible for AP site repair, while APE1L and APE2 prefer to repair 3'-blocked single-stranded DNA breaks. We further determined that APEs play an important role in DNA repair and the maintenance of genomic integrity in meiotic cells. The ape1l ape2 double mutant exhibited a greatly enhanced frequency of sporulation 1 (SPO11-1)-dependent and SPO11-1-independent double-stranded DNA breaks. The DNA damage response (DDR) was activated in ape1l ape2 to trigger pollen abortion. Our findings suggest functional divergence of plant APEs and reveal important roles of plant APEs during vegetative and reproductive development.

Advancing microRNA target site prediction with transformer and base-pairing patterns.

MicroRNAs (miRNAs) are short non-coding RNAs involved in various cellular processes, playing a crucial role in gene regulation. Identifying miRNA targets remains a central challenge and is pivotal for elucidating the complex gene regulatory networks. Traditional computational approaches have predominantly focused on identifying miRNA targets through perfect Watson-Crick base pairings within the seed region, referred to as canonical sites. However, emerging evidence suggests that perfect seed matches are not a prerequisite for miRNA-mediated regulation, underscoring the importance of also recognizing imperfect, or non-canonical, sites. To address this challenge, we propose Mimosa, a new computational approach that employs the Transformer framework to enhance the prediction of miRNA targets. Mimosa distinguishes itself by integrating contextual, positional and base-pairing information to capture in-depth attributes, thereby improving its predictive capabilities. Its unique ability to identify non-canonical base-pairing patterns makes Mimosa a standout model, reducing the reliance on pre-selecting candidate targets. Mimosa achieves superior performance in gene-level predictions and also shows impressive performance in site-level predictions across various non-human species through extensive benchmarking tests. To facilitate research efforts in miRNA targeting, we have developed an easy-to-use web server for comprehensive end-to-end predictions, which is publicly available at http://monash.bioweb.cloud.edu.au/Mimosa.

rAAV capsid mutants eliminate leaky expression from DNA donor template for homologous recombination.

Precise genomic editing through the combination of CRISPR/Cas systems and recombinant adeno-associated virus (rAAV)-delivered homology directed repair (HDR) donor templates represents a powerful approach. However, the challenge of effectively suppressing leaky transcription from the rAAV vector, a phenomenon associated to cytotoxicity, persists. In this study, we demonstrated substantial promoter activities of various homology arms and inverted terminal repeats (ITR). To address this issue, we identified a novel rAAV variant, Y704T, which not only yields high-vector quantities but also effectively suppresses in cis mRNA transcription driven by a robust promoter. The Y704T variant maintains normal functionality in receptor interaction, intracellular trafficking, nuclear entry, uncoating, and second-strand synthesis, while specifically exhibiting defects in transcription. Importantly, this inhibitory effect is found to be independent of ITR, promoter types, and RNA polymerases. Mechanistic studies unveiled the involvement of Valosin Containing Protein (VCP/p97) in capsid-mediated transcription repression. Remarkably, the Y704T variant delivers HDR donor templates without compromising DNA replication ability and homologous recombination efficiency. In summary, our findings enhance the understanding of capsid-regulated transcription and introduce novel avenues for the application of the rAAV-CRISPR/Cas9 system in human gene therapy.

HEI10 is subject to phase separation and mediates RPA1a degradation during meiotic interference-sensitive crossover formation.

Reciprocal exchanges of DNA between homologous chromosomes during meiosis, or crossovers (COs), shuffle genetic information in gametes and progeny. In many eukaryotes, the majority of COs (class I COs) are sensitive to a phenomenon called interference, which influences the occurrence of closely spaced double COs. Class I COs depend on a group of factors called ZMM (Zip, Msh, Mer) proteins including HEI10 (Human Enhancer of Invasion-10). However, how these proteins are recruited to class I CO sites is unclear. Here, we show that HEI10 forms foci on chromatin via a liquid-liquid phase separation (LLPS) mechanism that relies on residue Ser70. A HEI10S70F allele results in LLPS failure and a defect in class I CO formation. We further used immunoprecipitation-mass spectrometry to identify RPA1a (Replication Protein A 1) as a HEI10 interacting protein. Surprisingly, we find that RPA1a also undergoes phase separation and its ubiquitination and degradation are directly regulated by HEI10. We also show that HEI10 is required for the condensation of other class I CO factors. Thus, our results provide mechanistic insight into how meiotic class I CO formation is controlled by HEI10 coupling LLPS and ubiquitination.

Pharmacotherapy for adults with overweight and obesity: a systematic review and network meta-analysis of randomised controlled trials.

BACKGROUND: Pharmacotherapy provides an option for adults with overweight and obesity to reduce their bodyweight if lifestyle modifications fail. We summarised the latest evidence for the benefits and harms of weight-lowering drugs. METHODS: This systematic review and network meta-analysis included searches of PubMed, Embase, and Cochrane Library (CENTRAL) from inception to March 23, 2021, for randomised controlled trials of weight-lowering drugs in adults with overweight and obesity. We performed frequentist random-effect network meta-analyses to summarise the evidence and applied the Grading of Recommendations Assessment, Development, and Evaluation frameworks to rate the certainty of evidence, calculate the absolute effects, categorise interventions, and present the findings. The study was registered with PROSPERO, CRD 42021245678. FINDINGS: 14 605 citations were identified by our search, of which 132 eligible trials enrolled 48 209 participants. All drugs lowered bodyweight compared with lifestyle modification alone; all subsequent numbers refer to comparisons with lifestyle modification. High to moderate certainty evidence established phentermine-topiramate as the most effective in lowering weight (odds ratio [OR] of ≥5% weight reduction 8·02, 95% CI 5·24 to 12·27; mean difference [MD] of percentage bodyweight change -7·98, 95% CI -9·27 to -6·69) followed by GLP-1 receptor agonists (OR 6·33, 95% CI 5·00 to 8·00; MD -5·79, 95% CI -6·34 to -5·25). Naltrexone-bupropion (OR 2·69, 95% CI 2·10 to 3·44), phentermine-topiramate (2·40, 1·68 to 3·44), GLP-1 receptor agonists (2·22, 1·74 to 2·84), and orlistat (1·71, 1·42 to 2·05) were associated with increased adverse events leading to drug discontinuation. In a post-hoc analysis, semaglutide, a GLP-1 receptor agonist, showed substantially larger benefits than other drugs with a similar risk of adverse events as other drugs for both likelihood of weight loss of 5% or more (OR 9·82, 95% CI 7·09 to 13·61) and percentage bodyweight change (MD -11·40, 95% CI -12·51 to -10·29). INTERPRETATION: In adults with overweight and obesity, phentermine-topiramate and GLP-1 receptor agonists proved the best drugs in reducing weight; of the GLP-1 agonists, semaglutide might be the most effective. FUNDING: 1.3.5 Project for Disciplines of Excellence, West China Hospital, Sichuan University.

ProsperousPlus: a one-stop and comprehensive platform for accurate protease-specific substrate cleavage prediction and machine-learning model construction.

Proteases contribute to a broad spectrum of cellular functions. Given a relatively limited amount of experimental data, developing accurate sequence-based predictors of substrate cleavage sites facilitates a better understanding of protease functions and substrate specificity. While many protease-specific predictors of substrate cleavage sites were developed, these efforts are outpaced by the growth of the protease substrate cleavage data. In particular, since data for 100+ protease types are available and this number continues to grow, it becomes impractical to publish predictors for new protease types, and instead it might be better to provide a computational platform that helps users to quickly and efficiently build predictors that address their specific needs. To this end, we conceptualized, developed, tested and released a versatile bioinformatics platform, ProsperousPlus, that empowers users, even those with no programming or little bioinformatics background, to build fast and accurate predictors of substrate cleavage sites. ProsperousPlus facilitates the use of the rapidly accumulating substrate cleavage data to train, empirically assess and deploy predictive models for user-selected substrate types. Benchmarking tests on test datasets show that our platform produces predictors that on average exceed the predictive performance of current state-of-the-art approaches. ProsperousPlus is available as a webserver and a stand-alone software package at http://prosperousplus.unimelb-biotools.cloud.edu.au/.

Advancing mRNA subcellular localization prediction with graph neural network and RNA structure.

MOTIVATION: The asymmetrical distribution of expressed mRNAs tightly controls the precise synthesis of proteins within human cells. This non-uniform distribution, a cornerstone of developmental biology, plays a pivotal role in numerous cellular processes. To advance our comprehension of gene regulatory networks, it is essential to develop computational tools for accurately identifying the subcellular localizations of mRNAs. However, considering multi-localization phenomena remains limited in existing approaches, with none considering the influence of RNA's secondary structure. RESULTS: In this study, we propose Allocator, a multi-view parallel deep learning framework that seamlessly integrates the RNA sequence-level and structure-level information, enhancing the prediction of mRNA multi-localization. The Allocator models equip four efficient feature extractors, each designed to handle different inputs. Two are tailored for sequence-based inputs, incorporating multilayer perceptron and multi-head self-attention mechanisms. The other two are specialized in processing structure-based inputs, employing graph neural networks. Benchmarking results underscore Allocator's superiority over state-of-the-art methods, showcasing its strength in revealing intricate localization associations. AVAILABILITY AND IMPLEMENTATION: The webserver of Allocator is available at http://Allocator.unimelb-biotools.cloud.edu.au; the source code and datasets are available on GitHub (https://github.com/lifuyi774/Allocator) and Zenodo (https://doi.org/10.5281/zenodo.13235798).

MicroRNA397 promotes rice flowering by regulating the photorespiration pathway.

The precise timing of flowering plays a pivotal role in ensuring successful plant reproduction and seed production. This process is intricately governed by complex genetic networks that integrate internal and external signals. This study delved into the regulatory function of microRNA397 (miR397) and its target gene LACCASE-15 (OsLAC15) in modulating flowering traits in rice (Oryza sativa). Overexpression of miR397 led to earlier heading dates, decreased number of leaves on the main stem, and accelerated differentiation of the spikelet meristem. Conversely, overexpression of OsLAC15 resulted in delayed flowering and prolonged vegetative growth. Through biochemical and physiological assays, we uncovered that miR397-OsLAC15 had a profound impact on carbohydrate accumulation and photosynthetic assimilation, consequently enhancing the photosynthetic intensity in miR397-overexpressing rice plants. Notably, we identified that OsLAC15 is at least partially localized within the peroxisome organelle, where it regulates the photorespiration pathway. Moreover, we observed that a high CO2 concentration could rescue the late flowering phenotype in OsLAC15-overexpressing plants. These findings shed valuable insights into the regulatory mechanisms of miR397-OsLAC15 in rice flowering and provided potential strategies for developing crop varieties with early flowering and high-yield traits through genetic breeding.

Mefloquine targets NLRP3 to reduce lipopolysaccharide-induced systemic inflammation and neural injury.

The NLR family pyrin domain containing 3 (NLRP3) inflammasome plays an important role in the pathogenesis of a wide variety of human diseases. So far, drugs directly and specifically targeting the NLRP3 inflammasome are not available for clinical use since the safety and efficacy of new compounds are often unclear. A promising approach is thus to identify NLRP3 inhibitors from existing drugs that are already in clinical use. Here, we show that mefloquine, a well-known antimalarial drug, is a highly selective and potent NLRP3 inhibitor by screening a FDA-approved drug library. Mechanistically, mefloquine directly binds to the NLRP3 NACHT and LRR domains to prevent NLRP3 inflammasome activation. More importantly, mefloquine treatment attenuates the symptoms of lipopolysaccharide-induced systemic inflammation and Parkinson's disease-like neural damage in mice. Our findings identify mefloquine as a potential therapeutic agent for NLRP3-driven diseases and migth expand its clinical use considerably.

Artificial Neuron Devices.

Efforts to design devices emulating complex cognitive abilities and response processes of biological systems have long been a coveted goal. Recent advancements in flexible electronics, mirroring human tissue's mechanical properties, hold significant promise. Artificial neuron devices, hinging on flexible artificial synapses, bioinspired sensors, and actuators, are meticulously engineered to mimic the biological systems. However, this field is in its infancy, requiring substantial groundwork to achieve autonomous systems with intelligent feedback, adaptability, and tangible problem-solving capabilities. This review provides a comprehensive overview of recent advancements in artificial neuron devices. It starts with fundamental principles of artificial synaptic devices and explores artificial sensory systems, integrating artificial synapses and bioinspired sensors to replicate all five human senses. A systematic presentation of artificial nervous systems follows, designed to emulate fundamental human nervous system functions. The review also discusses potential applications and outlines existing challenges, offering insights into future prospects. We aim for this review to illuminate the burgeoning field of artificial neuron devices, inspiring further innovation in this captivating area of research.

PDIA3 orchestrates effector T cell program by serving as a chaperone to facilitate the non-canonical nuclear import of STAT1 and PKM2.

Dysregulated T cell activation underpins the immunopathology of rheumatoid arthritis (RA), yet the machineries that orchestrate T cell effector program remain incompletely understood. Herein, we leveraged bulk and single-cell RNA sequencing data from RA patients and validated protein disulfide isomerase family A member 3 (PDIA3) as a potential therapeutic target. PDIA3 is remarkably upregulated in pathogenic CD4 T cells derived from RA patients and positively correlates with C-reactive protein level and disease activity score 28. Pharmacological inhibition or genetic ablation of PDIA3 alleviates RA-associated articular pathology and autoimmune responses. Mechanistically, T cell receptor signaling triggers intracellular calcium flux to activate NFAT1, a process that is further potentiated by Wnt5a under RA settings. Activated NFAT1 then directly binds to the Pdia3 promoter to enhance the expression of PDIA3, which complexes with STAT1 or PKM2 to facilitate their nuclear import for transcribing T helper 1 (Th1) and Th17 lineage-related genes, respectively. This non-canonical regulatory mechanism likely occurs under pathological conditions, as PDIA3 could only be highly induced following aberrant external stimuli. Together, our data support that targeting PDIA3 is a vital strategy to mitigate autoimmune diseases, such as RA, in clinical settings.

DNA polymerase epsilon interacts with SUVH2/9 to repress the expression of genes associated with meiotic DSB hotspot in Arabidopsis.

Meiotic recombination is initiated by the SPORULATION 11 (SPO11)-triggered formation of double-strand breaks (DSBs) that usually occur in open chromatin with active transcriptional features in many eukaryotes. However, gene transcription at DSB sites appears to be detrimental for repair, but the regulatory mechanisms governing transcription at meiotic DSB sites are largely undefined in plants. Here, we demonstrate that the largest DNA polymerase epsilon subunit POL2A interacts with SU(VAR)3 to 9 homologs SUVH2 and SUVH9. N-SIM (structured illumination microscopy) observation shows that the colocalization of SUVH2 with the meiotic DSB marker γ-H2AX is dependent on POL2A. RNA-seq of male meiocytes demonstrates that POL2A and SUVH2 jointly repress the expression of 865 genes, which have several known characteristics associated with meiotic DSB sites. Bisulfite-seq and small RNA-seq of male meiocytes support the idea that the silencing of these genes by POL2A and SUVH2/9 is likely independent of CHH methylation or 24-nt siRNA accumulation. Moreover, pol2a suvh2 suvh9 triple mutants have more severe defects in meiotic recombination and fertility compared with either pol2a or suvh2 suvh9. Our results not only identify a epigenetic regulatory mechanism for gene silencing in male meiocytes but also reveal roles for DNA polymerase and SUVH2/9 beyond their classic functions in mitosis.

Histone demethylase IBM1-mediated meiocyte gene expression ensures meiotic chromosome synapsis and recombination.

Histone methylation and demethylation play important roles in plant growth and development, but the involvement of histone demethylation during meiosis is poorly understood. Here we show that disruption of Arabidopsis thaliana INCREASE IN BONSAI METHYLATION 1 (IBM1) causes incomplete synapsis, chromosome entanglement and reduction of recombination during meiosis, leading to sterility. Interestingly, these ibm1 meiotic defects are rescued by mutations in either SUVH4/KYP or CMT3. Using transcriptomic analyses we show that mutation of IBM1 down-regulates thousands of genes expressed in meiocytes, and that expression of about 38% of these genes are restored to wild type levels in ibm1 cmt3 double mutants. Changes in the expression of 437 of these, including the ARABIDOPSIS MEI2-LIKE AML3-5 genes, are correlated with a significant reduction of gene body CHG methylation. Consistently, the aml3 aml4 aml5 triple have defects in synapsis and chromosome entanglement similar to ibm1. Genetic analysis shows that aml3 aml4 aml5 ibm1 quadruple mutants resembles the ibm1 single mutant. Strikingly, over expression of AML5 in ibm1 can partially rescue the ibm1 meiotic defects. Taken together, our results demonstrate that histone demethylase IBM1 is required for meiosis likely via coordinated regulation of meiocyte gene expression during meiosis.

DNA polymerase epsilon binds histone H3.1-H4 and recruits MORC1 to mediate meiotic heterochromatin condensation.

Heterochromatin is essential for genomic integrity and stability in eukaryotes. The mechanisms that regulate meiotic heterochromatin formation remain largely undefined. Here, we show that the catalytic subunit (POL2A) of Arabidopsis DNA polymerase epsilon (POL ε) is required for proper formation of meiotic heterochromatin. The POL2A N terminus interacts with the GHKL adenosine triphosphatase (ATPase) MORC1 (Microrchidia 1), and POL2A is required for MORC1's localization on meiotic heterochromatin. Mutations affecting the POL2A N terminus cause aberrant morphology of meiotic heterochromatin, which is also observed in morc1. Moreover, the POL2A C-terminal zinc finger domain (ZF1) specifically binds to histone H3.1-H4 dimer or tetramer and is important for meiotic heterochromatin condensation. Interestingly, we also found similar H3.1-binding specificity for the mouse counterpart. Together, our results show that two distinct domains of POL2A, ZF1 and N terminus bind H3.1-H4 and recruit MORC1, respectively, to induce a continuous process of meiotic heterochromatin organization. These activities expand the functional repertoire of POL ε beyond its classic role in DNA replication and appear to be conserved in animals and plants.

Tankyrases inhibit innate antiviral response by PARylating VISA/MAVS and priming it for RNF146-mediated ubiquitination and degradation.

During viral infection, sensing of viral RNA by retinoic acid-inducible gene-I-like receptors (RLRs) initiates an antiviral innate immune response, which is mediated by the mitochondrial adaptor protein VISA (virus-induced signal adaptor; also known as mitochondrial antiviral signaling protein [MAVS]). VISA is regulated by various posttranslational modifications (PTMs), such as polyubiquitination, phosphorylation, O-linked ß-d-N-acetylglucosaminylation (O-GlcNAcylation), and monomethylation. However, whether other forms of PTMs regulate VISA-mediated innate immune signaling remains elusive. Here, we report that Poly(ADP-ribosyl)ation (PARylation) is a PTM of VISA, which attenuates innate immune response to RNA viruses. Using a biochemical purification approach, we identified tankyrase 1 (TNKS1) as a VISA-associated protein. Viral infection led to the induction of TNKS1 and its homolog TNKS2, which translocated from cytosol to mitochondria and interacted with VISA. TNKS1 and TNKS2 catalyze the PARylation of VISA at Glu137 residue, thereby priming it for K48-linked polyubiquitination by the E3 ligase Ring figure protein 146 (RNF146) and subsequent degradation. Consistently, TNKS1, TNKS2, or RNF146 deficiency increased the RNA virus-triggered induction of downstream effector genes and impaired the replication of the virus. Moreover, TNKS1- or TNKS2-deficient mice produced higher levels of type I interferons (IFNs) and proinflammatory cytokines after virus infection and markedly reduced virus loads in the brains and lungs. Together, our findings uncover an essential role of PARylation of VISA in virus-triggered innate immune signaling, which represents a mechanism to avoid excessive harmful immune response.

Toxoplasma gondii Causes Adverse Pregnancy Outcomes by Damaging Uterine Tissue-Resident NK Cells That Secrete Growth-Promoting Factors.

Vertical transmission of the intracellular parasite, Toxoplasma gondii can lead to adverse pregnancy outcomes especially when infection occurs in early pregnancy. Decidual natural killer (dNK) cells accumulate at the maternal-fetal interface in large numbers during early pregnancy. Their nutritional roles during infection with T. gondii remain poorly defined. In the present study, we demonstrated that a functional deficiency of the uterine tissue-resident NK (trNK) cells, a subset of dNK cells, contributes to the adverse pregnancy outcomes induced by T. gondii in early pregnancy. Adverse pregnancy outcomes could be ameliorated by adoptive transfer of trNK cells. Moreover, fetal growth restriction could be improved after supplementation of growth-promoting factors. In addition to the widely recognized disturbance of the immune balance at the interface between the mother and the fetus, our study reveals a novel mechanism in T. gondii that contributes to the adverse pregnancy outcomes.

Chimeric antigen receptors of HBV envelope proteins inhibit hepatitis B surface antigen secretion.

OBJECTIVES: Chronic hepatitis B (CHB) caused by HBV infection greatly increases the risk of liver cirrhosis and hepatocellular carcinoma. Hepatitis B surface antigen (HBsAg) plays critical roles in the pathogenesis of CHB. HBsAg loss is the key indicator for cure of CHB, but is rarely achieved by current approved anti-HBV drugs. Therefore, novel anti-HBV strategies are urgently needed to achieve sustained HBsAg loss. DESIGN: We developed multiple chimeric antigen receptors (CARs) based on single-chain variable fragments (scFvs, namely MA18/7-scFv and G12-scFv), respectively, targeting HBV large and small envelope proteins. Their impacts on HBsAg secretion and HBV infection, and the underlying mechanisms, were extensively investigated using various cell culture models and HBV mouse models. RESULTS: After secretory signal peptide mediated translocation into endoplasmic reticulum (ER) and secretory pathway, MA18/7-scFv and CARs blocked HBV infection and virion secretion. G12-scFv preferentially inhibited virion secretion, while both its CAR formats and crystallisable fragment (Fc)-attached versions blocked HBsAg secretion. G12-scFv and G12-CAR arrested HBV envelope proteins mainly in ER and potently inhibited HBV budding. Furthermore, G12-scFv-Fc and G12-CAR-Fc strongly suppressed serum HBsAg up to 130-fold in HBV mouse models. The inhibitory effect lasted for at least 8 weeks when delivered by an adeno-associated virus vector. CONCLUSION: CARs possess direct antiviral activity, besides the well-known application in T-cell therapy. Fc attached G12-scFv and G12-CARs could provide a novel approach for reducing circulating HBsAg.