Design, synthesis, and antitumor activity evaluation of potent fourth-generation EGFR inhibitors for treatment of Osimertinib resistant non-small cell lung cancer (NSCLC).

Epidermal growth factor receptor (EGFR) is one of the most studied drug targets for treating non-small-cell lung cancer (NSCLC). However, there are no approved inhibitors for the C797S resistance mutation caused by the third-generation EGFR inhibitor (Osimertinib). Therefore, the development of fourth-generation EGFR inhibitors is urgent. In this study, we clarified the structure-activity relationship of several synthesized compounds as fourth-generation inhibitors against human triple (Del19/T790M/C797S) mutation. Representative compound 52 showed potent inhibitory activity against EGFRL858R/T790M/C797S with an IC50 of 0.55 nM and significantly inhibited the proliferation of the Ba/F3 cell line harboring EGFRL858R/T790M/C797S with an IC50 of 43.28 nM. Moreover, 52 demonstrated good pharmacokinetic properties and excellent in vivo efficacy. Overall, the compound 52 can be considered a promising candidate for overcoming EGFR C797S-mediated mutations.

Transcriptomic Signature of 3D Hierarchical Porous Chip Enriched Exosomes for Early Detection and Progression Monitoring of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a highly lethal malignant tumor, and the current non-invasive diagnosis method based on serum markers, such as α-fetoprotein (AFP), and des-γ-carboxy-prothrombin (DCP), has limited efficacy in detecting it. Therefore, there is a critical need to develop novel biomarkers for HCC. Recent studies have highlighted the potential of exosomes as biomarkers. To enhance exosome enrichment, a silicon dioxide (SiO2) microsphere-coated three-dimensional (3D) hierarchical porous chip, named a SiO2-chip is designed. The features of the chip, including its continuous porous 3D scaffold, large surface area, and nanopores between the SiO2 microspheres, synergistically improved the exosome capture efficiency. Exosomes from both non-HCC and HCC subjects are enriched using an SiO2-chip and performed RNA sequencing to identify HCC-related long non-coding RNAs (lncRNAs) in the exosomes. This study analysis reveales that LUCAT-1 and EGFR-AS-1 are two HCC-related lncRNAs. To further detect dual lncRNAs in exosomes, quantitative real time polymerase chain reaction (qRT-PCR) is employed. The integration of dual lncRNAs with AFP and DCP significantly improves the diagnostic accuracy. Furthermore, the integration of dual lncRNAs with DCP effectively monitors the prognosis of patients with HCC and detects disease progression. In this study, a liquid biopsy-based approach for noninvasive and reliable HCC detection is developed.

HBV promotes its replication by up-regulating RAD51C gene expression.

Chronic hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC), pegylated-interferon-α(PEG-IFNα) and long-term nucleos(t)ide analogs (NUCs) are mainly drugs used to treat HBV infection, but the effectiveness is unsatisfactory in different populations, the exploration of novel therapeutic approaches is necessary. RAD51C is associated with DNA damage repair and plays an important role in the development and progression of tumors. Early cDNA microarray results showed that RAD51C expression was significantly increased in HBV-infected HCC cells, however, the relationship between HBV infection and abnormal expression of RAD51C has not been reported. Therefore, we conducted RT-PCR, western blot, Co-immunoprecipitation(Co-IP), and immunofluorescence(IF) to detect HBV-RAD51C interaction in RAD51C overexpression or interfering HCC cells. Our results showed that RAD51C and HBV X protein(HBX) produced a direct interaction in the nucleus, the HBV infection of HCC cells promoted RAD51C expression, and the increased expression of RAD51C promoted HBV replication. This indicated that RAD51C is closely related to the occurrence and development of HCC caused by HBV infection, and may bring a breakthrough in the the prevention and treatment study of HCC.

STAT3 palmitoylation initiates a positive feedback loop that promotes the malignancy of hepatocellular carcinoma cells in mice.

Constitutive activation of the transcription factor STAT3 (signal transducer and activator of transcription 3) contributes to the malignancy of many cancers such as hepatocellular carcinoma (HCC) and is associated with poor prognosis. STAT3 activity is increased by the reversible palmitoylation of Cys108 by the palmitoyltransferase DHHC7 (encoded by ZDHHC7). Here, we investigated the consequences of S-palmitoylation of STAT3 in HCC. Increased ZDHHC7 abundance in HCC cases was associated with poor prognosis, as revealed by bioinformatics analysis of patient data. In HepG2 cells in vitro, DHHC7-mediated palmitoylation enhanced the expression of STAT3 target genes, including HIF1A, which encodes the hypoxia-inducible transcription factor HIF1α. Inhibiting DHHC7 decreased the S-palmitoylation of STAT3 and decreased HIF1α abundance. Furthermore, stabilization of HIF1α by cyclin-dependent kinase 5 (CDK5) enabled it to promote the expression of ZDHHC7, which generated a positive feedback loop between DHHC7, STAT3, and HIF1α. Perturbing this loop reduced the growth of HCC cells in vivo. Moreover, DHHC7, STAT3, and HIF1α were all abundant in human HCC tissues. Our study identifies a pathway connecting these proteins that is initiated by S-palmitoylation, which may be broadly applicable to understanding the role of this modification in cancer.

Association between systemic immune-inflammation index and chronic obstructive pulmonary disease: a population-based study.

BACKGROUND: The Systemic Immune-Inflammation Index (SII) is a quantitative measurement of the systemic immune-inflammatory response in the human body. The SII has been shown to have prognostic value in various clinical settings, including critical illness, sepsis, and cancer. Its role in chronic obstructive pulmonary disease (COPD) remains unclear and requires further investigation. METHODS: We analyzed demographic data from 16,636 participants in the National Health and Nutrition Examination Survey. Logistic regression analysis was performed to assess the correlation between COPD, lung function, chronic respiratory symptoms and SII. We used Cox proportional hazards (PH) model to analyze the relationship between SII and mortality in COPD patients and healthy individuals. We used propensity score matching (PSM) method to match the COPD population with similar baseline levels with the normal population to further analyze the correlation between SII and COPD. RESULTS: We recruited 16,636 participants, ages 40 and above, for the study. A multivariable logistic regression analysis revealed that a higher SII level was independently associated with an elevated likelihood of COPD (Odds Ratio (OR) = 1.449; 95% Confidence Interval (CI): 1.252-1.676, P < 0.0001) after controlling for all other factors. Results of subgroup analysis showed a significant positive correlation between SII and COPD in different age groups, gender, Body Mass Index, smoking status, and those with a history of hypertension. The SII index had positive correlation with COPD after PSM (OR = 1.673; 95%CI: 1.443-1.938). After full adjustment, an increase in the SII is associated with a higher all-cause mortality rate. The hazard ratio (HR) with a 95% CI in the general population, COPD patients, and healthy individuals are 1.161 (1.088, 1.239), 1.282 (1.060, 1.550), and 1.129 (1.055, 1.207), respectively. CONCLUSIONS: Higher SII levels are linked to higher prevalence of COPD. COPD patients with a higher SII levels have a higher risk of all-cause mortality. Additional large-scale, long-term studies are necessary to confirm these results.

Human papillomavirus genotypic characteristics of 60,685 subjects under age-expansion vaccination of the nine-valent human papillomavirus vaccine: A cross-sectional study.

BACKGROUND: Human papillomavirus (HPV) vaccination is a key initiative to promote the WHO global strategy to accelerate the elimination of cervical cancer, and this study aimed to investigate the current status of HPV infection and genotypic characteristics of the population under the impact of age-expansion of nine-valent HPV vaccination policy in China. METHODS: The clinical data of 60,685 subjects who were admitted in the Renmin Hospital of Wuhan University and underwent HPV genotyping from January 2017 to October 2022 were retrospectively analyzed. RESULTS: The total number of positive HPV genotyping in the included population was 10,303, with a positivity rate of 17.0 %. The HPV positivity rate in the male and female populations increased slowly year by year, with a higher rate of positivity in men (32.7 %) than in women (16.7 %) (P < 0.001). HPV was predominantly single infection in all populations, with higher prevalence of high-risk HPV than low-risk HPV in females, while low-risk HPV infection was predominant in the male population. The age distribution of female subjects infected with HPV, with HPV52 as the most common type, showed a bimodal pattern. As for HPV infected male subjects, HPV6 was the main type, and there was no bimodal age distribution. The expanded age vaccination of the nine-valent HPV vaccine will result in 42.4 % efficiency of vaccine protection for 49.9 % of age-eligible women. If the nine-valent HPV vaccine were open to males in China, it would reduce HPV infections in men by 56.4 %. CONCLUSIONS: The HPV positivity rate in the population remains high and tends to increase, and the age-expansion of the nine-valent HPV vaccine would contribute to reducing the threat of disease caused by HPV infection for age-eligible women. Moreover, attention should be paid to enhancing HPV screening in males and opening up vaccination when appropriate.

Co-delivery of Cas9 mRNA and guide RNAs edits hepatitis B virus episomal and integration DNA in mouse and tree shrew models.

With 296 million chronically infected individuals worldwide, hepatitis B virus (HBV) causes a major health burden. The major challenge to cure HBV infection lies in the fact that the source of persistence infection, viral episomal covalently closed circular DNA (cccDNA), could not be targeted. In addition, HBV DNA integration, although normally results in replication-incompetent transcripts, considered as oncogenic. Though several studies evaluated the potential of gene-editing approaches to target HBV, previous in vivo studies have been of limited relevance to authentic HBV infection, as the models do not contain HBV cccDNA or feature a complete HBV replication cycle under competent host immune system. In this study, we evaluated the effect of in vivo codelivery of Cas9 mRNA and guide RNAs (gRNAs) by SM-102-based lipid nanoparticles (LNPs) on HBV cccDNA and integrated DNA in mouse and a higher species. CRISPR nanoparticle treatment decreased the levels of HBcAg, HBsAg and cccDNA in AAV-HBV1.04 transduced mouse liver by 53%, 73% and 64% respectively. In HBV infected tree shrews, the treatment achieved 70% reduction of viral RNA and 35% reduction of cccDNA. In HBV transgenic mouse, 90% inhibition of HBV RNA and 95% inhibition of DNA were observed. CRISPR nanoparticle treatment was well tolerated in both mouse and tree shrew, as no elevation of liver enzymes and minimal off-target was observed. Our study demonstrated that SM-102-based CRISPR is safe and effective in targeting HBV episomal and integration DNA in vivo. The system delivered by SM-102-based LNPs may be used as a potential therapeutic strategy against HBV infection.

Antibiotic-induced gut bacteria depletion has no effect on HBV replication in HBV immune tolerance mouse model.

Commensal microbiota is closely related to Hepatitis B virus (HBV) infection. Gut bacteria maturation accelerates HBV immune clearance in hydrodynamic injection (HDI) HBV mouse model. However, the effect of gut bacteria on HBV replication in recombinant adeno-associated virus (AAV)-HBV mouse model with immune tolerance remains obscure. We aim to investigate its role on HBV replication in AAV-HBV mouse model. C57BL/6 mice were administrated with broad-spectrum antibiotic mixtures (ABX) to deplete gut bacteria and intravenously injected with AAV-HBV to establish persistent HBV replication. Gut microbiota community was analyzed by fecal qPCR assay and 16S ribosomal RNA (rRNA) gene sequencing. HBV replication markers in blood and liver were determined by ELISA, qPCR assay and Western blot at indicated time points. Immune response in AAV-HBV mouse model was activated through HDI of HBV plasmid or poly(I:C) and then detected by quantifying the percentage of IFN-γ+/CD8+ T cells in the spleen via flow cytometry as well as the splenic IFN-γ mRNA level via qPCR assay. We found that antibiotic exposure remarkably decreased gut bacteria abundance and diversity. Antibiotic treatment failed to alter the levels of serological HBV antigens, intrahepatic HBV RNA transcripts and HBc protein in AAV-HBV mouse model, but contributed to HBsAg increase after breaking of immune tolerance. Overall, our data uncovered that antibiotic-induced gut bacteria depletion has no effect on HBV replication in immune tolerant AAV-HBV mouse model, providing new thoughts for elucidating the correlation between gut bacteria dysbiosis by antibiotic abuse and clinical chronic HBV infection.

Electrochemical signal amplification strategy based on trace metal ion modified WS2 for ultra-sensitive detection of miRNA-21.

Previous researches have suggested the potential correlation between the development of breast cancer and the concentration of miRNA-21 in serum. Theoretically the doping of multivalent metal ions in WS2 could bring higher electron transfer capacity, but this hasn't been proven. To fill this research gap, through one-pot method we prepared seven nanocomposite structures modified with different metal ions (Co2+, Ni2+, Mn2+, Zn2+, Fe3+, Cr3+, La3+). Characterization revealed that ammonia produced by hydrothermal urea exfoliated the multilayer graphene oxide (MGO) and provided a nitrogen source for doping reduction to form a 3D flower-like structure (NrGOF) with high specific surface area. Meanwhile, the modification of WS2 by Fe3+ not only enhanced its electrochemical conductivity but also gave the material an additional peroxidase activity centre. In the composite Fe3+-WS2/NrGOF-AgNPs, NrGOF is used as a conductive loading interface for WS2, while Fe3+ served as the catalytic and electron transfer centre for secondary amplification of the electrochemical signal. The experimental results showed that the sensing platform has a low limit of detection (LOD) of 1.18 aM for miRNA-21 in the concentration range of 10-17-10-12 M and has been successfully applied to the detection of real serum samples.

Hepatitis B Virus Core Protein Is Not Required for Covalently Closed Circular DNA Transcriptional Regulation.

Hepatitis B virus (HBV) infection is a major health burden worldwide, and currently there is no cure. The persistence of HBV covalently closed circular DNA (cccDNA) is the major obstacle for antiviral trement. HBV core protein (HBc) has emerged as a promising antiviral target, as it plays important roles in critical steps of the viral life cycle. However, whether HBc could regulate HBV cccDNA transcription remains under debate. In this study, different approaches were used to address this question. In synthesized HBV cccDNA and HBVcircle transfection assays, lack of HBc showed no effect on transcription of HBV RNA as well as HBV surface antigen (HBsAg) production in a hepatoma cell line and primary human hepatocytes. Reconstitution of HBc did not alter the expression of cccDNA-derived HBV markers. Similar results were obtained from an in vivo mouse model harboring cccDNA. Chromatin immunoprecipitation (ChIP) or ChIP sequencing assays revealed transcription regulation of HBc-deficient cccDNA chromatin similar to that of wild-type cccDNA. Furthermore, treatment with capsid assembly modulators (CAMs) dramatically reduced extracellular HBV DNA but could not alter viral RNA and HBsAg. Our results demonstrate that HBc neither affects histone modifications and transcription factor binding of cccDNA nor directly influences cccDNA transcription. Although CAMs could reduce HBc binding to cccDNA, they do not suppress cccDNA transcriptional activity. Thus, therapeutics targeting capsid or HBc should not be expected to sufficiently reduce cccDNA transcription. IMPORTANCE Hepatitis B virus (HBV) core protein (HBc) has emerged as a promising antiviral target. However, whether HBc can regulate HBV covalently closed circular DNA (cccDNA) transcription remains elusive. This study illustrated that HBc has no effect on epigenetic regulation of cccDNA, and it does not participate in cccDNA transcription. Given that HBc is dispensable for cccDNA transcription, novel cccDNA-targeting therapeutics are needed for an HBV cure.

Analysis of Common Respiratory Infected Pathogens in 3100 Children after the Coronavirus Disease 2019 Pandemic.

OBJECTIVE: To investigate the epidemiological features in children after the coronavirus disease 2019 (COVID-19) pandemic. METHODS: This study collected throat swabs and serum samples from hospitalized pediatric patients of Renmin Hospital of Wuhan University, Wuhan, Hubei province, China before and after the COVID-19 pandemic. Respiratory infected pathogens [adenovirus (ADV), influenza virus A/B (Flu A/B), parainfluenza virus 1/2/3 (PIV1/2/3), respiratory syncytial virus (RSV), Mycoplasma pneumoniae (MP), and Chlamydia pneumoniae (CP)] were detected. The pathogens, age, and gender were used to analyze the epidemiological features in children after the COVID-19 pandemic. RESULTS: The pathogen detection rate was significantly higher in females than in males (P<0.05), and the infection of PIV1 and MP was mainly manifested. After the COVID-19 pandemic, PIV1, PIV3, RSV, and MP had statistically different detection rates among the age groups (P<0.05), and was mainly detected in patients aged 0-6 years, 0-3 years, 0-3 years, and 1-6 years, respectively. When comparing before the COVID-19 pandemic, the total detection rate of common respiratory pathogens was lower (P<0.05). Except for the increase in the detection rate of PIV1 and CP, the infection rate of other pathogens had almost decreased. CONCLUSION: The prevention and control measures for the COVID-19 pandemic effectively changed the epidemiological features of common respiratory tract infectious diseases in pediatric children.

The Role of Membrane-Associated E3 Ubiquitin Ligases in Cancer.

The cell membrane system comprises the plasma membrane, endoplasmic reticulum, Golgi apparatus, lysosome, mitochondria, and nuclear membrane, which are essential for maintaining normal physiological functions of cells. The proteins associated with these membrane-organelles are frequently modified to regulate their functions, the most common of which is ubiquitin modification. So far, many ubiquitin E3 ligases anchored in the membrane system have been identified as critical players facilitating intracellular biofunctions whose dysfunction is highly related to cancer. In this review, we summarized membrane-associated E3 ligases and revealed their relationship with cancer, which is of great significance for discovering novel drug targets of cancer and may open up new avenues for inducing ubiquitination-mediated degradation of cancer-associated membrane proteins via small chemicals such as PROTAC and molecular glue.

Structure-based rational design enables efficient discovery of a new selective and potent AKT PROTAC degrader.

AKT and associated signaling pathways have been recognized as promising therapeutic targets for decades, and growing evidence indicates that inhibition or degradation of cellular AKT are viable strategies to treat cancer. Guided by an in silico modeling approach for rational linker design and based on our previous work in this field, we herein efficiently synthesized a small group of cereblon-recruiting AKT PROTAC molecules and identified a highly potent AKT degrader B4. Compared to the existing AKT degraders, B4 has a structurally unique AKT targeting warhead derived from the pyrazole-furan conjugated piperidine derivatives. It induces selective degradation of all three isoforms of AKT and exhibits efficacious anti-proliferation against several human hematological cancers. Notably, B4 demonstrates potent inhibition of AKT downstream signaling superior to its parental inhibitor. Together with its active analogs, B4 expands the arsenal of AKT chemical degraders as a valuable probe to uncover AKTs new functions and as a potential drug candidate to treat cancer.

Long-chain fatty acyl coenzyme A inhibits NME1/2 and regulates cancer metastasis.

SignificanceThe study provided a long-sought molecular mechanism that could explain the link between fatty acid metabolism and cancer metastasis. Further understanding may lead to new strategies to inhibit cancer metastasis. The chemical proteomic approach developed here will be useful for discovering other regulatory mechanisms of protein function by small molecule metabolites.

Noncovalent CDK12/13 dual inhibitors-based PROTACs degrade CDK12-Cyclin K complex and induce synthetic lethality with PARP inhibitor.

Cyclin-dependent kinase 12 (CDK12) plays a crucial role in DNA-damage response gene transcription and has recently been validated as a promising target in cancer therapy. However, existing CDK12 inhibitors potently inhibit its closest isoform CDK13, which could cause potential toxicity. Therefore, the development of CDK12 inhibitors with isoform-selectivity against CDK13 continues to be a challenge. By taking advantage of the emerging PROteolysis-TArgeting Chimeras (PROTACs) approach, we have synthesized a potent PROTAC degrader PP-C8 based on the noncovalent dual inhibitors of CDK12/13 and demonstrated its specificity for CDK12 over CDK13. Notably, PP-C8 induces profound degradation of cyclin K simultaneously and downregulates the mRNA level of DNA-damage response genes. Global proteomics profiling revealed PP-C8 is highly selective toward CDK12-cyclin K complex. Importantly, PP-C8 demonstrates profound synergistic antiproliferative effects with PARP inhibitor in triple-negative breast cancer (TNBC). The potent and selective CDK12 PROTAC degrader developed in this study could potentially be used to treat CDK12-dependent cancers as combination therapy.

A Novel Mouse Model Harboring Hepatitis B Virus Covalently Closed Circular DNA.

BACKGROUND AND AIMS: The persistence of viral covalently closed circular DNA (cccDNA) is the major obstacle for antiviral treatment against hepatitis B virus (HBV). Basic and translational studies are largely hampered due to the lack of feasible small animal models to support HBV cccDNA formation. The aim of this study is to establish a novel mouse model harboring cccDNA. METHODS: An adeno-associated virus (AAV) vector carrying a replication-deficient HBV1.04-fold genome (AAV-HBV1.04) was constructed. The linear HBV genome starts from nucleotide 403 and ends at 538, which results in the splitting of HBV surface and polymerase genes. Different HBV replication markers were evaluated for AAV-HBV1.04 plasmid-transfected cells, the AAV-HBV1.04 viral vector-transduced cells, and mice injected with the AAV-HBV1.04 viral vector. RESULTS: Compared with the previously reported AAV-HBV1.2 construct, direct transfection of AAV-HBV1.04 plasmid failed to produce hepatitis B surface antigen and progeny virus. Interestingly, AAV-HBV1.04 viral vector transduction could result in the formation of cccDNA and the production of all HBV replication markers in vitro and in vivo. The formation of cccDNA could be blocked by ATR (ataxia-telangiectasia and Rad3-related protein) inhibitors but not HBV reverse transcription inhibitor or capsid inhibitors. The AAV-HBV1.04 mouse supported long-term HBV replication and responded to antiviral treatments. CONCLUSIONS: This AAV-HBV1.04 mouse model can support HBV cccDNA formation through ATR-mediated DNA damage response. The de novo formed cccDNA but not the parental AAV vector can lead to the production of hepatitis B surface antigen and HBV progeny. This model will provide a unique platform for studying HBV cccDNA and developing novel antivirals against HBV infection.

Cyclin-dependent kinases-based synthetic lethality: Evidence, concept, and strategy.

Synthetic lethality is a proven effective antitumor strategy that has attracted great attention. Large-scale screening has revealed many synthetic lethal genetic phenotypes, and relevant small-molecule drugs have also been implemented in clinical practice. Increasing evidence suggests that CDKs, constituting a kinase family predominantly involved in cell cycle control, are synthetic lethal factors when combined with certain oncogenes, such as MYC, TP53, and RAS, which facilitate numerous antitumor treatment options based on CDK-related synthetic lethality. In this review, we focus on the synthetic lethal phenotype and mechanism related to CDKs and summarize the preclinical and clinical discoveries of CDK inhibitors to explore the prospect of CDK inhibitors as antitumor compounds for strategic synthesis lethality in the future.

High-Throughput Enzyme Assay for Screening Inhibitors of the ZDHHC3/7/20 Acyltransferases.

As enzymes that mediate the attachment of long-chain fatty acids to cysteine residues, ZDHHC proteins have been reported to be promising therapeutic targets for treating cancer and autoimmune diseases. Yet, due to the lack of potent selective inhibitors, scrutiny of the biological functions of ZDHHCs has been limited. The main hindrance for developing ZDHHC inhibitors is the lack of a facile high-throughput assay. Here, we developed a ZDHHC3/7/20 high-throughput assay based on the acylation-coupled lipophilic induction of polarization (Acyl-cLIP) method and screened several potential ZDHHC inhibitors. Furthermore, we demonstrated that in vitro results from the Acyl-cLIP assay are supported by the results from cell-based assays. We envision that this new ZDHHC3/7/20 Acyl-cLIP assay will accelerate the high-throughput screening of large compound libraries for improved ZDHHC inhibitors and provide therapeutic benefits for cancer and autoimmune diseases.

Novel function of SART1 in HNF4α transcriptional regulation contributes to its antiviral role during HBV infection.

BACKGROUND & AIMS: Our understanding of the interactions between HBV and its host cells is still quite limited. Spliceosome associated factor 1 (SART1) has recently been found to restrict HCV. Thus, we aimed to dissect its role in HBV infection. METHODS: SART1 was knocked down by RNA interference and over-expressed by lentiviral or adeno-associated virus (AAV) vectors in HBV-infected cell cultures and in vivo in HBV-infected mice. Luciferase reporter assays were used to determine viral or host factor promoter activities, and chromatin immunoprecipitation (ChIP) was used to investigate protein-DNA interactions. RESULTS: In HBV-infected cell cultures, downregulation of SART1 did not affect covalently closed circular HBV DNA but resulted in markedly enhanced HBV RNA, antigen expression and progeny virus production. On the other hand, HBV transcription and replication were significantly inhibited by overexpression of SART1. Similar results were observed in AAV-HBV-infected mice persistently replicating HBV. Inhibition of Janus kinases had no effect on SART1-mediated inhibition of HBV replication. HBV promoter assays revealed that SART1 reduced HBV core promoter activity. By screening known HBV transcription factors, we found that SART1 specifically suppressed the expression of hepatocyte nuclear factor 4α (HNF4α). Luciferase reporter and ChIP assays demonstrated a direct downregulation of HNF4α expression by association of SART1 with the HNF4α proximal P1 promoter element. CONCLUSIONS: We identify SART1 as a novel host factor suppressing HBV cccDNA transcription. Besides its effect on interferon-stimulated genes, SART1 exerts an anti-HBV activity by suppressing HNF4α expression, which is essential for transcription of HBV cccDNA. LAY SUMMARY: Hepatitis B virus (HBV) infects hepatocytes and persists in the form of covalently closed circular DNA (cccDNA), which remains a major obstacle to successful antiviral treatment. In this study, using various HBV models, we demonstrate that the protein SART1 restricts HBV cccDNA transcription by suppressing a key transcription factor, HNF4α.