Intracranial Phosphaturic Mesenchymal Tumor Detected by 68 Ga-DOTATATE PET/CT.

ABSTRACT: A 68 Ga-DOTATATE PET/CT scan was conducted to locate the causative tumor responsible for suspected tumor-induced osteomalacia in a 56-year-old woman. The PET/CT images showed a focus in the right occipital region. Subsequent MRI showed an extra-axial nodule in the right occipital region, mimicking a meningioma. Although rare, an intracranial phosphaturic mesenchymal tumor was still suspected because of the typical clinical settings. Finally, phosphaturic mesenchymal tumor was confirmed by the postoperative pathology.

Identification of novel tetrahydroquinoxaline derived phenyl ureas as modulators of the hepatitis B virus nucleocapsid assembly.

A key step of hepatitis B virus (HBV) replication is the selective packaging of pregenomic RNA (pgRNA) by core protein (Cp) dimers, forming a nucleocapsid where the reverse transcriptional viral DNA replication takes place. One approach in the development of new anti-HBV drugs is to disrupt the assembly of HBV nucleocapsids by misdirecting Cp dimers to assemble morphologically normal capsids devoid of pgRNA. In this study, we built upon our previous discovery of benzamide-derived HBV capsid assembly modulators by exploring fused bicyclic scaffolds with an exocyclic amide that is ß, γ to the fused ring, and identified 1,2,3,4-tetrahydroquinoxaline derived phenyl ureas as a novel scaffold. Structure-activity relationship studies showed that a favorable hydrophobic substitution can be tolerated at the 2-position of the 1,2,3,4-tetrahydroquinoxaline core, and the resulting compound 88 demonstrated comparable or improved antiviral potencies in mouse and human hepatocyte-derived HBV-replicating cell lines compared to our previously reported benzamide compound, 38017 (8). In addition, a novel bis-urea series based on 1,2,3,4-tetrahydroquinoxaline was also found to inhibit HBV DNA replication with sub-micromolar EC50 values. The mode of action of these compounds is consistent with specific inhibition of pgRNA encapsidation into nucleocapsids in hepatocytes.

Recombinant HBsAg-S and RFRP-3 DNA vaccine promotes reproduction hormone secretion in sheep.

RF-amide related peptides (RFRP) have been proposed as critical regulators of gonadotropin secretion in mammals. This study was designed to construct a DNA vaccine and investigate the effect of vaccine encoding RFRP-3 on reproduction physiology in ewe. A recombinant vaccine was constructed using two copies of the RFRP-3 gene and HBsAg-S that generate a fusion protein to induce an immunology response. Results showed this recombinant vaccine could produce a significant antibody titer in the treated animals (P < 0.05). The specific RFRP-3 antibody response induced by the vaccine was detected at week 2 with a peak at week 6 after the initial immunization. Furthermore, we found that ewes inoculated with pVAX-tPA-HBsAg-S-2RFRP-asd vaccine significantly raised the concentration of GnRH, LH and E2 in serum compared to the control group. LH and E2 concentration in the treated ewes (Group T) was significantly higher than that in control ewes (Group C) at weeks 10, 12 and 14 after the initial immunization, respectively (P < 0.05). Therefore, RFRP-3 can be used as a target for DNA immunization to promote reproductive hormone secretion in ewes and RFRP-3 gene immunization might be a candidate tool to regulate mammal reproduction.

Deep regression using 99mTc-DTPA dynamic renal imaging for automatic calculation of the glomerular filtration rate.

OBJECTIVES: To develop and evaluate an artificial intelligence (AI) system that can automatically calculate the glomerular filtration rate (GFR) from dynamic renal imaging without manually delineating the regions of interest (ROIs) of kidneys and the corresponding background. METHODS: This study was a single-center retrospective analysis of the data of 14,634 patients who underwent 99mTc-DTPA dynamic renal imaging. Two systems based on convolutional neural networks (CNN) were developed and evaluated: sGFRa predicts the radioactive counts of ROIs and calculates GFR using the Gates equation and sGFRb directly predicts GFR from dynamic renal imaging without using other information. The root-mean-square error (RMSE), mean absolute error (MAE), mean absolute percentage error (MAPE), and R2 were used to evaluate the performance of our approach. RESULTS: sGFRa achieved an RMSE of 5.05, MAE of 4.03, MAPE of 6.07%, and R2 of 0.93 for total GFR while sGFRb achieved an RMSE of 7.61, MAE of 5.92, MAPE of 8.92%, and R2 of 0.85 for total GFR. The accuracy of sGFRa and sGFRb in determining the stage of chronic kidney disease was 87.41% and 82.44%, respectively. CONCLUSIONS: The findings of sGFRa show that automatic GFR calculation based on CNN and using dynamic renal imaging is feasible and efficient and, additionally, can aid clinical diagnosis. Furthermore, the promising results of sGFRb demonstrate that CNN can predict GFR from dynamic renal imaging without additional information. KEY POINTS: • Our CNN-based AI systems can automatically calculate GFR from dynamic renal imaging without manually delineating the ROIs of kidneys and the corresponding background. • sGFRa accurately predicted the radioactive counts of ROIs and calculated GFR using the Gates method. • sGFRb-predicted GFR directly without any parameters related to the Gates equation.

FDG PET/CT Image of Soft Tissue Aneurysmal Bone Cyst.

ABSTRACT: Soft tissue aneurysmal bone cyst is very rare. Herein, we report FDG PET/CT findings of aneurysmal bone cyst in a 19-year-old man. On conventional image, it presented as a paravertebral soft tissue mass with heterogeneous enhancement and rim eggshell-like calcification. On PET/CT, this solitary lesion had intense FDG uptake with an SUV max of 10.33. The final pathology supported a diagnosis of aneurysmal bone cyst. Our case suggests that soft tissue aneurysmal bone cyst should be regarded as a differential diagnosis of solitary paravertebral mass with intense FDG uptake.

Amino acid residues at core protein dimer-dimer interface modulate multiple steps of hepatitis B virus replication and HBeAg biogenesis.

The core protein (Cp) of hepatitis B virus (HBV) assembles pregenomic RNA (pgRNA) and viral DNA polymerase to form nucleocapsids where the reverse transcriptional viral DNA replication takes place. Core protein allosteric modulators (CpAMs) inhibit HBV replication by binding to a hydrophobic "HAP" pocket at Cp dimer-dimer interfaces to misdirect the assembly of Cp dimers into aberrant or morphologically "normal" capsids devoid of pgRNA. We report herein that a panel of CpAM-resistant Cp with single amino acid substitution of residues at the dimer-dimer interface not only disrupted pgRNA packaging, but also compromised nucleocapsid envelopment, virion infectivity and covalently closed circular (ccc) DNA biosynthesis. Interestingly, these mutations also significantly reduced the secretion of HBeAg. Biochemical analysis revealed that the CpAM-resistant mutations in the context of precore protein (p25) did not affect the levels of p22 produced by signal peptidase removal of N-terminal 19 amino acid residues, but significantly reduced p17, which is produced by furin cleavage of C-terminal arginine-rich domain of p22 and secreted as HBeAg. Interestingly, p22 existed as both unphosphorylated and phosphorylated forms. While the unphosphorylated p22 is in the membranous secretary organelles and the precursor of HBeAg, p22 in the cytosol and nuclei is hyperphosphorylated at the C-terminal arginine-rich domain and interacts with Cp to disrupt capsid assembly and viral DNA replication. The results thus indicate that in addition to nucleocapsid assembly, interaction of Cp at dimer-dimer interface also plays important roles in the production and infectivity of progeny virions through modulation of nucleocapsid envelopment and uncoating. Similar interaction at reduced p17 dimer-dimer interface appears to be important for its metabolic stability and sensitivity to CpAM suppression of HBeAg secretion.

Automatic identification of suspicious bone metastatic lesions in bone scintigraphy using convolutional neural network.

BACKGROUND: We aimed to construct an artificial intelligence (AI) guided identification of suspicious bone metastatic lesions from the whole-body bone scintigraphy (WBS) images by convolutional neural networks (CNNs). METHODS: We retrospectively collected the 99mTc-MDP WBS images with confirmed bone lesions from 3352 patients with malignancy. 14,972 bone lesions were delineated manually by physicians and annotated as benign and malignant. The lesion-based differentiating performance of the proposed network was evaluated by fivefold cross validation, and compared with the other three popular CNN architectures for medical imaging. The average sensitivity, specificity, accuracy and the area under receiver operating characteristic curve (AUC) were calculated. To delve the outcomes of this study, we conducted subgroup analyses, including lesion burden number and tumor type for the classifying ability of the CNN. RESULTS: In the fivefold cross validation, our proposed network reached the best average accuracy (81.23%) in identifying suspicious bone lesions compared with InceptionV3 (80.61%), VGG16 (81.13%) and DenseNet169 (76.71%). Additionally, the CNN model's lesion-based average sensitivity and specificity were 81.30% and 81.14%, respectively. Based on the lesion burden numbers of each image, the area under the receiver operating characteristic curve (AUC) was 0.847 in the few group (lesion number n ≤ 3), 0.838 in the medium group (n = 4-6), and 0.862 in the extensive group (n > 6). For the three major primary tumor types, the CNN-based lesion identifying AUC value was 0.870 for lung cancer, 0.900 for prostate cancer, and 0.899 for breast cancer. CONCLUSION: The CNN model suggests potential in identifying suspicious benign and malignant bone lesions from whole-body bone scintigraphic images.

Identification of hepatitis B virus core protein residues critical for capsid assembly, pgRNA encapsidation and resistance to capsid assembly modulators.

Assembly of hepatitis B virus (HBV) capsids is driven by the hydrophobic interaction of core protein (Cp) at dimer-dimer interface. Binding of core protein allosteric modulators (CpAMs) to a hydrophobic "HAP" pocket formed between the inter-dimer interface strengths the dimer-dimer interaction and misdirects the assembly of Cp dimers into non-capsid Cp polymers or morphologically normal capsids devoid of viral pregenomic (pg) RNA and DNA polymerase. In this study, we performed a systematic mutagenesis analysis to identify Cp amino acid residues at Cp dimer-dimer interface that are critical for capsid assembly, pgRNA encapsidation and resistance to CpAMs. By analyzing 70 mutant Cp with a single amino acid substitution of 25 amino acid residues around the HAP pocket, our study revealed that residue W102 and Y132 are critical for capsid assembly. However, substitution of many other residues did not significantly alter the amount of capsids, but reduced the amount of encapsidated pgRNA, suggesting their critical roles in pgRNA packaging. Interestingly, several mutant Cp with a single amino acid substitution of residue P25, T33 or I105 supported high levels of DNA replication, but conferred strong resistance to multiple chemotypes of CpAMs. In addition, we also found that WT Cp, but not the assembly incompetent Cp, such as Y132A Cp, interacted with HBV DNA polymerase (Pol). This later finding implies that encapsidation of viral DNA polymerase may depend on the interaction of Pol with a capsid assembly intermediate, but not free Cp dimers. Taking together, our findings reported herein shed new light on the mechanism of HBV nucleocapsid assembly and mode of CpAM action.

Hepatitis B virus nucleocapsid uncoating: biological consequences and regulation by cellular nucleases.

Upon infection of hepatocyte, Hepatitis B virus (HBV) genomic DNA in nucleocapsid is transported into the nucleus and converted into a covalently closed circular (ccc) DNA to serve as the template for transcription of viral RNAs. Viral DNA in the cytoplasmic progeny nucleocapsid is another resource to fuel cccDNA amplification. Apparently, nucleocapsid disassembly, or viral genomic DNA uncoating, is an essential step for cccDNA synthesis from both de novo infection and intracellular amplification pathways, and has a potential to activate DNA sensors and induce an innate immune response in infected hepatocytes. However, where and how the nucleocapsid disassembly occurs is not well understood. The work reported herein showed that the enhanced disassembly of progeny mature nucleocapsids in the cytoplasm supported cccDNA intracellular amplification, but failed to activate the cGAS-STING-mediated innate immune response in hepatocytes. Interestingly, while expression of a cytoplasmic exonuclease TREX1 in human hepatoma cells supporting HBV replication significantly reduced the amounts of cccDNA as well as its precursor, deproteinized relaxed circular (rc) DNA, expression of TREX1 in sodium taurocholate cotransporting polypeptide-expressing human hepatoma cells did not inhibit cccDNA synthesis from de novo HBV infection. The results from this cytoplasmic nuclease protection assay imply that the disassembly of progeny mature nucleocapsids and removal of viral DNA polymerase covalently linked to the 5' end of minus strand of rcDNA take place in the cytoplasm. On the contrary, the disassembly of virion-derived nucleocapsids during de novo infection may occur at a different subcellular compartment and possibly via distinct mechanisms.

Inhibition of coronavirus infection by a synthetic STING agonist in primary human airway system.

The newly emerged severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) coronavirus initiated a pneumonia outbreak (COVID-19) that rapidly spread worldwide and quickly became a public health emergency of international concern; However to date, except Remdesivir, there are no clinically approved specific or effective medicines to prevent or treat COVID-19. Therefore, the development of novel treatments against coronavirus infections caused by the current SARS-CoV-2 virus, as well as other highly pathogenic human coronaviruses, represents an urgent unmet need. Stimulator of interferon genes (STING) plays a central role in host defense mechanisms against microbial infections. STING activation leads to the induction of both type I interferon and autophagy responses, which elicit strong inhibitory effect against the infections caused by a broad range of microbial pathogens. However, whether STING activation can impact infections from SARS-CoV-2 or other coronaviruses remains largely unknown. In this study, we investigated the anti-coronavirus activity triggered by STING activation. We discovered that dimeric amidobenzimidazole (diABZI), a synthetic small molecule STING receptor agonist, showed potent anti-coronavirus activity against both the common cold human coronavirus 229E (HCoV-229E) and SARS-CoV-2 in cell culture systems. In addition, we demonstrated that the antiviral activity of diABZI was dependent on the interferon pathway in HCoV-229E infected normal human fibroblast lung cells (MRC-5) and reconstituted primary human airway air-liquid interface (ALI) cultures. Furthermore, low-dose of diABZI treatment at 0.1 µM effectively reduced the SARS-CoV-2 viral load at the epithelial apical surface and prevented epithelial damage in the reconstituted primary human bronchial airway epithelial ALI system. Our findings have thus revealed the therapeutic potential of STING agonists, such as diABZI, as treatments for SARS-CoV-2 and other human coronavirus infections.

Recurrent Malignant Pheochromocytoma With Unusual Peritoneal Carcinomatosis Detected on 131I-MIBG SPECT/CT.

The most common sites of metastasis for pheochromocytoma are the lymph nodes, bones, lungs, and liver. Peritoneal metastasis or implantation is very rare. We present a case of recurrent malignant pheochromocytoma with unusual multiple peritoneal carcinomatosis on I-MIBG SPECT/CT after laparoscopic adrenalectomy.

Interferon Alpha Induces Multiple Cellular Proteins That Coordinately Suppress Hepadnaviral Covalently Closed Circular DNA Transcription.

Covalently closed circular DNA (cccDNA) of hepadnaviruses exists as an episomal minichromosome in the nucleus of an infected hepatocyte and serves as the template for the transcription of viral mRNAs. It had been demonstrated by others and us that interferon alpha (IFN-α) treatment of hepatocytes induced a prolonged suppression of human and duck hepatitis B virus cccDNA transcription, which is associated with the reduction of cccDNA-associated histone modifications specifying active transcription (H3K9ac or H3K27ac), but not the histone modifications marking constitutive (H3K9me3) or facultative (H3K27me3) heterochromatin formation. In our efforts to identify IFN-induced cellular proteins that mediate the suppression of cccDNA transcription by the cytokine, we found that downregulating the expression of signal transducer and activator of transcription 1 (STAT1), structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1), or promyelocytic leukemia (PML) protein increased basal level of cccDNA transcription activity and partially attenuated IFN-α suppression of cccDNA transcription. In contrast, ectopic expression of STAT1, SMCHD1, or PML significantly reduced cccDNA transcription activity. SMCHD1 is a noncanonical SMC family protein and implicated in epigenetic silencing of gene expression. PML is a component of nuclear domain 10 (ND10) and is involved in suppressing the replication of many DNA viruses. Mechanistic analyses demonstrated that STAT1, SMCHD1, and PML were recruited to cccDNA minichromosomes and phenocopied the IFN-α-induced posttranslational modifications of cccDNA-associated histones. We thus conclude that STAT1, SMCHD1, and PML may partly mediate the suppressive effect of IFN-α on hepadnaviral cccDNA transcription.IMPORTANCE Pegylated IFN-α is the only therapeutic regimen that can induce a functional cure of chronic hepatitis B in a small, but significant, fraction of treated patients. Understanding the mechanisms underlying the antiviral functions of IFN-α in hepadnaviral infection may reveal molecular targets for development of novel antiviral agents to improve the therapeutic efficacy of IFN-α. By a loss-of-function genetic screening of individual IFN-stimulated genes (ISGs) on hepadnaviral mRNAs transcribed from cccDNA, we found that downregulating the expression of STAT1, SMCHD1, or PML significantly increased the level of viral RNAs without altering the level of cccDNA. Mechanistic analyses indicated that those cellular proteins are recruited to cccDNA minichromosomes and induce the posttranslational modifications of cccDNA-associated histones similar to those induced by IFN-α treatment. We have thus identified three IFN-α-induced cellular proteins that suppress cccDNA transcription and may partly mediate IFN-α silencing of hepadnaviral cccDNA transcription.

Prominent Bone Marrow Metastases Without Concurrent Intra-Chest Metastasis in a Case of Cardiac Angiosarcoma.

Primary cardiac angiosarcoma is rare and often advances by dissemination. Lungs are the most common metastatic sites, especially when the tumor originates in the right side of the heart. Bone metastases from cardiac angiosarcoma very rarely occur without concurrent pulmonary metastases. We report a case of cardiac angiosarcoma having prominent bone metastases without concurrent pulmonary lesions, as demonstrated by FDG PET/CT scan.

Effects of Fever on 18F-FDG Distribution In Vivo: a Preliminary Study.

PURPOSE: Elevated body temperature might change glucose metabolism in human organs. The purpose of this study is to explore 18F-FDG distribution in febrile patients on the day of 18F-FDG PET/CT scanning and compare it with patients with a normal temperature. PROCEDURES: 18F-FDG PET/CT was performed on 69 febrile patients and 82 patients with a normal temperature. Patient sociodemographic data, blood glucose levels before PET/CT, body temperature on the day of the exam, and laboratory test results were collected. Maximal standard uptake values (SUVmax) in the brain, mediastinal blood pool, liver, spleen, and the bone marrow were compared. RESULTS: Compared with the controls, SUVmax of the febrile patients was significantly lower in the brain, mediastinal blood pool, and the liver (p < 0.01), and higher in the spleen and bone marrow (p < 0.01). In the febrile group, SUVmax was not significantly different between the FDG burden and non-FDG burden patients (p > 0.05). Body temperature was found negatively correlated with SUVmax in the brain (r = - 0.646), mediastinal blood pool (r = - 0.530), and the liver (r = - 0.384), and positively correlated with the SUVmax in the spleen (r = 0.592) and bone marrow (r = 0.651). Multivariate linear regression established body temperature on the day of PET/CT as an independent affecting factor (p < 0.01) for the SUVmax in the brain, mediastinal blood pool, liver, spleen, and bone marrow. The SUV in the brain, liver, and mediastinal blood pool remained different (p < 0.05) after corrected with the SUVmax in the blood pool or liver. CONCLUSIONS: Fever influences 18F-FDG distribution in multiple human tissues and organs. Altered 18F-FDG distribution in vivo might affect results of disease lesion detection and tumor therapy response assessment. Correction with blood pool or liver SUV fails to cancel the effects of fever. The day of fever should be avoided for PET/CT scan, especially in assessing tumor therapy response.

Erratum: Author correction to 'Up-regulation of glycolipid transfer protein by bicyclol causes spontaneous restriction of hepatitis C virus replication' [Acta Pharmaceutica Sinica B 9 (2019) 769-781].

[This corrects the article DOI: 10.1016/j.apsb.2019.01.013.].

Up-regulation of glycolipid transfer protein by bicyclol causes spontaneous restriction of hepatitis C virus replication.

Bicyclol is a synthetic drug for hepatoprotection in clinic since 2004. Preliminary clinical observations suggest that bicyclol might be active against hepatitis C virus (HCV) with unknown mechanism. Here, we showed that bicyclol significantly inhibited HCV replication in vitro and in hepatitis C patients. Using bicyclol as a probe, we identified glycolipid transfer protein (GLTP) to be a novel restrictive factor for HCV replication. The GLTP preferentially bound host vesicle-associated membrane protein-associated protein-A (VAP-A) in competition with the HCV NS5A, causing an interruption of the complex formation between VAP-A and HCV NS5A. As the formation of VAP-A/NS5A complex is essential for viral RNA replication, up-regulation of GLTP by bicyclol reduced the level of VAP-A/NS5A complex and thus inhibited HCV replication. Bicyclol also exhibited an inhibition on HCV variants resistant to direct-acting antiviral agents (DAAs) with an efficacy identical to that on wild type HCV. In combination with bicyclol, DAAs inhibited HCV replication in a synergistic fashion. GLTP appears to be a newly discovered host restrictive factor for HCV replication, Up-regulation of GLTP causes spontaneous restriction of HCV replication.

Discovery and Mechanistic Study of a Novel Human-Stimulator-of-Interferon-Genes Agonist.

Stimulator of interferon genes (STING) is an integral ER-membrane protein that can be activated by 2'3'-cGAMP synthesized by cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) upon binding of double-stranded DNA. It activates interferon (IFN) and inflammatory cytokine responses to defend against infection by microorganisms. Pharmacologic activation of STING has been demonstrated to induce an antiviral state and boost antitumor immunity. We previously reported a cell-based high-throughput-screening assay that allowed for identification of small-molecule cGAS-STING-pathway agonists. We report herein a compound, 6-bromo-N-(naphthalen-1-yl)benzo[d][1,3]dioxole-5-carboxamide (BNBC), that induces a proinflammatory cytokine response in a human-STING-dependent manner. Specifically, we showed that BNBC induced type I and III IFN dominant cytokine responses in primary human fibroblasts and peripheral-blood mononuclear cells (PBMCs). BNBC also induced cytokine response in PBMC-derived myeloid dendritic cells and promoted their maturation, suggesting that STING-agonist treatment could potentially regulate the activation of CD4+ and CD8+ T lymphocytes. As anticipated, treatment of primary human fibroblast cells with BNBC induced an antiviral state that inhibited the infection of several kinds of flaviviruses. Taken together, our results indicate that BNBC is a human-STING agonist that not only induces innate antiviral immunity against a broad spectrum of viruses but may also stimulate the activation of adaptive immune responses, which is important for the treatment of chronic viral infections and tumors.

Sirt2 epigenetically down-regulates PDGFRα expression and promotes CG4 cell differentiation.

We have previously found that Sirt2 enhanced the outgrowth of cellular processes and MBP expression in CG4 cells, where Sirt2 expression is suppressed by transcription factor Nkx2.2. However, the detailed mechanism of Sirt2 facilitating oligodendroglial cell differentiation remained unclear. In the present study, we observed that Sirt2 partially translocated into the nuclei when CG4 cells were induced to differentiate. Sirt2 was detected at the CpG island of PDGFRα promoter via ChIP assay during the cells differentiation process rather than during the state of growth. Sirt2 deacetylated protein(s) bound to the promoter of PDGFRα and simultaneously appeared to facilitate histone3 K27 tri-methylation, both of which are suppressive signatures on gene transcription activation. In bisulfate assay, we identified that Sirt2 significantly induced DNA methylation of PDGFRα promoter compared with the control. Consistently, Sirt2 overexpression down-regulated PDGFRα expression in CG4 cells. The knock-down of PDGFRα or Sirt2 over-expression repressed cell proliferation, but knock-down of Sirt2 promoted cell proliferation. Taken together, Sirt2 translocated into the nuclei while the cells initiated a differentiation process, facilitating CG4 cell differentiation partially through epigenetic modification and suppression of PDGFRα expression. The repression of PDGFRα expression mediated by Sirt2 appeared to facilitate a transition of cellular processes, i.e. from a proliferating progenitor state to a post-mitotic state in CG4 cells.

Cellular DNA Topoisomerases Are Required for the Synthesis of Hepatitis B Virus Covalently Closed Circular DNA.

In order to identify host cellular DNA metabolic enzymes that are involved in the biosynthesis of hepatitis B virus (HBV) covalently closed circular (ccc) DNA, we developed a cell-based assay supporting synchronized and rapid cccDNA synthesis from intracellular progeny nucleocapsid DNA. This was achieved by arresting HBV DNA replication in HepAD38 cells with phosphonoformic acid (PFA), a reversible HBV DNA polymerase inhibitor, at the stage of single-stranded DNA and was followed by removal of PFA to allow the synchronized synthesis of relaxed circular DNA (rcDNA) and subsequent conversion into cccDNA within 12 to 24 h. This cccDNA formation assay allows systematic screening of the effects of small molecular inhibitors of DNA metabolic enzymes on cccDNA synthesis but avoids cytotoxic effects upon long-term treatment. Using this assay, we found that all the tested topoisomerase I and II (TOP1 and TOP2, respectively) poisons as well as topoisomerase II DNA binding and ATPase inhibitors significantly reduced the levels of cccDNA. It was further demonstrated that these inhibitors also disrupted cccDNA synthesis during de novo HBV infection of HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). Mechanistic analyses indicate that whereas TOP1 inhibitor treatment prevented the production of covalently closed negative-strand rcDNA, TOP2 inhibitors reduced the production of this cccDNA synthesis intermediate to a lesser extent. Moreover, small interfering RNA (siRNA) knockdown of topoisomerase II significantly reduced cccDNA amplification. Taking these observations together, our study demonstrates that topoisomerase I and II may catalyze distinct steps of HBV cccDNA synthesis and that pharmacologic targeting of these cellular enzymes may facilitate the cure of chronic hepatitis B.IMPORTANCE Persistent HBV infection relies on stable maintenance and proper functioning of a nuclear episomal form of the viral genome called cccDNA, the most stable HBV replication intermediate. One of the major reasons for the failure of currently available antiviral therapeutics to cure chronic HBV infection is their inability to eradicate or inactivate cccDNA. We report here a chemical genetics approach to identify host cellular factors essential for the biosynthesis and maintenance of cccDNA and reveal that cellular DNA topoisomerases are required for both de novo synthesis and intracellular amplification of cccDNA. This approach is suitable for systematic screening of compounds targeting cellular DNA metabolic enzymes and chromatin remodelers for their ability to disrupt cccDNA biosynthesis and function. Identification of key host factors required for cccDNA metabolism and function will reveal molecular targets for developing curative therapeutics of chronic HBV infection.