A polypeptide D-TTK001 targeting HBx controls cccDNA against HBV

Abstract Chronic infection of HBV is a serious global health issue. However, the HBV covalently closed circular DNA (cccDNA) cannot be completely cleaned in liver. Here, we engineer a novel polypeptide, termed D-TTK001, targeting HBV X protein (HBx) against HBV cccDNA. D-TTK001 is a D-type polypeptide, which has a high binding affinity to HBx. Interestingly, it is able to against HBV efficiently in primary human hepatocytes (PHHs) from three donors. D-TTK001 displays high activities against HBV, remarkably reducing the levels of cccDNA in the HBV-infected human liver-chimeric mice. Mechanically, D-TTK001 depresses cccDNA transcription by regulating the epigenetic modification of cccDNA minichromosome and disturbing the interaction of HBx with DDB1 in the cells. D-TTK001 decreases the levels of cccDNA by down-regulating MSL2, leading to the increase of APOBEC3B in liver. When combined with entecavir or interferon-α, D-TTK001 shows additive antiviral effects. Clinically, D-TTK001 is a strong candidate to clear cccDNA against HBV.

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IFN-α confers epigenetic regulation of HBV cccDNA minichromosome by modulating GCN5-mediated succinylation of histone H3K79 to clear HBV cccDNA

Clinical Epigenetics ◽

10.1186/s13148-020-00928-z ◽

2020 ◽

Vol 12 (1) ◽

Author(s):

Ying Yuan ◽

Hongfeng Yuan ◽

Guang Yang ◽

Haolin Yun ◽

Man Zhao ◽

...

Keyword(s):

Epigenetic Regulation ◽

Human Liver ◽

De Novo ◽

Underlying Mechanism ◽

Chimeric Mice ◽

Circular Dna ◽

Hbv Cccdna ◽

Heparg Cells ◽

B Virus ◽

Histone Acetyltransferase Gcn5

Abstract Background Hepatitis B virus covalently closed circular DNA (HBV cccDNA) is assembled by histones and non-histones into a chromatin-like cccDNA minichromosome in the nucleus. The cellular histone acetyltransferase GCN5, displaying succinyltransferase activity, is recruited onto cccDNA to modulate HBV transcription in cells. Clinically, IFN-α is able to repress cccDNA. However, the underlying mechanism of IFN-α in the depression of cccDNA mediated by GCN5 is poorly understood. Here, we explored the effect of IFN-α on GCN5-mediated succinylation in the epigenetic regulation of HBV cccDNA minichromosome. Results Succinylation modification of the cccDNA minichromosome has been observed in HBV-infected human liver-chimeric mice and HBV-expressing cell lines. Moreover, histone H3K79 succinylation by GCN5 was identified in the system. Interestingly, the mutant of histone H3K79 efficiently blocked the replication of HBV, and interference with GCN5 resulted in decreased levels of HBV DNA, HBsAg, and HBeAg in the supernatant from de novo HBV-infected HepaRG cells. Consistently, the levels of histone H3K79 succinylation were significantly elevated in the livers of HBV-infected human liver-chimeric mice. The knockdown or overexpression of GCN5 or the mutant of GCN5 could affect the binding of GCN5 to cccDNA or H3K79 succinylation, leading to a change in cccDNA transcription activity. In addition, Southern blot analysis validated that siGCN5 decreased the levels of cccDNA in the cells, suggesting that GCN5-mediated succinylation of histone H3K79 contributes to the epigenetic regulation of cccDNA minichromosome. Strikingly, IFN-α effectively depressed histone H3K79 succinylation in HBV cccDNA minichromosome in de novo HepG2-NTCP and HBV-infected HepaRG cells. Conclusions IFN-α epigenetically regulates the HBV cccDNA minichromosome by modulating GCN5-mediated succinylation of histone H3K79 to clear HBV cccDNA. Our findings provide new insights into the mechanism by which IFN-α modulate the epigenetic regulation of HBV cccDNA minichromosome.

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Mechanism of Hepatitis B Virus cccDNA Formation

Viruses ◽

10.3390/v13081463 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1463

Author(s):

Lei Wei ◽

Alexander Ploss

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Medical Problem ◽

Circular Dna ◽

Critical Step ◽

Hbv Cccdna ◽

Double Stranded Dna ◽

Cellular Environment ◽

B Virus ◽

Comprehensive Picture

Hepatitis B virus (HBV) remains a major medical problem affecting at least 257 million chronically infected patients who are at risk of developing serious, frequently fatal liver diseases. HBV is a small, partially double-stranded DNA virus that goes through an intricate replication cycle in its native cellular environment: human hepatocytes. A critical step in the viral life-cycle is the conversion of relaxed circular DNA (rcDNA) into covalently closed circular DNA (cccDNA), the latter being the major template for HBV gene transcription. For this conversion, HBV relies on multiple host factors, as enzymes capable of catalyzing the relevant reactions are not encoded in the viral genome. Combinations of genetic and biochemical approaches have produced findings that provide a more holistic picture of the complex mechanism of HBV cccDNA formation. Here, we review some of these studies that have helped to provide a comprehensive picture of rcDNA to cccDNA conversion. Mechanistic insights into this critical step for HBV persistence hold the key for devising new therapies that will lead not only to viral suppression but to a cure.

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Hepatitis B Core-Related Antigen as Surrogate Biomarker of Intrahepatic Hepatitis B Virus Covalently-Closed-Circular DNA in Patients with Chronic Hepatitis B: A Meta-Analysis

Diagnostics ◽

10.3390/diagnostics11020187 ◽

2021 ◽

Vol 11 (2) ◽

pp. 187

Author(s):

Gian Paolo Caviglia ◽

Angelo Armandi ◽

Chiara Rosso ◽

Davide Giuseppe Ribaldone ◽

Rinaldo Pellicano ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Hepatitis B Surface Antigen ◽

Meta Analysis ◽

Circular Dna ◽

Non Invasive ◽

Hbv Cccdna ◽

B Virus ◽

Chronic Hbv ◽

Intrahepatic Hbv

Hepatitis B virus (HBV) covalently-closed-circular (ccc)DNA is the key molecule responsible for viral persistence within infected hepatocytes. The evaluation of HBV cccDNA is crucial for the management of patients with chronic HBV infection and for the personalization of treatment. However, the need for liver biopsy is the principal obstacle for the assessment of intrahepatic HBV cccDNA. In the last decade, several studies have investigated the performance of hepatitis B core-related antigen (HBcrAg) as a surrogate of HBV cccDNA amount in the liver. In this meta-analysis, we collected 14 studies (1271 patients) investigating the correlation between serum HBcrAg and intrahepatic HBV cccDNA. Serum HBcrAg showed a high correlation with intrahepatic HBV cccDNA (r = 0.641, 95% confidence interval (CI) 0.510–0.743, p < 0.001). In a head-to-head comparison, we observed that the performance of HBcrAg was significantly superior to that of hepatitis B surface antigen (r = 0.665 vs. r = 0.475, respectively, p < 0.001). Subgroup analysis showed that the correlation between HBcrAg and intrahepatic HBV cccDNA was high, both in hepatitis B e antigen-positive and -negative patients (r = 0.678, 95% CI 0.403–0.840, p < 0.001, and r = 0.578, 95% CI 0.344–0.744, p < 0.001, respectively). In conclusion, the measurement of serum HBcrAg qualifies as a reliable non-invasive surrogate for the assessment of an intrahepatic HBV cccDNA reservoir.

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How to Clear HBV cccDNA: CRISPR/Cas9 Might Be Promising

10.20944/preprints201710.0065.v1 ◽

2017 ◽

Author(s):

Yan Qiu ◽

Ying Liu ◽

Wen Ren ◽

Jing Ren

Keyword(s):

Chronic Hepatitis ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Chronic Hepatitis B ◽

Health Concern ◽

Liver Carcinoma ◽

Major Health ◽

Circular Dna ◽

Hbv Cccdna ◽

B Virus

BACKGROUND: Chronic hepatitis B infected with Hepatitis B virus remains a major health concern worldwide. Despite standard interferon-α and nucleotide analogues have been shown to reduce the deterioration of liver disease among chronic hepatitis B patients, covalently closed circular DNA was still difficult to eradicate. METHODS: A literature search of Pubmed and Web of science was performed with the following key words: ‘CRISPR’, ‘CRISPR/Cas9’, ‘hepatitis B’, ‘HBV’, ‘chronic hepatitis B’ and ‘HBV cccDNA’. The information about CRISPR/Cas9 for the treatment of HBV cccDNA or hepatitis B was reviewed. RESULTS: CRISPR/Cas9 could treat hepatitis B through suppressing or clearing HBV cccDNA with different gRNAs. CONCLUSION: With the emergence of CRISPR/Cas9 (the RNA-guided clustered regulatory interspaced short palindromic repeats, CRISPR) editing technology, clearance of hepatitis B virus and better prevention of liver carcinoma seemed to be possible.

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Identification of 20(S)-Protopanaxadiol Metabolites in Human Liver Microsomes and Human Hepatocytes

Drug Metabolism and Disposition ◽

10.1124/dmd.110.036723 ◽

2010 ◽

Vol 39 (3) ◽

pp. 472-483 ◽

Cited By ~ 48

Author(s):

Liang Li ◽

Xiaoyan Chen ◽

Dan Li ◽

Dafang Zhong

Keyword(s):

Human Liver ◽

Liver Microsomes ◽

Human Hepatocytes ◽

Human Liver Microsomes

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Human Liver Spheroids as a Model to Study Aetiology and Treatment of Hepatic Fibrosis

Cells ◽

10.3390/cells9040964 ◽

2020 ◽

Vol 9 (4) ◽

pp. 964 ◽

Cited By ~ 4

Author(s):

Tracey Hurrell ◽

Vlasia Kastrinou-Lampou ◽

Achilleas Fardellas ◽

Delilah F. G. Hendriks ◽

Åsa Nordling ◽

...

Keyword(s):

Human Liver ◽

Progressive Disease ◽

Human Hepatocytes ◽

Alcoholic Fatty Liver ◽

Drug Candidates ◽

Alk5 Inhibitor ◽

Hepatocyte Markers ◽

Parenchymal Cells ◽

Oxidant Effect ◽

Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease affects approximately one billion adults worldwide. Non-alcoholic steatohepatitis (NASH) is a progressive disease and underlies the advancement to liver fibrosis, cirrhosis, and hepatocellular carcinoma, for which there are no FDA-approved drug therapies. We developed a hetero-cellular spheroid system comprised of primary human hepatocytes (PHH) co-cultured with crude fractions of primary human liver non-parenchymal cells (NPC) from several matched or non-matched donors, to identify phenotypes with utility in investigating NASH pathogenesis and drug screening. Co-culture spheroids displayed stable expression of hepatocyte markers (albumin, CYP3A4) with the integration of stellate (vimentin, PDGFRβ), endothelial (vWF, PECAM1), and CD68-positive cells. Several co-culture spheroids developed a fibrotic phenotype either spontaneously, primarily observed in PNPLA3 mutant donors, or after challenge with free fatty acids (FFA), as determined by COL1A1 and αSMA expression. This phenotype, as well as TGFβ1 expression, was attenuated with an ALK5 inhibitor. Furthermore, CYP2E1, which has a strong pro-oxidant effect, was induced by NPCs and FFA. This system was used to evaluate the effects of anti-NASH drug candidates, which inhibited fibrillary deposition following 7 days of exposure. In conclusion, we suggest that this system is suitable for the evaluation of NASH pathogenesis and screening of anti-NASH drug candidates.

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Identification of Human Sulfotransferases Active towards Silymarin Flavonolignans and Taxifolin

Metabolites ◽

10.3390/metabo10080329 ◽

2020 ◽

Vol 10 (8) ◽

pp. 329

Author(s):

Jiří Vrba ◽

Barbora Papoušková ◽

Pavel Kosina ◽

Kateřina Lněničková ◽

Kateřina Valentová ◽

...

Keyword(s):

Human Liver ◽

High Performance ◽

Herbal Remedy ◽

Expression Patterns ◽

Human Hepatocytes ◽

Tandem Mass ◽

Recombinant Enzymes ◽

Metabolic Reactions ◽

Natural Phenolic Compounds

Natural phenolic compounds are known to be metabolized by phase II metabolic reactions. In this study, we examined the in vitro sulfation of the main constituents of silymarin, an herbal remedy produced from the fruits of the milk thistle. The study focused on major flavonolignan constituents, including silybin A, silybin B, isosilybin A, isosilybin B, silychristin, and silydianin, as well as the flavonoid taxifolin. Using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS), individual flavonolignans and taxifolin were found to be sulfated by human liver and human intestinal cytosols. Moreover, experiments with recombinant enzymes revealed that human sulfotransferases (SULTs) 1A1*1, 1A1*2, 1A2, 1A3, 1B1, 1C4, and 1E1 catalyzed the sulfation of all of the tested compounds, with the exception of silydianin, which was not sulfated by SULT1B1 and SULT1C4. The sulfation products detected were monosulfates, of which some of the major ones were identified as silybin A 20-O-sulfate, silybin B 20-O-sulfate, and isosilybin A 20-O-sulfate. Further, we also observed the sulfation of the tested compounds when they were tested in the silymarin mixture. Sulfates of flavonolignans and of taxifolin were produced by incubating silymarin with all of the above SULT enzymes, with human liver and intestinal cytosols, and also with human hepatocytes, even though the spectrum and amount of the sulfates varied among the metabolic models. Considering our results and the expression patterns of human sulfotransferases in metabolic tissues, we conclude that flavonolignans and taxifolin can potentially undergo both intestinal and hepatic sulfation, and that SULTs 1A1, 1A3, 1B1, and 1E1 could be involved in the biotransformation of the constituents of silymarin.

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