Inhibited Hepatitis B Virus DNA Research Articles

MicroRNA-642a-5p Targets DNA Damage-Inducible Transcript 4 to Suppress Hepatitis B Virus Hepatoma Carcinoma Cell

Background: Hepatocellular carcinoma (HCC) is one of the most prevalent malignant tumors in clinical practice, with hepatitis B virus (HBV) being the most common risk factor for HCC. MicroRNAs (miRNAs) have emerged as a new marker for disease diagnosis and molecularly targeted therapies; however, the mechanism of miR-642a-5p in HBV-associated HCC remains unclear. Objectives: The aim of this study was to investigate the expression of miR-642a-5p, which targets DNA damage-inducible transcript 4 (DDIT4), in HBV-associated HCC, and its effect on the proliferation, migration, and invasion of HBV-positive HCC cells. Methods: miR-642a-5p in the serum of patients with HBV-associated liver cancer (LC), as well as miR-642a-5p and DDIT4 mRNA in LC tissues and cells, and HBV DNA in HBV-positive cells were detected. The targeting of DDIT4 by miR-642a-5p and the progression of cells were also examined. All cell experiments were repeated five times. Results: The results indicated that levels of miR-642a-5p were decreased, while levels of DDIT4 were increased in the serum, tissues, and cells of HBV-positive HCC patients. Overexpression of miR-642a-5p inhibited the progression of HBV-positive HCC cells, suppressed HBV DNA replication, cell proliferation, and invasion, and promoted apoptosis in HepG2.2.15 cells. Conclusions: In addition, miR-642a-5p directly targeted DDIT4, and knockdown of DDIT4 reversed the effects of miR-642a-5p upregulation, promoting the progression of HBV-positive HCC cells. In conclusion, miR-642a-5p is expressed at low levels in HBV-associated HCC and inhibits HBV DNA replication and tumor progression in HBV-positive HCC by targeting DDIT4. This study provides a foundation for molecular targeted therapy in HBV-positive HCC.

Biophysics-Guided Lead Discovery of HBV Capsid Assembly Modifiers.

Hepatitis B virus (HBV) is the leading cause of chronic liver pathologies worldwide. HBV nucleocapsid, a key structural component, is formed through the self-assembly of the capsid protein units. Therefore, interfering with the self-assembly process is a promising approach for the development of novel antiviral agents. Applied to HBV, this approach has led to several classes of capsid assembly modulators (CAMs). Here, we report structurally novel CAMs with moderate activity and low toxicity, discovered through a biophysics-guided approach combining docking, molecular dynamics simulations, and a series of assays with a particular emphasis on biophysical experiments. Several of the identified compounds induce the formation of aberrant capsids and inhibit HBV DNA replication in vitro, suggesting that they possess modest capsid assembly modulation effects. The synergistic computational and experimental approaches provided key insights that facilitated the identification of compounds with promising activities. The discovery of preclinical CAMs presents opportunities for subsequent optimization efforts, thereby opening new avenues for HBV inhibition.

Oxymatrine Inhibition of Hepatitis B Virus Replication Through ERK1/2 Pathway and HNF1α and HNF4α Block in vitro.

To explore the molecular mechanism of oxymatrine (OM) by increasing the phosphorylation of ERK1/2 signal factor and blocking the transcription factors HNF1α and HNF4α expression against hepatitis B virus (HBV) antigen secretion and HBV DNA replication in HepG2.2.15 cells. An experimental study. Place and Duration of the Study: Department of Laboratory Medicine,First Affiliated Hospital of Gannan Medical University, Jiangxi, China, between May 2020 and December 2022. HepG2.2.15 cells, known for stably expressing HBV particles, were utilised as a cell-based model to explore potential pathways pertaining to the OM inhibition of HBV replication. An MTT assay was utilised to measure cytotoxicity. HBsAg or HBeAg content was measured using an enzyme-linked immunosorbent assay kit. HBV DNA in cell-free culture media was examined using a fluorescent quantitative PCR kit. Real-time PCR was utilised to analyse HNF1α and HNF4α mRNA expression, whereas Western blotting was performed to evaluate HNF1α, HNF4α, and ERK1/2 protein expression. OM inhibited HBV DNA copy number in the cell supernatant, 3.5-kb RNA gene expression in cells, and HBsAg and HBeAg secretion. OM upregulated p-ERK1/2 protein and significantly downregulated HNF1α and HNF4α gene transcription and protein translation. OM may inhibit the replication of HBV by inducing the phosphorylation of ERK1/2 and blocking the transcription factors HNF1α and HNF4α expression that are essential for viral replication. Oxymatrine, ERK1/2, Hepatocyte nuclear factor, Anti-HBV.

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 β, 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.

Lipid phosphatase SAC1 suppresses hepatitis B virus replication through promoting autophagic degradation of virions

Phosphatidylinositol lipids play vital roles in lipid signal transduction, membrane recognition, vesicle transport, and viral replication. Previous studies have revealed that SAC1-like phosphatidylinositol phosphatase (SACM1L/SAC1), which uses phosphatidylinositol-4-phosphate (PI4P) as its substrate, greatly affects the replication of certain bacteria and viruses in vitro. However, it remains unclear whether and how SAC1 modulates hepatitis B virus (HBV) replication in vitro and in vivo. In the present study, we observed that SAC1 silencing significantly increased HBV DNA replication, subviral particle (SVP) expression, and secretion of HBV virions, whereas SAC1 overexpression exerted the opposite effects. Moreover, SAC1 overexpression inhibited HBV DNA replication and SVP expression in a hydrodynamic injection-based HBV-persistent replicating mouse model. Mechanistically, SAC1 silencing increased the number of HBV-containing autophagosomes as well as PI4P levels on the autophagosome membrane. Moreover, SAC1 silencing blocked autophagosome-lysosome fusion by inhibiting the interaction between synaptosomal-associated protein 29 and vesicle-associated membrane protein 8. Collectively, our data indicate that SAC1 significantly inhibits HBV replication by promoting the autophagic degradation of HBV virions. Our findings support that SAC1-mediated phospholipid metabolism greatly modulates certain steps of the HBV life-cycle and provide a new theoretical basis for antiviral therapy.

ADAR1 Inhibits HBV DNA Replication via Regulating miR-122-5p in Palmitic Acid Treated HepG2.2.15 Cells.

Changes in living standards and diet structure, non-alcoholic fatty liver disease (NAFLD) is prevalent globally, including in Asia, where chronic hepatitis B (CHB) is endemic. As such, cooccurrence of NAFLD with CHB is common in Asia. However, the pathogenesis underlying the onset of fatty liver in CHB prognosis has not been fully elucidated. Therefore, we aimed to investigate the effects and mechanisms of lipotoxicity on hepatitis B virus (HBV) DNA replication. The expression of adenosine deaminase acting on RNA-1 (ADAR1) and miR-122 was evaluated in liver tissues from patients with CHB concurrent NAFLD. Palmitic acid-treated HepG2.2.15 cells were used as the cell model. The effect of lipotoxicity on HBV DNA replication was evaluated in vitro by transfecting the ADAR1 overexpression or knockdown lentiviral vector into HepG2.2.15 cells, respectively. qRT-PCR, western blotting and immunofluorescence were performed to determine ADAR1 expression. The expression of ADAR1 in the liver tissues of CHB patients with concurrent NAFLD was significantly down-regulated compared with that in CHB patients. Enforced expression of ADAR1 inhibited the HBV DNA replication, whereas ADAR1 knockdown resulted in increased HBV DNA expression in palmitic acid - treated HepG2.2.15 cells. Additionally, ADAR1 inhibited the HBV DNA replication by upregulating miR-122, which is most abundant in the liver and mainly inhibits HBV DNA replication. ADAR1 may act as a suppressor of HBV replication in palmitic acid -treated HepG2.2.15 cells by increasing miR-122 levels. Thus, ADAR1 may serve as a potential biomarker and therapeutic target for CHB with concurrent NAFLD.

Susceptibility of Drug Resistant Hepatitis B Virus Mutants to Besifovir.

Currently, interferon alpha and nucleos(t)ide analogues (NAs) are clinically available to treat hepatitis B virus (HBV) infection. Several NAs, including lamivudine (LMV), adefovir (ADV), entecavir (ETV) and tenofovir (TDF or TAF) have been approved and administered to chronic hepatitis B (CHB) patients. NAs inhibit HBV DNA synthesis by targeting the reverse transcriptase (RT) domain of HBV polymerase. Several mutations in the RT domain which lead to drug resistance against NAs have been reported, even for TDF and TAF which are highly potent with very low resistance rate. Besifovir (BFV) is a new antiviral dGMP analogue able to be used as a new NA drug for the control of CHB infection. Drug resistance to BFV is not well known due to its shorter duration of clinical use. Recently, we reported that rtL180M (M) and rtM204V (V) mutations, already resistant to LMV, are associated with BFV resistance. However, the susceptibility to BFV of previously known HBV mutants resistant to various drugs has not been studied. To investigate this, we performed in vitro drug susceptibility assays using natural and artificial mutants that are associated with resistance to LMV, ADV, ETV or TDF. As a result, LMV-resistant mutants were not susceptible to BFV and ETV-resistant clones showed partial resistance against BFV as well. However, ADV-resistant mutants were highly sensitive to BFV. In case of tenofovir-resistant mutations, the HBV mutants harboring primary mutations to tenofovir resistance were susceptible to BFV. Therefore, our study revealed that BSV may serve as an alternative drug for patients with ADV-, ETV-, TDF- or TAF-resistance.

(-)-Lariciresinol Isolated from the Roots of Isatis indigotica Fortune ex Lindl. Inhibits Hepatitis B Virus by Regulating Viral Transcription.

Chronic hepatitis induced by hepatitis B virus (HBV) infection is a serious public health problem, leading to hepatic cirrhosis and liver cancer. Although the currently approved medications can reliably decrease the virus load and prevent the development of hepatic diseases, they fail to induce durable off-drug control of HBV replication in the majority of patients. The roots of Isatis indigotica Fortune ex Lindl., a traditional Chinese medicine, were frequently used for the prevention of viral disease in China. In the present study, (−)-lariciresinol ((−)-LRSL), isolated from the roots of Isatis indigotica Fortune ex Lindl., was found to inhibit HBV DNA replication of both wild-type and nucleos(t)ide analogues (NUCs)-resistant strains in vitro. Mechanism studies revealed that (−)-LRSL could block RNA production after treatment, followed by viral proteins, and then viral particles and DNA. Promoter reporter assays and RNA decaying dynamic experiments indicated that (−)-LRSL mediated HBV RNA reduction was mainly due to transcriptional inhibition rather than degradation. Moreover, (−)-LRSL in a dose-dependent manner also inhibited other animal hepadnaviruses, including woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV). Combining the analysis of RNA-seq, we further found that the decrease in HBV transcriptional activity by (−)-LRSL may be related to hepatocyte nuclear factor 1α (HNF1α). Taken together, (−)-LRSL represents a novel chemical entity that inhibits HBV replication by regulating HNF1α mediated HBV transcription, which may provide a new perspective for HBV therapeutics.

S6K1 inhibits HBV replication through inhibiting AMPK-ULK1 pathway and disrupting acetylation modification of H3K27

AimsTo investigated the effect of S6K1 on the replication and transcription of HBV DNA using multiple cell models. Main methodsThe pgRNA, total HBV RNA and HBV DNA level were detected by Real-time PCR. The HBcAg expression by Western blot and the activity of four HBV promoters, such as preS1, preS2/S, core, and X promoters by using dual luciferase reporter assay. Moreover, we determined S6K1 interacted with HBcAg in both cytoplasm and nucleus through Immunofluorescence, co-immunoprecipitation (CoIP) and Western blot. Key findingsS6K1 inhibited HBV DNA replication and cccDNA-dependent transcription in HBV-expressing stable cell lines. The mechanistic study revealed that S6K1 suppressed HBV DNA replication by inhibiting AMPK-ULK1 autophagy pathway, and the nuclear S6K1 suppressed HBV cccDNA-dependent transcription by inhibiting the acetylation modification of H3K27. In addition, HBV capsid protein (HBcAg) suppressed the phosphorylation level of S6K1Thr389 by interacting with S6K1, indicating a viral antagonism of S6K1-mediated antiviral mechanism. SignificanceThe p70 ribosomal S6 kinase (S6K1) is a serine/threonine protein kinase, and it plays a significant role in different cellular processes. It has been previously reported that S6K1 affects hepatitis B virus (HBV) replication but the underlying mechanism remains unclear. In this study, our data suggested that the activation of S6K1 restricts HBV replication through inhibiting AMPK-ULK1 autophagy pathway and H3K27 acetylation. These findings indicated that S6K1 might be a potential therapeutic target for HBV infection.

Entecavir add-on Peg-interferon therapy plays a positive role in reversing hepatic fibrosis in treatment-naïve chronic hepatitis B patients: a prospective and randomized controlled trial.

BackgroundThe efficacy of entecavir (ETV) add-on peg-interferon therapy compared with ETV monotherapy in treatment-naïve hepatitis B virus (HBV) patients remains controversial. We investigated whether adding peg-interferon to ongoing ETV treatment leads to a better curative effect or not.MethodsAll patients have been recruited between August 2013 and January 2015 from the Shanghai Public Health Clinical Center and Zhongshan Hospital (China). Eligible HBV patients (n = 144) were randomly divided (1:1) to receive either ETV monotherapy (n = 70) or peg-interferon add-on therapy from week 26 to 52 (n = 74). Patients were followed-up for at least 2 years. Indexes including hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) seroconversion rate, sustained virologic response, transient elastography value, and histological scores were evaluated every 3 months until the end of the study. The rate of patients with HBsAg loss was defined as the primary endpoint criteria.ResultsAt week 26, no patient achieved HBsAg seroconversion in either group. At week 52, one patient in the monotherapy group was HBsAg-negative but there was none in the combination therapy group. The monotherapy group showed significantly better liver function recovery results than the combination therapy group. At week 78, one patient in the combination group had HBsAg seroconverted. At week 104, only three patients in the combination therapy group were HBsAg-negative compared with one patient in monotherapy. The mean alanine aminotransferase and aspartate aminotransferase levels and transient elastography values decreased significantly compared with baseline. Both groups showed a favorable decrease in alpha-fetoprotein (monotherapy: 4.5 [2.8, 7.1] vs. 2.2 [1.8, 3.1] ng/mL, P < 0.001; combination therapy: 5.7 [3.0, 18.8] vs. 3.2 [2.0, 4.3] ng/mL, P < 0.001) and an improved result of liver biopsy examination scores. The combination group showed a better improvement in histology compared with the monotherapy group (mean transient elastography value 6.6 [4.9, 9.8] vs. 7.8 [5.4, 11.1] kPa, P = 0.028). But there was no significant difference in HBsAg conversion rate (1.8% [1/56] vs. 4.1% [3/73], P = 0.809) and HBeAg conversion rate (12.5% [7/56] vs. 11.0% [8/73], P = 0.787), as well as HBV-DNA, sustained virologic response (93.2% vs. 98.5%, P = 0.150) between the two groups.ConclusionsBoth therapies supported liver function recovery and histology improvement. Combination therapy did not show better anti-viral efficacy in HBsAg or HBeAg seroconversion compared with monotherapy. However, combination therapy played a more positive role in reversing hepatic fibrosis compared with monotherapy.Trial registrationClinicalTrials.gov: NCT02849132; https://clinicaltrials.gov/ct2/show/NCT02849132

Assessment of quinazolinone derivatives as novel non-nucleoside hepatitis B virus inhibitors

Hepatitis B virus (HBV) infection is a worldwide public health issue. Search for novel non-nucleoside anti-HBV agents is of great importance. In the present study, a series of quinazolinones derivatives (4a-t and 5a-f) were synthesized and evaluated as novel anti-HBV agents. Among them, compounds 5e and 5f could significantly inhibit HBV DNA replication with IC50 values of 1.54 μM and 0.71 μM, respectively. Interestingly, the selective index values of 5f was higher than that of lead compound K284–1405, suggesting 5f possessed relatively safety profile than K284–1405. Notably, 5e and 5f exhibited remarkably anti-HBV activities against lamivudine and entecavir resistant HBV strain with IC50 values of 1.90 and 0.84 μM, confirming their effectiveness against resistant HBV strain. In addition, molecular docking studies indicated that compounds 5e and 5f could well fit into the dimer-dimer interface of HBV core protein dominated by hydrophobic interactions. Notably, their binding modes were different from the lead compound K284–1405, which may be attributed to the additional substituent groups in the quinazolinone scaffold. Taken together, 5e and 5f possessed novel chemical structure and potent anti-HBV activity against both drug sensitive and resistant HBV strains, thus warranting further research as potential non-nucleoside anti-HBV candidates.

Direct Inhibition of Hepatitis B e Antigen by Core Protein Allosteric Modulator.

Hepatitis B e antigen (HBeAg) is an important immunomodulator for promoting host immune tolerance during chronic hepatitis B (CHB) infection. In patients with CHB, HBeAg loss and seroconversion represent partial immune control of CHB infection and are regarded as valuable endpoints. However, the current approved treatments have only a limited efficacy in achieving HBeAg seroconversion in HBeAg‐positive patients. Hepatitis B virus (HBV) core protein has been recognized as an attractive antiviral target, and two classes of core protein allosteric modulator (CpAM) have been discovered: the phenylpropenamides (PPAs) and the heteroaryldihydropyrimidines (HAPs). However, their differentiation and potential therapeutic benefit beyond HBV DNA inhibition remain to be seen. Here, we show that in contrast to PPA series compound AT‐130, a HAP CpAM, HAP_R01, reduced HBeAg levels in multiple in vitro and in vivo HBV experimental models. Mechanistically, we found that HAP_R01 treatment caused the misassembly of capsids formed by purified HBeAg in vitro. In addition, HAP_R01 directly reduces HBeAg levels by inducing intracellular precore protein misassembly and aggregation. Using a HAP_R01‐resistant mutant, we found that HAP_R01‐mediated HBeAg and core protein reductions were mediated through the same mechanism. Furthermore, HAP_R01 treatment substantially reduced serum HBeAg levels in an HBV mouse model. Conclusion: Unlike PPA series compound AT‐130, HAP_R01 not only inhibits HBV DNA levels but also directly reduces HBeAg through induction of its misassembly. HAP_R01, as well as other similar CpAMs, has the potential to achieve higher anti‐HBeAg seroconversion rates than currently approved therapies for patients with CHB. Our findings also provide guidance for dose selection when designing clinical trials with molecules from HAP series.

Application of clustered regularly interspaced short palindromic repeats- associated protein 9 gene editing technology for treatment of HBV infection

A lack of effective drugs and technical means to eradicate hepatitis B virus (HBV) is a bottleneck that limits the ability to fully cure HBV infection. Recently, genome-editing technology based on clustered regularly interspaced short palindromic repeats -associated protein 9 is an emerging technique for editing specific gene loci, which can specifically target HBV covalently closed circular DNA, effectively inhibits HBV DNA replication and regulates HBV functional protein expression, and is expected to become a powerful gene therapy tool for the complete eradication of HBV. Considering this, it has become the focus of attention for scholars at home and abroad that how to use clustered regularly interspaced short palindromic repeats -associated protein 9 to accomplish modification of HBV genomes for complete eradication of HBV. This paper summarizes the latest progress based on the latest research results at home and abroad in the application of clustered regularly interspaced short palindromic repeats -associated protein 9 gene editing technology in anti-HBV infection treatment, and expounds its potential and challenges as a radical cure for HBV infection.

Interleukin-34 inhibits hepatitis B virus replication in vitro and in vivo.

BackgroundThe hepatitis B virus (HBV) infection could activate the immune system and induce extensive inflammatory response. As the most important inflammatory factor, interleukins are critical for anti-viral immunity. Here we investigated whether interleukin-34 (IL-34) play a role in HBV infection.Methodology/Principal findingsIn this study, we first found that both serum IL-34 and IL-34 mRNA in PBMCs in chronic HBV patients was significantly decreased compared to the healthy controls. Furthermore, both IL-34 protein and mRNA levels were declined hepatoma cells expressing HBV. In addition, the clinical parameters analysis found that serum IL-34 was significantly associated with HBV DNA (P = 0.0066), ALT (P = 0.0327), AST (P = 0.0435), TB (P = 0.0406), DB (P = 0.0368) and AFP (P = 0.0225). Correlation analysis also found that serum IL-34 negatively correlated with HBV DNA copies, ALT and AST. In vitro studies found that IL-34 treatment in HepAD38 and HepG2.2.15 cells markedly inhibited HBV DNA, total RNA, 3.5kb mRNA and HBc protein. In vivo studies further demonstrated IL-34 treatment in HBV transgenic mice exhibited greater inhibition on HBV DNA, total RNA, 3.5kb mRNA and HBc protein, suggesting the effect to IL-34 on HBV is likely due to host innate or adaptive immune response.Conclusions/SignificanceOur study identified a novel interleukin, IL-34, which has anti-viral activity in HBV replication in hepatocytes in vitro and in vivo. These data suggest a rationale for the use of IL-34 in the HBV treatment.

Complete Spectrum of CRISPR/Cas9-induced Mutations on HBV cccDNA.

Hepatitis B virus (HBV) causes chronic infections that cannot yet be cured. The virus persists in infected hepatocytes, because covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs, is stable in nondividing cells. Antiviral therapies with nucleoside analogues inhibit HBV DNA synthesis in capsids in the cytoplasm of infected hepatocytes, but do not destroy nuclear cccDNA. Because over 200 million people are still infected, a cure for chronic hepatitis B (CHB) has become one of the major challenges in antiviral therapy. As a first step toward the development of curative therapies, we previously demonstrated that the CRISPR/Cas9 system can be used to functionally inactivate cccDNA derived from infectious HBV. Moreover, some evidence suggests that certain cytokines might induce an APOBEC-mediated cascade leading to the destruction of cccDNA. In this report we investigated whether a combination of the two mechanisms could act synergistically to inactivate cccDNA. Using next generation sequencing (NGS), we determined the complete spectrum of mutations in cccDNA following Cas9 cleavage and repair by nonhomologous end joining (NHEJ). We found that over 90% of HBV DNA was cleaved by Cas9. In addition our results showed that editing of HBV DNA after Cas9 cleavage is at least 15,000 times more efficient that APOBEC-mediated cytosine deamination following treatment of infected cells with interferon alpha (IFNα). We also found that a previously used method to detect cytosine deaminated DNA, termed 3D-PCR, overestimates the amount and frequency of edited HBV DNA. Taken together, our results demonstrated that the CRISPR/Cas9 system is so far the best method to functionally inactivate HBV cccDNA and provide a cure for CHB.

Hepatitis B Virus Stimulated Fibronectin Facilitates Viral Maintenance and Replication through Two Distinct Mechanisms.

Fibronectin (FN) is a high molecular weight extracellular matrix protein that functions in cell adhesion, growth, migration, and embryonic development. However, little is known about the role of FN during viral infection. In the present study, we found significantly higher levels of FN in sera, and liver tissues from hepatitis B virus (HBV) patients relative to healthy individuals. HBV expression enhanced FN mRNA and protein levels in the hepatic cell lines Huh7 and HepG2. HBV infection of susceptible HepG2-sodium taurocholate co-transporting polypeptide cells also increased FN expression. We also found that transcriptional factor specificity protein 1 was involved in the induction of FN by HBV. Knockdown of FN expression significantly inhibited HBV DNA replication and protein synthesis through activating endogenous IFN-α production. In addition, FN interacted with the transforming growth factor β-activated protein kinase 1 (TAK1) and TAK1-binding protein complex and attenuated interferon signaling by inhibiting TAK1 phosphorylation. Furthermore, the nuclear translocation of NF-κB/p65 was found to be inhibited by FN. We also observed that FN promoted HBV enhancers to support HBV expression. These results suggest novel functions of endogenous FN involved in immune evasion and maintenance of HBV replication.

ZEB2 inhibits HBV transcription and replication by targeting its core promoter.

Hepatitis B virus (HBV) infection is a major cause of liver diseases, especially liver cirrhosis and hepatocellular carcinoma. However, the interaction between host and HBV has not been fully elucidated. ZEB2 is a Smad-interacting, multi-zinc finger protein that acts as a transcription factor or repressor for several signaling pathways. This study found that the expression of ZEB2 was decreased in HBV-expressing cells. Overexpression of ZEB2 inhibited HBV DNA replicative intermediates, 3.5kb mRNA, core protein level, and the secretion of HBsAg and HBeAg. In contrast, ZEB2 knockdown promoted HBV replication. Furthermore, ZEB2 could bind to HBV core promoter and inhibit its promoter activity. Mutation at the ZEB2 binding site in HBV core promoter eradicated ZEB2-mediated inhibition of HBV replication. This study identifies ZEB2 as a novel host restriction factor that inhibits HBV replication in hepatocytes. These data may shed light on development of new antiviral strategies.

Inhibition of hepatitis B virus replication by targeting ribonucleotide reductase M2 protein

Chronic hepatitis B virus (HBV) infection is a key factor for hepatocellular carcinoma worldwide. Ribonucleotide reductase (RR) regulates the deoxyribonucleoside triphosphates biosynthesis and serves as a target for anti-cancer therapy. Here, we demonstrate that RR is essential for HBV replication and the viral covalently-closed-circular DNA (cccDNA) synthesis in host liver cells. By performing computer-assisted virtual screening against the crystal structure of RR small subunit M2 (RRM2), osalmid, was identified as a potential RRM2-targeting compound. Osalmid was shown to be 10-fold more active in inhibiting RR activity than hydroxyurea, and significantly inhibited HBV DNA and cccDNA synthesis in HepG2.2.15 cells. In contrast, hydroxyurea and the RR large subunit (RRM1)-inhibitory drug gemcitabine showed little selective activity against HBV replication. In addition, osalmid also was shown to possess potent activity against a 3TC-resistant HBV strain, suggesting utility in treating drug-resistant HBV infections. Interestingly, osalmid showed synergistic effects with lamivudine (3TC) in vitro and in vivo without significant toxicity, and was shown to inhibit RR activity in vivo, thus verifying its in vivo function. Furthermore, 4-cyclopropyl-2-fluoro-N-(4-hydroxyphenyl) benzamide (YZ51), a novel derivative of osalmid, showed higher efficacy than osalmid with more potent RR inhibitory activity. These results suggest that RRM2 might be targeted for HBV inhibition, and the RRM2-targeting compound osalmid and its derivative YZ51 could be a novel class of anti-HBV candidates with potential use for hepatitis B and HBV-related HCC treatment.

Cleaved c-FLIP mediates the antiviral effect of TNF-α against hepatitis B virus by dysregulating hepatocyte nuclear factors

Cytokines are key molecules implicated in the defense against virus infection. Tumor necrosis factor-alpha (TNF-α) is well known to block the replication of hepatitis B virus (HBV). However, the molecular mechanism and the downstream effector molecules remain largely unknown. In this study, we investigated the antiviral effect and mechanism of p22-FLIP (FLICE-inhibitory protein) by ectopic expression in vitro and in vivo. In addition, to provide the biological relevance of our study, we examined that the p22-FLIP is involved in TNF-α-mediated suppression of HBV in primary human hepatocytes. We found that p22-FLIP, a newly discovered c-FLIP cleavage product, inhibited HBV replication at the transcriptional level in both hepatoma cells and primary human hepatocytes, and that c-FLIP conversion to p22-FLIP was stimulated by the TNF-α/NF-κB pathway. p22-FLIP inhibited HBV replication through the upregulation of HNF3β but downregulation of HNF4α, thus inhibiting both HBV enhancer elements. Finally, p22-FLIP potently inhibited HBV DNA replication in a mouse model of HBV replication. Taken together, these findings suggest that the anti-apoptotic p22-FLIP serves a novel function of inhibiting HBV transcription, and mediates the antiviral effect of TNF-α against HBV replication.

Inhibitory effect of Phyllanthus urinaria L. extract on the replication of lamivudine-resistant hepatitis B virus in vitro.

BackgroundLong-term treatment of chronic hepatitis B (CHB) with nucleos(t)ide analogs results in the emergence of drug-resistant hepatitis B virus (HBV) harboring mutations in the polymerase (P) gene. The Phyllanthus extract has anti-HBV activity; however, its antiviral activity against lamivudine (LMV)-resistant mutants has not been examined.MethodsHBV harboring LMV-resistant mutations (rtM204I, rtM204V, and rtM204S) in the P gene at the YMDD (203tyrosine-methionine-aspartate-aspartate206) reverse transcriptase (RT) active site were generated and their sensitivity to Phyllanthus urinaria koreanis extract examined. Southern blotting and real-time PCR were used to determine the concentration of plant extract required to inhibit HBV DNA synthesis by 50 and 90 % (EC50 and EC90, respectively). An enzyme-linked immunosorbent assay was used to measure the EC50 of HBV surface antigen (HBsAg) and HBV core antigen (HBcAg) secretion, and the 50 % cytotoxic concentration of the extract was measured in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Real-time RT-PCR was used to measure mRNA expression levels.ResultsThe expression of intracellular HBV DNAs in HBV WT- or mutant-transfected HepG2 cells decreased upon treatment with Phyllanthus extract. The secretion of HBsAg and HBcAg also fell in a dose-dependent manner. Phyllanthus extract induced interferon-beta (IFN-β), cyclooxygenase-2 (COX-2), and interleukin-6 (IL-6) mRNA expression in HBV WT-transfected HepG2 cells, possibly via activation of extracellular signal-regulated kinases and c-jun N-terminal kinases and the induction of retinoic acid inducible gene-I, toll-like receptor 3, myeloid differentiation primary response gene 88, and/or tumor necrosis factor receptor-associated factor 6 gene expression. HBV transfection in the absence of extract or exposure of cells to extract alone did not trigger these signaling cascades.ConclusionsPhyllanthus extract inhibited HBV DNA synthesis and HBsAg and HBcAg secretion by replicating cells harboring HBV wild-type and LMV-resistant mutants, likely by inducing the expression of IFN-β, COX-2, and IL-6. These data indicate that Phyllanthus extract may be useful as an alternative therapeutic agent for the treatment of drug-resistant CHB patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-015-0792-3) contains supplementary material, which is available to authorized users.