Evidence for Dual Effects of DNA-Reactive Bile Acid Derivatives (Bamets) on Hepatitis B Virus Life Cycle in an In Vitro Replicative System

2002 ◽  
Vol 13 (6) ◽  
pp. 371-380 ◽  
Author(s):  
Marta R Romero ◽  
Maria C Martinez-Diez ◽  
Monica G Larena ◽  
Rocio IR MacIas ◽  
Mercedes Dominguez ◽  
...  
Keyword(s):  
Life Cycle ◽  
Bile Acid ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Hbv Dna ◽  
Drug Uptake ◽  
Host Cells ◽  
B Virus ◽  
Bile Acid Derivatives ◽  
Hepg2 2.2.15

A liver targeting strategy to direct antiviral drugs toward hepatitis B virus (HBV) was investigated. As model drugs we used cisplatin-bile acid derivatives (Bamets) to determine the production of virions by HBV-transfected hepatoblastoma cells (HepG2 2.2.15). Drug uptake was determined using flameless atomic absorption spectrometry to measure platinum cell contents. Cytotoxic effect was determined by formazan formation and neutral red uptake tests. The release of viral surface protein was evaluated by ELISA. The abundance of HBV-DNA in the medium was determined by quantitative real-time PCR and its structure by Southern blot analysis. The uptake of Bamets by HepG2 2.2.15 cells was higher than that of cisplatin. At concentrations lower than 10 μM, distinct Bamets have no toxic effect on host cells, whereas cisplatin dramatically reduced cell viability at concentrations higher than 1 μM. All the drugs tested inhibited the release of viral proteins to the medium, but induced a marked and progressive dose-dependent increase in the amount of viral DNA in the medium. This was mainly due to the release of short fragments of HBV-DNA in the case of cisplatin. On the contrary, Bamets induced an enhanced release of circular forms of HBV-DNA. These findings suggest the existence of a dual effect of Bamets on HBV life-cycle by enhancing the production of DNA replicative intermediates but reducing the secretion of complete virions. Altogether these characteristics recommend consideration of these compounds as a useful experimental tool in the investigation of novel liver targeted therapeutic agents based on bile acid derivatives for the treatment of HBV infections, or to carry out further studies on the HBV life cycle.

NTCP oligomerization occurs downstream of the NTCP-EGFR interaction during hepatitis B virus internalization

2021 ◽  
Author(s):  
Kento Fukano ◽  
Mizuki Oshima ◽  
Senko Tsukuda ◽  
Hideki Aizaki ◽  
Mio Ohki ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Bile Acid ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Entry ◽  
Sodium Taurocholate ◽  
Growth Factor Receptor ◽  
Host Cells ◽  
B Virus ◽  
Epidermal Growth

Sodium taurocholate cotransporting polypeptide (NTCP) is a receptor that is essential for hepatitis B virus (HBV) entry into the host cell. A number of HBV entry inhibitors targeting NTCP have been reported to date; these inhibitors have facilitated a mechanistic analysis of the viral entry process. However, the mechanism of HBV internalization into host cells after interaction of virus with NTCP remains largely unknown. Recently, we reported that troglitazone, a thiazolidinedione derivative, specifically inhibits both HBV internalization and NTCP oligomerization, resulting in inhibition of HBV infection. Here, using troglitazone as a chemical probe to investigate entry process, the contribution of NTCP oligomerization to HBV internalization was evaluated. Using surface plasmon resonance and transporter kinetics, we found that troglitazone directly interacts with NTCP and non-competitively interferes with NTCP-mediated bile acid uptake, suggesting that troglitazone allosterically binds to NTCP, rather than to the bile acid-binding pocket. Additionally, alanine scanning mutagenesis showed that a mutation at phenylalanine 274 of NTCP (F274A) caused a loss of HBV susceptibility and disrupted both the oligomerization of NTCP and HBV internalization without affecting viral attachment to the cell surface. An inhibitor of the interaction between NTCP and epidermal growth factor receptor (EGFR), another host cofactor essential for HBV internalization, impeded NTCP oligomerization. Meanwhile, co-immunoprecipitation analysis revealed that neither troglitazone nor the F274A mutation in NTCP affect the NTCP-EGFR interaction. These findings suggest that NTCP oligomerization is initiated downstream of the NTCP-EGFR interaction, and then triggers HBV internalization. This study provides significant insight into the HBV entry mechanisms. Importance Hepatitis B virus (HBV) infection is mediated by a specific interaction with sodium taurocholate cotransporting polypeptide (NTCP), a viral entry receptor. Although the virus-receptor interactions are believed to trigger viral internalization into host cells, the exact molecular mechanisms of HBV internalization are not understood. In this study, we revealed the mode of action whereby troglitazone, a specific inhibitor of HBV internalization, impedes NTCP oligomerization, and identified NTCP phenylalanine 274 as a residue essential for this oligomerization. We further analyzed the association between NTCP oligomerization and HBV internalization, a process that is mediated by epidermal growth factor receptor (EGFR), another essential host cofactor for HBV internalization. Our study provides critical information on the mechanism of HBV entry, and suggests that oligomerization of the viral receptor serves as an attractive target for drug discovery.

scholarly journals Antiviral Properties and Mechanism of Action Studies of the Hepatitis B Virus Capsid Assembly Modulator JNJ-56136379

2020 ◽  
Vol 64 (5) ◽  
Author(s):  
Jan Martin Berke ◽  
Pascale Dehertogh ◽  
Karen Vergauwen ◽  
Wendy Mostmans ◽  
Koen Vandyck ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Core Protein ◽  
Hbv Dna ◽  
Size Exclusion ◽  
Capsid Assembly ◽  
Phase Ii Trials ◽  
B Virus

ABSTRACT Capsid assembly is a critical step in the hepatitis B virus (HBV) life cycle, mediated by the core protein. Core is a potential target for new antiviral therapies, the capsid assembly modulators (CAMs). JNJ-56136379 (JNJ-6379) is a novel and potent CAM currently in phase II trials. We evaluated the mechanisms of action (MOAs) and antiviral properties of JNJ-6379 in vitro. Size exclusion chromatography and electron microscopy studies demonstrated that JNJ-6379 induced the formation of morphologically intact viral capsids devoid of genomic material (primary MOA). JNJ-6379 accelerated the rate and extent of HBV capsid assembly in vitro. JNJ-6379 specifically and potently inhibited HBV replication; its median 50% effective concentration (EC50) was 54 nM (HepG2.117 cells). In HBV-infected primary human hepatocytes (PHHs), JNJ-6379, when added with the viral inoculum, dose-dependently reduced extracellular HBV DNA levels (median EC50 of 93 nM) and prevented covalently closed circular DNA (cccDNA) formation, leading to a dose-dependent reduction of intracellular HBV RNA levels (median EC50 of 876 nM) and reduced antigen levels (secondary MOA). Adding JNJ-6379 to PHHs 4 or 5 days postinfection reduced extracellular HBV DNA and did not prevent cccDNA formation. Time-of-addition PHH studies revealed that JNJ-6379 most likely interfered with postentry processes. Collectively, these data demonstrate that JNJ-6379 has dual MOAs in the early and late steps of the HBV life cycle, which is different from the MOA of nucleos(t)ide analogues. JNJ-6379 is in development for chronic hepatitis B treatment and may translate into higher HBV functional cure rates.

scholarly journals DOCK11 and DENND2A play pivotal roles in the maintenance of hepatitis B virus in host cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0246313
Author(s):  
Shinichi Hashimoto ◽  
Takayoshi Shirasaki ◽  
Taro Yamashita ◽  
Sadahiro Iwabuchi ◽  
Yutaka Suzuki ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Limit Of Detection ◽  
Low Frequency ◽  
Hbv Dna ◽  
Small Gtpase ◽  
Host Cells ◽  
Sequencing Analysis ◽  
Circular Dna ◽  
B Virus

Human hepatitis B virus (HBV) infection remains a serious health problem worldwide. However, the mechanism for the maintenance of HBV in a latent state within host cells remains unclear. Here, using single-cell RNA sequencing analysis, we identified four genes linked to the maintenance of HBV in a liver cell line expressing HBV RNA at a low frequency. These genes included DOCK11 and DENND2A, which encode small GTPase regulators. In primary human hepatocytes infected with HBV, knockdown of these two genes decreased the amount of both HBV DNA and covalently closed circular DNA to below the limit of detection. Our findings reveal a role for DOCK11 and DENND2A in the maintenance of HBV.

scholarly journals Small Interfering RNA Screening for the Small GTPase Rab Proteins Identifies Rab5B as a Major Regulator of Hepatitis B Virus Production

2019 ◽  
Vol 93 (15) ◽  
Author(s):  
Jun Inoue ◽  
Masashi Ninomiya ◽  
Teruyuki Umetsu ◽  
Takuya Nakamura ◽  
Takayuki Kogure ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Hbv Dna ◽  
Vesicular Trafficking ◽  
Hbv Infection ◽  
Rab Proteins ◽  
Early Endosomes ◽  
B Virus ◽  
Interfering Rna

ABSTRACTViruses are considered to use vesicular trafficking in infected cells, but the details of assembly/release pathways of hepatitis B virus (HBV) are still unknown. To identify key regulators of HBV production, we performed short interfering RNA (siRNA) screening for Rab proteins, which are considered to act as molecular switches in vesicular trafficking using HepG2.2.15 cells. Among 62 Rab proteins, the suppression of Rab5B most significantly increased HBV DNA in the culture supernatant. Surprisingly, 5 days after the transfection of Rab5B siRNA, HBV DNA in the supernatant was increased more than 30-fold, reflecting the increase of infectious HBV particles. Northern blotting showed that transcription of 2.4/2.1-kb mRNA coding envelope proteins containing large hepatitis B surface protein (LHBs) was increased. Analysis of hepatocyte nuclear factors (HNFs) showed that transcription of HNF4α, which is known to enhance 2.4-kb mRNA transcription, was regulated by Rab5B. Also, it was revealed that LHBs had accumulated in the endoplasmic reticulum (ER) after Rab5B depletion but not in the multivesicular body (MVB), which is thought to be an organelle utilized for HBV envelope formation. Therefore, it was considered that Rab5B is required for the transport of LHBs from the ER to MVB. Immunofluorescent microscopy showed that HBs proteins, including LHBs, colocalized with HBc in the ER of Rab5B-depleted cells, suggesting that HBV envelopment occurs not only in the MVB but also in the ER. In conclusion, Rab5B is a key regulator of HBV production and could be a target of antiviral therapy.IMPORTANCEHBV infection is a worldwide health problem, but the mechanisms of how HBV utilizes cellular machinery for its life cycle are poorly understood. In particular, it has been unclear how the viral components and virions are transported among the organelles. The HBV budding site has been reported to be the ER or MVB, but it has not been clearly determined. In this study, siRNA-based screening of Rab proteins using HBV-expressing cells showed that Rab5B, one of the Rab5 isoforms, has important roles in late steps of the HBV life cycle. Although Rab5 is known to work on early endosomes, this study showed that Rab5B plays a role in the transport of LHBs between the ER and MVB. Furthermore, it affects the transcription of LHBs. This is the first report on the mechanisms of HBV envelope protein transport among the organelles, and the results provide important insights into the therapeutic control of HBV infection.

scholarly journals The Anti-Hepatitis B Virus Activity ofBoehmeria niveaExtract in HBV-Viremia SCID Mice

2010 ◽  
Vol 7 (2) ◽  
pp. 189-195 ◽  
Author(s):  
Jia-Ming Chang ◽  
Kai-Ling Huang ◽  
Thomas Ta-Tung Yuan ◽  
Yiu-Kay Lai ◽  
Le-Mei Hung
Keyword(s):  
Animal Model ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Tumor Growth ◽  
Killer Cell ◽  
Intraperitoneal Administration ◽  
Cell Activity ◽  
Hbv Dna ◽  
B Virus ◽  
Hepg2 2.2.15

Boehmeria niveaextract (BNE) is widely used in southern Taiwan as a folk medicine for hepato-protection and hepatitis treatment. In previous studies, we demonstrated that BNE could reduce the supernatant hepatitis B virus (HBV) DNA in HBV-producing HepG2 2.2.15 cells. In the present study, we established an animal model of HBV viremia and used it to validate the efficacy of BNEin vivo. In this animal model, serum HBV DNA and HBsAg were elevated in accordance with tumor growth. To evaluate the anti-HBV activity of BNE, HBV-viremia mice were built up after one subcutaneous inoculation of HepG2 2.2.15 tumor cells in severe combined immunodeficiency mice over 13 days. The levels of serum HBV DNA were elevated around 105–106copies per milliliter. Both oral and intraperitoneal administration of BNE were effective at inhibiting the production of HBsAg and HBV DNA, whereas tumor growth was not affected by all test articles. Intraperitoneal administration of BNE appeared to have greater potential to inhibit serum HBV DNA levels compared with oral administration under the same dosage. Notably, reduced natural killer cell activity was also observed after high dosage of BNE administration, and this correlated with reduced serum HBV DNA. In conclusion, BNE exhibited potential anti-HBV activity in an animal model of HBV viremia.

Bile acid derivatives inhibit hepatitis B virus infection in vitro and in vivo

2020 ◽  
Vol 73 ◽  
pp. S869
Author(s):  
Kiyoaki Ito ◽  
Akinori Okumura ◽  
Koichi Watashi ◽  
Masashi Yoneda
Keyword(s):  
Bile Acid ◽  
Hepatitis B Virus ◽  
Virus Infection ◽  
Hepatitis B ◽  
Hepatitis B Virus Infection ◽  
B Virus ◽  
Bile Acid Derivatives

272 Use of LioCyx-M, autologous hepatitis B virus (HBV)-Specific T cell receptor (TCR) T-cells, in advanced HBV-related hepatocellular carcinoma (HCC)

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A297-A297
Author(s):  
Fu-Sheng Wang ◽  
Fanping Meng ◽  
Jiehua Jin ◽  
Yuanyuan Li ◽  
Regina Wanju Wong ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
Hepatitis B Virus ◽  
T Cell ◽  
Hepatitis B ◽  
T Cell Receptor ◽  
Dose Level ◽  
Cell Receptor ◽  
Hbv Dna ◽  
B Virus

BackgroundWe have demonstrated the ability of Hepatitis-B-virus (HBV)-specific T cell receptor (TCR) bioengineered T cells to recognize and lyse Hepatocellular carcinoma (HCC) cells expressing HBV antigens derived from HBV-DNA integration in patients with liver transplant.1 LioCyx-M is an immunotherapeutic product composing of autologous T cells transiently modified with in-vitro transcribed mRNA encoding HBV-specific TCR. The transient TCR expression makes LioCyx -M amenable to a dose escalating posology.MethodsThe primary endpoint of this phase 1 trial is to assess the safety and tolerability of LioCyx-M in patients with advanced HBV-HCC without curative treatment options. Eligible patients were diagnosed with Barcelona clinic liver cancer stage B or C HCC (Child-Pugh < 7 points), receiving >1 year antiviral treatment prior to enrollment. These patients had matching HLA class I genotypes which present HBV encoded antigen. Peripheral blood was collected from each patient prior to each dose for LioCyx-M manufacturing. Patients received 4 escalating doses of 1×104 cells/kg, 1×105 cells/kg, 1×106 cells/kg, 5×106 cells/kg bodyweight (BW) in the first treatment cycle, each intravenously administered weekly. Patients underwent 1-month safety assessment post the 4th infusion, according to Common Terminology NCI CTCAE Version 4.0.3. If there were no dose associated toxicities, patients were eligible to continue administration of LioCyx-M at dose of 5 × 106 cells/kg BW weekly. Tumor response per RECIST 1.1 criteria and survival time were assessed.ResultsAt data cutoff (30 April 2020), eight patients were enrolled, with a median age of 53 (range: 49 - 67). These patients received a median number of 6 (range: 4 - 12) infusions of LioCyx-M. 1 patient developed Grade 3 elevations in alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST) and bilirubin after receiving LioCyx-M at dose level of 1×105 cells/kg BW. Another patient had Grade 1 transient fever after receiving LioCyx-M at dose level 5×106 cells/kg BW in the 4th, 5th and 6th infusions. No treatment-related adverse events (trAEs) such as cytokine release syndrome or neurotoxicity were observed. No fatal trAEs were observed. The median time to progression was 1.9 months (range: 0.2 - 9.5 months). The median overall survival was 34 months (range: 3 - 38.2 months).ConclusionsThe encouraging clinical outcome and tolerable safety highlight the good benefit-risk profile of LioCyx-M. Therefore, further exploration of efficacy of LioCyx-M treatment for advanced HBV-HCC is warranted in a Phase 2 proof-of-concept clinical study.AcknowledgementsFunding: Lion TCR.Trial RegistrationNCT03899415Ethics ApprovalThe study was approved by Fifth Medical Center of Chinese PLA General Hospital’s Ethics Board, approval number R2016185DI010.ReferenceTan AT, Yang N, Lee Krishnamoorthy T, et al. Use of Expression Profiles of HBV-DNA Integrated Into Genomes of Hepatocellular Carcinoma Cells to Select T Cells for Immunotherapy. Gastroenterology 2019;156(6):1862–1876.e9.

scholarly journals A Comparison Research of Hepatitis B Virus Large Surface Protein with HBV DNA Detecting

2008 ◽  
Vol 12 ◽  
pp. e415
Author(s):  
Z.L. Wu ◽  
X.D. Lu ◽  
X.Q. Zhong ◽  
L.F. Ling ◽  
G. Lin ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Surface Protein ◽  
Hbv Dna ◽  
Comparison Research ◽  
B Virus

scholarly journals Occult Hepatitis B Virus Infection among HIV Positive Patients in Nigeria

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Oluyinka Oladele Opaleye ◽  
Adeolu Sunday Oluremi ◽  
Adetona Babatunde Atiba ◽  
Moses Olubusuyi Adewumi ◽  
Olatunji Victor Mabayoje ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Virus Infection ◽  
Hepatitis B ◽  
Hbv Dna ◽  
Hiv Positive ◽  
Hepatitis B Virus Infection ◽  
Occult Hepatitis ◽  
B Virus ◽  
Occult Hepatitis B ◽  
Detectable Hbsag

HIV has been known to interfere with the natural history of hepatitis B virus (HBV) infection. In this study we investigate the prevalence of occult hepatitis B virus infection (OBI) among HIV-infected individuals in Nigeria. Overall, 1200 archived HIV positive samples were screened for detectable HBsAg using rapid technique, in Ikole Ekiti Specialist Hospital. The HBsAg negative samples were tested for HBsAg, anti-HBc, and anti-HCV by ELISA. Polymerase chain reaction was used for HBV DNA amplification and CD4 counts were analyzed by cytometry. Nine hundred and eighty of the HIV samples were HBsAg negative. HBV DNA was detected in 21/188 (11.2%) of patients without detectable HBsAg. CD4 count for the patients ranged from 2 to 2,140 cells/μL of blood (mean = 490 cells/μL of blood). HCV coinfection was detected only in 3/188 (1.6%) of the HIV-infected patients (P>0.05). Twenty-eight (29.2%) of the 96 HIV samples screened were positive for anti-HBc. Averagely the HBV viral load was <50 copies/mL in the OBI samples examined by quantitative PCR. The prevalence of OBI was significantly high among HIV-infected patients. These findings highlight the significance of nucleic acid testing in HBV diagnosis in HIV patients.

scholarly journals Phosphorothioate Di- and Trinucleotides as a Novel Class of Anti-Hepatitis B Virus Agents

2004 ◽  
Vol 48 (6) ◽  
pp. 2199-2205 ◽  
Author(s):  
Radhakrishnan P. Iyer ◽  
Yi Jin ◽  
Arlene Roland ◽  
John D. Morrey ◽  
Samir Mounir ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Nucleoside Analogs ◽  
Hbv Dna ◽  
Parallel Synthesis ◽  
Hbv Replication ◽  
Long Term Treatment ◽  
B Virus ◽  
Dna Strand

ABSTRACT Several nucleoside analogs are under clinical development for use against hepatitis B virus (HBV). Lamivudine (3TC), a nucleoside analog, and adefovir dipivoxil (ADV), an acyclonucleotide analog, are clinically approved. However, long-term treatment can induce viral resistance, and following the cessation of therapy, viral rebound is frequently observed. There continues to be a need for new antiviral agents with novel mechanisms of action. A library of more than 600 di- and trinucleotide compounds synthesized by parallel synthesis using a combinatorial strategy was screened for potential inhibitors of HBV replication using the chronically HBV-producing cell line 2.2.15. Through an iterative process of synthesis, lead optimization, and screening, three analogs were identified as potent inhibitors of HBV replication: dinucleotides ORI-7246 (drug concentration at which a 10-fold reduction of HBV DNA was observed [EC90], 1.4 μM) and ORI-9020 (EC90, 1.2 μM) and trinucleotide ORI-7170 (EC90, 7.2 μM). These analogs inhibited the replication of both strands of HBV DNA. No suppression of HBV protein synthesis or intracellular core particle formation by these analogs was observed. No inhibition of HBV DNA strand elongation by the analogs or their 5′-triphosphate versions was apparent in in vitro polymerase assays. Although the exact mechanism of action is not yet identified, present data are consistent with an inhibition of the HBV reverse transcriptase-directed priming step prior to elongation of the first viral DNA strand. In transient-transfection assays, these analogs inhibited the replication of 3TC-resistant HBV. Synergistic interactions in combination treatments between the analogs and either 3TC or ADV were observed. These compounds represent a novel class of anti-HBV molecules and warrant further investigation as potential therapeutic agents.

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