scholarly journals Effects of Moloney Leukemia Virus 10 Protein on Hepatitis B Virus Infection and Viral Replication

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 651 ◽  
Author(s):  
Maritza Puray-Chavez ◽  
Mahmoud Farghali ◽  
Vincent Yapo ◽  
Andrew Huber ◽  
Dandan Liu ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Cell Lines ◽  
Core Protein ◽  
Leukemia Virus ◽  
Hbv Dna ◽  
Genomic Rna ◽  
Over Expression ◽  
B Virus ◽  
Moloney Leukemia Virus

Moloney leukemia virus 10 (MOV10) is an RNA helicase that has been shown to affect the replication of several viruses. The effect of MOV10 on Hepatitis B virus (HBV) infection is not known and its role on the replication of this virus is poorly understood. We investigated the effect of MOV10 down-regulation and MOV10 over-expression on HBV in a variety of cell lines, as well as in an infection system using a replication competent virus. We report that MOV10 down-regulation, using siRNA, shRNA, and CRISPR/Cas9 gene editing technology, resulted in increased levels of HBV DNA, HBV pre-genomic RNA, and HBV core protein. In contrast, MOV10 over-expression reduced HBV DNA, HBV pre-genomic RNA, and HBV core protein. These effects were consistent in all tested cell lines, providing strong evidence for the involvement of MOV10 in the HBV life cycle. We demonstrated that MOV10 does not interact with HBV-core. However, MOV10 binds HBV pgRNA and this interaction does not affect HBV pgRNA decay rate. We conclude that the restriction of HBV by MOV10 is mediated through effects at the level of viral RNA.

scholarly journals A Novel Pyridazinone Derivative Inhibits Hepatitis B Virus Replication by Inducing Genome-Free Capsid Formation

2015 ◽  
Vol 59 (11) ◽  
pp. 7061-7072 ◽  
Author(s):  
Ya-Juan Wang ◽  
Dong Lu ◽  
Yi-Bin Xu ◽  
Wei-Qiang Xing ◽  
Xian-Kun Tong ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Core Protein ◽  
Cell Model ◽  
Morphological Changes ◽  
Gradient Centrifugation ◽  
Hbv Dna ◽  
Capsid Formation ◽  
B Virus ◽  
Cell Model System

ABSTRACTHere we first identified a novel pyridazinone derivative, compound 3711, as a nonnucleosidic hepatitis B virus (HBV) inhibitor in a cell model system. 3711 decreased extracellular HBV DNA levels by 50% (50% inhibitory concentration [IC50]) at 1.5 ± 0.2 μM and intracellular DNA levels at 1.9 ± 0.1 μM, which demonstrated antiviral activity at levels far below those associated with toxicity. Both the 3TC/ETV dually resistant L180M/M204I mutant and the adefovir (ADV)-resistant A181T/N236T mutant were as susceptible to 3711 as wild-type HBV. 3711 treatment induced the formation of genome-free capsids, a portion of which migrated faster on 1.8% native agarose gel. The induced genome-free capsids sedimented more slowly in isopycnic CsCl gradient centrifugation without significant morphological changes. 3711 treatment decreased levels of HBV DNA contained in both secreted enveloped virion and naked virus particles in supernatant. 3711 could interfere with capsid formation of the core protein (Cp) assembly domain. A Cp V124W mutant, which strengthens capsid interdimer interactions, recapitulated the effect of 3711 on capsid assembly. Pyridazinone derivative 3711, a novel chemical entity and HBV inhibitor, may provide a new opportunity to combat chronic HBV infection.

scholarly journals Subtype-Independent Immature Secretion and Subtype-Dependent Replication Deficiency of a Highly Frequent, Naturally Occurring Mutation of Human Hepatitis B Virus Core Antigen

1999 ◽  
Vol 73 (12) ◽  
pp. 10122-10128 ◽  
Author(s):  
Thomas Ta-Tung Yuan ◽  
Pei-Ching Tai ◽  
Chiaho Shih
Keyword(s):  
Amino Acid ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Core Protein ◽  
Hbv Dna ◽  
Core Antigen ◽  
Wild Type ◽  
Virion Assembly ◽  
Human Hepatitis ◽  
B Virus

ABSTRACT The most frequent mutation of the human hepatitis B virus (HBV) core antigen occurs at amino acid 97. Recently, a phenylalanine (F)-to-leucine (L) mutation at this position (mutant F97L) in HBV surface antigen subtype ayw has been shown to result in an immature secretion phenotype, which is characterized by the nonselective export of an excessive amount of virions containing minus-strand, single-stranded HBV DNA. While subtype aywmutant F97L has been found in Europe, the major reservoir of HBV resides in Asia and Africa. We report here that the immature secretion phenotype indeed can be found in an HBV strain (subtypeadr) prevalent in Asia, changing from an isoleucine (I) to a leucine (mutant I97L). Despite its immature secretion phenotype, theadr variant I97L replicates as well as its parentaladr wild-type I97I, supporting the conclusion that the extracellular phenotype of immature secretion is not a consequence of the intracellular HBV DNA replication defect. Further studies demonstrated that it is the acquisition of a leucine, rather than the loss of a wild-type amino acid at codon 97, that is important for immature secretion. We conclude that immature secretion is a subtype-independent phenotype and deficiency in intracellular DNA synthesis is a subtype-dependent phenotype. The former is caused by thetrans-acting effect of a mutant core protein, while the latter by a cis-acting effect of a mutated nucleotide on the ayw genome. These immature secretion variants provide an important tool for studying the regulation of HBV virion assembly and secretion.

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 Hepatitis B Virus Capsid Assembly Modulators, but Not Nucleoside Analogs, Inhibit the Production of Extracellular Pregenomic RNA and Spliced RNA Variants

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Angela M. Lam ◽  
Suping Ren ◽  
Christine Espiritu ◽  
Mollie Kelly ◽  
Vincent Lau ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Particle Production ◽  
Core Protein ◽  
Antiviral Agents ◽  
Nucleoside Analogs ◽  
Hbv Dna ◽  
Capsid Assembly ◽  
Virion Production ◽  
B Virus

ABSTRACT The hepatitis B virus (HBV) core protein serves multiple essential functions in the viral life cycle, and antiviral agents that target the core protein are being developed. Capsid assembly modulators (CAMs) are compounds that target core and misdirect capsid assembly, resulting in the suppression of HBV replication and virion production. Besides HBV DNA, circulating HBV RNA has been detected in patient serum and can be associated with the treatment response. Here we studied the effect of HBV CAMs on the production of extracellular HBV RNA using infected HepaRG cells and primary human hepatocytes. Representative compounds from the sulfonamide carboxamide and heteroaryldihydropyrimidine series of CAMs were evaluated and compared to nucleos(t)ide analogs as inhibitors of the viral polymerase. The results showed that CAMs blocked extracellular HBV RNA with efficiencies similar to those with which they blocked pregenomic RNA (pgRNA) encapsidation, HBV DNA replication, and Dane particle production. Nucleos(t)ide analogs inhibited viral replication and virion production but not encapsidation or production of extracellular HBV RNA. Profiling of HBV RNA from both culture supernatants and patient serum showed that extracellular viral RNA consisted of pgRNA and spliced pgRNA variants with an internal deletion(s) but still retained the sequences at both the 5′ and 3′ ends. Similar variants were detected in the supernatants of infected cells with and without nucleos(t)ide analog treatment. Overall, our data demonstrate that HBV CAMs represent direct antiviral agents with a profile differentiated from that of nucleos(t)ide analogs, including the inhibition of extracellular pgRNA and spliced pgRNA.

scholarly journals Hepatitis B virus maturation is affected by the incorporation of core proteins having a C-terminal substitution of arginine or lysine stretches

1999 ◽  
Vol 80 (10) ◽  
pp. 2661-2671 ◽  
Author(s):  
Eric Ka-Wai Hui ◽  
Kun-Lin Chen ◽  
Szecheng J. Lo
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Core Protein ◽  
Hbv Dna ◽  
Wild Type ◽  
Terminal Portion ◽  
Virus Maturation ◽  
The Core ◽  
Core Proteins ◽  
B Virus

Assembly of replication-competent hepadnavirus nucleocapsids requires interaction of core protein, polymerase and encapsidation signal (ϵ) with viral pregenomic RNA. The N-terminal portion (aa 1–149) of the core protein is able to self-assemble into nucleocapsids, whereas the C-terminal portion (aa 150–183) is known to interact with pregenomic RNA. In this study, two hepatitis B virus (HBV) core mutants (C144Arg and C144Lys) in which the C-terminal SPRRR (Ser-Pro-Arg-Arg-Arg) motif was replaced by a stretch of arginine or lysine residues were generated to test their role in pregenome encapsidation and virus maturation. Mutant or wild-type core-expression plasmids were co-transfected with a core-negative plasmid into human hepatoma HuH-7 cells to compare trans-complementation efficiency for virus replication. Both low- and high-density capsids were present in the cytoplasm and culture medium of HuH-7 cells in all transfections. Nucleocapsids formed by C144Arg and C144Lys, however, lost the endogenous polymerase activity to repair HBV DNA. Furthermore, in co-transfection of pHBVC144Arg or pHBVC144Lys with a plasmid which produces replication-competent nucleocapsids, the HBV DNA repairing signal was reduced 40- to 80-fold. This is probably due to formation of mosaic particles of wild-type and mutant cores. Results indicated that the SPRRR motif at the core protein C terminus is important for HBV DNA replication and maturation. Additionally, triple-plasmid transfection experiments showed that nucleocapsids containing various amounts of C144Arg and wild-type core proteins exhibited a bias in selecting a shorter pregenome for encapsidation and DNA replication. It is therefore suggested that unknown factors are also involved in HBV pregenome packaging.

The role of Moloney leukemia virus 10 in hepatitis B virus expression in hepatoma cells

2015 ◽  
Vol 197 ◽  
pp. 85-91 ◽  
Author(s):  
Yan-Xiu Ma ◽  
Di Li ◽  
Li-Juan Fu ◽  
Bao-qing Fu ◽  
Si-Jia Chen ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Leukemia Virus ◽  
Hepatoma Cells ◽  
B Virus ◽  
Moloney Leukemia Virus ◽  
Virus Expression

scholarly journals Hepatitis B Virus DNA-negative Dane Particles Lack Core Protein but Contain a 22-kDa Precore Protein without C-terminal Arginine-rich Domain

2005 ◽  
Vol 280 (23) ◽  
pp. 21713-21719 ◽  
Author(s):  
Tatsuji Kimura ◽  
Nobuhiko Ohno ◽  
Nobuo Terada ◽  
Akinori Rokuhara ◽  
Akihiro Matsumoto ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Core Protein ◽  
Hbv Dna ◽  
Core Antigen ◽  
Western Blots ◽  
Rich Domain ◽  
Core Proteins ◽  
B Virus ◽  
Dane Particles

DNA-negative Dane particles have been observed in hepatitis B virus (HBV)-infected sera. The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail. In the present study, the protein composition of the particles was examined using new enzyme immunoassays for the HBV core antigen (HBcAg) and for the HBV precore/core proteins (core-related antigens, HBcrAg). HBcrAg were abundant in fractions slightly less dense than HBcAg and HBV DNA. Three times more Dane-like particles were observed in the HBcrAg-rich fraction than in the HBV DNA-rich fraction by electron microscopy. Western blots and mass spectrometry identified the HBcrAg as a 22-kDa precore protein (p22cr) containing the uncleaved signal peptide and lacking the arginine-rich domain that is involved in binding the RNA pregenome or the DNA genome. In sera from 30 HBV-infected patients, HBcAg represented only a median 10.5% of the precore/core proteins in enveloped particles. These data suggest that most of the Dane particles lack viral DNA and core capsid but contain p22cr. This study provides a model for the formation of the DNA-negative Dane particles. The precore proteins, which lack the arginine-rich nucleotide-binding domain, form viral RNA/DNA-negative capsid-like particles and are enveloped and released as empty particles.

scholarly journals Inhibition of Hepatitis B Virus Gene Expression and Replication by Helioxanthin and its Derivative

2005 ◽  
Vol 16 (3) ◽  
pp. 193-201 ◽  
Author(s):  
Ying Li ◽  
Lei Fu ◽  
Hosup Yeo ◽  
Ju-Liang Zhu ◽  
Chen-Kung Chou ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Protein Expression ◽  
Core Protein ◽  
Hbv Dna ◽  
Interferon Α ◽  
Dependent Manner ◽  
Therapeutic Drugs ◽  
Chronic Hepatitis B Virus ◽  
B Virus

Chronic hepatitis B virus (HBV) infection continues to be an important worldwide cause of morbidity and mortality. All the currently approved therapeutic drugs have their limitations: interferon-α (IFN-α) has limited efficacy and a high incidence of adverse effects; nucleoside analogues are very efficient HBV DNA inhibitors, but resistance occurs eventually. Therefore, it is important to develop new non-nucleoside/nucleotide agents with different modes of action that can be used for antiviral combination therapy. Here, we report on a novel class of compounds, helioxanthin and its derivative 5-4-2, which had potent anti-HBV activities in HepG2.2.15 cells, with the EC50s of 1 and 0.08 μM, respectively. The lamivudine-resistant HBV, L526M/M550V double mutant strain, was also sensitive to helioxanthin and 5-4-2. This class of compounds not only inhibited HBV DNA, but also decreased HBV mRNA and HBV protein expression. The EC50 of HBV DNA inhibition was consistent with the EC50 of HBV 3.5 Kb transcript inhibition, which was 1 and 0.09 μM for helioxanthin and 5-4-2 respectively. Western blot analysis of cell lysate from HepG2.2.15 cells showed that the core protein expression decreased in a dose-dependent manner after drug treatment. In conclusion, helioxanthin and 5-4-2 are potentially unique new anti-HBV agents, which possess a different mechanism of action from existing therapeutic drugs. Both compounds inhibited HBV RNA and protein expression in addition to inhibiting HBV DNA.

scholarly journals Coinfection of Hepatic Cell Lines with Human Immunodeficiency Virus and Hepatitis B Virus Leads to an Increase in Intracellular Hepatitis B Surface Antigen

2010 ◽  
Vol 84 (12) ◽  
pp. 5860-5867 ◽  
Author(s):  
David M. Iser ◽  
Nadia Warner ◽  
Peter A. Revill ◽  
Ajantha Solomon ◽  
Fiona Wightman ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Hiv Infection ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Hepatitis B Surface Antigen ◽  
Hbv Dna ◽  
Hepatic Cell ◽  
B Virus ◽  
Hepatic Cell Lines

ABSTRACT Liver-related mortality is increased in the setting of HIV-hepatitis B virus (HBV) coinfection. However, interactions between HIV and HBV to explain this observation have not been described. We hypothesized that HIV infection of hepatocytes directly affects the life cycle of HBV. We infected human hepatic cell lines expressing HBV (Hep3B and AD38 cells) or not expressing HBV (Huh7, HepG2, and AD43 cells) with laboratory strains of HIV (NL4-3 and AD8), as well as a vesicular stomatitis virus (VSV)-pseudotyped HIV expressing enhanced green fluorescent protein (EGFP). Following HIV infection with NL4-3 or AD8 in hepatic cell lines, we observed a significant increase in HIV reverse transcriptase activity which was infectious. Despite no detection of surface CD4, CCR5, and CXCR4 by flow cytometry, AD8 infection of AD38 cells was inhibited by maraviroc and NL4-3 was inhibited by AMD3100, demonstrating that HIV enters AD38 hepatic cell lines via CCR5 or CXCR4. High-level infection of AD38 cells (50%) was achieved using VSV-pseudotyped HIV. Coinfection of the AD38 cell line with HIV did not alter the HBV DNA amount or species as determined by Southern blotting or nucleic acid signal amplification. However, coinfection with HIV was associated with a significant increase in intracellular HBsAg when measured by Western blotting, quantitative HBsAg, and fluorescence microscopy. We conclude that HIV infection of HBV-infected hepatic cell lines significantly increased intracellular HBsAg but not HBV DNA synthesis and that increased intrahepatic HBsAg secondary to direct infection by HIV may contribute to accelerated liver disease in HIV-HBV-coinfected individuals.

scholarly journals The CCCTC-binding factor CTCF represses hepatitis B virus Enhancer I and regulates viral transcription

2020 ◽  
Author(s):  
V D’Arienzo ◽  
J Ferguson ◽  
G Giraud ◽  
F Chapus ◽  
JM Harris ◽  
...  
Keyword(s):  
Liver Disease ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Genome ◽  
Hbv Dna ◽  
Ctcf Binding ◽  
Viral Transcription ◽  
Genomic Rna ◽  
Binding Factor ◽  
B Virus

ABSTRACTHepatitis B virus (HBV) infection is of global importance with over 2 billion people exposed to the virus during their lifetime and at risk of progressive liver disease, cirrhosis and hepatocellular carcinoma. HBV is a member of the hepadnaviridae family that replicates via episomal copies of a covalently closed circular DNA (cccDNA) genome. The chromatinization of this small viral genome, with overlapping open reading frames and regulatory elements, suggests an important role for epigenetic pathways to regulate viral transcription. The chromatin-organising transcriptional insulator protein CCCTC-binding factor (CTCF) has been reported to regulate transcription in a diverse range of viruses. We identified two conserved CTCF binding sites in the HBV genome within Enhancer I and chromatin immunoprecipitation (ChIP) analysis demonstrated an enrichment of CTCF binding to integrated or episomal copies of the viral genome. siRNA knockdown of CTCF results in a significant increase in pre-genomic RNA levels in de novo infected HepG2 cells and those supporting episomal HBV DNA replication. Furthermore, mutation of these sites in HBV DNA minicircles abrogated CTCF binding and increased pre-genomic RNA levels, providing evidence of a direct role for CTCF in repressing HBV transcription.IMPORTANCEHepatitis B virus (HBV) is a global cause of liver disease. At least 300 million individuals are chronically infected with HBV, frequently leading to life-threatening liver cirrhosis and cancer. Following viral entry, HBV DNA enters the nucleus and is bound by histones that are subject to epigenetic modification. The HBV genome contains two enhancer elements that stimulate viral transcription but the interplay between the viral enhancers and promoters is not fully understood. We have identified the host cell protein CCCTC binding factor (CTCF) as a repressor of HBV gene expression. CTCF binds to the HBV genome within Enhancer I and represses transcription of pre-genomic RNA. These findings provide new insights into how HBV transcription is regulated and show a new role for CTCF as a transcriptional insulator by associating with the viral genome between Enhancer I and the downstream basal core promoter.

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