scholarly journals The Heteroaryldihydropyrimidine Bay 38-7690 Induces Hepatitis B Virus Core Protein Aggregates Associated with Promyelocytic Leukemia Nuclear Bodies in Infected Cells

mSphere ◽  
2018 ◽  
Vol 3 (2) ◽  
Author(s):  
Andrew D. Huber ◽  
Jennifer J. Wolf ◽  
Dandan Liu ◽  
Anna T. Gres ◽  
Jing Tang ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Replication ◽  
Promyelocytic Leukemia ◽  
Nuclear Bodies ◽  
Capsid Assembly ◽  
B Virus ◽  
Infected Cells ◽  
Dose Dependent

ABSTRACTHeteroaryldihydropyrimidines (HAPs) are compounds that inhibit hepatitis B virus (HBV) replication by modulating viral capsid assembly. While their biophysical effects on capsid assemblyin vitrohave been previously studied, the effect of HAP treatment on capsid protein (Cp) in individual HBV-infected cells remains unknown. We report here that the HAP Bay 38-7690 promotes aggregation of recombinant Cpin vitroand causes a time- and dose-dependent decrease of Cp in infected cells, consistent with previously studied HAPs. Interestingly, immunofluorescence analysis showed Cp aggregating in nuclear foci of Bay 38-7690-treated infected cells in a time- and dose-dependent manner. We found these foci to be associated with promyelocytic leukemia (PML) nuclear bodies (NBs), which are structures that affect many cellular functions, including DNA damage response, transcription, apoptosis, and antiviral responses. Cp aggregation is not an artifact of the cell system used, as it is observed in HBV-expressing HepAD38 cells, in HepG2 cells transfected with an HBV-expressing plasmid, and in HepG2-NTCP cells infected with HBV. Use of a Cp overexpression vector without HBV sequences shows that aggregation is independent of viral replication, and use of an HBV-expressing plasmid harboring a HAP resistance mutation in Cp abrogated the aggregation, demonstrating that the effect is due to direct compound-Cp interactions. These studies provide novel insight into the effects of HAP-based treatment at a single-cell level.IMPORTANCEDespite the availability of effective vaccines and treatments, HBV remains a significant global health concern, with more than 240 million individuals chronically infected. Current treatments are highly effective at controlling viral replication and disease progression but rarely cure infections. Therefore, much emphasis is being placed on finding therapeutics with new drug targets, such as viral gene expression, covalently closed circular DNA formation and stability, capsid formation, and host immune modulators, with the ultimate goal of an HBV cure. Understanding the mechanisms by which novel antiviral agents act will be imperative for the development of curative HBV therapies.

scholarly journals A Kinase Chaperones Hepatitis B Virus Capsid Assembly and Captures Capsid Dynamics in vitro

2011 ◽  
Vol 7 (11) ◽  
pp. e1002388 ◽  
Author(s):  
Chao Chen ◽  
Joseph Che-Yen Wang ◽  
Adam Zlotnick
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Capsid Assembly ◽  
Virus Capsid ◽  
B Virus

scholarly journals Lack of Interaction Between Elbasvir/Grazoprevir and Approved HIV and Hepatitis B Virus Drugs in Viral Replication In Vitro

2016 ◽  
Vol 3 (suppl_1) ◽  
Author(s):  
Robert Chase ◽  
Stephanie Curry ◽  
Min Xu ◽  
Lei Ba ◽  
Carolyn Bahnck-Teets ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Replication ◽  
B Virus

Phosphorylation of hepatitis B virus core C-terminally truncated protein (Cp149) by PKC increases capsid assembly and stability

2008 ◽  
Vol 416 (1) ◽  
pp. 47-54 ◽  
Author(s):  
Hang Kang ◽  
Jaehoon Yu ◽  
Guhung Jung
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Core Protein ◽  
Virus Titre ◽  
Capsid Assembly ◽  
Human Hepatoma Cells ◽  
Virus Core ◽  
C Terminus ◽  
B Virus

The HBV (hepatitis B virus) core is a phosphoprotein whose assembly, replication, encapsidation and localization are regulated by phosphorylation. It is known that PKC (protein kinase C) regulates pgRNA (pregenomic RNA) encapsidation by phosphorylation of the C-terminus of core, which is a component packaged into capsid. Neither the N-terminal residue phosphorylated by PKC nor the role of the C-terminal phosphorylation have been cleary defined. In the present study we found that HBV Cp149 (core protein C-terminally truncated at amino acid 149) expressed in Escherichia coli was phosphorylated by PKC at Ser106. PKC-mediated phosphorylation increased core affinity, as well as assembly and capsid stability. In vitro phosphorylation with core mutants (S26A, T70A, S106A and T114A) revealed that the Ser106 mutation inhibited phosphorylation of core by PKC. CD analysis also revealed that PKC-mediated phosphorylation stabilized the secondary structure of capsid. When either pCMV/FLAG-Cp149[WT (wild-type)] or pCMV/FLAG-S106A Cp149 was transfected into Huh7 human hepatoma cells, mutant capsid level was decreased by 2.06-fold with the S106A mutant when compared with WT, although the same level of total protein was expressed in both cases. In addition, when pUC1.2x and pUC1.2x/S106A were transfected, mutant virus titre was decreased 2.31-fold compared with WT virus titre. In conclusion, PKC-mediated phosphorylation increased capsid assembly, stability and structural stability.

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 Preclinical Characterization of NVR 3-778, a First-in-Class Capsid Assembly Modulator against Hepatitis B Virus

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Angela M. Lam ◽  
Christine Espiritu ◽  
Robert Vogel ◽  
Suping Ren ◽  
Vincent Lau ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Antiviral Activity ◽  
Viral Replication ◽  
De Novo ◽  
Hbv Dna ◽  
Cross Resistance ◽  
Capsid Assembly ◽  
B Virus

ABSTRACT NVR 3-778 is the first capsid assembly modulator (CAM) that has demonstrated antiviral activity in hepatitis B virus (HBV)-infected patients. NVR 3-778 inhibited the generation of infectious HBV DNA-containing virus particles with a mean antiviral 50% effective concentration (EC50) of 0.40 µM in HepG2.2.15 cells. The antiviral profile of NVR 3-778 indicates pan-genotypic antiviral activity and a lack of cross-resistance with nucleos(t)ide inhibitors of HBV replication. The combination of NVR 3-778 with nucleos(t)ide analogs in vitro resulted in additive or synergistic antiviral activity. Mutations within the hydrophobic pocket at the dimer-dimer interface of the core protein could confer resistance to NVR 3-778, which is consistent with the ability of the compound to bind to core and to induce capsid assembly. By targeting core, NVR 3-778 inhibits pregenomic RNA encapsidation, viral replication, and the production of HBV DNA- and HBV RNA-containing particles. NVR 3-778 also inhibited de novo infection and viral replication in primary human hepatocytes with EC50 values of 0.81 µM against HBV DNA and between 3.7 and 4.8 µM against the production of HBV antigens and intracellular HBV RNA. NVR 3-778 showed favorable pharmacokinetics and safety in animal species, allowing serum levels in excess of 100 µM to be achieved in mice and, thus, enabling efficacy studies in vivo. The overall preclinical profile of NVR 3-778 predicts antiviral activity in vivo and supports its further evaluation for safety, pharmacokinetics, and antiviral activity in HBV-infected patients.

An in vitro fluorescence screen to identify antivirals that disrupt hepatitis B virus capsid assembly

2006 ◽  
Vol 24 (3) ◽  
pp. 358-362 ◽  
Author(s):  
Stephen J Stray ◽  
Jennifer M Johnson ◽  
Benjamin G Kopek ◽  
Adam Zlotnick
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Capsid Assembly ◽  
Virus Capsid ◽  
B Virus

scholarly journals Stimulation of Cellular Proliferation by Hepatitis B Virus X Protein

2001 ◽  
Vol 17 (3) ◽  
pp. 153-157 ◽  
Author(s):  
Charles R. Madden ◽  
Betty L. Slagle
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Cellular Proliferation ◽  
X Protein ◽  
Hepatitis Viruses ◽  
B Virus ◽  
Infected Cells ◽  
Proliferation In Vitro

Chronic infection with the hepatitis B virus (HBV) is a known risk factor in the development of human hepatocellular carcinoma (HCC). The HBV-encoded X protein, HBx, has been investigated for properties that may explain its cancer cofactor role in transgenic mouse lines. We discuss here recent data showing that HBx is able to induce hepatocellular proliferation in vitro and in vivo. This property of HBx is predicted to sensitize hepatocytes to other HCC cofactors, including exposure to carcinogens and to other hepatitis viruses. Cellular proliferation is intimately linked to the mechanism(s) by which most tumor-associated viruses transform virus-infected cells. The HBx alteration of the cell cycle provides an additional mechanism by which chronic HBV infection may contribute to HCC.

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