A New Role for Capsid Assembly Modulators To Target Mature Hepatitis B Virus Capsids and Prevent Virus Infection

ABSTRACT Hepatitis B virus (HBV) is a major human pathogen, killing an estimated 887,000 people per year. Therefore, potentially curative therapies are of high importance. Following infection, HBV deposits a covalently closed circular DNA (cccDNA) in the nucleus of infected cells that serves as a transcription template and is not affected by current therapies. HBV core protein allosteric modulators (CpAMs) prevent correct capsid assembly but may also affect early stages of HBV infection. In this study, we aimed to determine the antiviral efficacy of a novel, structurally distinct heteroaryldihydropyrimidine (HAP)-type CpAM, HAP_R01, and investigated whether and how HAP_R01 prevents the establishment of HBV infection. HAP_R01 shows a significant inhibition of cccDNA formation when applied during the first 48 h of HBV infection. Inhibiting cccDNA formation, however, requires >1-log10-higher concentrations than inhibition of the assembly of newly forming capsids (half-maximal effective concentration [EC50], 345 to 918 nM versus 26.8 to 43.5 nM, respectively). Biophysical studies using a new method to detect the incoming capsid in de novo infection revealed that HAP_R01 can physically change mature capsids of incoming virus particles and affect particle integrity. Treating purified HBV virions with HAP_R01 reduced their infectivity, highlighting the unique antiviral activity of CpAMs to target the capsid within mature HBV particles. Accordingly, HAP_R01 shows an additive antiviral effect in limiting de novo infection when combined with viral entry inhibitors. In summary, HAP_R01 perturbs capsid integrity of incoming virus particles and reduces their infectivity and thus inhibits cccDNA formation in addition to preventing HBV capsid assembly.

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Regulation of HBV Replication by Cyclin Docking Motifs in Core Protein

Journal of Virology ◽

10.1128/jvi.00230-21 ◽

2021 ◽

Author(s):

Haitao Liu ◽

Ji Xi ◽

Jianming Hu

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Protein Phosphorylation ◽

Capsid Protein ◽

Core Protein ◽

Hbv Infection ◽

Capsid Assembly ◽

Cyclin Dependent Kinase ◽

Terminal Domain ◽

B Virus

Hepatitis B virus (HBV) capsid or core protein (HBc) consists of an N-terminal domain (NTD) and C-terminal domain (CTD) connected by a short linker peptide. Dynamic phosphorylation and dephosphorylation of HBc regulate its multiple functions in capsid assembly and viral replication. The cellular cyclin-dependent kinase 2 (CDK2) plays a major role in HBc phosphorylation and furthermore, is incorporated into the viral capsid, accounting for most of the “endogenous kinase” activity associated with the capsid. The packaged CDK2 is thought to play a role in phosphorylating HBc to trigger nucleocapsid disassembly (uncoating), an essential step during viral infection. However, little is currently known on how CDK2 is recruited and packaged into the capsid. We have now identified three RXL motifs, in the HBc NTD, known as cyclin docking motifs (CDMs), which mediates the interactions of various CDK substrates/regulators with CDK/cyclin complexes. Mutations of the CDMs in the HBc NTD reduced CTD phosphorylation and diminished CDK2 packaging into the capsid. Also, the CDM mutations showed little effects on capsid assembly and pregenomic RNA (pgRNA) packaging but impaired the integrity of mature nucleocapsids. Furthermore, the CDM mutations blocked covalently closed circular DNA (CCC DNA) formation during infection while having no effect on or enhancing CCC DNA formation via intracellular amplification. These results indicate that the HBc NTD CDMs play a role in CDK2 recruitment and packaging, which, in turn, is important for productive infection. Author Summary Hepatitis B virus (HBV) is an important global human pathogen and persistently infects hundreds of millions of people, who are at high risk of cirrhosis and liver cancer. HBV capsid packages a host cell protein kinase, the cyclin-dependent kinase 2 (CDK2), which is thought to be required to trigger disassembly of the viral nucleocapsid during infection by phosphorylating the capsid protein, a prerequisite for successful infection. We have identified docking sites on the capsid protein for recruiting CDK2, in complex with its cyclin partner, to facilitate capsid protein phosphorylation and CDK2 packaging. Mutations of these docking sites reduced capsid protein phosphorylation, impaired CDK2 packaging into HBV capsids, and blocked HBV infection. These results provide novel insights regarding CDK2 packaging into HBV capsids and the role of CDK2 in HBV infection and should facilitate the development of antiviral drugs that target the HBV capsid protein.

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Heteroaryldihydropyrimidines Alter Capsid Assembly By Adjusting the Binding Affinity and Pattern of the Hepatitis B Virus Core Protein

Journal of Chemical Information and Modeling ◽

10.1021/acs.jcim.9b01010 ◽

2019 ◽

Vol 59 (12) ◽

pp. 5104-5110 ◽

Cited By ~ 2

Author(s):

Huihui Liu ◽

Susumu Okazaki ◽

Wataru Shinoda

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Binding Affinity ◽

Core Protein ◽

Capsid Assembly ◽

Virus Core ◽

B Virus

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

Biochemical Journal ◽

10.1042/bj20080724 ◽

2008 ◽

Vol 416 (1) ◽

pp. 47-54 ◽

Cited By ~ 24

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.

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Hepatitis B Virus Core Protein Domains Essential for Viral Capsid Assembly in a Cellular Context

Journal of Molecular Biology ◽

10.1016/j.jmb.2020.04.026 ◽

2020 ◽

Vol 432 (13) ◽

pp. 3802-3819 ◽

Cited By ~ 3

Author(s):

Virgile Rat ◽

Xavier Pinson ◽

Florian Seigneuret ◽

Stéphanie Durand ◽

Charline Herrscher ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Core Protein ◽

Protein Domains ◽

Viral Capsid ◽

Capsid Assembly ◽

Virus Core ◽

B Virus ◽

Cellular Context

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02439-19 ◽

2020 ◽

Vol 64 (5) ◽

Cited By ~ 6

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01734-18 ◽

2018 ◽

Vol 63 (1) ◽

Cited By ~ 10

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.

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Performance Evaluation of Four Dominant Anti-Hepatitis B Core Antigen (HBcAb) Kits in Japan for Preventing de novo Hepatitis B Virus (HBV) Infection

Clinical Laboratory ◽

10.7754/clin.lab.2014.140504 ◽

2015 ◽

Vol 61 (01+02/2015) ◽

Cited By ~ 2

Author(s):

Eiji Kobayashi ◽

Matsuo Deguchi ◽

Masanori Kagita ◽

Nori Yoshioka ◽

Mifumi Kita ◽

...

Keyword(s):

Performance Evaluation ◽

Hepatitis B Virus ◽

Hepatitis B ◽

De Novo ◽

Hbv Infection ◽

Core Antigen ◽

B Virus ◽

De Novo Hepatitis B ◽

Hepatitis B Core Antigen

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00680-17 ◽

2017 ◽

Vol 61 (8) ◽

Cited By ~ 31

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.

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