Host-regulated Hepatitis B Virus Capsid Assembly in a Mammalian Cell-free System

ABSTRACTMultiple subunits of the hepatitis B virus (HBV) core protein (HBc) assemble into an icosahedral capsid that packages the viral pregenomic RNA (pgRNA). The N-terminal domain (NTD) of HBc is sufficient for capsid assembly, in the absence of pgRNA or any other viral or host factors, under conditions of high HBc and/or salt concentrations. The C-terminal domain (CTD) is deemed dispensable for capsid assembly although it is essential for pgRNA packaging. We report here that HBc expressed in a mammalian cell lysate, rabbit reticulocyte lysate (RRL), was able to assemble into capsids when (low-nanomolar) HBc concentrations mimicked those achieved under conditions of viral replicationin vivoand were far below those used previously for capsid assemblyin vitro. Furthermore, at physiologically low HBc concentrations in RRL, the NTD was insufficient for capsid assembly and the CTD was also required. The CTD likely facilitated assembly under these conditions via RNA binding and protein-protein interactions. Moreover, the CTD underwent phosphorylation and dephosphorylation events in RRL similar to those seenin vivowhich regulated capsid assembly. Importantly, the NTD alone also failed to accumulate in mammalian cells, likely resulting from its failure to assemble efficiently. Coexpression of the full-length HBc rescued NTD assembly in RRL as well as NTD expression and assembly in mammalian cells, resulting in the formation of mosaic capsids containing both full-length HBc and the NTD. These results have important implications for HBV assembly during replication and provide a facile cell-free system to study capsid assembly under physiologically relevant conditions, including its modulation by host factors.IMPORTANCEHepatitis B virus (HBV) is an important global human pathogen and the main cause of liver cancer worldwide. An essential component of HBV is the spherical capsid composed of multiple copies of a single protein, the core protein (HBc). We have developed a mammalian cell-free system in which HBc is expressed at physiological (low) concentrations and assembles into capsids under near-physiological conditions. In this cell-free system, as in mammalian cells, capsid assembly depends on the C-terminal domain (CTD) of HBc, in contrast to other assembly systems in which HBc assembles into capsids independently of the CTD under conditions of nonphysiological protein and salt concentrations. Furthermore, the phosphorylation state of the CTD regulates capsid assembly and RNA encapsidation in the cell-free system in a manner similar to that seen in mammalian cells. This system will facilitate detailed studies on capsid assembly and RNA encapsidation under physiological conditions and identification of antiviral agents that target HBc.

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

PLoS Pathogens ◽

10.1371/journal.ppat.1002388 ◽

2011 ◽

Vol 7 (11) ◽

pp. e1002388 ◽

Cited By ~ 44

Author(s):

Chao Chen ◽

Joseph Che-Yen Wang ◽

Adam Zlotnick

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Capsid Assembly ◽

Virus Capsid ◽

B Virus

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Safety, tolerability, and pharmacokinetics of the novel hepatitis B virus capsid assembly modulator GST-HG141 in healthy Chinese subjects: a first-in-human single- and multiple-dose escalation trial

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01220-21 ◽

2021 ◽

Author(s):

Cuiyun Li ◽

Min Wu ◽

Hong Zhang ◽

Jiajia Mai ◽

Lizhi Yang ◽

...

Keyword(s):

Steady State ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Maximum Plasma ◽

Capsid Assembly ◽

Virus Capsid ◽

B Virus ◽

The Mean ◽

Healthy Chinese ◽

Chinese Subjects

Background: Hepatitis B virus capsid assembly modulators (HBV CAMs) are promising, clinically validated therapeutic agents for the treatment of chronic hepatitis B (CHB). The safety, tolerability, and pharmacokinetic (PK) profiles of GST-HG141, a novel HBV CAM, were evaluated in healthy Chinese volunteers. Method: This phase Ia study included two parts: a double-blinded, randomized, placebo-controlled single-ascending-dose (SAD) (50, 100, 200, 300, 400, or 500 mg) study comprising a food-effect investigation (300 mg), and a multiple-ascending-dose (MAD) (100 or 200 mg BID) study. Result: GST-HG141 reached the maximum plasma concentration (C max ) at 1.25–3.00 h (median T max ). The exposure exhibited a linear increase, while the mean half-life (t 1/2 ) ranged from 13.096 h to 22.121 h. The exposure of GST-HG141 (300 mg) was higher after food intake by about 2.4-fold. In the MAD study, steady-state was reached at around day 5, and the mean trough steady-state concentrations were 423 and 588 ng/mL for 50 and 100mg cohorts, respectively. The ratios of GST-HG141 accumulation were <1.5. GST-HG141 was well tolerated in healthy Chinese subjects. The rates of adverse events (AEs) in the GST-HG141 cohort did not differ from those of the placebo cohort. Conclusion: GST-HG141 was tolerated in healthy Chinese subjects. The safety and PK profiles of GST-HG141 support the further evaluation of its efficacy in individuals with CHB.

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Discovery of New Hepatitis B Virus Capsid Assembly Modulators by an Optimal High-Throughput Cell-Based Assay

ACS Infectious Diseases ◽

10.1021/acsinfecdis.9b00030 ◽

2019 ◽

Vol 5 (5) ◽

pp. 778-787 ◽

Cited By ~ 4

Author(s):

Yameng Pei ◽

Chunting Wang ◽

Haijing Ben ◽

Lei Wang ◽

Yao Ma ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

High Throughput ◽

Capsid Assembly ◽

Virus Capsid ◽

B Virus

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Pharmacokinetics, safety and tolerability of single- and multiple-ascending doses of JNJ-64530440, a novel hepatitis B virus capsid assembly modulator, in healthy volunteers

Antiviral Therapy ◽

10.1177/13596535211044331 ◽

2021 ◽

pp. 135965352110443

Author(s):

Thomas N Kakuda ◽

Jeysen Z Yogaratnam ◽

Christopher Westland ◽

Edward J Gane ◽

Christian Schwabe ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Antiviral Activity ◽

Healthy Volunteers ◽

Second Generation ◽

Plasma Concentrations ◽

Pharmacokinetic Parameters ◽

Capsid Assembly ◽

Virus Capsid ◽

B Virus

Background Pharmacokinetics and safety of JNJ-64530440, a hepatitis B virus capsid assembly modulator producing normal empty capsids (CAM-N), in healthy volunteers were evaluated. Methods This Phase I study (NCT03439488) was a double-blind, randomised, placebo-controlled study. Adults ( n = 10/cohort, five Asian/five non-Asian), randomised 4:1, received single-ascending doses of oral JNJ-64530440 (first- and second-generation formulations) or placebo under fasted (50, 150, 300 and 900 mg) or fed (300, 750, 1,000, 2000 and 4000 mg) conditions. Multiple-ascending doses of 750 or 2000 mg once daily and 750 mg twice daily JNJ-64530440 (second-generation formulation) for 7 days were evaluated. Pharmacokinetic parameters were estimated from plasma concentrations. Safety was assessed throughout. Results Less than dose-proportional increases in maximum plasma concentrations (Cmax) and area under the plasma concentration–time curves (AUCs) were observed across the doses. Mean plasma half-lives ranged from 9.3 to 14.5 h. Cmax and AUC were ∼two fold higher under fed versus fasting conditions and slightly higher in Asians versus Caucasians. JNJ-64530440 doses ≥750 mg achieved plasma levels higher than protein-binding adjusted concentrations demonstrating in vitro antiviral activity. No serious adverse events (AEs), treatment discontinuations or dose-limiting toxicities were seen. AE frequency/severity did not increase with dose. Conclusions Single (up to 4000 mg) and multiple doses (up to 2000 mg for 7 days) of JNJ-64530440 were well tolerated in healthy volunteers. Multiple doses ≥750 mg/day achieved plasma concentrations expected to have antiviral activity that may lower hepatitis B surface antigen. No clinically relevant differences in tolerability or pharmacokinetic parameters were seen between Asians versus Caucasians.

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