Identification and characterization of a novel hepatitis B virus pregenomic RNA encapsidation inhibitor

Hepatitis B virus (HBV) chronically infects millions of people worldwide, which underscores the importance of discovering and designing novel anti-HBV therapeutics to complement current treatment strategies. An underexploited but attractive therapeutic target is ε, a cis-acting regulatory stem-loop RNA situated within the HBV pregenomic RNA (pgRNA). The binding of ε to the viral polymerase protein (P) is pivotal, as it triggers the packaging of pgRNA and P, as well as the reverse transcription of the viral genome. Consequently, small molecules capable of disrupting this interaction hold the potential to inhibit the early stages of HBV replication. The rational design of such ligands necessitates high-resolution structural information for the ε–P complex or its individual components. While these data are currently unavailable for P, our recent structural elucidation of ε through solution nuclear magnetic resonance spectroscopy marks a significant advancement in this area. In this review, we provide a brief overview of HBV replication and some of the therapeutic strategies to combat chronic HBV infection. These descriptions are intended to contextualize our recent experimental efforts to characterize ε and identify ε-targeting ligands, with the ultimate goal of developing novel anti-HBV therapeutics.

Section: Discussionmentioning

confidence: 99%

Section: Tackling Hbv: Insights Into Viral Replication and Evolving T...mentioning

confidence: 99%

Section: Tackling Hbv: Insights Into Viral Replication and Evolving T...mentioning

confidence: 99%

See 1 more Smart Citation

Hepatitis B Virus Epsilon (ε) RNA Element: Dynamic Regulator of Viral Replication and Attractive Therapeutic Target

Olenginski,

Attionu,

Henninger

et al. 2023

“…Techniques for drug development include 3D in silico modeling that attempts to dock libraries of chemicals to viral proteins. Candidates may be chosen for cell-based or cell-free assays [27][28][29][30]. Cell based assays are more physiologically relevant, have the ability to test toxicity, and usually involve cells permissive to HBV (or transfected HBV DNA) such as primary human hepatocytes, HepG2, Huh7, HepaRG, or others [8,31].…”

Section: Of 19mentioning

confidence: 99%

Molecular, Evolutionary, and Structural Analysis of the Terminal Protein Domain of Hepatitis B Virus Polymerase, a Potential Drug Target

Buhlig

Bowersox

Braun

et al. 2020

Approximately 250 million people are living with chronic hepatitis B virus (HBV) infections, which claim nearly a million lives annually. The target of all current HBV drug therapies (except interferon) is the viral polymerase; specifically, the reverse transcriptase domain. Although no high-resolution structure exists for the HBV polymerase, several recent advances have helped to map its functions to specific domains. The terminal protein (TP) domain, unique to hepadnaviruses such as HBV, has been implicated in the binding and packaging of the viral RNA, as well as the initial priming of and downstream synthesis of viral DNA—all of which make the TP domain an attractive novel drug target. This review encompasses three types of analysis: sequence conservation analysis, secondary structure prediction, and the results from mutational studies. It is concluded that the TP domain of HBV polymerase is comprised of seven subdomains (three unstructured loops and four helical regions) and that all three loop subdomains and Helix 5 are the major determinants of HBV function within the TP domain. Further studies, such as modeling inhibitors of these critical TP subdomains, will advance the TP domain of HBV polymerase as a therapeutic drug target in the progression towards a cure.

“…HBV DNA was analyzed by qPCR as previously described using a ViiA 7 system (Life Technology, Carlsbad, CA, USA) [6]. In brief, Premix Ex Taq (Cat No.…”

Section: Quantitative Real-time Pcr For Dna Quantificationmentioning

confidence: 99%

A Simple and Cost-Effective DNA Preparation Method Suitable for High-Throughput PCR Quantification of Hepatitis B Virus Genomes

Yang

Koenig

et al. 2020

Self Cite

Hepatitis B virus (HBV) is a para-retrovirus that reverse transcribes its pregenomic RNA into relaxed circular DNA inside viral nucleocapsids. The number of HBV genomes produced in vitro is typically quantified using commercial silica-membrane-based nucleic acid purification kits to isolate total DNA followed by HBV-specific quantitative PCR (qPCR). However, despite the convenience of commercial kits, this procedure is costly and time-consuming due to multiple centrifugation steps, which produce unnecessary waste. Here, we report a rapid, cost-effective, and environmentally friendly total DNA preparation method. The assay is based on the simple incubation of detergent and proteinase K with cells or cell-free supernatants to permeabilize cells and disrupt viral particles. After heat inactivation and subsequent centrifugation to clear the lysates, DNA samples are directly subjected to qPCR to quantify HBV genomes. As a proof of concept, the assay was developed in 12-well plates to assess intra- and extracellular HBV genome equivalents (GEqs) of stably viral-replicating cell lines (e.g., HepAD38) and HBV-infected HepG2-NTCP cells, both treated with lamivudine (LMV), an HBV replication inhibitor. Viral DNA was also prepared from the serum of patients chronically infected with HBV. To validate the assay, a representative commercial DNA isolation kit was used side-by-side to isolate intra- and extracellular HBV DNA. Both methods yielded comparable amounts of HBV GEqs with comparable LMV 50% efficient concentration (EC50) values. The assay was subsequently adapted to 96- and 384-well microtiter plates using HepAD38 cells. The EC50 values were comparable to those obtained in 12-well plates. In addition, the calculated coefficient of variation, Z’ values, and assay window demonstrated high reproducibility and quality. We devised a novel, robust, reproducible, high-throughput microtiter plate DNA preparation method suitable for quantifying HBV GEqs by qPCR analysis. This strategy enables rapid and convenient quantitative analysis of multiple viral DNA samples in parallel to investigate intracellular HBV replication and the secretion of DNA-containing viral particles.