ArticleJournal of virology2014
Large-scale production and structural and biophysical characterizations of the human hepatitis B virus polymerase.
Article in Journal of virology, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed, 26 citations in OpenAlex.
- Deviated binding of anti-HBV nucleoside analog E-CFCP-TP to the reverse transcriptase active site attenuates the effect of drug-resistant mutations.Scientific reports · 2024Article
- Structural basis of HIV inhibition by L-nucleosides: Opportunities for drug development and repurposing.Drug discovery today · 2022Review
- Relaxing the restricted structural dynamics in the human hepatitis B virus RNA encapsidation signal enables replication initiation in vitro.PLoS pathogens · 2022Article
- Preparation and functional evaluation of monoclonal antibodies targeting Hepatitis B Virus Polymerase.Virulence · 2021Article
- Article
- Review
- Non-nucleoside hepatitis B virus polymerase inhibitors identified by an in vitro polymerase elongation assay.Journal of gastroenterology · 2020Article
- Structural features in common of HBV and HIV-1 resistance against chirally-distinct nucleoside analogues entecavir and lamivudine.Scientific reports · 2020Article
- HIV-1 with HBV-associated Q151M substitution in RT becomes highly susceptible to entecavir: structural insights into HBV-RT inhibition by entecavir.Scientific reports · 2018Article
- Mapping of Functional Subdomains in the Terminal Protein Domain of Hepatitis B Virus Polymerase.Journal of virology · 2017Article
- Toward Elimination of Hepatitis B Virus Using Novel Drugs, Approaches, and Combined Modalities.Clinics in liver disease · 2016Review
- Structure of the HIV-1 reverse transcriptase Q151M mutant: insights into the inhibitor resistance of HIV-1 reverse transcriptase and the structure of the nucleotide-binding pocket of Hepatitis B virus polymerase.Acta crystallographica. Section F, Structural biology communications · 2015Article
- The Impact of Mass Spectrometry-Based Proteomics on Fundamental Discoveries in Virology.Annual review of virology · 2014Article
- Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses.Proceedings of the National Academy of Sciences of the United States of America · 2014Article
Corrections and comments
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Authors and funding
7 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
unlabelledHepatitis B virus (HBV) is a major human pathogen that causes serious liver disease and 600,000 deaths annually. Approved therapies for treating chronic HBV infections usually target the multifunctional viral polymerase (hPOL). Unfortunately, these therapies--broad-spectrum antivirals--are not general cures, have side effects, and cause viral resistance. While hPOL remains an attractive therapeutic target, it is notoriously difficult to express and purify in a soluble form at yields appropriate for structural studies. Thus, no empirical structural data exist for hPOL, and this impedes medicinal chemistry and rational lead discovery efforts targeting HBV. Here, we present an efficient strategy to overexpress recombinant hPOL domains in Escherichia coli, purifying them at high yield and solving their known aggregation tendencies. This allowed us to perform the first structural and biophysical characterizations of hPOL domains. Apo-hPOL domains adopt mainly α-helical structures with small amounts of β-sheet structures. Our recombinant material exhibited metal-dependent, reverse transcriptase activity in vitro, with metal binding modulating the hPOL structure. Calcomine orange 2RS, a small molecule that inhibits duck HBV POL activity, also inhibited the in vitro priming activity of recombinant hPOL. Our work paves the way for structural and biophysical characterizations of hPOL and should facilitate high-throughput lead discovery for HBV. IMPORTANCE: The viral polymerase from human hepatitis B virus (hPOL) is a well-validated therapeutic target. However, recombinant hPOL has a well-deserved reputation for being extremely difficult to express in a soluble, active form in yields appropriate to the structural studies that usually play an important role in drug discovery programs. This has hindered the development of much-needed new antivirals for HBV. However, we have solved this problem and report here procedures for expressing recombinant hPOL domains in Escherichia coli and also methods for purifying them in soluble forms that have activity in vitro. We also present the first structural and biophysical characterizations of hPOL. Our work paves the way for new insights into hPOL structure and function, which should assist the discovery of novel antivirals for HBV.
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