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

Nature Biotechnology volume 24, pages 358–362 (2006)Cite this article

Abstract

Virus assembly has not been routinely targeted in the development of antiviral drugs, in part because of the lack of tractable methods for screening in vitro. We have developed an in vitro assay of hepatitis B virus (HBV) capsid assembly, based on fluorescence quenching of dye-labeled capsid protein, for testing potential inhibitors. This assay is adaptable to high-throughput screening and can identify small-molecule inhibitors of virus assembly that prevent, inappropriately accelerate and/or misdirect capsid formation to yield aberrant particles. An in vitro primary screen has the advantage of identifying promising lead compounds affecting assembly without the requirement that they be taken up by cells in culture and be nontoxic. Our approach may facilitate the identification of antivirals targeting viruses other than HBV, such as avian influenza and HIV.

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Figure 1: Strong correlation of real-time light scattering and fluorescence signals for assembly of BODIPY-FL–labeled C150.
Figure 2: Fluorescence quenching correlates with size-exclusion chromatography at equilibrium.
Figure 3: Quenching is dominated by interactions between dimers in trans, and occurs across vertices.
Figure 4: Fluorescence quenching in a microtiter plate format is sensitive to assembly misdirection and inhibition.

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Acknowledgements

This work was supported by a Research Scholar Grant (RSG-99-339-04-MBC) from the American Cancer Society and National Institutes of Health Grant R01-AI067417. We thank Sheryl Christofferson of the Oklahoma Medical Research Foundation Sequencing Core Facility, Bruce Baggenstoss of the OUHSC EPSCOR Mass Spectrometry Facility, Pablo Ceres for assistance with design of mutagenic primers, Christina Bourne for distance determinations from structural data and Laura Buford, Brian Firestone and Quincie Phan for excellent technical assistance. We thank Gillian Air for critical reading of the manuscript.

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Author notes

  1. Benjamin G Kopek

    Present address: McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 975 NE 10th St. BRC 456, Oklahoma City, 73104, Oklahoma, USA

    Stephen J Stray, Jennifer M Johnson, Benjamin G Kopek & Adam Zlotnick

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  1. Stephen J Stray
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  2. Jennifer M Johnson
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Correspondence to Adam Zlotnick.

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A provisional patent has been issued based on this work.

Supplementary information

Supplementary Fig. 1

Assembly of C150BO. (PDF 930 kb)

Supplementary Fig. 2

Absorbance of assembled and unassembled C150:C150BO mixtures. (PDF 124 kb)

Supplementary Table 1

Comparison of capsid stability (KDapp) of Cp149 wild-type and C150BO. (PDF 66 kb)

Supplementary Table 2

Mutagenic primers. (PDF 40 kb)

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Stray, S., Johnson, J., Kopek, B. et al. An in vitro fluorescence screen to identify antivirals that disrupt hepatitis B virus capsid assembly. Nat Biotechnol 24, 358–362 (2006). https://doi.org/10.1038/nbt1187

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