Dataset of high-throughput ligand screening against the RNA Packaging Signals regulating Hepatitis B Virus nucleocapsid formation

Fardokht Abulwerdi; Farzad Fatehi; Iain W Manfield; Stuart F J Le Grice; John S Schneekloth; Reidun Twarock; Peter G Stockley; Nikesh Patel

doi:10.1016/j.dib.2022.108206

Dataset of high-throughput ligand screening against the RNA Packaging Signals regulating Hepatitis B Virus nucleocapsid formation

Data Brief. 2022 Apr 26:42:108206. doi: 10.1016/j.dib.2022.108206. eCollection 2022 Jun.

Authors

Fardokht Abulwerdi¹, Farzad Fatehi^{2

3}, Iain W Manfield⁴, Stuart F J Le Grice¹, John S Schneekloth¹, Reidun Twarock^{2

3

5}, Peter G Stockley⁴, Nikesh Patel⁴

Affiliations

¹ Center for Cancer Research, National Cancer Institute, 21702-1201, Frederick, MD, USA.
² Department of Mathematics, University of York, York, YO10 5DD, UK.
³ York Cross-disciplinary Centre for Systems Analysis, University of York, York, YO10 5GE, UK.
⁴ Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
⁵ Department of Biology, University of York, York, YO10 5DD, UK.

Abstract

Multiple ssRNA viruses which infect bacteria, plants or humans use RNA Packaging Signal (PS)-mediated regulation during assembly to package their genomes faithfully and efficiently. PSs typically comprise short nucleotide recognition motifs, most often presented in the unpaired region of RNA stem-loops, and often bind their cognate coat proteins (CPs) with nanomolar affinity. PSs identified to date are resilient in the face of the typical error prone replication of their virus-coded polymerases, making them potential drug targets. An immobilised array of small molecular weight, drug-like compounds was panned against a fluorescently-labelled oligonucleotide encompassing the most conserved Hepatitis B Virus (HBV) PS, PS1, known to be a major determinant in nucleocapsid formation. This identified > 70 compounds that bind PS1 uniquely in the array. The commercially available 66 of these were tested for their potential effect(s) on HBV nucleocapsid-like particle (NCP) assembly in vitro, which identified potent assembly inhibitors. Here, we describe a high-throughput screen for such effects using employing fluorescence anisotropy in a 96-well microplate format. HBV genomic RNAs (gRNA) and short oligonucleotides encompassing PS1 were 5' labelled with an Alexa Fluor 488 dye. Excess (with respect to stoichiometric T = 4 NCP formation) HBV core protein (Cp) dimers were titrated robotically into solutions containing each of these RNAs stepwise, using a Biomek 4000 liquid handling robot. The anisotropy values of these mixtures were monitored using a POLARstar microplate reader. NCP-like structures were challenged with RNase A to identify reactions that did not result in complete NCP formation. The results imply that ∼50% of the compounds prevent complete NCP formation, highlighting both PS-meditated assembly and the PS-binding compounds as potential directly-acting anti-virals with a novel molecular target. Importantly, this method allows high-throughput in vitro screening for assembly inhibitors in this major human pathogen.

Keywords: Assembly inhibitors; Hepatitis B Virus nucleocapsid assembly; High-throughput screening; RNA Packaging Signal-mediated virus assembly.

Abstract

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