Autor: |
Fardokht Abulwerdi, Farzad Fatehi, Iain W. Manfield, Stuart F.J. Le Grice, John S. Schneekloth, Reidun Twarock, Peter G. Stockley, Nikesh Patel |
Jazyk: |
angličtina |
Rok vydání: |
2022 |
Předmět: |
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Zdroj: |
Data in Brief, Vol 42, Iss , Pp 108206- (2022) |
Druh dokumentu: |
article |
ISSN: |
2352-3409 |
DOI: |
10.1016/j.dib.2022.108206 |
Popis: |
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. |
Databáze: |
Directory of Open Access Journals |
Externí odkaz: |
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