Rapid HIV-1 capsid interaction screening using fluorescence fluctuationspectroscopy

Lau, D.; Walsh, J. C.; Dickson, C. F.; Tuckwell, A.; Stear, J. H.; Hunter, D. J. B.; Bhumkar, A.; Shah, V.; Turville, S. G.; Sierecki, E.; Gambin, Y.; Jacques, D. A.; Böcking, T.

doi:10.1101/2020.11.13.382242

bioRxiv · 10.1101/2020.11.13.382242

Rapid HIV-1 capsid interaction screening using fluorescence fluctuationspectroscopy

Lau, D.·Walsh, J. C.·Dickson, C. F.·Tuckwell, A.·Stear, J. H.·Hunter, D. J. B.·Bhumkar, A.·Shah, V.·Turville, S. G.·Sierecki, E.·Gambin, Y.·Jacques, D. A.·Böcking, T.

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Abstract

The HIV capsid is a multifunctional protein capsule for delivery of the viral genetic material into the nucleus of the target cell. Host cell proteins bind to a number of repeating binding sites on the capsid to regulate steps in the replication cycle. Here we develop a fluorescence fluctuation spectroscopy method using self-assembled capsid particles as the bait to screen for fluorescence-labelled capsid-binding analytes ( prey molecules) in solution. The assay capitalizes on the property of the HIV capsid as a multivalent interaction platform, facilitating high sensitivity detection of multiple prey molecules that have accumulated onto capsids as spikes in fluorescence intensity traces. By using a scanning stage, we reduced the measurement time to 10 s without compromising on sensitivity, providing a rapid binding assay for screening libraries of potential capsid interactors. The assay can also identify interfaces for host molecule binding by using capsids with defects in known interaction interfaces. Two-color coincidence detection using fluorescent capsid as bait further allows quantification of binding levels and determination of binding affinities. Overall, the assay provides new tools for discovery and characterization of molecules used by HIV capsid to orchestrate infection. The measurement principle can be extended for the development of sensitive interaction assays utilizing natural or synthetic multivalent scaffolds as analyte-binding platforms.

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Lau, D., Walsh, J. C., Dickson, C. F., Tuckwell, A., Stear, J. H., Hunter, D. J. B., Bhumkar, A., Shah, V., Turville, S. G., Sierecki, E., Gambin, Y., Jacques, D. A., Böcking, T.. 2020-11-16. Rapid HIV-1 capsid interaction screening using fluorescence fluctuationspectroscopy. https://doi.org/10.1101/2020.11.13.382242

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