Rapid HIV-1 capsid interaction screening using fluorescence fluctuation spectroscopy

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.

Selectivity of Lewy body protein interactions along the aggregation pathway of α-synuclein

“…To test binding to these unlabelled objects (Fig. 4d), we observed changes in the aggregation behaviour of the interactors and quantified the brightness of the GFP-labelled interactor before and after addition of unlabelled oligomers 15 . Binding to oligomers was detected by the apparition of fluorescent bursts in the GFP channel shortly after mixing (Fig.…”

“…This enables the detection of slowly diffusing particles and the scanning of a larger volume of sample, as in ref. 15 . To avoid measuring the same particles multiple times, the well is translated in x-direction for 15 s, then shifted by 200 µm in the y-direction, scanned in x in the reverse direction for 15 s, moved another 200 µm in the same y-direction and so on.…”

“…In all, 2.5 µL of purified oligomers were then added to the diluted interactors, incubated for 1 min, then 3 fluorescent traces of 100 s were acquired and B values were calculated as above. Upon binding to oligomers, multiple interactors are brought together in the same complex, creating brighter diffusing particles, therefore, brightness is a sensitive parameter to detect binding to unlabelled particles 15,88 . Monomeric GFP was used as a control, validating that brightness values were unchanged upon mixing with the oligomers.…”

Recognition of HIV-1 capsid by PQBP1 licenses an innate immune sensing of nascent HIV-1 DNA

“…Here, we sought to delineate the binding interface between capsid and PQBP1 using quantitative two-color coincidence detection (TCCD). This technique measures fluorescence intensity fluctuations from cross-linked CA (A204C) self-assembled particles labeled with AF568, and capsid-binding proteins labeled with AF488, diffusing through the confocal volume (Figure 2A, orange and green traces, respectively; Figure S2A; Lau et al, 2019Lau et al, , 2021. Accumulation of the binding proteins on the capsid results in fluorescence peaks in the binder trace that coincide with the capsid peak (Figure 2A, middle; Figure S2B).…”

“…The TCCD fluorescence fluctuation spectroscopy approach has been described previously (Lau et al, 2021). Binding reactions were performed in 20 mM Tris-HCl (pH 8.0) and 75 mM NaCl containing 8 mM of CA (monomeric equivalent) that were assembled under high salt conditions with binders as follows: 100 nM GFP-tagged PQBP1, 20 nM PQBP1-AF488, 50 nM CypA-AF488, 10 nM CPSF6 313-327 -AF488, and 10 nM of fluorescein-12-dATP (PerkinElmer, NEL465001EA).…”