Fluorescence biosensor for real-time interaction dynamics of host proteins with HIV-1 capsid tubes

Abstract

The human immunodeficiency virus 1 (HIV-1) capsid serves as a binding platform for proteins and small molecules from the host cell that regulate various steps in the virus life cycle. However, there are currently no quantitative methods that use assembled capsid lattices for measuring host-pathogen interaction dynamics. Here we developed a single molecule fluorescence biosensor using self-assembled capsid tubes as biorecognition elements and imaged capsid binders using total internal reflection fluorescence microscopy in a microfluidic setup. The method is highly sensitive in its ability to observe and quantify binding, obtain dissociation constants, extract kinetics with an extended application of using more complex analytes that can accelerate characterisation of novel capsid binders.

Recognition of HIV-1 Capsid Licenses Innate Immune Response to Viral Infection

“…We then sought to delineate the binding interface between capsid and PQBP1 utilizing two-color coincidence detection (TCCD). This technique measures fluorescence intensity fluctuations from both 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 S2;(Lau et al, 2021; Lau et al, 2019)). Accumulation of binders on the capsid results in the appearance of fluorescence peaks in the binder trace that coincide with the capsid peak (Figure 2A, middle; Figure S2B), whereby the variation in the peak amplitudes is due to the heterogeneity in the size of the in vitro assembled capsid particles.…”

“…Accumulation of binders on the capsid results in the appearance of fluorescence peaks in the binder trace that coincide with the capsid peak (Figure 2A, middle; Figure S2B), whereby the variation in the peak amplitudes is due to the heterogeneity in the size of the in vitro assembled capsid particles. As a control, we assessed the binding of a CPSF6 peptide comprising the capsid-binding residues (Bhattacharya et al, 2014; Lau et al, 2019; Price et al, 2014) to CA A204C particles (Figure 2B). Consistent with previously published data, we observed a significant reduction in CPSF6 binding to the N74D capsid mutant that disables the FG binding site in comparison to the strong binding to WT and R18G capsid mutant (Mallery et al, 2018).…”

“…The copyright holder for this preprint this version posted January 10, 2022. ; https://doi.org/10.1101/2022.01.10.472699 doi: bioRxiv preprint Leishmania tarentolae cell-free expression mix to 60 nM DNA and expressed for 2.5 hrs at 28 ˚C. (Lau et al, 2019) The expressed PQBP1 fragments were bound to Ni-NTA beads (BIORAD) for 30 min on ice and washed with 300 µL of 20 mM Tris-HCl (pH 8.0) and 100 mM NaCl before eluting bound protein with 40 µL of the same buffer containing 0.5 M imidazole. Purified PQBP1 constructs were then dialyzed into 20 mM Tris-HCl (pH 8.0) and 75 mM NaCl for 1 hr and were immediately used for TCCD measurements.…”

Prospects and Opportunities for Microsystems and Microfluidic Devices in the Field of Otorhinolaryngology

“…Since microchannels have very small volumes, the amount of solutions required for each assay is reduced. This will help in reducing the mortality rate and in controlling the propagation of life-threatening diseases such as malaria, HIV and AIDS (human immunodeficiency virus infection and acquired immune deficiency syndrome), measles, tuberculosis, lower respiratory conditions and so on [33][34][35].…”

Advances in Continuous Microfluidics-Based Technologies for the Study of HIV Infection

“…The biosensor assays showed detection limits for weak interactors with Kd = 1–100 µM (such as CPSF6). This system could accelerate characterization of novel capsid binders [ 75 , 76 ].…”