Fluorescence Biosensor for Real-Time Interaction Dynamics of Host Proteins with HIV-1 Capsid Tubes

Abstract

Supporting Materials and MethodsPurification of recombinant CA. Constructs for expression of untagged wild type CA and CA A14C/E45C in a pET11a vector were a gift from Owen Pornillos. The construct for expression of CA K158C was generated from the plasmid containing wild type CA using site directed mutagenesis. CA was expressed in E. coli BL21 Rosetta (DE3, pLysS RARE) grown in Luria-Bertani medium supplemented with ampicillin (100 μg/mL) and chloramphenicol (34 μg/mL) at 37 °C with shaking. Protein expression was induced by adding IPTG to a final concentration of 0.4 mM when the optical density at 600 nm reached 0.5 and allowed to proceed at 20 °C for 16 hours with shaking. Cells were harvested by centrifugation, resuspended in lysis buffer (50 mM Tris, pH 8, 2 mM DTT, 0.02% NaN3, 1 mg/mL lysozyme, Complete protease inhibitor) and lysed by sonication. For purification of CA A14C/E45C, the lysis buffer contained 200 mM of 2-mercaptoethanol to prevent disulfide crosslinking. For purification, the cell lysate was clarified by centrifugation (43150 g, 1 hour, 4°C). CA assembly was induced by adding solid NaCl (2.5 M) and the solution was incubated for 15 min on ice and then pelleted (3900 g, 45 min, 4°C). The supernatant was removed and the pellet was redissolved in buffer A (50 mM Tris, pH 8, 2 mM DTT, 0.02% NaN3) to induce disassembly of CA. CA assembly was repeated by adding NaCl (2.5 M), the assembled protein was pelleted and redissolved in buffer A as above. The solution was then filtered (0.22 μm) and CA was further purified by subtractive anion exchange chromatography using a HiTrap Q HP column (GE Healthcare, 17115301). The column was washed with 5 column volumes (CV) of buffer A, followed by 5 CV of buffer B (buffer A containing 1 M NaCl) and then equilibrated with 5 CV of buffer A at 5 mL/min before injecting the sample at 1 mL/min. CA was collected in the flowthrough and fractions containing CA were identified using reducing SDS-PAGE. Protein concentrations were determined using the Bradford assay (Thermo Fisher Scientific, 23236) with BSA as a standard. CA protein solutions in buffer A were concentrated to a concentration of ≥ 150 μM (3.8 mg/mL) using centrifugal ultrafiltration devices (Amicon Ultra-15, 10 MWCO, Merck, UFC901008), snap frozen in liquid nitrogen and stored at -80 °C.

Rapid HIV-1 capsid interaction screening using fluorescence fluctuation spectroscopy

“…33 For two-color experiments, capsid particles were rendered fluorescent by incorporating a small fraction of the AF568labelled CA K158C into the CA A204C lattice during self-assembly. 23 The binding assay was carried out by adding a mixture of capsid particles and labelled analyte into a well of a 192-well polydimethylsiloxane device adhered to a glass coverslip. We then measured fluorescence intensity (photon counting) traces as a function of time using an inverted confocal microscope with custom-built two-color excitation and two-channel single-molecule detection modules ( Fig 1A).…”

“…Indeed, oligomerization of CPSF6 may contribute to its physiological role in HIV infection by increasing avidity and mediating capsid disassembly. 23,38 Finally, we synthesized a tris-NTA derivative of AF488 to label recombinant proteins with hexahistidine tag via formation of a noncovalent complex (Supporting Fig 3A) with a half-life of about 30 min. 39 His-tagged CypA labelled with this compound showed the expected inhibitor-reversible increase in brightness in the presence of capsid while no increase was observed for a tris-NTA-AF488labelled control protein of bacterial origin (LcrV) (Supporting Fig 3B).…”

“…23 CA K158C was labelled with Alexa Fluor 568-C5maleimide (AF568, Thermo Fisher Scientific, A20341) as previously described. 23 Untagged recombinant CypA was expressed, purified and labelled with Alexa Fluor 488-C5 maleimide (AF488, Thermo Fisher Scientific, A10254) as previously described. 16 Labelling of peptides.…”

“…Recently, we also described fluorescence biosensors to quantify binding of fluorescence-labelled analytes to surface-immobilized CA tubes or spheres. 22,23 Here we develop an analytical method to facilitate rapid screening of fluorescence-labelled analyte binding to capsid in solution, a capability that complements existing approaches. The method builds on single molecule spectroscopy approaches for detection of protein oligomerization and aggregation [24][25][26] that monitor in real time the light emitted from fluorescence-tagged protein assemblies diffusing through a confocal excitation volume (~1 fL).…”

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].…”