nanoDSF: In vitro Label-Free Method to Monitor Picornavirus Uncoating and Test Compounds Affecting Particle Stability

Abstract

Thermal shift assays measure the stability of macromolecules and macromolecular assemblies as a function of temperature. The Particle Stability Thermal Release Assay (PaSTRy) of picornaviruses is based on probes becoming strongly fluorescent upon binding to hydrophobic patches of the protein capsid (e.g., SYPRO Orange) or to the viral RNA genome (e.g., SYTO-82) that become exposed upon heating virus particles. PaSTRy has been exploited for studying the stability of viral mutants, viral uncoating, and the effect of capsid-stabilizing compounds. While the results were usually robust, the thermal shift assay with SYPRO Orange is sensitive to surfactants and EDTA and failed at least to correctly report the effect of excipients on an inactivated poliovirus 3 vaccine. Furthermore, interactions between the probe and capsid-binding antivirals as well as mutual competition for binding sites cannot be excluded. To overcome these caveats, we assessed differential scanning fluorimetry with a nanoDSF device as a label-free alternative. NanoDSF monitors the changes in the intrinsic tryptophan fluorescence (ITF) resulting from alterations of the 3D-structure of proteins as a function of the temperature. Using rhinovirus A2 as a model, we demonstrate that nanoDFS is well suited for recording the temperature-dependence of conformational changes associated with viral uncoating with minute amounts of sample. We compare it with orthogonal methods and correlate the increase in viral RNA exposure with PaSTRy measurements. Importantly, nanoDSF correctly identified the thermal stabilization of RV-A2 by pleconaril, a prototypic pocket-binding antiviral compound. NanoDFS is thus a label-free, high throughput-customizable, attractive alternative for the discovery of capsid-binding compounds impacting on viral stability.

Targeting G-quadruplexes in the rhinovirus genome by Pyridostatin inhibits uncoating and highlights a critical role for sodium ions.

“…Virus pre-incubated with PDS at 34°C exhibited a striking 4.7°C earlier onset of genomic RNA accessibility to SYTO 82 compared to the control condition, i.e., incubation with PDS at 4°C (to prevent diffusion of this compound through the protein shell) (T on of 37.7°C vs. 42.4°C). However, the temperatures T max at the peak of the SYTO 82 signal (indicative of the complete conversion of all native virions into permanently porous A-particles) and the peak of the rst derivative (at T 50 , corresponding to 50 % RNA accessibility) 73 remained practically unaltered.…”

“…Puri ed RV-A2 was incubated with PDS under conditions of strongly diminished capsid breathing (at 4°C) and a capsid breathing-promoting temperature (at 34°C). Unbound PDS was removed, and a Particle Stability Thermal Release Assay (PaSTRy) 72,73 was performed to determine a possible impact of PDS on temperature-dependent uncoating, a commonly used model for in vitro uncoating of picornaviruses 74 . Figure 3a shows the temperatures where the genomic RNA becomes accessible to SYTO 82 as deduced from the temperature vs uorescent emission curves 73 (Supplementary Fig.…”

Lactoferrin affects rhinovirus B-14 entry into H1-HeLa cells

“…PaSTRy was performed as described [ 22 ] using a Bio-Rad CFX Connect Real-Time PCR instrument to detect the SYTO-82 (Thermo Fisher Scientific, USA) signal, indicating viral RNA accessibility. Purified RV-B14 (0.5 mg ml −1 ) was incubated with SYTO-82 (5 µM) in PBS +/- BLf (0.25, 0.5, and 1.0 mg ml −1 ) in 70 µl at the given final concentrations.…”

“…The generated subviral particles were washed five times with PBS using Amicon Ultra Filters (Sigma-Aldrich, USA) for reneutralisation. The B particles were generated by heating purified RV-B14 (10 µg) to 56 °C for 10 min [ 22 ]. After washing with 1% BSA in PBS-T (PBS containing 0.05% Tween-20), plates were incubated with rabbit anti-RV-B14 serum [ 18 ] diluted (1:200) in PBS-T containing 1% BSA at 34 °C for 1 h. The plates were then washed and incubated with DyLight 800-conjugated anti-rabbit antibody (Thermo Fisher Scientific, USA) diluted 1:500 in PBS-T containing 1% BSA at 34 °C for 1 h. After washing, the plates were scanned using an Odyssey CLx Imaging System for fluorescence emission using the 800 nm channel.…”

Periplasmic Expression of SpyTagged Antibody Fragments Enables Rapid Modular Antibody Assembly

“…Particularly for these constructs, the improved versions of SpyCatcher are helpful to keep the reaction time short. To further characterize the coupling reaction, we measured melting curves of uncoupled and coupled FcCatchers via nanoscale differential scanning fluorimetry 26 and compared them to the analogous wildtype full-length immunoglobulins. For uncoupled FcCatcher, a first melting point of 45°C could be detected ( Figure 2E), similar to isolated SpyCatcher2 (Supplementary figure S8).…”