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.

The ~ 2.4 µm long rhinovirus ss(+)RNA genome consists of roughly 7,200 nucleotides. It is tightly folded to fit into the ~ 22 nm diameter void in the protein capsid. In addition to previously predicted secondary structural elements in the RNA, using the QGRS mapper, we revealed the presence of multiple quadruplex forming G-rich sequences (QGRS) in the RV-A, B, and C clades, with four of them being exquisitely conserved. The biophysical analyses of ribooligonucleotides corresponding to selected QGRS demonstrate G-quadruplex (GQ) formation in each instance and resulted in discovering another example of an unconventional, two-layer zero-nucleotide loop RNA GQ stable at physiological conditions. By exploiting the temperature-dependent viral breathing to allow diffusion of small compounds into the virion, we demonstrate that the GQ-binding compounds PhenDC3 and pyridostatin (PDS) uniquely interfere with viral uncoating. Remarkably, this inhibition was entirely prevented in the presence of K+ but not Na+, despite the higher GQ stabilising effect of K+. Based on virus thermostability studies combined with ultrastructural imaging of isolated viral RNA, we propose a mechanism where Na+ keeps the encapsidated genome loose, allowing its penetration by PDS to promote the transition of QGRS sequestered in alternative metastable structures into GQs. The resulting conformational change then materialises in a severely compromised RNA release from the proteinaceous shell. Targeting extracellularly circulating RVs with GQ-stabilisers might thus become a novel way of combating the common cold.