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

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... GRS 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.