Fig. 4

In vitro pentamer binding to the predicted PSs. In vitro analysis of PS: pentamer interactions by MST, HPeV1 pentamers were titrated into fixed amounts of 5ʹ-Alexa 488-labelled short RNAs encompassing PSs 6, 7, 9, 14 and 21 and their variants (labelled m). The binding data were assessed semi-quantitatively (+/−) noting the amplitude of the signal change over the range of pentamer concentrations and where significant binding began to occur. These were compared to a known PS-CP interaction, that of the 19 nt bacteriophage MS2 TR (translational operator) stem-loop and its CP⁴⁰ (K _d ~1–4 nM, depending on binding assay used), which was designated as +++++, i.e. low nano-molar affinity. The highest protein concentration samples from these assays were recovered, negatively-stained and examined by electron microscopy (top row shows the PS and the bottom row shows its mutant). Scale bar is 25 nm