Biophysical characterization of the interaction of the M2-1 protein from human respiratory syncytial virus (HRSV) with RNA and with the steroidal alkaloids cyclopamine and solasodine.

Human respiratory syncytial virus (HRSV) is the major causative agent of chronic lower respiratory tract diseases in children and elderly worldwide. The M2-1 protein from HRSV acts as a transcription antitermination factor, enabling the transcription of all genes. This protein is divided into four domains: a zinc finger domain; the oligomerization domain; the core domain that interacts with the viral RNA; and unstructured C-terminal domain. In 2016, a study published by Bailly and colleagues showed that steroidal alkaloids had significant antiviral activity against HRSV and that the main target of the compound was the M2-1 protein core domain (cdM2-1). Then, the present study aimed to answer two questions: i) what are the thermodynamic parameters and the non-covalent interactions involved in the formation and stabilization of the cdM2-1/RNA complex? ii) do solasodine and cyclopamine really interact in the core domain? Which regions of these molecules are involved in the interaction with the protein? To achieve the results, we measured the fluorescence quenching of cdM2-1 by RNA under different conditions of temperature, ionic strength and pH. The determined dissociation constant for the complex was on the range of hundreds nanomolar and the binding stoichiometry is of two proteins per RNA molecule. The effect of temperature revealed a negative binding enthalpy change, whereas the increase either in pH or in saline concentration confirmed that electrostatic forces drive the interaction. The results also indicate that it is spontaneous, with hydrogen bonds and van der Waals forces playing a key role on the stabilization of the complex. Regarding to the steroidal alkaloids, it was found that both interact at the AMP binding site and the complex formed was visualized by molecular docking, which demonstrated that the interaction occurs at the cdM2-1 / zinc finger interdomain interface region and it depends on hydrogen bonds and hydrophobic contacts to occur. These findings require further investigation and are important for understanding the mechanism of interaction between HRSV M2-1 protein with RNA and with promising candidates for the development of antivirals. (AU)