CHARACTERIZATION OF THE INTERACTION INTERFACE BETWEEN THE M2-1 PROTEIN AND THE PHOSPHOPROTEIN P OF HUMAN METAPNEUMOVIRUS

Human metapneumovirus (hMPV) is a major etiological agent of respiratory tract infections, associated with bronchiolitis and pneumonia, primarily affecting children, older adults, and immunocompromised individuals. Despite its global epidemiological significance, no specific antiviral therapies for hMPV infection are currently available. In this context, the M2-1 protein functions as an essential transcriptional cofactor during viral replication by interacting with the phosphoprotein P and RNA through its core domain, thereby promoting the assembly of the transcriptional complex. Therefore, the present study aims to characterize the interaction interface between the core domain of the hMPV M2-1 protein (cdM2-1) and the phosphoprotein P using P-derived peptides P135–158, P127–148, and P127–158. The cdM2-1 protein was expressed in Escherichia coli BL21 (DE3) and purified by affinity chromatography followed by size-exclusion chromatography. The interaction between cdM2-1 and the P127–158 peptide was investigated by chemical shift perturbation experiments using two-dimensional ¹H–¹⁵N HSQC spectra acquired by Nuclear Magnetic Resonance (NMR) spectroscopy, allowing the identification of residues located in the α2, α4, α5 and α6 helices of cdM2-1 as significant for complex formation, resulting in a dissociation constant (Kd) of 2.0 ± 0.6 µM. Fluorescence anisotropy experiments further revealed an increase in anisotropy upon complex formation, yielding a Kd value of 9.6 ± 0.3 µM. Next steps include evaluating the interaction between cdM2-1 and the P135–158 and P127–148 peptides by fluorescence and NMR spectroscopy, aiming to refine the identification of the residues directly involved in the interaction interface. The results obtained are expected to contribute to the structural and biophysical understanding of the hMPV M2-1/P interaction, providing a foundation for further studies focused on the development of antivirals targeting the hMPV transcriptional complex.