POLYANIONIC MOLECULES AS POTENTIAL COMPETITORS FOR THE HMPV M2-1 CORE DOMAIN

The human Metapneumovirus (hMPV) is a respiratory virus associated with acute respiratory infections and the development of severe bronchiolitis and pneumonia, particularly in neonates, children, older adults, and immunocompromised individuals. In early 2025, hMPV gained attention due to a marked increase in reported cases in the Northern Hemisphere, especially in China. Although first isolated in 2001, no licensed vaccines or specific antiviral therapies are currently available against hMPV. Viral replication depends on positive-sense RNA synthesis and the transcription of polyadenylated mRNAs, processes mediated by the transcription complex composed of the P, N, and L proteins. The P protein organizes this complex by interacting with N and L, thereby positioning the RNA polymerase on the viral genome. During transcription and replication, the M2-1 protein is recruited to the complex and functions as a transcription antitermination factor. Through its core domain (cdM2-1), it simultaneously interacts with the P protein and the nascent RNA, stabilizing the transcription complex, promoting the expression of genes located near the 5′ end, and favoring the synthesis of full-length RNA transcripts. In vivo studies have demonstrated that M2-1 is essential for viral infectivity. Therefore, the present study aims to experimentally characterize the interaction between the hMPV cdM2-1 and polyanionic molecules—yeast RNA, suramin, heparin, and enoxaparin—evaluated as potential competitive inhibitors of the protein interaction sites for both the P protein and the nascent RNA. The interaction between cdM2-1 and polyanions was characterized using recombinant cdM2-1 and spectroscopic techniques, including Nuclear Magnetic Resonance, fluorescence spectroscopy, and circular dichroism (CD). The fluorescence experiments confirmed interactions of different magnitudes between cdM2-1 and the polyanions, establishing the following order of binding affinity: yeast RNA > suramin > heparin > enoxaparin; 15N HSQC spectra mapped the residues of the binding site in cdM2-1; and CD measurements showed that the cdM2-1/polyanion interaction didn’t promote significant change in the secondary structure of the core domain. Electrostatic interactions were shown to play a major role, although the structural specificities of the ligands resulted in distinct affinity profiles, providing a basis for the rational design of antiviral molecules capable of disrupting the biological interactions of M2-1.