Recombinant production and detailed structural characterization of hMPV M2-1 core domain

The human Metapneumovirus (hMPV) is recognized as one of the leading etiological agents of acute respiratory infections, such as bronchiolitis and pneumonia, particularly affecting children, elderly, and immunocompromised individuals. Despite its clinical relevance, no licensed vaccines or effective treatments are currently available. The hMPV M2-1 protein functions as a transcriptional antitermination factor, preventing premature dissociation of the transcriptional complex and promoting the synthesis of 5’-end genes and viral replication. Its interactions with biological partners are mediated by electrostatic forces, notably through the highly positively charged core domain (cdM2-1). During viral transcription and replication, M2-1 interacts with the nascent viral mRNA and with the viral phosphoprotein P. These molecular interactions, essential for the viral replication cycle, make cdM2-1 a promising target for the development of viral replication inhibitors and potential antiviral drugs. This study aims to characterize the interaction between cdM2-1 and polyanionic molecules (enoxaparin, heparin, and suramin), seeking high-affinity ligands capable of binding to biologically relevant interaction sites on the protein. For that, the recombinant production of hMPV cdM2-1 was performed for the first time. This production was carried out by transforming E. coli BL21 cells with a pET28a(+) vector containing the gene sequence of cdM2-1 with a histidine tag and a protease cleavage site for purification. Bacterial cultures were grown and induced in both LB and M9 minimal media supplemented with the 15N isotope. Protein purification included extraction steps, affinity chromatography, proteolytic cleavage (TEV), and size-exclusion chromatography. Following, the sample was subjected to circular dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopy for preliminary structural analysis. The CD spectrum revealed a typical profile of alpha-helix secondary structure, and the 15N HSQC experiment showed a spectrum with homogeneous and dispersed resonance intensities, indicating a well-structured protein. The next steps involve physicochemical experiments to characterize the structural stability of the hMPV cdM2-1 in solution and its interaction with polyanions by using fluorescence and NMR spectroscopy. Therefore, it is expected to identify ligands capable of competitively binding to cdM2-1 and potentially attenuating the hMPV replication cycle.