LIQUID–LIQUID PHASE SEPARATION OF THE M2-1 PROTEIN FROM PNEUMOVIRIDAE: BIOPHYSICOCHEMICAL INSIGHTS AND THE ROLE OF POLYANIONS

Human Respiratory Syncytial Virus (hRSV) and Human Metapneumovirus (hMPV) are among the leading etiological agents of acute respiratory tract infections, particularly affecting pediatric, elderly, and immunocompromised populations. Both viruses belong to the Pneumoviridae family and share the expression of the M2-1 protein, a pivotal transcriptional regulatory factor. Emerging evidence suggests that M2-1 plays a central role in the formation of biomolecular condensates via liquid-liquid phase separation (LLPS), which function as membraneless viral compartments orchestrating RNA transcription and replication. This study aims to elucidate the biophysicochemical mechanisms governing the LLPS behavior of the M2-1 protein from both hRSV and hMPV, focusing on the effects of physicochemical parameters — including protein concentration, pH, and ionic strength — as well as the impact of polyanionic modulators such as yeast RNA, heparin, and suramin. Recombinant M2-1 was heterologously expressed in Escherichia coli and purified using affinity and size exclusion chromatography, ensuring high purity and functional integrity. The characterization of the condensates was carried out using complementary and robust methodologies: UV-Vis spectrophotometry (for turbidity measurements), bright-field microscopy, and fluorescence microscopy, allowing morphological and dynamic assessment of LLPS under different experimental conditions. These approaches supported the construction of phase diagrams and provided information on the modulatory role of polyanions in condensate stability. The results indicated that acidic pH and high ionic strength negatively affect condensate integrity, while polyanions modulate size and morphology, likely through specific electrostatic interactions. Bioinformatic analyses reveal high structural conservation between hRSV and hMPV M2-1 proteins, supporting the hypothesis of a conserved LLPS mechanism among pneumoviruses. Altogether, this work advances our understanding of M2-1’s functional role in the viral replication cycle and highlights the potential of targeting phase-separated viral condensates as a novel antiviral strategy for hRSV and hMPV — as exemplified by the in vivo efficacy of condensate-disrupting agents such as cyclopamine.