BIOPHYSICAL-CHEMICAL CHARACTERIZATION OF THE LIQUID-LIQUID PHASE TRANSITION PROCESS OF THE M2-1 PROTEIN FROM HUMAN RESPIRATORY SYNCYTIAL VIRUS

Human Respiratory Syncytial Virus (hRSV) is one of the primary agents responsible for acute respiratory illnesses such as bronchiolitis and pneumonia in children, the elderly, and immunocompromised individuals, as well as those with cardiorespiratory diseases. The main risk group associated with hRSV comprises children with congenital immunodeficiency, bronchopulmonary dysplasia, heart disease, hypertension, prematurity, and low birth weight. The hRSV M2-1 protein plays a crucial role in viral replication by participating in transcription and the development of viral factories in the inclusion bodies. These are biomolecular condensates formed by liquid-liquid phase separation (LLPS), where M2-1 interacts with other viral proteins (phosphoprotein, nucleoprotein, and L protein) and viral RNA to facilitate efficient viral replication and transcription. The present work aims to investigate the formation of liquid condensates by hRSV M2-1 protein in response to controlled physicochemical changes. The methodology involves recombinant expression of M2-1, followed by purification through affinity chromatography and molecular exclusion, to monitor LLPS formation under different conditions of polyethylene glycol (PEG, crowded agent), temperature, ionic strength, and pH through turbidity experiments and bright light microscopy. Initially, the recombinant production of M2-1 protein was performed in the LB medium, resulting in a protein with a significant degree of purity. From microscopy measurement, using an inverted trinocular biological microscope with a 40x objective, it was observed that the formation of droplets in a 50 mM Bis-Tris buffer with 5 μM M2-1 protein increases in increment weight/volume percentage of PEG (from 0 to 15%), suggesting that the presence of the crowded agent favored the LLPS process of this protein. It was also seen that in increased concentrations of M2-1, the formation of droplets is higher in the presence of 15% (w/v) PEG than in its absence. The next steps of this study include the investigation of the effect of temperature, ionic strength, and pH on the LLPS process. This study can contribute to understanding the mechanisms behind the formation of viral factories by hRSV in which M2-1 protein is involved, providing insights into how physicochemical conditions can modulate inclusion bodies and consequently the viral replication and transcription processes.