Modulation of hRSV M2-1 Liquid Condensates by Polyanionic Ligands: Stabilization and Disruption

Human Respiratory Syncytial Virus is a major cause of acute respiratory infections such as bronchiolitis and pneumonia. These conditions are hazardous in infants during their first year of life and in elderly individuals with pre-existing health issues. Among the viral proteins, M2-1 plays a key role in supporting the transcription process by stabilizing RNA polymerase, making it essential for viral gene expression. Replication of hRSV takes place within cytoplasmic structures called inclusion bodies. These structures are formed through a process known as liquid-liquid phase separation (LLPS), in which viral proteins and RNA come together to create a highly concentrated environment that favors transcription and replication. The M2-1 protein is capable of forming LLPS on its own and, in the presence of RNA, can generate protein and RNA coacervates. It also interacts with the N and Phosphoprotein of the virus, adjusting the size of the condensates through its binding with the phosphoprotein. This study aims to examine how M2-1 undergoes phase separation and how different polyanionic molecules influence this behavior, these being RNA, heparin, suramin, chondroitin A, and enoxaparin. Recombinant M2-1 was successfully produced in Escherichia coli using LB culture medium and was purified with high yield and purity. The phase separation process was studied using UV-visible spectroscopy at 370 nm along with bright-field microscopy. Initial results showed that increasing concentrations of M2-1 led to higher turbidity, which was significantly enhanced in the presence of 5% (w/v) polyethylene glycol. This result suggests that molecular crowding promotes the LLPS of M2-1. Polyanionic molecules enhance LLPS formation up to an equimolar M2-1/polyanion ratio, beyond which phase separation declines sharply—less so with suramin and enoxaparin—suggesting disruption of the liquid condensates. Therefore, these findings offer novel insights into the molecular mechanisms underlying LLPS of hRSV M2-1 in the context of ligand binding, underscoring the potential of polyanionic molecules to disrupt the formation of viral inclusion bodies. A deeper understanding of these processes could inform the development of innovative antiviral strategies aimed at targeting the physical organization of the viral replication machinery.