Abstract It was shown previously that the Matrix (M), Phosphoprotein (P), and the Fusion (F) proteins of Respiratory syncytial virus (RSV) are sufficient to produce virus-like particles (VLPs) that resemble the RSV infection-induced virions. However, the exact mechanism and interactions among the three proteins are not known. This work examines the interaction between P and M during RSV assembly and budding. We show that M interacts with P in the absence of other viral proteins in cells using a Split Nano Luciferase assay. By using recombinant proteins, we demonstrate a direct interaction between M and P. By using Nuclear Magnetic Resonance (NMR) we identify three novel M interaction sites on P, namely site I in the α N2 region, site II in the 115-125 region, and the oligomerization domain (OD). We show that the OD, and likely the tetrameric structural organization of P, is required for virus-like filament formation and VLP release. Although sites I and II are not required for VLP formation, they appear to modulate P levels in RSV VLPs. Importance Human RSV is the commonest cause of infantile bronchiolitis in the developed world and of childhood deaths in resource-poor settings. It is a major unmet target for vaccines and anti-viral drugs. The lack of knowledge of RSV budding mechanism presents a continuing challenge for VLP production for vaccine purpose. We show that direct interaction between P and M modulates RSV VLP budding. This further emphasizes P as a central regulator of RSV life cycle, as an essential actor for transcription and replication early during infection and as a mediator for assembly and budding in the later stages for virus production.

Tetramerization of Phosphoprotein is essential for Respiratory Syncytial virus budding while its N terminal region mediates direct interactions with the Matrix protein