Tetramerization Of Phosphoprotein Is Essential For Respiratory Syncytial Virus Budding While Its N-Terminal Region Mediates Direct Interactions With The Matrix Protein

It was shown previously that the matrix (M), phosphoprotein (P), and 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 by 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 aN2 region, site II in the 115 to 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 respiratory syncytial virus (RSV) is the leading 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 antiviral drugs. The lack of knowledge of the RSV budding mechanism presents a continuing challenge for virus-like particle (VLP) production for vaccine purposes. We show that direct interaction between P and M modulates RSV VLP budding. This further emphasizes P as a central regulator of the 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.