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 αN2 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.