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

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 αN2 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.ImportanceHuman 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.

Importance of RNA length for in vitro encapsidation by the nucleoprotein of human respiratory syncytial virus

“…Importantly, the P protein (241 residues long), which forms tetramers through its central oligomerisation domain (residues 131-151) flanked by highly disordered N and C-terminal domains (P NTD and P CTD respectively) that are involved in multiple protein-protein interactions, plays a central role in regulating polymerase activity as well as in IBs formation. The P NTD interacts with N 0 (37), M2-1 (41), the cellular phosphatase PP1 (41) and the viral matrix protein M responsible for virion assembly (42), whereas P CTD is involved in the interaction with L (43,44) and NC (45,46), and is critical for IBs formation (39). Both the length of the P CTD and its interaction with N complexed to RNA are required for efficient IBs morphogenesis.…”

Respiratory syncytial virus matrix protein assembles as a lattice with local and extended order that coordinates the position of the fusion glycoprotein

“…Influenza matrix (M1) associates with, but is not required for VLP formation driven by Hemagglutinin and Neuraminidase. Purified RSV M can assemble into a uniform helical filament in the presence of specific lipids [59], but requires expression of P and the F cytoplasmic tail for assembly of filamentous particles in cells [36, 37]. Many viral matrix proteins have also been shown to interact with and/or coordinate the positioning of nucleoprotein [30-32, 34, 52, 53].…”

“…RSV P is a non-catalytic phosphoprotein that is essential for viral RNA synthesis. It’s exact role in virion or VLP assembly remains unknown, but it is known to interact directly with M [37]. P interacts with other structural proteins as well, including L and M2-1, and may be involved in mediating interaction between M and other proteins [36-38].…”

In Vitro Study of Rna Encapsidation by the Nucleoprotein of Human Respiratory Syncytial Virus

“…Importantly, the P protein (241 residues long), which forms tetramers through its central oligomerisation domain (residues 131-151) flanked by highly disordered N and C-terminal domains (P NTD and P CTD respectively) that are involved in multiple protein-protein interactions, plays a central role in regulating polymerase activity as well as in IBs' formation. The P NTD interacts with N 0 (35), M2-1 (39), the cellular phosphatase PP1 (39) and the viral matrix protein M responsible for virion assembly (40), whereas P CTD is involved in the interaction with L (41, 42) and NC (43,44), and is critical for IBs' formation (37). Both the length of the P CTD and its interaction with N complexed to RNA are required for efficient IBs' morphogenesis.…”