Stability and conformational transitions of the RSV nucleocapsid.

ESPERANTE S; ALVAREZ PAGGI D; DE PRAT GAY, GONZALO; SALGUEIRO M

ASHEVILLE

Simposio; 11th International Respiratory Syncytial Virus Symposium; 2018

International Society for Influenza and other Respiratory Viruses Diseases

The RSV genome is tightly bound to the N protein, conforming the nucleocapsid, a rod-shaped structure with helicoidal symmetry. It is known that for the RNA-dependent RNA polymerase to function, the RNA must remain bound to the N protein. However, structural data shows that bound RNA adopts a specific conformation occluding around 50% of the bases, precluding Watson-Crick base pairing. These results strongly suggest the notion of alternative conformations of the nucleocapsid for replication and transcription.Recombinant expression of RSV nucleoprotein (and other members of the Mononegavirales) commonly yields ring-like structures of ca. 10 nm consisting of 10 or 11 protomers of the N protein bound to an RNA molecule of around 70nt, constituting an ideal minimal unit for assessing conformational transitions in the nucleocapsid.We assessed the stability and alternative conformation of the N rings by biophysical and spectroscopic methods and molecular dynamics simulations. The secondary and tertiary structure was studied by circular dichroism and MD calculations. Fluorescence spectroscopy allowed for probing protein conformation and RNA binding, monitored by the quenching of the intrinsic tryptophan fluorescence by the RNA molecule. RNA release and conformational changes were also monitored by dynamic light scattering (DLS) and size exclusion chromatography (SEC).Temperature, pH, ionic strength, addition of detergents and of denaturing agents all perturb the dynamics of N, revealing varying degrees of RNA compactness, and different secondary and tertiary structures of the nucleocapsid. We observed reversible and irreversible aggregation of N rings, and conformational changes of the RNA that range from loosening to full detachment of RNA, associated with protein secondary structure changes that vary depending on the experimental conditions. Our results suggest the existence of finely-tuned conformational and functional transitions, and that some of the characteristics of the RNA-N binding can only be accessed on higher order structures. These results may be of relevance to other viruses of the Mononegavirales order.