Single-stranded RNA viruses comprise a number of human pathogens (measles, mumps, influenza), expressing their own machinery for replication in infected cells. The proteins involved in this process, including the nucleoprotein (N) which assembles and protects newly synthesized RNA genomes, are important potential targets in the conception of inhibitors to treat infected individuals. Many of these proteins exhibit extensive disorder, and do not fold into a stable three-dimensional structure, but as highly flexible polymers constantly interchange between different conformations. The presence of this level of disorder, in viruses whose genetic information is normally so parsimoniously exploited, remains unexplained.
This extensive flexibility is challenging for classical structural approaches, placing their functional mechanisms at the centre of a contemporary paradigm of molecular biology – how to develop a molecular understanding of highly dynamic assemblies. Due to their high flexibility, such proteins are able to exploit diverse functional mechanisms that are inaccessible to folded proteins, for example the formation of membraneless organelles, transient sub-compartments that appear to provide optimal conditions to enhance essential molecular interactions, while maintaining immiscibility with respect to the host immune system. The molecular origin of this phenomenon remained poorly understood prior to this project.
The elaboration of time-resolved, atomic resolution description of their interaction trajectories requires the development of adapted methodologies that can describe both the intrinsically disordered regions as well as the folded domains at atomic resolution. To describe the functional modes of these dynamic assemblies, we use NMR, in combination with fluorescence spectroscopy and imaging, X-ray scattering and molecular simulation, we follow the assembly process upon binding RNA to N, during the formation of the replication complex and essential interactions with host and viral partners. This integration of complementary state-of-the-art technology leads to new understanding of the replication process over a broad range of length and timescales and can be transferred to a large number of viral replication paradigms comprising both structured and intrinsically disordered components.
