Periodic Reporting for period 2 - DynamicAssemblies (Conformational studies of highly dynamic viral replication complexes)
Periodo di rendicontazione: 2021-05-01 al 2022-10-31
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.
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.
1) Investigation of the role of the disordered domain of Measles virus phosphoprotein in nucleocapsid assembly.
2) Description of the formation of membraneless organelles by the nucleoprotein and phosphoprotein of Measles virus, identifying the amino acids in the disordered domains of the nucleo- and phosphoproteins that are responsible for phase separation using NMR and observation of accelerated encapsidation of viral RNA by the nucleoprotein within the droplets.
3) Combination of NMR relaxation and molecular simulation to investigate the dynamic properties the disordered domain of Measles nucleoprotein in the dilute and condensed phase at atomic resolution.
4) Description of the dynamic assembly of the intrinsically disordered nucleoprotein from SARS-CoV-2 that folds upon binding to its viral partner nsp3, identifying important new targets for viral inhibition.
2) Description of the formation of membraneless organelles by the nucleoprotein and phosphoprotein of Measles virus, identifying the amino acids in the disordered domains of the nucleo- and phosphoproteins that are responsible for phase separation using NMR and observation of accelerated encapsidation of viral RNA by the nucleoprotein within the droplets.
3) Combination of NMR relaxation and molecular simulation to investigate the dynamic properties the disordered domain of Measles nucleoprotein in the dilute and condensed phase at atomic resolution.
4) Description of the dynamic assembly of the intrinsically disordered nucleoprotein from SARS-CoV-2 that folds upon binding to its viral partner nsp3, identifying important new targets for viral inhibition.
During the course of the first half of the funding period, we characterised phase separation of measles virus proteins that form liquid droplets upon mixing, and observed the acceleration of processes that are essential for viral replication within these droplets allows us fascinating insight into the mechanisms that viruses use to protect against the host immune system by forming so-called viral factories within the cell. We were also able to identify the role of the disordered phosphoprotein and to compare the dynamics of measles proteins under phase-separated and non-phase-separated conditions.
The characterisation of the role of the disordered domain of SARS CoV2 nucleoprotein in the formation of an essential complex with the viral partner nsp3 provided new insight into the mechanism of viral replication and provides new targets for the development of methods to treat the infection.
In the next periods we will finalize the description of the dynamics of viral IDPs inside droplets using NMR relaxation and molecular dynamics simulation. We will also continue to describe the role of the phosphoprotein in measles virus nucleocapsid assembly. We will describe the interaction between viral RNA and the nucleocapsid protein of SARS-CoV-2, describe the role of phosphorylation and mutations associated with variants of concern, and investigate the interaction between nucleocapsid proteins and host proteins in influenza.
The characterisation of the role of the disordered domain of SARS CoV2 nucleoprotein in the formation of an essential complex with the viral partner nsp3 provided new insight into the mechanism of viral replication and provides new targets for the development of methods to treat the infection.
In the next periods we will finalize the description of the dynamics of viral IDPs inside droplets using NMR relaxation and molecular dynamics simulation. We will also continue to describe the role of the phosphoprotein in measles virus nucleocapsid assembly. We will describe the interaction between viral RNA and the nucleocapsid protein of SARS-CoV-2, describe the role of phosphorylation and mutations associated with variants of concern, and investigate the interaction between nucleocapsid proteins and host proteins in influenza.