VIRAL_IDP

Revealing the role of intrinsically disordered proteins in transcription and replication of measles and sendai paramyxoviruses using nuclear magnetic resonance spectroscopy

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 Obiettivo del progetto (Objective)

'Over the last decade it has become increasingly clear that a large fraction (up to 40%) of the proteins encoded by the human genome are disordered. Intrinsically disordered proteins (IDPs) remain functional despite a lack of a well-defined structure, such that the classical structure-function paradigm breaks down, and new insight into the relationship between primary sequence and molecular function is necessary. The object of this proposal is to study the role of IDPs in the replication and transcription of Measles (MeV) and Sendai viruses (SeV). Replication and transcription of the viral RNA in these related paramyxoviridiae is initiated by an interaction between the intrinsically disordered C-terminal domain, NTAIL, of the nucleoprotein and the highly flexible C-terminal domain, PX, of the phosphoprotein. Upon binding to PX, both NTAIL proteins undergo alpha-helical folding of the molecular recognition element, positioned at the C-terminal end of the unfolded domain of the nucleoprotein. To fully understand the molecular basis of this interaction, and to determine the role of the disordered domains, atomic resolution models of the proteins are necessary in their free, pre-recognition states and in complex with their partner proteins. In order to achieve this aim I will use high field solution state NMR, small angle neutron and X-ray scattering and molecular modeling. Due to its inherent flexibility solution state NMR studies of the NTAIL domain will also be performed in the context of the entire 13C/15N labeled nucleocapsid (particles whose size ranges from 10-500MD). These particles will be further studied using solid state NMR and electron microscopy. This system represents a paradigm of intermolecular interaction involving highly flexible proteins and will therefore reveal features of transient folding upon binding in IDPs, while shedding new light on viral replication molecular mechanisms whose comprehension is crucial for the design of new antiviral drugs.'

Introduzione (Teaser)

Measles virus constitutes the main cause of childhood mortality in developing countries. Elucidation of viral replication is fundamental for the design of effective antiviral treatment strategies.

Descrizione progetto (Article)

Despite its medical importance and the extensive vaccination campaigns, measles has not been eradicated yet. Efforts to design antiviral therapies against the virus replication complex have been unsuccessful as its structure and formation have not been characterised in detail.

The measles virus is an RNA virus containing a genome transcribed through the concerted action of the viral phosphoprotein P and nucleoprotein N (NTAIL). Scientists on the EU-funded VIRAL_IDP project worked on delineating the structure and the mechanism underlying the association of the replication complex proteins. Accumulating evidence suggests that the C-terminal domain of NTAIL is intrinsically disordered. Upon binding to phosphoprotein P, it undergoes helical folding, essentially changing its shape.

To fully understand the molecular basis of this interaction, scientists had to construct atomic resolution models of the proteins before and after replication complex formation. To this end, they employed spectroscopic methods in order to describe the proteins that present high flexibility.

They validated previous observations that NTAIL consisted of multiple helical structures and it was possible to further characterise the dynamics of the inter-conversion between the different populations. This information helped project researchers to elucidate the mechanism by which NTAIL controls the initiation of viral transcription and replication.

Overall, the work by the VIRAL_IDP project shed light into the basic biochemical mechanisms that govern genome replication and transcription in the measles virus. The methods developed during the study for the biophysical characterisation of the measles virus could find applications elsewhere.