Soutenance de thèse : Structural characterization of Orthoparamyxovirinae P and C proteins encoded by overlapping sequences


Salle des séminaires IBS

par Ada Roy (IBS/Groupe Machines de Réplication Virale)

The Paramyxoviridae family is a large family of non-segmented negative RNA viruses, some of which are pathogenic to humans or domestic animals. Their small genome comprises six transcription units, including the one coding for the phosphoprotein, which is polycistronic and allows the production of both the phosphoprotein, an essential component for the functioning of the RNA synthesis machinery, and one or more accessory proteins, notably involved in the escape from the immune system. In particular, the genome of most members of the Orthoparamyxovirinae subfamily contains a superimposed gene in the sequence of the gene coding for the phosphoprotein but in a different reading frame. Cells infected with these viruses thus express an accessory protein C by a mechanism of alternative translation initiation. This protein is multi-functional and is involved in regulating viral RNA synthesis, antagonising the innate immune response and budding new viral particles. For some viruses, this protein is a virulence factor. However, we lack information on the structure and mechanisms of action of protein C.
The aim of this work was to characterise the structure of the C protein of two paramyxoviruses in order to better understand how this protein can act in infected cells. We resolved the structure of the C protein of Tupaia paramyxovirus (TupV). Despite low sequence similarity, the three-dimensional structure indicates a folding similar to that of the Sendai virus C-terminal domain. In collaboration, we have shown that the TupV C protein inhibits the RNA synthesis machinery of Nipah virus. It has been shown, in the case of measles virus, that protein C inhibits RNA synthesis through an interaction with the phosphoprotein (P). With this in mind, we undertook the structural characterisation of the TupV P and investigated whether it interacts with the protein C.
We used different strategies to express and purify the Nipah virus C protein, alone or in complex with cellular partners. We defined several soluble constructs and used one of these proteins to generate llama nanobodies to use as crystallisation chaperones. We co-expressed the protein C with the ESCRT-I subcomplex and showed that the protein C associates with these proteins.
In conclusion, we demonstrated that the C proteins of the Orthoparamyxovirinae subfamily share a common structure and can cross-act with species belonging to different genera suggesting that they have a common origin.

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