Etude structurale et fonctionnelle de la protéine ORF3 du VHE par RMN en solution

Hepatitis E Virus (HEV) is the most common cause of acute viral hepatitis worldwide with over 20 million infections and around 44,000 deaths recorded annually. Until today, 8 HEV genotypes are reported with only the first four genotypes infecting humans. In developing countries, HEV is transmitted via the fecal-oral route through contaminated drinking water, whereas in developed countries, the transmission occurs by consumption of uncooked or undercooked meat from infected animals. There is no specific treatment for HEV infection, apart from a vaccine which is available only in China. Therefore, HEV represents a public health problem which is constantly growing around the world. HEV is small, icosahedral virus with 27 to 34 nm diameter and classified in the Hepeviridae family. Virions can be found as quasi-enveloped, by host-cell-derived membranes, in bloodstream or as non-enveloped in the faeces and bile of infected individuals. The virus contains a ~7.2 kb positive-sense, single-stranded RNA genome which comprises three open reading frames: ORF1, ORF2 and ORF3. ORF1 encodes a non-structural polyprotein that includes multiple functional domains responsible for the replication of the viral genome. ORF2 encodes the viral capsid protein that assembles to make the viral particles. ORF3 encodes a small regulatory multifunctional protein which is poorly characterized. Previous studies have shown that ORF3 is mainly involved in the release of the infectious viral particles and interacts with other viral and host proteins inside the cell. This small protein is thought to interact with the human Ubiquitin E2 variant (UEV) domain of Tsg101 protein (Tsg101 UEV), a member of endosomal sorting complex required for transport-I (ESCRT-I), which has been involved in the release of HIV, Ebola and other viruses. Previous studies have also shown that ORF3 protein is associated with the cellular membranes either via its oligomerization and transmembrane insertion, or via palmitoylation of its N-terminal Cysteine-rich region.In this study, a detailed molecular characterization of the ORF3 protein in order to decipher its functional role(s) during the HEV life cycle is achieved using NMR spectroscopy and other biophysical techniques. Firstly, we performed HEV ORF3 recombinant expression in. E. coli and purified the protein. Its structural characterization by solution-state NMR spectroscopy shown that it is an intrinsically disordered protein devoid of any stable 3D structure organization. Secondly, for the further determination of the ORF3 protein membrane association, the Nanodisc (ND) technology is used which mimics a bilayer membrane and thus, is closed to the native environment of membrane proteins. Although the attachment of the protein into a ND assembly was successful, a reliable conclusion about the membrane attachment of the protein cannot be drawn based on our experimental results. Finally, we in-depth characterized the molecular interaction between HEV ORF3 and Tsg101 UEV proteins. We identified the binding sites in both ORF3 and Tsg101 UEV proteins by NMR spectroscopy, we measured the affinity of the interaction using Isothermal Titration Calorimetry (ITC) and finally a high-resolution atomic structure of the complex between Tsg101 UEV protein and a 10-residues ORF3-derived peptide was solved by X-ray crystallography. In addition, we used our biochemical and structural data to start the setup of an experimental assay that could be used to detect potential antiviral compounds targeting the ORF3-Tsg101 interaction.