Study of the role of Cyclophilins in the viral cycle of Coronaviruses and host restriction

Coronaviruses (CoVs) are viruses causing serious illness in humans and animals. The latest emergence in humans of SARS-CoV and then MERS-CoV are the result of interspecific transmissions between wildlife and humans. Due to their genomic plasticity and their ability to infect multiple animal species, mammals or birds, CoVs represent an emergence threat. Inter-species viral transmission is a problem that involves multiple stages. Beyond the cellular receptor barrier, CoVs infecting a potential new host species must be able to interact with the cellular factors necessary for their replication. Recent studies have shown that cyclophilins (CyP) are proteins essential for the replicative cycle of CoVs, but the precise mechanisms of interaction are not known. In particular, it has not been identified the viral proteins, and their subdomains, interacting with these CyPs. The first objective of the project is therefore to study the involvement of CyP in the viral cycle of different CoVs and to verify that they are indeed necessary for the replication of CoVs (Tasks 1.1 and 2). To meet this objective, small molecules (<500 g / mol) which inhibit cyclophilins generated by partner 2 will be used in particular. Their inhibitory effect has already been verified on HCoV-229E and their use has already made it possible to dissect the mechanism of action of CyPA in HIV replication. The second objective will be to identify the main CyPs involved (Task 1.2). Different strategies will be considered such as the use of siRNAs from different CyPs and the establishment of knock out (KO) lines for the most interesting CyPs. Identifying the main CyPs necessary for the CoV cycle will be necessary in order to then study the interactions between the viral proteins and the Cyps (Tasks 3 and 4). The third objective is to determine the viral proteins of CoVs interacting with cellular CyPs (Task 3). To achieve this objective, an original methodology developed by Y. Jacob (Institut Pasteur), called GPCA (Gaussia princeps Luciferase-based protein-fragment Complementation Assay) will be implemented. It makes it possible to quantitatively assess the level of interaction between two partner proteins. This approach is already used for other projects at partner 1. This work is essential to achieve task 4. The fourth objective is to assess the potential for inter-species transmission of different CoVs in wildlife (Task 4). The emergence of SARS-CoV and then MERS-CoV taught us that CoVs in wildlife could represent a public health risk due to their ability to change host species. The EPICOREM program funded by the ANR from 2013 to 2017 demonstrated the circulation of CoVs in wildlife in France in bats, rabbits, hedgehogs and rodents. If it was not possible to isolate and cultivate these CoVs, the laboratories of ANSES (E. Monchatre-Leroy, ANSES Nancy) and the University of Caen (M. Le Gouil and and A. Vabret , University of Caen) have the sequences of these CoVs and genomic material allowing the sequencing and cloning of genes encoding the viral proteins of interest identified in the previous objective. The work will consist in evaluating the interactions between the viral proteins of interest of these new CoVs and the CyP of homologous and heterologous hosts.