Characterization of the activity of an enzyme and its inhibitors involved in the replication of SARS-CoV-2 by study on the individual molecule

Nsp13 helicase is the molecular motor of SARS-CoV-2 RNA unwinding and is essential for its replication. Although it is one of the best conserved proteins of the coronovirus family (100% helicase sequence similarity between SARS-CoV-1, SARS-CoV-2 and Mers-CoV) , its functional properties are still poorly understood, the main reason being that standard bulk experiments provide poor characterization of this type of molecular motors of DNA / RNA unwinding. This is because when a helicase translocates onto a duplex nucleic acid (DNA), the duplex can fold behind the helicase, thus preventing observation of the dynamics of the enzyme with standard electrophoresis gels. Such methods allow the action of the enzyme to be observed only on very small substrates (about 20 bp) which cannot fold after passage of the enzyme, and therefore do not accurately describe the dynamics of the enzyme. motor (processivity, speed, pauses induced by the sequence, change of strand, etc.). In addition, their conclusions are often ambiguous because the observed and quantified dehybridization of short duplexes can occur spontaneously during helicase binding by the fusion of a few bases, and is therefore not a completely reliable indicator of the activity of unfolding. By relying on the strong expertise of our consortium in the field of monomolecular helicase studies (and in particular on Upf1, a human helicase structurally very similar to Nsp13), we wish to provide the community working on the virus with a precise functional characterization of the enzyme. By describing the functional properties of the helicase of the virus, which can only be observed through measurements on the single molecule, this project therefore aims to: - quickly reveal a poorly understood component of the virus proteome, essential for the dissemination of the virus. - validate ~ 10 potential therapeutic pathways targeting helicase.