The new virus discovered in December 2019, is a coronavirus causing an outbreak of pulmonary disease. Its rapid spreading over the world led on March 11, the World Health Organization to declare the outbreak a pandemic. The new coronavirus is a positive single stranded RNA molecule that is immediately translated upon its entry into the cell. The first protein to be translated, NSP1, binds to the host small ribosomal subunit 40S and recruits a yet unidentified cellular nuclease that triggers the degradation of the host mRNAs. In this way, the virus hijacks the host translation machinery to its own and exclusive use. Evidence suggests that an internal ribosomal entry site (IRES), immune to the NSP1 inactivating effect, initiates the COVID-19 RNA translation. The current proposal addresses the molecular mechanisms of translation initiation of the viral RNA during the COVID-19 infectious process. We will focus on the role of the 5’UTR of the viral genome in order to determine whether it uses a novel IRES element and to identify the RNA regions implicated. We will also investigate the function of NSP1 protein and characterize the mode of action of translation inhibition of cellular mRNAs. Overall, the main goal of this proposal is to understand the fundamental mechanism of viral translation during COVID-19 infection in order to develop a functional screen for testing specific inhibitors for therapeutic purposes. In preliminary investigations, we have compared the predicted RNA structure of the 5’UTR of COVID-19 with the IRES structures of HCV and CrPV and predicted its 3D structure (coll. Zhichao Miao, Newcastle University). In 3D structure comparisons, we find internal loops as potential function sites and inhibitory elements that would prevent binding of eIF4E, essential component of the canonical translation initiation machinery. In translation assays we have already showed that translation is essentially mediated by a cap-independent mechanism. In this proposal we will characterize the putative IRES by chemical probing and mutagenesis in order to determine its structure. The binding of human 40S particles will be explored by sedimentation assays on sucrose gradients and the scanning ability evaluated by AUG shift assays. The activity of the IRES variants will be further measured by translation assays of IRES-reporters constructs. Altogether, these assays should lead to a comprehensive understanding of the IRES structure and mechanism of ribosome recruitment. In parallel, the cellular trans-acting factors, such as the unknown nuclease recruited by NSP1, will be identified by Mass Spectrometry analysis using purification protocols developed in the lab to purify huge ribosome-mRNP complexes. Later, we will set up a complete pipeline from the NSP1 gene cloning to the atomic structure of NSP1 in complex with the human 40S ribosomal subunit programmed with the IRES RNA by cryo-EM. As COVID-19 spreading is extremely fast, a protocol based on cell-free translation assay will be immediately implemented for high-throughput screening of drugs that have already been shown to be active on RNA IRES elements as well as against available chemical libraries. We are planning to recapitulate the NSP1-mediated specific inhibition using two reporters, a reporter containing 5’UTR from ß-globin that is targeted by NSP1 and another immune reporter containing the COVID-19 IRES. This will allow isolating specific inhibitors that have exclusive effects on NSP1, namely, the inhibitor should specifically target the COVID-19 and be a potential drug candidate against COVID-19 disease. For this part, we will collaborate with two spin-off companies of the University of Strasbourg, NovAliX and Urania Therapeutics, that have expertise in functional and in silico high-throughput screening techniques.