SUMMARY The use of viral enzymes as targets for antiviral development has led to many antiviral therapies of use in the clinic. Several viral polymerase and/or protease inhibitors have been developed for HIV, HCV and HSV, among others. Less common is the development of antivirals against viral products with unknown enzymatic properties, although some exceptions include the NS5b inhibitors used for the treatment of HCV infections. As many viral products lack clear enzymatic activities that can be used to develop in vitro screen assays for inhibitors, the use of viral enzymes as targets for therapeutic intervention limits the number of available viral targets. In project 4 – Development of CoV Inhibitors Against Non-Enzymatic Targets of this antiviral development consortium, we explore SARS-CoV-2 targets with unknown enzymatic activities and not covered by the additional SARS-CoV-2 projects of this proposal. We have chosen as targets both a viral protein required for virulence, Nsp1, as well as the viral RNA. Both the viral Nsp1 and the viral RNA have specific unique sequences and structures that distinguish them from host molecules. In aim 1, we combine biochemical, genetic, structural and in silico approaches to identify therapeutic molecules that prevent the inhibition of host responses exerted by Nsp1 in infected cells. In aim 2 we use advanced antisense strategies to target specific conserved functional sequences of the viral RNA involved in replication, transcription and/or translation. In aim 3, we plan to identify molecules that bind and prevent the function of conserved RNA structures that guide viral RNA replication and transcription. For this purpose, we have assembled a team of investigators with ample expertise in the use of RNA-based therapies, virology, cell biology, RNA biology and host-virus interactions. This team interacts closely with the Cores of this consortium to benefit of the structural, ADME and PK expertise, as well as of the antiviral assays, animal models and chemical libraries provided by these Cores. While the Project is mainly in an exploratory phase, several hits that target RNA sequences (Aim 2) or RNA structures (Aim 3), have already been identified and will move forward in the antiviral pipeline of our consortium. For others, like Nsp1, there is enough biochemical and cell biology available information, with many of this information generated by the members of our team, that justifies the proposed screen for Nsp1 inhibitors. Advancement of such classes of inhibitors against unconventional viral targets will open the door for the development of new therapies against other viruses and viral products with unknown enzymatic function, expanding our druggable space for the treatment of viral infections.