The goal of this project is to identify and characterize novel targets for the development of therapeutics against the newly discovered coronavirus that causes Severe Acute Respiratory Syndrome (SARS-CoV). SARS-CoV emerged in 2002 and infected over 8000 people world-wide, causing 774 deaths. Currently, there are no FDA approved vaccines or anti-viral agents directed against this virus. SARS-CoV is classified in the order Nidovirales, which are enveloped, positive-strand RNA viruses that generate a nested set of mRNAs during viral replication. The RNA replicase that mediates transcription and replication is encoded in the 5'-most 21 kb of the 29.7 kb RNA genome. The replicase gene is translated to generate polyproteins pp1a and pp1ab, which are processed by viral proteases SCLpro and PLpro, and assembled onto intracellular membranes to generate the viral replication complex. We hypothesize that SARS-CoV SCLpro and PLpro are potential targets for the development of antiviral agents. Our preliminary data demonstrates that novel SCLpro inhibitors designed and synthesized by our collaborators effectively block SARS-CoV replication. To identify and characterize a second therapeutic target, we expressed the SARS-CoV PLpro domain and established frans-cleavage assays to characterize protease activity. In this proposal, we describe experiments to further characterize SARS-CoV PLpro activity, and experiments to assess the activity of second generation SCLpro inhibitors. The results of these experiments will provide critical information for the further development of protease inhibitors that may be used as therapeutic agents to reduce SARS-CoV replication and pathogenesis.