Characterization of SARS coronavirus polymerase and helicase activities

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The joint Chinese-German research proposal focusses on the characterization of coronavirus RNA-dependent RNA polymerase (RdRp) and helicase activities, two attractive targets for anti-SARS therapy. Despite their key role in coronavirus replication, there is only very limited functional and structural information on both the RdRp and helicase, which, thus far, does not allow to develop selective enzyme inhibitors (or even drugs) suitable for the treatment of coronavirus infections including SARS. We therefore decided to take a broad biochemical and molecular approach to obtain information on the enzymatic and structural properties of the two enzymes. For this purpose, we will first produce a broad range of monoclonal and polyclonal antibodies specific for the SARS coronavirus (SARS-CoV) RdRp and helicase proteins as well as other SARS-CoV pp1a/pp1ab proteins presumed to be involved in viral RNA synthesis. Second, using a yeast-two-hybrid (Y2H) approach, we will try to indentify interactions of the SARS-CoV RdRp with other proteins of the replication complex. Third, potential protein-protein interactions identified in the Y2H screen will be confirmed by co-immunoprecipitation experiments using metabolically labelled SARS-CoVinfected cells and characterized in further detail by GST pull-down experiments. Fourth, recombinant forms of the RdRp of SARS-CoV and other coronaviruses will be characterized biochemically to establish their activities, to elucidate their substrate requirements and to determine their (sub)domain organization. Finally (fifth), recombinant forms of the SARSCoV helicase will be characterized in terms of substrate specificity, cofactor requirements, processivity and active-site residues. The combined data will be used to develop assays suitable for high-throughput screening of compound libraries to identify candidate inhibitors. The projects will, for the first time, provide infomation on the activities of coronavirus polymerases and their interactions with other components of the multi-subunit replication complex of coronaviruses. Experiments summarized under #1 through #3 will be carried out mainly by the Chinese group, whereas the German group will focus on topics #4 and #5. It is planned that one of the Chinese collaborators spends at least 6 months (if possible, up to one year) in Würzburg. He will be involved in the heterologous expression, purification and biochemical characterization of coronavirus polymerases and helicases.