Characterization of SARS coronavirus polymerase and helicase activities

中德联合研究计划的重点是表征冠状病毒RNA依赖性RNA聚合酶（RdRp）和解旋酶活性，这是抗sars治疗的两个有吸引力的靶点。尽管它们在冠状病毒复制中起着关键作用，但关于RdRp和解旋酶的功能和结构信息非常有限，到目前为止，还无法开发出适合治疗包括SARS在内的冠状病毒感染的选择性酶抑制剂（甚至药物）。因此，我们决定采取广泛的生化和分子方法来获得关于这两种酶的酶和结构特性的信息。为此，我们将首先生产广泛的针对SARS冠状病毒（SARS- cov） RdRp和解旋酶蛋白以及其他被认为参与病毒RNA合成的SARS- cov pp1a/pp1ab蛋白的单克隆和多克隆抗体。其次，使用酵母-双杂交（Y2H）方法，我们将尝试确定SARS-CoV RdRp与复制复合体的其他蛋白质的相互作用。第三，在Y2H筛选中发现的潜在蛋白-蛋白相互作用将通过使用代谢标记的sars - cov感染细胞的共免疫沉淀实验加以证实，并通过GST下拉实验进一步详细表征。第四，将对SARS-CoV和其他冠状病毒的RdRp重组形式进行生化表征，以确定其活性，阐明其底物需求并确定其（亚）结构域组织。最后（第五部分），SARSCoV解旋酶的重组形式将从底物特异性、辅因子需求、加工性和活性位点残基方面进行表征。合并后的数据将用于开发适合高通量筛选化合物文库的检测方法，以确定候选抑制剂。这些项目将首次提供有关冠状病毒聚合酶活性及其与冠状病毒多亚基复制复合体其他组分相互作用的信息。在#1到#3中总结的实验将主要由中国小组进行，而德国小组将专注于主题#4和#5。计划安排一名中国合作者在维尔纽斯堡呆至少6个月（如果可能的话，最多一年）。他将参与冠状病毒聚合酶和解旋酶的异源表达、纯化和生化表征。

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.