Characterization of SARS coronavirus polymerase and helicase activities

SARS 冠状病毒聚合酶和解旋酶活性的表征

• 批准号: 5437785
• 负责人: Professor Dr. John Ziebuhr
• 金额: --
• 依托单位: Department of Molecular Virology
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2004
• 资助国家: 德国
• 起止时间: 2003-12-31 至 2007-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5437785?language=en
• 关键词: Characterization; SARS; coronavirus; polymerase; helicase

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.

中德联合研究计划的重点是冠状病毒RNA依赖的RNA聚合酶（RdRp）和解旋酶活性的表征，这是抗SARS治疗的两个有吸引力的靶点。尽管RdRp和解旋酶在冠状病毒复制中起着关键作用，但它们的功能和结构信息非常有限，到目前为止，还不能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物化学和分子方法来获得这两种酶的酶和结构特性的信息。为此目的，我们将首先生产广泛的单克隆和多克隆抗体，特异性的SARS冠状病毒（SARS-CoV）RdRp和解旋酶蛋白，以及其他SARS-CoV pp 1a/pp 1ab蛋白，推测参与病毒RNA合成。第二，使用酵母双杂交（Y2 H）的方法，我们将试图确定SARS冠状病毒RdRp与复制复合物的其他蛋白质的相互作用。第三，在Y2 H筛选中鉴定的潜在蛋白质-蛋白质相互作用将通过使用代谢标记的SARS-CoV感染细胞的免疫共沉淀实验来证实，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重组形式进行生物化学表征，以确定其活性，阐明其底物要求并确定其（子）结构域组织。最后（第五），重组形式的SARSCoV解旋酶的特点是底物特异性，辅因子的要求，持续合成能力和活性位点残基。组合的数据将用于开发适合于化合物文库的高通量筛选的测定，以鉴定候选抑制剂。这些项目将首次提供有关冠状病毒聚合酶的活动及其与冠状病毒多亚基复制复合物其他组分相互作用的信息。在#1至#3中总结的实验将主要由中国组进行，而德国组将专注于主题#4和#5。根据计划，其中一名中国合作者将在维尔茨堡至少停留6个月（如有可能，最长可达一年）。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。