EFFECTORS AND IINHIBITORS OF SARS VIRUS POLYMERASE

SARS病毒聚合酶的效应物和抑制剂

• 批准号: 7028852
• 负责人: Neerja Kaushik-Basu
• 金额: $ 22.78万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-15 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7028852
• 关键词: DNA directed RNA polymerase; SARS virus; antiviral agents; drug design /synthesis /production; drug discovery /isolation; drug screening /evaluation; emerging infectious disease; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; gene expression; molecular cloning; nucleic acid biosynthesis; posttranslational modifications; protein purification; protein structure function; severe acute respiratory syndrome; transfection /expression vector; virus RNA; virus genetics; virus protein; virus replication

DESCRIPTION (provided by applicant): Replication of positive stranded RNA viruses in virus-infected cells is believed to be catalyzed by viral replicase complexes, which may consist of various virally encoded nonstructural proteins including the RNA dependent RNA polymerases (RdRp), and host factors. The multi-domain replicase gene 1 of the SARS coronavirus (SARS-CoV), the recently identified causative agent of Severe Acute Respiratory Syndrome (SARS) encompasses nearly two-thirds of its genome (approximately 22 kb) and is predicted to encode two overlapping polyproteins, ppla (replicase la) and pplab (replicase lab), which undergo cotranslational proteolytic processing to yield a number of functional polypeptides required for viral replication and transcription. Non-structural protein 9 (nsp9), a putative proteolytic product of replicase 1ab is proposed to encode the putative viral RNA dependent RNA polymerase (RdRp) or POL domain, the protein responsible for synthesizing the viral positive strand RNA genome via (-) strand intermediate and therefore represents an attractive target for therapeutic intervention. The goal of this application is to identify inhibitors of this protein. To achieve this, we propose to generate a functionally active SARS-CoV RdRp and establish its biochemical characteristics pertaining to RNA synthesis, substrate utilization and other enzymatic attributes via analogy to other members of the RNA dependent RNA polymerase family. Given the complexity of the replicase gene and the lack of enzymatic and biochemical data on any Coronavirus RdRp, our attempt to generate a functionally active SARS-CoV polymerase capable of replicating the entire 30 kb viral genome remains highly exploratory in nature. We propose to clone the putative polymerase domain (nsp9) of the SARS-CoV in both prokaryotic and eukaryotic expression vectors (in case it requires post-translational modification) and purify the active protein. Another important aspect of these studies is to identify the essential structural elements and domains required to generate a functionally active polymerase. Towards this objective, we have identified a "conserved RdRP domain" of 560 amino acids spanning residues 373 to 932 in the putative SARS-POL domain including the characteristic XDD signature motif of RdRp. Based on secondary structure predictions, we will construct three N-terminal truncation clones of SARS-CoV RdRp, and delineate the contribution of the alpha-helices and beta-sheets formed by the initial N terminal 372 residues. SARS-POL domain contains five "XDD motifs". We propose to experimentally elucidate its catalytic "XDD motif". This will establish similarities and differences between the SARS-CoV RdRp versus all other coronavirus RdRps. To establish the enzymatic activity of the various constructs generated by this approach, specific conditions such as those used by hepatitis C virus replicase, poliovirus replicase, HIV-1 RT and other related RdRp will be utilized. It is possible that SARS-CoV RdRp may exhibit complete variance in its enzymatic characteristic that will also be examined. The proposed investigation will thus establish a new knowledge base for the SARSCoV RdRp in particular and members of the Coronoviridiae RdRp family in general. Further, analysis of the sensitivity of the SARS-CoV RdRp towards a panel of known and novel inhibitors of polymerases will provide new leads in the quest for specific antiviral agents against the SARS-CoV RdRp.

描述（由申请人提供）：据信病毒感染细胞中阳性滞留的RNA病毒的复制被病毒复制酶复合物催化，该复制酶复合物可能由各种病毒编码的非依赖RNA依赖性RNA聚合酶（RDRP）（RDRP）和宿主因子组成。 SARS冠状病毒（SARS-COV）的多域复制酶基因1，最近确定的严重急性呼吸系统综合征（SARS）的病因包括其近三分之二的基因组（约22 kb）（约22 kb）蛋白水解处理以产生病毒复制和转录所需的许多功能多肽。提出了一种非结构蛋白9（NSP9），一种复制酶1AB的推定蛋白水解产物来编码推定的病毒RNA依赖RNA依赖RNA的RNA聚合酶（RDRP）或POL结构域，该蛋白质负责合成病毒正链RNA RNA基因组通过（ - ）链（ - ）链链中的链链中的链链RNA基因组，因此代表了具有诱人的目标。该应用的目的是鉴定该蛋白质的抑制剂。为了实现这一目标，我们建议通过类似于RNA依赖RNA聚合酶家族的其他成员，生成功能活性的SARS-COV RDRP，并建立与RNA合成，底物利用和其他酶促属性有关的生化特征。鉴于复制酶基因的复杂性以及对任何冠状病毒RDRP缺乏酶促和生化数据，我们试图生成能够复制整个30 KB病毒基因组的功能活性SARS-COV聚合酶的尝试仍然具有很高的探索性。我们建议在原核和真核表达载体中克隆SARS-COV的推定聚合酶结构域（NSP9）（如果需要翻译后修饰）并净化活性蛋白。这些研究的另一个重要方面是确定产生功能活性聚合酶所需的基本结构元素和域。为了实现这一目标，我们已经确定了一个560个氨基酸的“保守的RDRP结构域”，这些氨基酸在推定的SARS-POL域中跨越残基373至932，包括RDRP的特征XDD签名基序。基于二级结构预测，我们将构建SARS-COV RDRP的三个N末端截断克隆，并描述由初始N末端372残基形成的α-螺旋和β-片的贡献。 SARS-POL域包含五个“ XDD图案”。我们建议通过实验阐明其催化“ XDD基序”。这将建立SARS-COV RDRP与所有其他冠状病毒RDRP之间的相似性和差异。为了建立该方法产生的各种构建体的酶活性，将利用丙型肝炎病毒复制酶，脊髓灰质炎病毒复制酶，HIV-1 RT和其他相关RDRP的特定条件。 SARS-COV RDRP可能会在其酶促特征上表现出完全的差异，这也将被检查。因此，拟议的调查将为Sarscov RDRP尤其是Coronoviridiae RDRP家族的成员建立一个新的知识基础。此外，对SARS-COV RDRP对一系列已知和新型聚合酶抑制剂的敏感性的分析将为寻求针对SARS-COV RDRP的特定抗病毒剂提供新的潜在客户。