EFFECTORS AND IINHIBITORS OF SARS VIRUS POLYMERASE

SARS病毒聚合酶的效应物和抑制剂

• 批准号: 6873098
• 负责人: Neerja Kaushik-Basu
• 金额: $ 19.34万
• 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 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6873098
• 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（由申请方提供）：认为病毒感染细胞中正链RNA病毒的复制由病毒复制酶复合物催化，病毒复制酶复合物可能由各种病毒编码的非结构蛋白（包括RNA依赖性RNA聚合酶（RdRp））和宿主因子组成。SARS冠状病毒多结构域复制酶基因1 SARS冠状病毒（SARS-CoV）是最近发现的严重急性呼吸系统综合征（SARS）的病原体，其基因组约占其基因组的三分之二（约22 kb），并预测编码两个重叠的多聚蛋白，ppla（复制酶la）和pplab（复制酶实验室），其经历共翻译蛋白水解加工以产生病毒复制和转录所需的许多功能性多肽。非结构蛋白9（Non-structural protein 9，nsp 9）是复制酶1ab的蛋白水解产物，编码病毒RNA依赖性RNA聚合酶（RdRp）或POL结构域，该蛋白负责通过（-）链中间体合成病毒正链RNA基因组，因此是一个有吸引力的治疗靶点。本申请的目的是鉴定该蛋白的抑制剂。为了实现这一点，我们建议产生一个功能活性的SARS冠状病毒RdRp和建立其生化特性有关的RNA合成，底物利用和其他酶的属性通过类比的其他成员的RNA依赖的RNA聚合酶家族。鉴于复制酶基因的复杂性和缺乏任何冠状病毒RdRp的酶和生化数据，我们试图产生一个功能活性的SARS冠状病毒聚合酶能够复制整个30 kb的病毒基因组仍然是高度探索性的性质。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（nsp 9）（如果它需要翻译后修饰）并纯化活性蛋白。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件和结构域。为了实现这一目标，我们已经确定了一个“保守的RdRP域”的560个氨基酸跨越残基373至932在推定的SARS-POL域，包括特征XDD签名基序的RdRP。在二级结构预测的基础上，我们将构建三个SARS-CoV RdRp的N端截短克隆，并描述由初始N端372个残基形成的α-螺旋和β-折叠的贡献。SARS-POL结构域包含5个“XDD基序”。我们建议实验阐明其催化“XDD模体”。这将确定SARS-CoV RdRp与所有其他冠状病毒RdRp之间的相似性和差异。为了确定通过该方法产生的各种构建体的酶活性，将利用特定条件，例如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所使用的条件。SARS-CoV RdRp可能在其酶特性方面表现出完全的差异，这也将被检查。因此，拟议的调查将建立一个新的知识基础，特别是SARSCoV RdRp和冠状病毒RdRp家族的成员一般。此外，SARS-CoV RdRp对一组已知和新型聚合酶抑制剂的敏感性分析将为寻求针对SARS-CoV RdRp的特异性抗病毒剂提供新的线索。