EFFECTORS AND IINHIBITORS OF SARS VIRUS POLYMERASE

描述(申请人提供)：正链RNA病毒在病毒感染细胞中的复制被认为是由病毒复制酶复合体催化的，该复合体可能由各种病毒编码的非结构蛋白组成，包括RNA依赖的RNA聚合酶(RdRp)和宿主因子。SARS冠状病毒(SARS-CoV)是新近发现的严重急性呼吸综合征(SARS)的病原体，其多结构域复制酶基因1包含近三分之二的基因组(约22kb)，并被预测编码两个重叠的多蛋白，PPLA(复制酶1a)和Pplab(复制酶Lab)，它们经过共翻译蛋白水解酶加工产生许多病毒复制和转录所需的功能多肽。非结构蛋白9(NSP9)是复制酶1AB的蛋白水解物，编码病毒RNA依赖的RNA聚合酶(RdRp)或POL结构域，负责通过(-)链中间产物合成病毒正链RNA基因组，因此是治疗干预的一个有吸引力的靶点。这项应用的目标是确定这种蛋白质的抑制剂。为了实现这一目标，我们建议通过与依赖RNA的RNA聚合酶家族的其他成员类比来构建具有功能活性的SARS-CoV RdRp，并建立其与RNA合成、底物利用和其他酶属性有关的生化特性。鉴于复制酶基因的复杂性和任何冠状病毒RdRp的酶和生化数据的缺乏，我们试图产生功能活跃的SARS-CoV聚合酶，能够复制整个30kb的病毒基因组，本质上仍然是高度探索性的。我们建议在原核和真核表达载体中克隆SARS-CoV的假定聚合酶结构域(Nsp9)(以防需要翻译后修饰)，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。为此，我们在SARS-POL结构域中发现了一个由560个氨基酸组成的“保守的RdRP结构域”，跨越373到932个残基，其中包括RdRp的XDD特征基序。在二级结构预测的基础上，我们将构建SARS冠状病毒RdRp的三个N末端截断克隆，并描述由最初的N末端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”。我们建议从实验上阐明它的催化“XDD基序”。这将确定SARS冠状病毒RdRp与所有其他冠状病毒RdRp的异同。为了确定由这种方法产生的各种构建体的酶活性，将利用特定的条件，例如丙型肝炎病毒复制酶、脊髓灰质炎病毒复制酶、HIV-1RT和其他相关RdRp所使用的条件。SARS-CoV RdRp有可能在其酶特性上表现出完全不同，这也将被检测。因此，拟议的调查将特别为SARSCoV RdRp以及冠状病毒科RdRp家族的一般成员建立一个新的知识库。此外，对SARS冠状病毒RdRp对一组已知和新型聚合酶抑制剂的敏感性的分析将为寻找针对SARS冠状病毒RdRp的特定抗病毒药物提供新的线索。

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.