Novel Nucleic Acid-Binding Proteins in Emerging Viruses

摘要 SARS和MERS是具有大流行潜力的人类呼吸道冠状病毒。的MERS 该病毒影响全球25个国家，病死率为30- 40%。没有 针对冠状病毒的有效药物或疫苗只有少数冠状病毒蛋白（10-35%）具有 解决结构，使得很难理解病毒蛋白质与结合伴侣的相互作用。 我们研究的长期目标是确定不寻常的结构生物化学 核酸结构，包括聚（ADP-核糖），以及识别和 处理这些生物分子。对冠状病毒（CoV）基因组的分析表明，许多 冠状病毒蛋白具有潜在的核酸结合和加工功能。鸟嘌呤 四链体有助于病毒基因的转录和翻译调节。这 调节允许病毒逃避免疫系统并控制病毒基因表达。的 鸟嘌呤四链体结合蛋白的作用才刚刚开始被认识， 鸟嘌呤四链体结合的结构基础知之甚少。 另一种不寻常的核酸，聚（ADP-核糖），是一种翻译后修饰， 调节超过20种生物化学途径。PAR和PAR合成酶 抗病毒活性和病毒与宿主的相互作用。目前还不知道 这种PTM对肽和蛋白质，以及PAR本身的构象知之甚少。 我们的研究采用溶液核磁共振，生物化学和计算技术， 研究这些病毒蛋白。我们工作的中心假设是， 四链体结合和PAR结合的宏结构域蛋白存在于非结构蛋白中 冠状病毒。我们的工作将发展这些蛋白质的结构-功能关系， 其中许多相对于已知蛋白质是高度不同的。我们专注于新兴病毒 如MERS、SARS和蝙蝠冠状病毒，它们是主要的动物宿主， 冠状病毒进化我们将使用凝胶迁移试验、酶试验和SELEX来鉴定病毒 和作为病毒蛋白相互作用靶的宿主序列。我们将使用大规模的 生物化学筛选以鉴定这些蛋白质的其他生物化学功能。另外我们 将检查不寻常的核酸PAR的构象，并确定构象 使用分子模拟、快速采集NMR和 酶促合成以产生新的同位素标记的PAR低聚物。构象变化 在PAR化后可能有助于酶的激活或抑制，并且具有重要的 对抑制剂和抗病毒药物设计的影响。

Abstract SARS and MERS are human respiratory coronaviruses with pandemic potential. The MERS virus affects 25 countries worldwide and has a case fatality rate of 30-40%. There is no effective drug or vaccine against a coronavirus. Only a few coronavirus proteins (10-35%) have solved structures, making it difficult to understand viral protein interactions with binding partners. The long-term goal of our research is to define the structural biochemistry of unusual nucleic acid structures, including poly(ADP-ribose), and the viral proteins that recognize and process these biomolecules. Analysis of the coronavirus (CoV) genome shows that many coronavirus proteins have potential nucleic acid binding and processing functions. Guanine quadruplexes contribute to transcriptional and translational regulation of viral genes. This regulation allows the virus to evade the immune system and control viral gene expression. The roles of guanine quadruplex-binding proteins are just beginning to be recognized, and the structural basis of guanine quadruplex binding is poorly understood. Another unusual nucleic acid, poly(ADP-ribose), is a post-translational modification that regulates more than 20 biochemical pathways. PAR and PAR-synthesizing enzymes contribute to antiviral activity and virus-host interactions. There is presently no knowledge of the effect of this PTM on peptides and proteins, and little knowledge of the conformation of PAR itself. Our research employs solution NMR, biochemical and computational techniques to investigate these viral proteins. The central hypothesis of our work is that both guanine quadruplex-binding and PAR-binding macrodomain proteins occur in the nonstructural proteins of coronaviruses. Our work will develop structure-function relationships for these proteins, many of which are highly divergent relative to known proteins. We focus on emerging viruses such as MERS, SARS, and bat coronaviruses, which are primary animal reservoirs for coronavirus evolution. We will use gel shift assays, enzyme assays and SELEX to identify viral and host sequences that are targets of viral protein interaction. We will use large-scale biochemical screens to identify other biochemical functions for these proteins. In addition, we will examine conformations of the unusual nucleic acid PAR and determine the conformational effects of PARylation on proteins using molecular simulations, rapid acquisition NMR, and enzymatic synthesis to produce new isotope-labeled PAR oligomers. Conformational changes upon PARylation may contribute to enzymatic activation or inhibition, and have important implications for the design of inhibitors and antivirals.