Coronavirus antiviral nucleoside analogs: inhibition and reduced susceptibility

项目总结 冠状病毒(CoV)是一类能引起人类呼吸道疾病的正义型rna病毒。 从普通感冒到严重而致命的疾病。SARS冠状病毒(CoV)在中国的出现 2002年和MERS冠状病毒的持续传播强调了冠状病毒引起新的人畜共患病的能力 具有大流行潜力的感染。尽管这些感染的死亡率很高，但没有治疗方法或 目前已有针对任何CoV的疫苗可用。广谱抗病毒核苷类似物，如 利巴韦林(RBV)对冠状病毒无效。这一局限归因于独特的校对 非结构蛋白14(nsp14-exon)中的外显子，帮助依赖RNA的RNA 编码在非结构蛋白12(nsp12-RdRp)中的聚合酶(RdRp)在这些大的 正链RNA病毒。我们已经鉴定了两个抗病毒核苷类似物GS-5734和EIDD-1931，在 分别与吉列德科学公司和埃默里药物开发研究所合作，这两家机构 对多种冠状病毒具有广泛的活性，细胞毒性最小。此外，我们还发现了两种突变 在NSP12-RdRp的预测指状域内，降低对GS-5734的易感性，一种C-腺苷 核苷类似物。该提案的目标是定义这些抗病毒药物通过哪些机制 化合物抑制冠状病毒复制并确定抗药性突变对病毒适合性、复制的影响 保真度和核苷酸选择性。在特定目标1中，GS-5734和EIDD-1931介导的抑制的基础 将使用深度测序、RT-qPCR和Northern印迹分析来确定CoV复制的 核苷类似物显示的两种最常见的抗病毒作用机制之间：链 终止和致命性突变。在具体目标2中提出的实验将确定 降低对GS-5734和EIDD-1931易感性的变异在冠状病毒复制保真度、病毒适应性、 以及对其他核苷类似物的敏感性。总之，这些研究将探索GS-5734和 EIDD-1931抑制冠状病毒复制并探索这些抗病毒核苷类似物治疗冠状病毒的潜力 单独和合作作为针对现有和新出现的CoV的有效疗法。这项研究 还将为开发广泛活跃和互补的抗病毒方法提供信息，以对抗冠状病毒 感染。最后，这些研究将利用GS-5734和EIDD-1931作为更好地理解机制的工具 以及冠状病毒复制效率和保真度的病毒介体。

PROJECT SUMMARY Coronaviruses (CoVs) are a family of positive-sense RNA viruses that cause respiratory illnesses in humans ranging from the common cold to severe and lethal disease. The emergence of SARS-coronavirus (CoV) in 2002 and the continued circulation of MERS-CoV emphasize the capacity of CoVs to cause new zoonotic infections with pandemic potential. Despite the high mortality rates of these infections, no therapeutics or vaccines against any CoVs are currently available. Broadly active antiviral nucleoside analogs such as Ribavirin (RBV) are ineffective against CoVs. This limitation is attributed to a unique proofreading exoribonuclease (ExoN) in nonstructural protein 14 (nsp14-ExoN) that aids the RNA-dependent RNA polymerase (RdRp) encoded in nonstructural protein 12 (nsp12-RdRp) in high fidelity replication of these large positive-strand RNA viruses. We have identified two antiviral nucleoside analogs, GS-5734 and EIDD-1931, in collaboration with Gilead Sciences and the Emory Institute for Drug Development, respectively, which are broadly active against multiple CoVs with minimal cytotoxicity. In addition, we have identified two mutations within the predicted fingers domain of the nsp12-RdRp that reduce susceptibility to GS-5734, a C-adenosine nucleoside analog. The goals of this proposal are to define mechanisms through which these antiviral compounds inhibit CoV replication and determine the impact of resistance mutations on viral fitness, replication fidelity, and nucleotide selectivity. In Specific Aim 1, the basis of GS-5734 and EIDD-1931-mediated inhibition of CoV replication will be defined using deep sequencing, RT-qPCR, and Northern blot analysis to distinguish between the two most common mechanisms of antiviral action displayed by nucleoside analogs: chain termination and lethal mutagenesis. Experiments proposed in Specific Aim 2 will determine the impact of mutations that reduce susceptibility to GS-5734 and EIDD-1931 on coronavirus replication fidelity, viral fitness, and susceptibility to other nucleoside analogs. Together, these studies will probe mechanisms of GS-5734 and EIDD-1931 inhibition of CoV replication and explore the potential for these antiviral nucleoside analogs to individually and cooperatively serve as potent therapies against existing and emerging CoVs. This research also will inform the development of broadly active and complementary antiviral approaches to combat CoV infections. Finally, these studies will utilize GS-5734 and EIDD-1931 as tools to better understand mechanisms and viral mediators of CoV replication efficiency and fidelity.