Revealing the mechanism of nucleotide selection, addition and proofreading of the SARS-coronavirus replication transcription complex at the single molecule level

RNA病毒是一组非常多样化的病原体。这一点特别强调了它们的复制转录复合体（RTC）中涉及的各种各样的因素以及它们在信使和基因组RNA合成模式中的艺术性。病毒RNA的合成由RTC保证，RTC也通过核苷酸错配结合和促进病毒基因组重组来保证进化。然而，对于长基因组单链阳性RNA病毒，如冠状病毒（CoV） （~ 30kb）， RTC的突变率过高，无法产生感染性病毒粒子。因此，冠状病毒通过编码一种RNA校对酶（即3 ‘至5 ’外切酶）来纠正过多的突变，从而解决了这一问题，这也保护了人类致病性冠状病毒，例如中东呼吸综合征（MERS）、严重急性呼吸综合征（SARS）和新型冠状病毒2019 （COVID-19）免受靶向RTC的抗病毒核苷酸类似物的影响。了解冠状病毒外切酶在掺入后如何感知核苷酸错配和类似物仍然是开发更有效的抗病毒药物的重要研究课题。为了表征核苷酸错配和类似物掺入的动力学并推导动力学模型，标准的散装生物化学分析使用短模板（~10 nt）并且没有竞争核苷酸，这是不现实的条件。使用高通量磁镊子和千碱基长模板，我将能够在单分子水平上以接近单碱基分辨率监测SARS-CoV RTC核苷酸错配和类似物结合的动力学特征，并在饱和浓度下与四种天然核苷酸竞争。SARS-CoV RTC可以说是最著名的冠状病毒RTC， CoV RTC的高度保守性将使我的提议未来的结果可扩展到其他冠状病毒，如MERS和COVID-19。我将得出一个完整的动力学模型，描述核苷酸错配结合的动力学，并利用SARS-CoV RTC表征市售核苷酸类似物的作用机制。

RNA viruses are a very diverse group of pathogens. This is particularly underlined by the large variety of factors involved in their replication transcription complex (RTC) and their artfulness in modes of messenger and genomic RNA synthesis. Viral RNA synthesis is ensured by the RTC, which also ensures evolution through nucleotide mismatch incorporations and by promoting viral genome recombination. However, the mutation rate of the RTC is too high to generate infectious virions for long genome single stranded positive RNA viruses, such as coronavirus (CoV) (~30 kb). Therefore, CoV have solved this problem by encoding an RNA proofreading enzyme, i.e. a 3’ to 5’ exonuclease to correct the excessive numbers of mutations, which also protects human pathogenic CoV’s, e.g. the Middle East respiratory syndrome (MERS), severe acute respiratory syndrome (SARS) and new coronavirus 2019 (COVID-19) from antiviral nucleotide analogues targeting the RTC. Understanding how CoV exonuclease senses nucleotide mismatch and analogues after incorporation remains an important research topic to develop more efficient antiviral drugs. To characterize the kinetics of nucleotide mismatch and analogues incorporation and derive a kinetic model, standard bulk biochemistry assays use short templates (~10 nt) and no competing nucleotides, which are not realistic conditions. Using high throughput magnetic tweezers and kilobases long templates, I will be able to monitor the kinetic signature of SARS-CoV RTC nucleotide mismatch and analogue incorporation at the single molecule level with near single base resolution and in competition with the four natural nucleotides at saturating concentrations. SARS-CoV RTC is arguably the best-known coronavirus RTC, and the high degree of conservation of CoV RTC will make the future results of my proposal extendable to other CoVs, such as MERS and COVID-19. I will derive a complete kinetic model describing the kinetics of nucleotide mismatch incorporation and characterize the mechanism of action of commercially available nucleotide analogues using SARS-CoV RTC.