In situ functional genomics to understand transcriptional regulation

项目摘要 COVID-19疫情在短短数月内夺走了全球近50万人的生命。 最近，一种携带刺突蛋白点突变（D 614 G）的SARS-CoV-2病毒新分离株， 出现并迅速超过其他病毒，包括来自武汉的SARS-CoV-2原始分离株， 中国这种刺突变异是SARS-CoV-2基因组中最普遍的进化枝（A2 a）的一个定义特征 最近，我们和其他人已经证明这种变异导致病毒体增加约8倍 在人类细胞转导中。这是SARS-CoV-2群体变异体作用于 功能增益方式。 虽然现在有数百种Spike变体在流通，但我们缺乏高通量的工具。 这些变异体的特征及其毒力。在这里，我们建议发展一个平行的，高- 通量方法，使用合并正向遗传筛选检测所有刺突变体，检查 这些突变对刺突蛋白水解切割和ACE 2受体结合动力学的影响，并验证了这些变化 通过一种创新的反式互补分析，用活SARS-CoV-2进行病毒转导。 我们提出的研究旨在了解刺突蛋白变体与宿主（人）之间的相互作用。 细胞感染及其潜在的生化机制。这项研究将使我们能够预测 特定的Spike变体可能导致更严重的COVID-19爆发。

PROJECT SUMMARY The COVID-19 pandemic has taken the lives of nearly 500,000 people worldwide in the span of a few months. Recently, a novel isolate of the SARS-CoV-2 virus carrying a point mutation in the Spike protein (D614G) has emerged and rapidly surpassed others in prevalence, including the original SARS-CoV-2 isolate from Wuhan, China. This Spike variant is a defining feature of the most prevalent clade (A2a) of SARS-CoV-2 genomes worldwide and, recently, we and others have demonstrated this variant leads to virions with an ~8-fold increase in human cell transduction. This is the first experimental evidence of a SARS-CoV-2 population variant acting in a gain-of-function manner. Although there are hundreds of Spike variants now in circulation, we lack tools for high-throughput characterization of these variants and their virulence. Here, we propose to develop a massively-parallel, high- throughput approach to test all Spike variants using a pooled forward genetic screen, examine the impact of these mutations on proteolytic cleavage of Spike and on ACE2 receptor binding kinetics, and validate changes in viral transduction with live SARS-CoV-2 via an innovative trans-complementation assay. Our proposed studies aim to understand the interactions between Spike protein variants and host (human) cell infection and their underlying biochemical mechanisms. This research will enable us to predict whether particular Spike variants can drive more serious COVID-19 outbreaks.