In situ functional genomics to understand transcriptional regulation

项目总结 新冠肺炎疫情在短短几个月内就夺走了全球近50万人的生命。 最近，一种新的SARS-CoV-2病毒分离株在Spike蛋白(D614G)上带有点突变， 出现并迅速超过其他流行株，包括来自武汉的最初的SARS-CoV-2毒株， 中国。这种尖峰变异是SARS-CoV-2基因组中最流行的分支(A2a)的一个决定性特征 世界范围内，最近，我们和其他人已经证明了这种变异会导致病毒粒子增加约8倍 在人类细胞转导中。这是首次有实验证据表明SARS-CoV-2群体变异在 一种功利性的方式。 尽管现在有数百种Spike变种在流通，但我们缺乏实现高吞吐量的工具 这些变种的特征和它们的毒力。在这里，我们建议开发一种大规模并行的、高度的 吞吐量方法，使用汇集的正向遗传筛选来测试所有Spike变体，检查 这些突变对SPEKE的蛋白水解性切割和对ACE2受体结合动力学的影响，并验证变化 通过创新的反式互补试验与活体SARS-CoV-2进行病毒转导。 我们建议的研究旨在了解Spike蛋白变体与宿主(人类)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 特定的尖峰变种可以导致更严重的新冠肺炎爆发。

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.