In situ functional genomics to understand transcriptional regulation

项目总结

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.