RAPID: Learning about Coronavirus Genome Replication by Interfering with It

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Due to the global pandemic caused by SARS-CoV-2, there is an urgent need to understand how the virus reproduces. After the virus infects a cell, the viral replicase enzyme makes thousands of copies of the viral genome. This project will determine the molecular features of the SARS-CoV-2 RNA genome that enable it to be copied by the coronavirus replicase complex. Having determined those features, the PIs will use that knowledge to design genome-like molecules that may interfere with replication of normal genomes and thus have potential as novel therapeutic agents. The PIs will evaluate the effectiveness of the genome-like molecules at blocking virus replication in cells and in more complicated tissue culture that includes a variety of different cell types meant to mimic the conditions in the human lung. Additionally, the project will broaden participation in STEM by funding a Latina post-doctoral scholar to perform most of the experiments.The PIs will use a combination of biochemistry and culture to determine the minimal features of a SARS-CoV-2 replicon that enable it to be replicated. Next, the PIs will apply that knowledge to design defective-interfering RNA that can be replicated very efficiently yet does not encode infectious virus particles. Extending from natural defective-interfering particles in other viruses, this RNA will likely serve as a template for the coronavirus replicase. When an intact virus penetrates a cell that contains defective-interfering RNA, the defective interfering RNA will serve as a template for the virus replicase. Doing so will interfere with viral replication by occupying the replicase and consuming nucleotides that could otherwise be incorporated into new intact virus genomes. The interfering RNAs will be delivered using a virus-like particle system designed to enter cells expressing the SARS-CoV-2 receptor and the protease necessary for SARS-CoV-2 entry. The PIs will determine the extent to which defective-interfering particles block replication in simple tissue culture or in human airway epithelial organoids, which have multiple differentiated cell types iorganized similarly to those found in explanted tissue. The epithelial organoids are derived from healthy human tissue donors and represent some of the genetic diversity in human populations. Broader impacts include the potential for basic understanding of the interfering particles to contribute to developing similar particles to treat or prevent COVID-19.This RAPID award is made by the Genetic Mechanisms Program in the Division of Molecular and Cellular Biosciences to respond to the COVID-19 pandemic.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.