RAPID: Learning about Coronavirus Genome Replication by Interfering with It

由于SARS-CoV-2引起的全球大流行，迫切需要了解病毒如何繁殖。病毒感染细胞后，病毒复制酶会复制数千份病毒基因组。 该项目将确定SARS-CoV-2 RNA基因组的分子特征，使其能够被冠状病毒复制酶复合物复制。 在确定了这些特征之后，PI将利用这些知识来设计可能干扰正常基因组复制的基因组样分子，从而具有作为新型治疗剂的潜力。 PI将评估基因组样分子在细胞和更复杂的组织培养中阻断病毒复制的有效性，其中包括旨在模拟人类肺部条件的各种不同细胞类型。 此外，该项目还将扩大STEM的参与范围，资助一名拉丁裔博士后学者进行大部分实验。PI将使用生物化学和培养相结合的方法来确定SARS-CoV-2复制子的最小特征，使其能够被复制。 接下来，PI将应用这些知识来设计可以非常有效地复制但不编码感染性病毒颗粒的缺陷干扰RNA。 从其他病毒中的天然缺陷干扰颗粒延伸而来，这种RNA可能会作为冠状病毒复制酶的模板。当一个完整的病毒穿透含有缺陷干扰RNA的细胞时，缺陷干扰RNA将作为病毒复制酶的模板。 这样做将通过占据复制酶和消耗核苷酸来干扰病毒复制，否则这些核苷酸可以并入新的完整病毒基因组中。 干扰RNA将使用病毒样颗粒系统进行递送，该系统设计用于进入表达SARS-CoV-2受体和SARS-CoV-2进入所必需的蛋白酶的细胞。 PI将确定缺陷干扰颗粒在简单组织培养物或人气道上皮类器官中阻断复制的程度，所述类器官具有多种分化的细胞类型，其组织类似于在组织中发现的细胞类型。 上皮类器官来源于健康的人类组织供体，代表了人类群体中的一些遗传多样性。更广泛的影响包括对干扰颗粒的基本了解的潜力，以有助于开发类似的颗粒来治疗或预防COVID-19。该奖项反映了NSF的法定使命，并通过使用基金会的知识产权进行评估，优点和更广泛的影响审查标准。

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.