Transcriptional regulation of ACE2 and the adaption of SARS-CoV-2

摘要 严重急性呼吸综合征冠状病毒2(SARS-CoV-2)，冠状病毒病的病原体 2019年(新冠肺炎)，引发了一场全球健康危机。目前有有效的疫苗可用于预防SARS- CoV-2感染，但我们治疗SARS-CoV-2感染的选择仍然有限。要通知 开发旨在对抗SARS-CoV-2感染的创新干预措施，我们需要更好的 了解SARS-CoV-2感染的分子细节，特别是宿主依赖因素 病毒感染所需的。我们已经进行了全基因组CRISPR基因敲除筛查，并鉴定出PCBD1， TRAF3和RAD54L2是SARS-CoV-2感染所需的新宿主因子。我们的初步数据显示 血管紧张素转换酶2的转录需要PCBD1、TRAF3和RAD54L2 (ACE2)。ACE2是SARS-CoV-2进入细胞的主要受体，在病毒感染中起关键作用 和发病机制。血管紧张素转换酶2在多种人体组织中差异表达。然而，分子 ACE2在不同组织中转录调控的基础尚不清楚，这构成了一个明确的 SARS-CoV-2研究中的知识缺口。在目标1中，我们将确定PCBD1、TRAF3和 RAD54L2在不同组织来源的Vero E6细胞系ACE2转录调控中的作用 CALU-3(人肺)、C2BBe1(人大肠)和HK-2(人肾)。SARS-CoV-2 通过基因突变不断进化以适应宿主。我们发现了一系列的突变 可能促进SARS-CoV-2在细胞内复制的刺激物(S)和核衣壳(N)在低水平表达 ACE2。一些突变(如N S194L)在印度和墨西哥早期流行， 这表明这些变异可能有助于病毒对宿主的适应。在目标2中，我们将分析其功能 刺突病毒(N81S、L242P、E484D和P1079T)和核衣壳变异体(S194L)在病毒粒子生产和细胞中的作用 SARS-CoV-2的进入。我们建议的研究将提供关于转录调控的新信息 ACE2和SARS-CoV-2的适应。从长远来看，这些研究将提供新的目标和战略。 用于开发抗病毒药物。

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19), has caused a global health crisis. Currently effective vaccines are available to prevent SARS- CoV-2 infection, but our options for the treatment of SARS-CoV-2 infection remain limited. To inform the development of innovative interventions designed to counteract SARS-CoV-2 infection, we need a better understanding of the molecular details of SARS-CoV-2 infection, especially the host dependency factors that are required for virus infection. We have performed a genome-wide CRISPR knockout screen and identified PCBD1, TRAF3, and RAD54L2 as novel host factors required for SARS-CoV-2 infection. Our preliminary data showed that PCBD1, TRAF3, and RAD54L2 are required for the transcription of angiotensin converting enzyme 2 (ACE2). ACE2 is the primary receptor for the cellular entry of SARS-CoV-2, and plays key roles in virus infection and pathogenesis. ACE2 is differentially expressed in a wide variety of human tissues. However, the molecular basis for the transcriptional regulation of ACE2 in different tissues remains unexplored, which constitutes a clear knowledge gap in SARS-CoV-2 research. In Aim 1, we will determine the function of PCBD1, TRAF3, and RAD54L2 in transcriptional regulation of ACE2 in four cell lines derived from different tissues: Vero E6 (monkey kidney), Calu-3 (human lung), C2BBe1 (human large intestine), and HK-2 (human kidney). SARS-CoV-2 continuously evolves by genetic mutations for the adaption to the host. We identified a number of mutations in Spike (S) and Nucleocapsid (N) that may enhance the replication of SARS-CoV-2 in cells expressed low levels of ACE2. Some mutation (such as N S194L) was prevalent in India and Mexico during early circulation, suggesting these variants may contribute to the viral adaption to the host. In Aim 2, we will analyze the function of Spike (N81S, L242P, E484D, and P1079T) and Nucleocapsid (S194L) variants in virion production and cell entry of SARS-CoV-2. Our proposed studies will provide new information regarding the transcriptional regulation of ACE2 and the adaption of SARS-CoV-2. In the long term, these studies will provide new targets and strategies for the development of antiviral drugs.