LAUSR

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a coronavirus identified as the cause of an outbreak of coronavirus disease (COVID-19), which now causes death in over 6% of infected individuals worldwide (1–5). Patients with confirmed infection have reported respiratory illness, such as fever, cough, and shortness of breath (6). Once contacted with the human airway, the spike proteins of this virus can associate with the surface receptors of sensitive cells, which mediate the entrance of the virus into target cells for further replication. Xu and colleagues first modeled the spike protein to identify the receptor for SARS-CoV-2 and indicated that ACE2 (angiotensin-converting enzyme 2) could be the receptor for this virus (7). ACE2 is previously known as the receptor for severe acute respiratory syndrome coronavirus (SARSCoV) and human coronavirus NL63 (HCoV-NL63) (8–10). Studies focusing on the genome sequence and structure of the receptorbinding domain of the spike proteins further confirmed that the new coronavirus can efficiently use ACE2 as a receptor for cellular entry, with an estimated 10to 20-fold higher affinity to ACE2 than SARS-CoV (11, 12). Zhou and colleagues conducted virus infectivity studies and showed that ACE2 is essential for SARSCoV-2 to enter HeLa cells (13). These data indicate that ACE2 is the receptor for SARS-CoV-2. The tissue expression and distribution of the receptor decide the tropism of the virus infection, which has a major implication for understanding its pathogenesis and designing therapeutic strategies. Previous studies have investigated the RNA expression of ACE2 in 72 human tissues and demonstrated its expression in lung and other organs (14). The lung is a complex organ with multiple types of cells, so such real-time PCR RNA profiling based on bulk tissue could mask the ACE2 expression in each type of cell in the human lung. The ACE2 protein level was also investigated by immunostaining in lung and other organs (14, 15). These studies showed that in the normal human lung, ACE2 is mainly expressed by type II alveolar (AT2) and type I alveolar (AT1) epithelial cells. Endothelial cells were also reported to be ACE2 positive. Immunostaining detection is a reliable method for the identification of protein distribution, yet accurate quantification remains a challenge for such analysis. The recently developed single-cell RNA-sequencing technology enables us to study the ACE2 expression in each cell type and provides quantitative information at a single-cell resolution. Previous work has built up the online database for single-cell RNA-sequencing analysis of eight normal human lung transplant donors (16). In the current work, we used the updated bioinformatics tools to analyze the data. Some of the results of these studies have been previously reported in the form of a preprint (https://doi.org/10.1101/2020.01.26.919985) (16). We analyzed 43,134 cells derived from the normal lung tissue of eight adult donors (Figure 1A). We performed unsupervised graphbased clustering (Seurat version 2.3.4), and for each individual, we identified 8–11 transcriptionally distinct cell clusters based on their marker gene expression profile. Typically, the clusters include AT2 cells, AT1 cells, airway epithelial cells (ciliated cells and club cells), fibroblasts, endothelial cells, and various types of immune cells. The cell cluster map of a representative donor (a 55-yr-old Asian man) was visualized using t-distributed stochastic neighbor embedding (tSNE), as shown in Figure 1B. Next, we analyzed the cell type–specific expression pattern of ACE2 in each individual. For all donors, ACE2 is expressed in 0.64% of all human lung cells. The majority of the ACE2expressing cells (83% in average) are AT2 cells. Other ACE2expressing cells include AT1 cells, airway epithelial cells, fibroblasts, endothelial cells, and macrophages. However, their ACE2-expressing cell ratio is relatively low and is variable among individuals. For the representative donor (Asian male, 55 yr old), the expressions of ACE2 and cell type–specific markers in each cluster are demonstrated in Figure 2A. There are 1.46 0.4% of AT2 cells expressing ACE2. To further understand the special population of ACE2-expressing AT2, we performed a gene ontology (GO) enrichment analysis to study which biological processes are involved with this cell population by comparing them with the AT2 cells not expressing ACE2. Surprisingly, we found that multiple viral life cycle–related functions are significantly overrepresented in ACE2-expressing AT2 cells, including those relevant to viral replication and transmission (Figure 2B). We found an upregulation of CAV2 and ITGB6 genes in ACE2-expressing AT2. These genes are components of caveolae, which is a special subcellular structure on the plasma membrane critical to the internalization of various viruses, including SARS-CoV (17–19). We also found an enrichment of multiple ESCRT (endosomal sorting complex required for transport) machinery gene members (including CHMP3, CHMP5, CHMP1A, and VPS37B) in ACE2-expressing AT2 cells that were related to virus budding and release (20, 21). These data showed that this small population of ACE2-expressing AT2 cells is particularly prone to SARS-CoV-2 infection. We further analyzed each donor and their ACE2-expressing patterns. As the sample size was very small, no significant association was detected between the ACE2-expressing cell number and any characteristics of the individual donors. But we did notice that one donor had a five-fold higher ACE2-expressing cell ratio than average. The observation on this case suggested that ACE2expressing profile heterogeneity might exist between individuals, which could make some individuals more vulnerable to SARS-CoV-2 than others. However, these data need to be interpreted very cautiously because of the very small sample size of the current dataset, and a larger cohort study is necessary to draw conclusions. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http:// creativecommons.org/licenses/by-nc-nd/4.0/). For commercial usage and reprints, please contact Diane Gern ([email protected]).