A Single-Cell RNA Expression Map of Human Coronavirus Entry Factors

Abstract

To predict the tropism of human coronaviruses, we profile 28 SARS-CoV-2 and coronavirus-associated receptors and factors (SCARFs) using single-cell transcriptomics across various healthy human tissues. SCARFs include cellular factors both facilitating and restricting viral entry. Intestinal goblet cells, enterocytes, and kidney proximal tubule cells appear highly permissive to SARS-CoV-2, consistent with clinical data. Our analysis also predicts non-canonical entry paths for lung and brain infections. Spermatogonial cells and prostate endocrine cells also appear to be permissive to SARS-CoV-2 infection, suggesting male-specific vulnerabilities. Both pro- and anti-viral factors are highly expressed within the nasal epithelium, with potential age-dependent variation, predicting an important battleground for coronavirus infection. Our analysis also suggests that early embryonic and placental development are at moderate risk of infection. Lastly, SCARF expression appears broadly conserved across a subset of primate organs examined. Our study establishes a resource for investigations of coronavirus biology and pathology.

Keywords: COVID-19; SARS-CoV-2; coronaviruses; restriction factors; scRNA-seq; viral receptors.

Graphical abstract

Figure 1

SARS-CoV-2 and SCARFs A cartoon illustration of the infection cycle of SARS-CoV-2 and its cellular interaction with entry factors (cell surface receptors, proteases, and RFs) and post-entry factors (replication and assembly/trafficking factors) considered in this study.

Figure 2

SCARF Expression in Preimplantation Embryo and at the MFI (A) Heatmap of SCARF transcript levels in each stage of human preimplantation development (n = 20 oocytes and embryos, 124 single cells). RPKM, reads per kilobase of transcript per million mapped reads; ICM, inner cell mass; EPI, epiblast; PE, primitive endoderm; TE, trophectoderm; ESC, embryonic stem cell. (B) Uniform Manifold Approximation and Projection (UMAP) plot illustrating cell clusters identified at the MFI (n = 22 donors, 64,054 single cells). (C) RNA transcript intensity and density of SCARFs across the major cell types of the MFI. The dot color scales from blue to red, corresponding to lower and higher expression, respectively. The size of the dot is directly proportional to the percentage of cells expressing the gene in a given cell type. Star represents the significant enrichment of the percentage of positive cells calculated using one-sided Fisher’s exact test and adjusted by Bonferroni correction. (D) Feature plots displaying unsupervised identification of cells expressing ACE2, DPP4, BSG, and ANPEP over the UMAP of the MFI. Cells are colored according to expression levels, from light blue (low expression) to red (high expression). (E) Boxplots showing the percentages of ACE2⁺TMPRSS2⁺, DPP4⁺TMPRSS2⁺, and BSG⁺TMPRSS2⁺ cells in the major cell types of the MFI (see also Table S4). Each dot represents an individual sample. Stars represent the adjusted p value obtained by Fisher’s exact test adjusted by Bonferroni correction.

Figure 3

SCARF Expression in Reproductive Organs (A) SCARF expression in the adult testis (n = 2, 6,572 single cells). ST, spermatid; SPG, spermatogonia; SSC, spermatogonial stem cell. (B) Heatmap of the fraction of double-positive cells for different receptor-protease combinations in each of the major cell types of the testis. Color scheme includes white for the lowest fraction (<0.05% of cells), gold for medium (0.05%–2%), purple for high (2%–6%), and dark blue for the highest fraction (>6%) of double-positive cells per cell type. (C) Boxplots showing average single-cell expression of SCARFs in different testis cell types. (D) SCARF expression in the adult ovary (n = 5, 12,160 single cells). Note that TMPRSS2 and TMPRSS4 transcripts were not detected (UMI = 0) in any single cell of the ovary.

Figure 4

SCARF Expression across 14 Adult Tissues (A) UMAP resolving ~200,000 single cells from 14 adult tissues obtained from the HCL into 33 different cell types. These cell types were consolidated from 78 cell clusters defined from the initial clustering (see also STAR Methods and Figure S5 for tissues corresponding to each cell type). Number of samples (n = 21 donors) is labeled on the plot. (B) Expression intensity and density of selected SCARFs in each of the 33 cell types defined in Figure 4A. Different dot size scales are used for genes with a low fraction of positive cells (group 1: up to 10%) and those with higher fractions (group 2: up to 60%). (C) Heatmap of the fraction of double-positive cells for different receptor-protease combinations in each of the 33 cell types.

Figure 5

SCARF Expression in Nasal Brushing Samples (A) Left panel: UMAP shows the cell clustering of 6 nasal epithelial scRNA-seq samples from 3 independent studies (n = 18,227 single cells). Right panel: feature plots showing expression of hCoV receptors (ACE2, ANPEP, and BSG), proteases (TMPRSS2, CTSB, and TMPRSS4), and RFs (LY6E and IFITM3). (B) Bar plots comparing the percent of double-positive cells for different receptor/protease combinations in ciliated and secretory cells of the young group (n = 3) and old group (n = 3) nasal samples. Error bars denote the standard error from mean value. Each dot represents an individual sample. Data are represented as mean ± SEM. (C) Venn diagrams illustrating shared and unique differentially expressed genes (DEGs) between ciliated and secretory cells within young group (n = 3) and old group (n = 3) samples. The upper panel indicates genes upregulated in ciliated cells, while the lower panel indicates genes upregulated in secretory cells. (D) Volcano plots showing DEGs between ciliated and secretory cells identified independently for each of the three studies.

Figure 6

Cross-species Analysis of SCARF Expression (A) UMAP clustering of 47 individual RNA-seq samples derived from four tissues (heart, lung, liver, and kidney) of three species (human, chimpanzee, and rhesus macaque) based on 11,929 orthologous RefSeq genes that were most variable in expression across the samples (see STAR Methods). (B) Scatterplot of normalized mean expression in CPM (counts per million) of each orthologous gene (x axis) and scaled dispersion (y axis) across the 47 samples. Every point corresponds to a single gene. The most 1,987 variable genes across the samples (log₂ scaled dispersion > 1) are indicated as pink dots. Red triangles indicate SCARFs. (C) Violin plot showing normalized expression of ACE2 and DPP4 per tissue. Each dot comes from a different sample. (D) Heatmap of scaled expression levels (TMM [trimmed mean of M]-normalized CPM) of ACE2, TMPRSS2, ANPEP, and DPP4 for each of the individual samples (n = 47) grouped by tissues and species. (E) Violin plots showing log₂-normalized expression profiles of ACE2, TMPRSS2, and ANPEP in the preimplantation blastocyst of cynomolgus macaque obtained by scRNA-seq.