bioRxiv preprint doi: https://doi.org/10.1101/2021.01.07.425801; this version posted January 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Fibrinolysis influences SARS-CoV-2 infection in ciliated cells 2 3 Yapeng Hou1, Yan Ding1, Hongguang Nie1, *, Hong-Long Ji2 4 5 1Department of Stem Cells and Regenerative Medicine, College of Basic Medical Science, China Medical 6 University, Shenyang, Liaoning 110122, China. 2Department of Cellular and Molecular Biology, University 7 of Texas Health Science Center at Tyler, Tyler, TX 75708, USA. 8 9 *Address correspondence to [email protected] 10 11 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.07.425801; this version posted January 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 12 Abstract 13 Rapid spread of COVID-19 has caused an unprecedented pandemic worldwide, and an inserted furin site 14 in SARS-CoV-2 spike protein (S) may account for increased transmissibility. Plasmin, and other host 15 proteases, may cleave the furin site of SARS-CoV-2 S protein and subunits of epithelial sodium channels ( 16 ENaC), resulting in an increment in virus infectivity and channel activity. As for the importance of ENaC in 17 the regulation of airway surface and alveolar fluid homeostasis, whether SARS-CoV-2 will share and 18 strengthen the cleavage network with ENaC proteins at the single-cell level is urgently worthy of consideration. 19 To address this issue, we analyzed single-cell RNA sequence (scRNA-seq) datasets, and found the PLAU 20 (encoding urokinase plasminogen activator), SCNN1G (ENaC), and ACE2 (SARS-CoV-2 receptor) were co- 21 expressed in alveolar epithelial, basal, club, and ciliated epithelial cells. The relative expression level of PLAU, 22 TMPRSS2, and ACE2 were significantly upregulated in severe COVID-19 patients and SARS-CoV-2 infected 23 cell lines using Seurat and DESeq2 R packages. Moreover, the increments in PLAU, FURIN, TMPRSS2, and 24 ACE2 were predominately observed in different epithelial cells and leukocytes. Accordingly, SARS-CoV-2 25 may share and strengthen the ENaC fibrinolytic proteases network in ACE2 positive airway and alveolar 26 epithelial cells, which may expedite virus infusion into the susceptible cells and bring about ENaC associated 27 edematous respiratory condition. 28 Keywords: SARS-CoV-2; plasmin; ENaC; COVID-19; furin bioRxiv preprint doi: https://doi.org/10.1101/2021.01.07.425801; this version posted January 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 29 Introduction 30 The SARS-CoV-2 infection leads to COVID-19 with pathogenesis and clinical features similar to those 31 of SARS and shares the same receptor, angiotensin-converting enzyme 2 (ACE2), with SARS-CoV to enter 32 host cells (Zhou et al. 2020, Li and Zheng 2020). By comparison, the transmission ability of SARS-CoV-2 is 33 much stronger than that of SARS-CoV, owning to diverse affinity to ACE2 (Wrapp and Wang 2020). The 34 fusion capacity of coronavirus via the spike protein (S protein) determines infectivity (Wrapp and Wang 2020, 35 Kam et al. 2009b). Highly virulent avian and human influenza viruses bearing a furin site (RxxR) in the 36 haemagglutinin have been described (Coutard et al. 2020). Cleavage of the furin site enhances the entry ability 37 of Ebola, HIV, and influenza viruses into host cells (Claas et al. 1998). Consisting of receptor-binding (S1) 38 and fusion domains (S2), coronavirus S protein needs to be primed through the cleavage at S1/S2 site and S2’ 39 site for membrane fusion (Jaimes et al. 2020, Huggins 2020). The newly inserted furin site in SARS-CoV-2 S 40 protein significantly facilitated the membrane fusion, leading to enhanced virulence and infectivity (Xia et al. 41 2020, Wang, Qiu, et al. 2020). 42 Plasmin cleaves the furin site in SARS-CoV S protein (Kam et al. 2009b), which is upregulated in the 43 vulnerable populations of COVID-19 (Ji et al. 2020). However, whether plasmin cleaves the newly inserted 44 furin site in the SARS-CoV-2 S protein remains obscure. Plasmin cleaves the furin site of human subunit of 45 epithelial sodium channels (ENaC) as demonstrated by LC-MS and functional assays (Zhao, Ali, and Nie 46 2020, Sheng et al. 2006). Very recently, it has been proposed that the global pandemic of COVID-19 may 47 partially be driven by the targeted mimicry of ENaC α subunit by SARS-CoV-2 (Gentzsch and Rossier 2020, 48 Muhanna et al. 2020). ENaC are located at the apical side of the airway and alveolar cells, acting as a critical 49 system to maintain the homeostasis of airway surface and alveolar fluid homeostasis (Ji et al. 2006, Matalon, 50 Bartoszewski, and Collawn 2015). The luminal fluid is required for keeping normal ciliary beating to expel 51 inhaled pathogens, allergens, and pollutants and for migration of immune cells that release pro-inflammatory 52 cytokines and chemokines (Hou et al. 2019a). The plasmin family and ACE2 are expressed in the respiratory 53 epithelium (Nie et al. 2009, Hanukoglu and Hanukoglu 2016, Kam et al. 2009a). However, if the plasmin 54 system and ENaC are involved in the fusion of SARS-CoV-2 into host cells is unknown. 55 This study aims to determine whether PLAU, SCNN1G, and ACE2 are co-expressed in the airway and 56 lung epithelial cells and whether SARS-CoV-2 infection alters their expression at the single-cell level. We 57 found that these genes, especially the PLAU was significantly upregulated in epithelial cells of 58 severe/moderate COVID-19 patients and SARS-CoV-2 infected cell lines, mainly owning to ciliated cells. We 59 conclude that the most susceptible cells for SARS-CoV-2 infection could be the ones co-expressing these 60 genes and sharing plasmin-mediated cleavage. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.07.425801; this version posted January 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 61 62 Results 63 Furin sites are identified in both virus and host ENaC proteins 64 A furin site was located at the S proteins of SARS-CoV-2 from Arginine-683 to Serine-687 (RRAR|S), 65 and similar site was also seen in the S protein of HCoV-OC43, MERS, and HCoV-HKU1 coronavirus (Fig. 66 1A). In addition, the highly conserved RxxR motif existed in the hemagglutinin protein of influenza H3N2, 67 Herpes, Ebola, HIV, Dengue, hepatitis B, West Nile, Marburg, Zika, Epstein-Barr, and respiratory syncytial 68 virus (RSV). The furin site (RKRR|E) was found in the gating relief of inhibition by proteolysis (GRIP) 69 domain of the extracellular loop of the mouse, rat, and human ENaC (Fig. 1B). The similarity of these furin 70 sites is 40-80%. 71 Respiratory cells co-express PLAU, SCNN1G, and ACE2 72 To identify subpopulations of cells co-expressing PLAU, SCNN1G, and ACE2, we analyzed 11 scRNA- 73 seq datasets by nferX scRNA-seq platform (https://academia.nferx.com/) (Supplementary Table 1). All three 74 genes were co-expressed in the following cells ranked by the expression level of PLAU from high to low: club 75 cells, goblets, basal cells, AT1 cells, ciliated cells, fibroblasts, mucous cells, deuterosomal cells, and AT2 cells 76 (Fig. 1C), which were supported by previous studies (Sungnak et al. 2020, Wang et al. 2008, Hanukoglu and 77 Hanukoglu 2016). These results suggest that these cell populations co-expressing PLAU-ENaC-ACE2 may 78 be more susceptible to the SARS-CoV-2 infection compared with others. In addition, the top ten ranked cell 79 sub-populations expressing PLAU, SCNN1G, or ACE2 alone were listed in Supplementary Table 2. To 80 compare the transcript of the proteases in different lung epithelial cells, we analyzed the lung dataset from 81 Gene Expression Omnibus (GEO) by Seurat, and the cells were annotated by their specific markers 82 (Supplementary Fig. 1A). The data showed that all these proteases were expressed in AT2 cells, including 83 PLAU, FURIN, PRSS3 (Trypsin), ELANE (Elastase), PRTN3 (Myeloblastin), CELA1 (Elastase-1), CELA2A 84 (Elastase-2A), CTRC (Chymotrypsin-C), TMPRSS4 (Transmembrane protease serine 4), and TMPRSS2 85 (Transmembrane protease serine 2) (Supplementary Fig. 1B). In AT2 cells, the proteases expression level in 86 order is: TMPRSS2 > FURIN > TMPRSS4 > PLAU > CELA1 > ELANE > PRSS3 > PRTN3 > CTRC > 87 CLEA2A. For PLAU, the high to low order is Basal > Club > Ciliated > AT1 > AT2. 88 The expression levels of proteases (PLAU, FURIN, TMPRSS2, PLG), ACE2, and SCNN1G in 11 cell 89 types co-expressing ACE2, SCNN1G, and PLAU were compared in Fig. 2. The club cells showed the highest 90 expression level of PLAU, and the ACE2, SCNN1G, TMPRSS2, FURIN, and PLG showed a higher 91 expression level in club cells compared with other cell types. Of note, the ciliated cell was the second and 92 seventh highest expression cell type of PLAU and ACE2, respectively. bioRxiv preprint doi: https://doi.org/10.1101/2021.01.07.425801; this version posted January 8, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 93 Expression levels of PLAU, SCNN1G, and ACE2 in SARS-CoV-2 infection 94 To detect the potential changes in the cell populations that co-express PLAU, SCNN1G, and ACE2, we 95 analyzed the scRNA-seq datasets of bronchoalveolar lavage fluid (BALF) cells, which are mainly composed 96 of epithelial cells and leukocytes.