key: cord-0840886-e8ejokmw
authors: Hou, Yapeng; Ding, Yan; Nie, Hongguang; Ji, Hong-Long
title: Fibrinolysis influences SARS-CoV-2 infection in ciliated cells
date: 2021-01-08
journal: bioRxiv
DOI: 10.1101/2021.01.07.425801
sha: e5fda362150cc1dc9a6890b94c3dbc5a9e106405
doc_id: 840886
cord_uid: e8ejokmw

Rapid spread of COVID-19 has caused an unprecedented pandemic worldwide, and an inserted furin site in SARS-CoV-2 spike protein (S) may account for increased transmissibility. Plasmin, and other host proteases, may cleave the furin site of SARS-CoV-2 S protein and γ subunits of epithelial sodium channels (γ ENaC), resulting in an increment in virus infectivity and channel activity. As for the importance of ENaC in the regulation of airway surface and alveolar fluid homeostasis, whether SARS-CoV-2 will share and strengthen the cleavage network with ENaC proteins at the single-cell level is urgently worthy of consideration. To address this issue, we analyzed single-cell RNA sequence (scRNA-seq) datasets, and found the PLAU (encoding urokinase plasminogen activator), SCNN1G (γENaC), and ACE2 (SARS-CoV-2 receptor) were co-expressed in alveolar epithelial, basal, club, and ciliated epithelial cells. The relative expression level of PLAU, TMPRSS2, and ACE2 were significantly upregulated in severe COVID-19 patients and SARS-CoV-2 infected cell lines using Seurat and DESeq2 R packages. Moreover, the increments in PLAU, FURIN, TMPRSS2, and ACE2 were predominately observed in different epithelial cells and leukocytes. Accordingly, SARS-CoV-2 may share and strengthen the ENaC fibrinolytic proteases network in ACE2 positive airway and alveolar epithelial cells, which may expedite virus infusion into the susceptible cells and bring about ENaC associated edematous respiratory condition.

The SARS-CoV-2 infection leads to COVID-19 with pathogenesis and clinical features similar to those 30 of SARS and shares the same receptor, angiotensin-converting enzyme 2 (ACE2), with SARS-CoV to enter 31 host cells (Zhou et al. 2020 , Li and Zheng 2020). By comparison, the transmission ability of SARS-CoV-2 is 32 much stronger than that of SARS-CoV, owning to diverse affinity to ACE2 (Wrapp and Wang 2020). The This study aims to determine whether PLAU, SCNN1G, and ACE2 are co-expressed in the airway and 55 lung epithelial cells and whether SARS-CoV-2 infection alters their expression at the single-cell level. We 56 found that these genes, especially the PLAU was significantly upregulated in epithelial cells of 57 severe/moderate COVID-19 patients and SARS-CoV-2 infected cell lines, mainly owning to ciliated cells. We 58 conclude that the most susceptible cells for SARS-CoV-2 infection could be the ones co-expressing these 59 genes and sharing plasmin-mediated cleavage.

Furin sites are identified in both virus and host ENaC proteins 63 A furin site was located at the S proteins of SARS-CoV-2 from Arginine-683 to Serine-687 (RRAR|S), 64 and similar site was also seen in the S protein of HCoV-OC43, MERS, and HCoV-HKU1 coronavirus (Fig.   65   1A ). In addition, the highly conserved RxxR motif existed in the hemagglutinin protein of influenza H3N2, 66 Herpes, Ebola, HIV, Dengue, hepatitis B, West Nile, Marburg, Zika, Epstein-Barr, and respiratory syncytial 67 virus (RSV). The furin site (RKRR|E) was found in the gating relief of inhibition by proteolysis (GRIP) 68 domain of the extracellular loop of the mouse, rat, and human ENaC (Fig. 1B) . The similarity of these furin 69 sites is 40-80%. To detect the potential changes in the cell populations that co-express PLAU, SCNN1G, and ACE2, we 94 analyzed the scRNA-seq datasets of bronchoalveolar lavage fluid (BALF) cells, which are mainly composed 95 of epithelial cells and leukocytes. There were three groups to be studied: 4 healthy controls, 3 moderate, and 96 6 severe COVID-19 patients. The expression level and the percentage of total cells expressing PLAU and 97 FURIN were significantly upregulated in the severe group compared with controls (P < 0.001), as well as the 98 expression levels of ACE2, TMPRSS2, SCNN1G, and PLG were also slightly upregulated ( Fig. 3A and B) . 99 The expression levels of PLAU, Furin, TMPRSS2, and ACE2 and the number of cells were profiled in 100 Fig. 4A . The data showed that these genes were upregulated in COVID-19 patients, and the number of cells 101 expressing these upregulated genes almost increased in a severity-dependent manner. PLAU was significantly 102 elevated in severe group (P < 0.001), and the other genes also showed an increasing trend (Fig. 4B) . 191 Acquisition, filtering, and processing of scRNA-seq data 192 The dataset downloaded from the Gene Expression Omnibus was filtered for integration. Lung scRNA-193 seq dataset (8 healthy controls in GSE122960) were filtered by total number of reads (nreads > 1,000), number 194 of detected genes (50 < ngenes < 7,500), and mitochondrial percentage (mito.pc < 0.2). BALF scRNA-seq 195 dataset was composed of 3 healthy controls, 3 moderate and 6 severe COVID-19 patients in GSE145926, and 196 1 healthy control in GSM3660650. These datasets were filtered by total number of reads (nreads > 1,000), 197 number of detected genes (20 < ngenes < 6,000), and mitochondrial percentage (mito.pc < 0.1). Finally, a In physiological conditions, the urokinase activates the plasminogen to plasmin, which will cleave the γENaC, leading to its activation. After infected by SARS-CoV-2, the PLAU (urokinase) expression level is significantly upregulated, which may help other viruses' invasion by activating the plasminogen to cleave the S protein. The green solid line represents the urokinase, plasminogen, ENaC mRNA transcripts and activation by plasmin under physiological conditions. The red solid line represents the activation process under infection conditions, while the grey dotted line denotes the repression effects.

Is amiloride a promising cardiovascular medication to persist in the 231

COVID-19 crisis?

SARS-CoV-2 strategically mimics proteolytic activation 233 of human ENaC

Thrombolysis restores perfusion in COVID-19 hypoxia

Potential of heparin and nafamostat combination therapy for COVID-19

Rescue 240 therapy for severe COVID-19-associated acute respiratory distress syndrome with tissue plasminogen activator: A case 241 series

Drives Development of COVID-19

Systemic fibrinolysis for acute pulmonary embolism complicating acute respiratory distress syndrome in 247 severe COVID-19: a case series

Regulation of epithelial 249 sodium channels in urokinase plasminogen activator deficiency

Early outcomes with utilization of tissue plasminogen activator in COVID-253 19-associated respiratory distress: A series of five cases

Tissue plasminogen activator (tPA) treatment for COVID-19 associated acute respiratory distress syndrome (ARDS): A 342 case series

A Unique Protease Cleavage Site Predicted in the Spike 344

Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

The role of furin cleavage site in

SARS-CoV-2 spike protein-mediated membrane fusion in the presence or absence of trypsin

SARS-CoV-2 induces transcriptional signatures in human lung 352 epithelial cells that promote lung fibrosis

Plasmin improves blood-gas barrier function in oedematous lungs by cleaving epithelial 354 sodium channels

A pneumonia outbreak associated with a new coronavirus 358 of probable bat origin