key: cord-0786975-2l717xiu
authors: Yamamoto, Mizuki; Gohda, Jin; Kobayashi, Ayako; Tomita, Keiko; Hirayama, Youko; Koshikawa, Naohiko; Seiki, Motoharu; Semba, Kentaro; Akiyama, Tetsu; Kawaguchi, Yasushi; Inoue, Jun-ichiro
title: Metalloproteinase-dependent and TMPRSS2-independnt cell surface entry pathway of SARS-CoV-2 requires the furin-cleavage site and the S2 domain of spike protein
date: 2021-12-15
journal: bioRxiv
DOI: 10.1101/2021.12.14.472513
sha: 0e4a61b4e20af8e0f9929ea0aad12ca7c2e5d52e
doc_id: 786975
cord_uid: 2l717xiu

The ongoing global vaccination program to prevent SARS-CoV-2 infection, the causative agent of COVID-19, has had significant success. However, recently virus variants have emerged that can evade the immunity in a host achieved through vaccination. Consequently, new therapeutic agents that can efficiently prevent infection from these new variants, and hence COVID-19 spread are urgently required. To achieve this, extensive characterization of virus-host cell interactions to identify effective therapeutic targets is warranted. Here, we report a cell surface entry pathway of SARS-CoV-2 that exists in a cell type-dependent manner is TMPRSS2-independent but sensitive to various broad-spectrum metalloproteinase inhibitors such as marimastat and prinomastat. Experiments with selective metalloproteinase inhibitors and gene-specific siRNAs revealed that a disintegrin and metalloproteinase 10 (ADAM10) is partially involved in the metalloproteinase pathway. Consistent with our finding that the pathway is unique to SARS-CoV-2 among highly pathogenic human coronaviruses, both the furin cleavage motif in the S1/S2 boundary and the S2 domain of SARS-CoV-2 spike protein are essential for metalloproteinase-dependent entry. In contrast, the two elements of SARS-CoV-2 independently contributed to TMPRSS2-dependent S2 priming. The metalloproteinase pathway is involved in SARS-CoV-2-induced syncytia formation and cytopathicity, leading us to theorize that it is also involved in the rapid spread of SARS-CoV-2 and the pathogenesis of COVID-19. Thus, targeting the metalloproteinase pathway in addition to the TMPRSS2 and endosome pathways could be an effective strategy by which to cure COVID-19 in the future. Author Summary To develop effective therapeutics against COVID-19, it is necessary to elucidate in detail the infection mechanism of the causative agent, SARS-CoV-2, including recently emerging variants. SARS-CoV-2 binds to the cell surface receptor ACE2 via the Spike protein, and then the Spike protein is cleaved by host proteases to enable entry. Selection of target cells by expression of these tissue-specific proteases contributes to pathogenesis. Here, we found that the metalloproteinase-mediated pathway is important for SARS-CoV-2 infection, variants included. This pathway requires both the prior cleavage of Spike into two domains and a specific sequence in the second domain S2, conditions met by SARS-CoV-2 but lacking in the related human coronavirus SARS-CoV. The contribution of several proteases, including metalloproteinases, to SARS-CoV-2 infection was cell type dependent, especially in cells derived from kidney, ovary, and endometrium, in which SARS-CoV-2 infection was metalloproteinase-dependent. In these cells, inhibition of metalloproteinases by treatment with marimastat or prinomastat, whose safety was previously confirmed in clinical trials, was important in preventing cell death. Our study provides new insights into the complex pathogenesis unique to COVID-19 and relevant to the development of effective therapies.

(ADAM-17 inhibitor) and BK-1361[37] (ADAM8 inhibitor) did not (Fig 5a) . This 282 suggests that ADAM10 may be involved in the ADAM family. MMP408 (Fig 5a) , indicating that the catalytic 289 activity of ACE2, to which the S protein directly binds as a receptor, is not involved. To 290 further confirm the involvement of ADAM10 in the metalloproteinase-dependent 291 pathway, ADAM10 was depleted by siRNA in HEC50B cells. Three independent siRNAs 292 effectively suppressed the expression of both the precursor and active forms of ADAM10 293 (Fig 5b) . An ADAM10 knockdown significantly inhibited SARS-CoV-2 pseudovirus 294 entry, while the entry of SARS-CoV, MERS-CoV, and VSVG pseudoviruses were not 295 affected (Fig 5c) , indicating that ADAM10 plays a role unique to SARS-CoV-2 in viral 296 entry. Furthermore, we examined the effects of the ADAM10 knockdown on the entry 297 pathway patterns by treating siRNA-transfected cells with either E-64d, marimastat, or a 298 18 combination of both. The combination treatment with E-64d and marimastat led to an 299 additive effect for the single treatments, resulting in the complete inhibition of viral entry 300 in both cells with normal ADAM10 expression and those with reduced ADAM10 301 expression (Fig 5d) . These results indicated that E-64d-resistant viral entry is a indicating that ADAM10 is involved in the metalloproteinase-dependent entry pathway 307 of SARS-CoV-2. 308

Recently, it was reported that ACE2 shedding by ADAM17 promotes SARS-309

CoV-2 infection [42] . While a CRISPR/Cas9-mediated knockout of ADAM17 enhanced 310 the accumulation of cellular ACE2 in HEC50B cells due to the inhibition of ACE2 the endosome pathway coexists with the metalloproteinase pathway to contribute to 331 authentic SARS-CoV-2 infection in these cells. Combination treatments with E-332 64d/marimastat or NH4Cl/marimastat showed much stronger inhibitory effects than the 333 treatment with each drug alone (Fig 6b) . Similarly, combination treatments with 334 20 nafamostat and marimastat or nafamostat and E-64d showed stronger inhibitory effects 335 than the nafamostat treatment alone in the HEC50B-TMPRSS2 cells (Fig 6c) . 336

Furthermore, when all three drugs were combined, they had a much stronger inhibitory 337 effect on viral infection when compared with the two-drug combinations (Fig 6c) 17 inhibitor) did not (Fig 6d) . Moreover, the ADAM10 knockdown by siRNA suppressed 344 SARS-CoV-2 infection by approximately 40% (Fig 6e) , indicating that ADAM10 is 345 partially involved. This effect was smaller than that for the various metalloproteinase 346 inhibitors (Fig 6a,d) , suggesting that metalloproteinases other than ADAM10 are also 347 involved in this pathway. 348

The ability of SARS-CoV-2 to form syncytia and induce cytopathicity is thought 349 to be related to its pathogenesis [44, 45] . To determine whether the metalloproteinase-350 dependent pathway is involved in syncytia formation, we first used HEC50B cells as a 351

representative for cells that predominantly use the metalloproteinase and endosome 352 21 pathways. Interestingly, the SARS-CoV-2-induced syncytia formation in HEC50B cells 353 24 h after infection was significantly blocked by 500 nM of marimastat and prinomastat 354 but not notably affected by 25 M E-64d (Fig 6f and S10 Fig). These results indicate that 355 the metalloproteinase-dependent pathway, but not the endosome pathway, is crucial for 356 syncytium formation, although both pathways similarly reduce viral infection (Fig 6b) . 357

Given that the metalloproteinases are normally localized at the cell surface, we used 358 HEC50B-TMPRSS2 cells, which have cell surface TMPRSS2 and metalloproteinase 359 pathways, to investigate their involvement in syncytia formation when they coexist. The 360 SARS-CoV-2-induced syncytia formation in the HEC50B-TMPRSS2 cells was not 361 significantly inhibited by marimastat or nafamostat alone, but was clearly inhibited by 362 the combined treatment (Fig 6g) . These results suggest that the metalloproteinase and 363 TMPRSS2 pathways cooperate to form syncytia. Next, we addressed the role of the 364 metalloproteinase pathway in SARS-CoV-2-induced cytotoxicity. SARS-CoV-2-induced 365 cytopathicity of HEC50B cells 3 d after infection was not inhibited by either E-64d, 366 marimastat, or prinomastat alone, but was significantly blocked when cells were treated 367 with E-64d in combination with either marimastat or prinomastat (Fig 6h) . In addition, 368 SARS-CoV-2-induced cytopathicity of the HEC50B-TMPRSS2 cells was not inhibited 369

by either E-64d, nafamostat, or marimastat alone, but was significantly blocked when 370 22 cells were treated with a combination of all three drugs (Fig 6i) . These results strongly 371

suggest that the inhibition of the metalloproteinase pathway is crucial to block syncytia 372 formation and cytopathicity in vivo, and consequently, that the metalloproteinase pathway 373 is likely to be involved in the pathogenesis of COVID-19. The S1/S2 boundary of SARS-CoV-2 contains the furin cleavage motif (Arg-X-402 X-Arg), while that of SARS-CoV contains only a single Arg. It has been reported that the 403 motif greatly increases the efficiency of S1/S2 cleavage [15, 16] , leading to enhanced viral 404 transmission both in vitro [15, 16, 23] and in vivo [54, 55] . This may be partially due to 405 the enhanced availability of S2 to TMPRSS2, due to the dissociation of S1[56, 57]. We 406 24 have shown that the furin cleavage motif is required for the metalloproteinase pathway, 407 and we propose that the induction of metalloproteinase-induced S2 priming is another 408 role of furin-mediated S1/S2 cleavage in enhanced viral transmission. Therefore, the 409 metalloproteinase-dependent entry pathway, which is unique among highly pathogenic 410 coronaviruses, is likely to be associated with the rapid spread of SARS-CoV-2. 411

Interestingly, experiments using pseudoviruses bearing chimeric S proteins between 412 SARS-CoV (without the metalloproteinase pathway) and SARS-CoV-2 (with the 413 metalloproteinase pathway) revealed that both the S1/S2 boundary of SARS-CoV-2 and 414 the S2 domain of SARS-CoV-2 S are essential for metalloproteinase-dependent entry. In 415 contrast, the two domains of SARS-CoV-2 independently contributed to TMPRSS2-416 dependent S2 priming. This discrepancy may be partially due to the difference in the 417 substrate recognition properties of the priming proteases in the two pathways. The S112 418 pseudovirus (a VSV pseudovirus bearing SARS-CoV S mutant, in which the S2 region 419 was replaced with the corresponding domain of SARS-CoV-2) can use the TMPRSS2 420 pathway more efficiently than the SARS-CoV S pseudovirus, which suggests that 421 TMPRSS2 may be partially accessible to the priming site in SARS-CoV-2 (C-terminal of 422 Arg815) but not to that in SARS-CoV (C-terminal of Arg797) without S1 dissociation. 423

In contrast, the putative priming protease in the metalloproteinase pathway, which may 424 25 not be a metalloproteinase but a protease activated by metalloproteinases, can access the 425 priming site only when the site occurs within the contextual characteristics of SARS-426

CoV-2 S2, and S1/S2 is cleaved to allow S1 dissociation. Determination of the priming 427 site in the metalloproteinase pathway and identification of the critical amino acid residues 428 generating the structural characteristics of SARS-CoV-2 S2 that allow metalloproteinase-429 dependent priming are required to understand its molecular mechanisms for the two 430 in study design, data collection and analysis, decision to publish, or preparation of the 976 manuscript. 977

A pneumonia outbreak 686 associated with a new coronavirus of probable bat origin

Epidemiology and cause 690 of severe acute respiratory syndrome (SARS) in Guangdong, People's Republic of China

Identification of a novel coronavirus in patients with severe acute respiratory syndrome

Isolation 698 of a novel coronavirus from a man with pneumonia in Saudi Arabia

Mechanisms of SARS-CoV-2 Transmission and 701 Pathogenesis

Characteristics of SARS-CoV-2 and COVID-19

Progress of the COVID-708 19 vaccine effort: viruses, vaccines and variants versus efficacy, effectiveness and escape

Epub 20210809

Impact of vaccination on new SARS-CoV-2 infections in the United Kingdom

Reduced sensitivity of SARS-CoV-2 variant Delta to antibody neutralization

The 720 biological and clinical significance of emerging SARS-CoV-2 variants

Epub 20210917

Neutralization of the SARS-CoV-2 Mu Variant by Convalescent and Vaccine Serum

Current Strategies of Antiviral Drug Discovery for COVID-19

Identification 730 and Development of Therapeutics for COVID-19

Mechanisms of SARS-CoV-2 entry into 734 cells

Cleavage Site in the Spike 737 Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells

Structural basis for the recognition of 744

SARS-CoV-2 by full-length human ACE2

Structure of the SARS-CoV-2 spike 748 receptor-binding domain bound to the ACE2 receptor

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically 752

Cathepsin L plays 756 a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target 757 for new drug development

SARS-CoV-2 tropism, entry, replication, and propagation: Considerations for drug discovery 762 and development

TMPRSS2 766 expression dictates the entry route used by SARS-CoV-2 to infect host cells

TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human 771 airway cells

Fusion in a Cell Fusion Assay System and Viral Infection In Vitro in a Cell-Type-Dependent

Identification of Nafamostat as a Potent Inhibitor of Middle East Respiratory Syndrome 780

To bind zinc or not to bind 49

Epub 20210420

Persistence of 857 viral RNA, pneumocyte syncytia and thrombosis are hallmarks of advanced

Nafamostat mesylate blocks activation of SARS-CoV-2: New treatment option for COVID-19

Cause Kidney and Testis Infection in COVID-19 Patients

SARS-CoV-2 cell tropism and 869 multiorgan infection

Loss 893 of furin cleavage site attenuates SARS-CoV-2 pathogenesis

Epub 20210125

The furin 897 cleavage site in the SARS-CoV-2 spike protein is required for transmission in ferrets

Receptor 901 binding and priming of the spike protein of SARS-CoV-2 for membrane fusion

Proteases and variants: context matters for 905 SARS-CoV-2 entry assays

Efficacy of the TMPRSS2 inhibitor camostat mesilate in patients hospitalized with Covid-19-930 a double-blind randomized controlled trial

A Randomized Trial of Hydroxychloroquine as Postexposure Prophylaxis for Covid-19

Epub 20200603. doi: 10.1056/NEJMoa2016638. PubMed 936 PMID: 32492293; PubMed Central PMCID: PMCPMC7289276. 937 66. Self WH

Hydroxychloroquine on Clinical Status at 14 Days in Hospitalized Patients With COVID-19: 939 A Randomized Clinical Trial

Enhanced 942 isolation of SARS-CoV-2 by TMPRSS2-expressing cells

Co-expression of 946 foreign proteins tethered to HIV-1 envelope glycoprotein on the cell surface by introducing 947 an intervening second membrane-spanning domain

Syrian hamsters as a small animal model for SARS-CoV-2 infection and countermeasure 952 development

TRAF6 956 maintains mammary stem cells and promotes pregnancy-induced mammary epithelial cell 957 expansion

Involvement of 960 ceramide in the propagation of Japanese encephalitis virus

PubMed Central 66 activity for each condition to the FL activity of the cells infected with pseudovirus in the 1161 presence of DMSO alone

Cont: cells infected with pseudovirus without S protein. E-64d: 25 M E-1163 64d, marima: 1 M marimastat, nafamo: 10 M nafamostat. (a, b) Effects of E-64d and 1164 marimastat on the entry of pseudoviruses bearing SARS-CoV S, SARS-CoV-2 S

A704 (a) and VeroE6 (b) cells. (c, d) Effects of E-64d and 1166 marimastat on the entry of pseudoviruses bearing chimeric S proteins in VeroE6 cells

Effects of E-64d and nafamostat on the entry of pseudoviruses bearing SARS-CoV S

E-64d and nafamostat on the entry of pseudoviruses bearing chimeric S proteins in 1170

VeroE6-TMPRSS2 cells. To establish VeroE6 cells expressing TMPRSS2 (VeroE6

recombinant pseudotype lentivirus expressing TMPRSS2 was produced 1172 using 293T cells with a VSV-G-expressing plasmid. Cells infected with pseudotype 1173 viruses were selected with 300 g/mL hygromycin for at least 1 week

Patterns of entry pathways were conserved in various variants of SARS-1176

Expression of WT or mutant SARS-CoV-2 S proteins with mutations precent in 1178

617.2 variants in the pseudoviruses. S proteins were 1179 detected using an anti-Flag-tag antibody that binds to a Flag-tag on the C-terminus of S 1180 proteins (top). Detection of vesicular stomatitis virus matrix protein (VSV M) served as 1181 a control (bottom). S0: uncleaved S protein

Effects of E-64d and marimastat on the entry of pseudoviruses bearing SARS-CoV-2 S 1183 in VeroE6 cells. E-64d: 25 M E-64d, marima: 1 M marimastat. (c) Effects of 1184 nafamostat on the entry of pseudovirus bearing SARS-CoV-2 S in VeroE6-TMPRSS2

Effects of E-64d and marimastat on the entry of pseudoviruses bearing SARS-1186

CoV-2 S in HEC50B cells. E-64d: 25 M E-64d, marima: 1 M marimastat. (e) Effects 1187 of marimastat on the entry of pseudoviruses bearing SARS-CoV-2 S in A704 cells. (f)

Effects of nafamostat on the entry of pseudoviruses bearing SARS-CoV-2 S

The relative pseudovirus entry was calculated by normalizing the FL activity for 1190 each condition to the FL activity of cells infected with pseudovirus in the presence of 1191 DMSO alone, which was set to 100%. Values are means ± SD (n = 3/group in b-f)

SARS-CoV-2 S in the presence of DMSO alone. * p < 0.05, ** p < 0.01. Cont: cells 1194 infected with a pseudovirus without S

ADAM10 #3) for the 1234 establishment of ADAM17-knockout cells. Pooled HEC50B cells infected with 1235 pseudotype viruses were selected with 1 g/mL puromycin for 1 week. 1236 1237 S9 Fig. Effects of drugs on SARS-CoV-2 infection. 1238 (a) Effects of the nafamostat on the SARS-CoV-2

Values are means ± SD (n = 3/group). ** p < 0.01. (b) Effects of the 1240

E-64d on the SARS-CoV-2 infection in HEC50B, A704, and VeroE6 cells. Values are

CoV-2 infection in HEC50B cells. Values are means (n = 2/group). The relative amount 1243 of viral RNA in the cells was normalized to cellular Rpl13a mRNA expression

The metalloproteinase-dependent entry pathway of authentic SARS-CoV-1246 2 is involved in syncytia formation

Phase contrast images of syncytia formation 24 h after SARS-CoV-2 infection in the 1248 presence of inhibitors. Red arrowheads indicate syncytia formation Scale bars, 100 m

the RL activity for each co-culture to that of the co-culture with cells expressing both 998 57 receptor and TMPRSS2 in the presence of DMSO, which was set to 100%. Values are 999 means ± SD (n = 3/group). ** p < 0.01. (d) Effects of the nafamostat on the TMPRSS2-1000 independent or -dependent cell fusion. Target cells expressing ACE2 alone or together 1001 with TMPRSS2 were used for co-culturing with effector cells expressing SARS-CoV-2 1002 S. Relative cell-fusion value was calculated by normalizing the RL activity for each co-1003 culture to that of the co-culture with cells expressing both ACE2 and TMPRSS2 in the 1004 presence of DMSO, which was set to 100%. Values are means ± SD (n = 3/group). nafamo, 1005 nafamostat. HCoV-NL63 S and WIV1-CoV S pseudovirus in HEC50B cells. c, Schematic illustration 1053 of C-terminally Flag-tagged chimeric S proteins in which the S1, S1/S2 boundary, and 1054 S2 domain from SARS-CoV S (red) or SARS-CoV-2 S (yellow) are indicated (top). 1055Amino acid sequences of the residues around the S1/S2 boundary of the coronaviruses 1056 HCoV-NL63 and amino acid sequences of the residues around the S1/S2 boundary of the 1199 coronaviruses (bottom). Numbers refer to amino acid residues. F: Flag tag. Arginine 1200 residues in the S1/S2 cleavage site and furin cleavage motif are highlighted in red.