REVIEWS Priming of SARS-CoV-2 S protein by several membrane-bound serine proteinases could explain enhanced viral infectivity and systemic COVID-19 infection Received for publication, September 10, 2020, and in revised form, November 30, 2020 Published, Papers in Press, December 2, 2020, https://doi.org/10.1074/jbc.REV120.015980 Pablo Fuentes-Prior* From the Molecular Bases of Disease, Biomedical Research Institute (IIB) Sant Pau, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain Edited by George DeMartino The ongoing COVID-19 pandemic has already caused over a enzyme 2 (ACE2), and a serine proteinase known as million deaths worldwide, and this death toll will be much TMPRSS2/TMPS2 or epitheliasin. (TMPRSS stands for higher before effective treatments and vaccines are available. “Transmembrane Protease, Serine”). The protruding spike (S) The causative agent of the disease, the coronavirus SARS-CoV- glycoproteins of SARS-CoVs interact first with ACE2 and are 2, shows important similarities with the previously emerged then proteolytically cleaved by TMPRSS2 to trigger fusion of SARS-CoV-1, but also striking differences. First, SARS-CoV-2 the viral envelope with the host cell membrane (1–9). (See possesses a significantly higher transmission rate and infec- Fig. 1A for the domain organization of the viral S protein and tivity than SARS-CoV-1 and has infected in a few months over Fig. 1, B–C for its three-dimensional (3D) structure). 60 million people. Moreover, COVID-19 has a systemic char- TMPRSS2 plays a dual role in the infection process as it also acter, as in addition to the lungs, it also affects the heart, liver, cleaves ACE2, which increases uptake of SARS-CoV and likely and kidneys among other organs of the patients and causes also SARS-CoV-2 virions (3). In addition, TMPRSS2 also ac- frequent thrombotic and neurological complications. In fact, tivates other human pathogenic coronaviruses that cause the the term “viral sepsis” has been recently coined to describe the common cold as well as several strains of influenza A viruses, clinical observations. Here I review current structure–function although these pathogens use unrelated attachment receptors information on the viral spike proteins and the membrane for host cell entry (10). fusion process to provide plausible explanations for these ob- The extraordinarily rapid spread of the COVID-19 servations. I hypothesize that several membrane-associated pandemic caused by SARS-CoV-2, the absence of a vaccine serine proteinases (MASPs), in synergy with or in place of and of pharmacological drugs capable of inhibiting viral TMPRSS2, contribute to activate the SARS-CoV-2 spike pro- infection and its large economic impact have spurred intense tein. Relative concentrations of the attachment receptor, ACE2, research on the cell entry mechanism, a key factor in viral MASPs, their endogenous inhibitors (the Kunitz-type trans- pathogenesis. Among several unresolved issues, there are two membrane inhibitors, HAI-1/SPINT1 and HAI-2/SPINT2, as questions of particular clinical relevance. First, why does the well as major circulating serpins) would determine the infec- virus show a much higher transmission rate and infectivity tion rate of host cells. The exclusive or predominant expression than SARS-CoV, despite highly similar protein structures, of major MASPs in specific human organs suggests a direct role including about 90% conserved residues of their spike pro- of these proteinases in e.g., heart infection and myocardial teins? Second, why is SARS-CoV-2 capable of infecting injury, liver dysfunction, kidney damage, as well as neurological different human organs, in contrast to the rather restricted cell complications. Thorough consideration of these factors could tropism of SARS-CoV? A plausible answer to these questions have a positive impact on the control of the current COVID-19 follows, based on a careful analysis of available structure– pandemic. function information regarding coronavirus spike proteins and the membrane fusion process. Coronaviruses cause important diseases in humans, most Furin cleavage preactivates SARS-CoV-2 spike proteins notably severe acute respiratory syndrome (SARS) and Middle The spike proteins of SARS-CoV-1 and 2 are activated East respiratory syndrome (MERS). According to the currently (“primed”) upon proteolysis of two peptide bonds: the first one, accepted mechanism of viral infection, both SARS-CoV (also corresponding to Arg685-Ser686 in the SARS-CoV-2 protein, termed SARS-CoV-1) and the related, recently emerged SARS- separates the so-called S1 and S2 subunits (Fig. 1A) (2). The N- CoV-2 rely on two membrane-bound host peptidases for entry terminal S1 subunit contains the receptor-binding domain into target cells: the carboxypeptidase, angiotensin-converting (RBD) responsible for ACE2 binding (8, 11, 12), while the C- terminal S2 subunit remains attached to the viral envelope after proteolysis at the S1/S2 site and is ultimately responsible * For correspondence: Pablo Fuentes-Prior, pfuentes@santpau.cat. for its fusion with the host cell membrane. This initial cleavage J. Biol. Chem. (2021) 296 100135 1 © 2020 THE AUTHOR. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). JBC REVIEWS: On the mechanism of SARS-CoV-2 spike protein activation Figure 1. The SARS-CoV-2 spike (S) protein is responsible for viral attachment to host cells and membrane fusion. A, schematic representation of domain organization. B, 3D structure of trimeric S glycoprotein in the prefusion conformation (after PDB 6VSB). The RBD of one monomer is in the “up” conformation, i.e., exposed for interaction with the human ACE2 ectodomain (blue), while the two other monomers are depicted as green and light-orange solid surfaces. Note that carbohydrate chains (color-coded spheres) are distributed all over the surface of the trimer. C, postfusion conformation of the viral S protein. After priming, the heptad repeats and central helix from each S2’ monomer adopt extended, tightly packed continuous α-helical structures that generate a 180-Å-long, cone-shaped arrangement with a prominent triple-helical core. (Here, the structure of the highly related murine hepatitis virus is represented; PDB 6B3O, ref. (161)). Residues topologically equivalent to Leu922 and Ser1196 of each monomer are located less than 20 Å apart. In this manner, the rearranged S trimer brings viral and host cell membranes into close proximity to allow their fusion and infection. D, S2’ cleavage site (Arg815- Ser816, red arrow), highlighting the solvent-protected conformation of these residues, which is incompatible with proteinase binding and proteolysis: the Arg815 side chain is clamped by acidic residues (Asp820, Asp867) and contacts with Phe823, while the side chain of Ser816 points inward and is fixed by those of Glu819 and Gln1054. E, comparison of S2’ priming sites in SARS-CoV-1 and 2. Several critical residues around the Arg815 side chain (Arg797 in SARS-CoV-1) are color-coded (carbon atoms are orange and green in SARS-CoV-1 and 2, respectively). Different conformations for most of the activation loop (upper right part of the panel) leads to significant changes in the shielding of the Arg815 side chain; Ser813→Thr797 and Glu868→Asp850 replacements in particular generate a “cage” better suited to hold the Arg815 side chain in its protected conformation. F, structure of the Arg815-binding pocket in MERS- CoV S protein (after PDB 5X59). As a major difference with SARS-CoV-1 and 2, the connector topologically equivalent to Val826-Gln836 in SARS-CoV-2 is defined and forms a lid that would sterically clash with an approaching proteinase. This lid projects the side chains of Ile828, Pro831 and Tyr833 toward Arg815, which, together with the replacement of Asp867 by an aliphatic valine, makes the Arg815 cage much more apolar in MERS-CoV. Conceivably, these subtle sequence and structure variations facilitate exposure and cleavage of the Arg815-Ser816 peptide bond SARS-CoV-2 and thus contribute to its in- crease infectivity compared with other related coronaviruses. (To facilitate comparisons, residues are numbered according to their topological equivalences in SARS-CoV-2). is followed by proteolysis at the S2’ site (Arg815-Ser816 in As a major difference with SARS-CoV, the S1/S2 peptide SARS-CoV-2; Fig. 1D), which exposes the highly antigenic bond in SARS-CoV-2 S proteins is efficiently severed by Ser816-Phe833 fusion peptide, and elicits major, irreversible another host cell proteinase, the proprotein convertase furin, conformational changes. This results in a radically different within the secretory pathway of infected cells (7). (For a review conformation of the S protein, which is competent for mem- on the roles of furin in homeostasis and disease, see ref. (14)). brane fusion and infection (see e.g., refs. (2, 13) and Fig. 1C). Because of furin-mediated proteolysis, the spike proteins in the 2 J. Biol. Chem. (2021) 296 100135 JBC REVIEWS: On the mechanism of SARS-CoV-2 spike protein activation Figure 2. Membrane-associated serine proteinases (MASPs): mosaic proteinases for cleavage of transmembrane and membrane-proximal sub- strates. Structural domains (approximate scale) are color-coded. Note the variety of noncatalytic scaffolds found in these mosaic proteinases, most notably in the matriptases (TMPRSS6, -7 and -14), corin/TMPRSS10, and enterokinase/TMPRSS15. In addition to type II transmembrane serine proteinases (TTSPs), type I or glycosylphosphatidylinositol (GPI)-anchored proteins that possess similar catalytic domains are also included (TMPRSS12/TMPSD, PRSS8/prostasin, PRSS21/testisin, PRS41/TESSP-1, and PRS55/T-SP1). CUB, complement C1r/C1s, Uegf, Bmp1; FRZ, frizzled; LDLR, low-density lipoprotein receptor; MAM, meprin, A-5 protein, and receptor protein-tyrosine phosphatase mu; SEA, sea urchin sperm protein, enterokinase, agrin; SRCR, scavenger receptor cysteine- rich. SARS-CoV-2 virions are found in a preactivated state and androgen-induced TMPRSS2 (23). These and other MASPs require cleavage of a single bond, Arg815-Ser816, for activa- are predicted to function as initiators of cascades of trans- and tion of the fusion machinery. This unique feature of SARS- coactivating proteases (16), which signal through important CoV-2 is essential for S protein-mediated cell–cell fusion physiological substrates such as the G-protein-coupled and entry into human cells and thus, an important determi- receptor, protease-activated receptor 2 (PAR2), the epithelial nant of viral infectivity and pathogenesis (6). sodium channel (ENaC), several matrix metalloproteinases (MMPs), and prohepatocyte growth factor (pro-HGF). This is Several membrane-associated serine proteinases might in addition to only poorly understood cross talk between synergize with or replace TMPRSS2 as cellular activator MASPs and the blood coagulation and fibrinolytic cascades, of SARS-CoV-2 including the activation of urokinase-type plasminogen acti- The canonical activator of SARS-CoV-1/-2, TMPRSS2, be- vator (uPA) (see e.g., refs. (24, 25) and below). longs to a subfamily of multidomain, transmembrane serine proteinases with important roles in development and ho- Colocalization of ACE2 and priming serine proteinases is meostasis (15–19). (The domain organization of TMPRSS2 essential for SARS-CoV-2 infection and related, membrane-associated serine proteinases (MASPs) ACE2 and TMPRSS2 colocalize on the surface of certain cell is schematically represented in Fig. 2; relevant information on types and act, de facto, as an entry platform for SARS-CoV-1 these enzymes is summarized in Table 1). Notably, most and -2 (6). ACE2 and TMPRSS2 are coexpressed at particu- MASPs are exclusively or predominantly expressed in specific larly high levels in ciliated and secretory cells of the nasal human organs, including the heart (corin/TMPRSS10), liver cavity, which probably function as portals for initial infection (hepsin/TMPRSS1 and matriptase-2/TMPRSS6), brain by SARS-CoV-2 (26). The two genes and FURIN are primarily (spinesin/TMPRSS5), esophagus (TMPRSS4 and TMPRSS11A coexpressed in the lungs in a subset of bronchial cells differ- to F), prostate (TMPRSS2 and prostasin/PRSS8), and testes entiating from secretory to ciliated identity (27), in line with (matriptase-3/TMPRSS7, TMPRSS12, testisin/PRSS21, the more frequent and potentially life-threatening complica- TESSP-1/PRSS41, and T-SP1/PRSS55) (Figs. 3A and 4A). tion of COVID-19, pneumonia. Finally, high ACE2 expression Most MASPs possess basal activity in their zymogenic, levels in differentiated enterocytes, the intestinal absorptive single-chain forms and could generate full proteolytic activity cells, might also explain the fact that the intestine is another after clustering on the cell membrane, which triggers autoac- important viral target (28). tivation in trans. Prostasin, which does not autoactivate (20), However, it has now become evident that SARS-CoV-2 is has been shown to form an evolutionary conserved proteolytic capable of infecting not only cells of the respiratory and in- cascade with matriptase essential for epithelial development testinal tracts but several other human organs as well, and (see e.g., refs. (21, 22)). Further, matriptase is also activated by COVID-19 is currently considered a systemic disease, for J. Biol. Chem. (2021) 296 100135 3 JBC REVIEWS: On the mechanism of SARS-CoV-2 spike protein activation Table 1 Summary of information regarding human membrane-associated serine proteinases (MASPs) Length Expression pattern and relevance for Recommended MASP namea/Other names EC/MEROPSb Gene name (residues) pathology Hepsin 3.4.21.106/ HPN (TMPRSS1) 417 Expressed at highest levels in the liver. Also TMPRSS1 S01.224 expressed at high levels in the kidneys and pancreas. Other expression organs: stomach and thyroid gland. Overexpressed in several primary tumors, in particular in prostate and ovarian cancers. TMPS2 3.4.21.-/S01.247 TMPRSS2 (PRSS10) 492 Highest expression in the prostate, stomach, TMPRSS2, epitheliasin, serine protease 10 small intestine, and colon. Expressed also in (PRSS10) the airway and alveolar cells. Overexpressed in most prostate cancer patients. Proteolytically cleaves and activates SARS-CoV-1 and 2 as well as MERS-CoV S proteins, which triggers virus–cell membrane fusion. TMPS3 3.4.21.-/S01.079 TMPRSS3 (ECHOS1, 454 TTSP also reported as an endothelium reticu- TMPRSS3, tumor-associated differentially TADG12) lum protein. Expressed in the inner ear and expressed gene 12 protein (TADG-12) linked to deafness. Expressed also at low levels in the stomach and breast, among other organs. TMPS4 3.4.21.-/S01.034 TMPRSS4 437 Highest expression levels in the esophagus. TMPRSS4, channel-activating protease 2 Broadly expressed in the kidney, bladder, co- (CAPH2), membrane-type serine protease 2 lon, small intestine, prostate, and vagina, (MT-SP2) among other tissues. Upregulated in prostate, colon, and gastric cancers. TMPS5 3.4.21.-/S01.313 TMPRSS5 457 Predominantly expressed in neurons, their TMPRSS5, spinal cord–enriched trypsin-like axons, and at the synapses of motor neurons protease (spinesin) in the spinal cord. Expressed at highest levels in the tibial nerve. Expressed throughout the brain. TMPS6 3.4.21.-/S01.308 TMPRSS6 811 Highest expression levels in the liver. Also TMPRSS6, matriptase-2 (MT2) expressed in the testis and in the pituitary gland. Plays a critical role in the regulation of iron homeostasis through cleavage of the cell surface BMP coreceptor, hemojuvelin. TMPS7 3.4.21.-/S01.072 TMPRSS7 843 Highest expression levels in the testis. Expressed TMPRSS7, matriptase-3 (MT3) also at significant levels in the skin, brain, lungs, as well as pituitary, thyroid, and minor salivary glands. TMPS9 3.4.21.-/S01.357 TMPRSS9 1059 Highest expression levels in the testis, but also TMPRSS9, polyserine protease 1, expressed in the spleen and liver. polyserase-1 Corin 3.4.21.-/S01.019 CORIN (CRN, TMPRSS10) 1042 Highly expressed in the heart (left ventricle and TMPRSS10, atrial natriuretic peptide- atrial appendage). Also expressed in the skin, converting enzyme, heart-specific serine pro- uterus, and vagina, among other organs. Ac- teinase ATC2 tivates the cardiac hormone, atrial natriuretic peptide, and thus involved in blood pressure regulation. TM11A 3.4.21.-/S01.292 TMPRSS11A (HATL1, ECRG1, 421 Expressed at highest levels in the esophagus, TMPRSS11A, airway trypsin-like protease 1, HESP) vagina, and cervix. Also expressed in the (HATL1), esophageal cancer-susceptibility lungs. gene 1 protein, epidermal type-II trans- membrane serine protease TM11B 3.4.21.-/S01.365 TMPRSS11B 416 Expressed at highest levels in the cervix, TMPRSS11B, airway trypsin-like protease 5 esophagus, and oral cavity. Significantly (HATL5) decreased in cervical, esophageal, and head and neck carcinomas. Highly upregulated in the lung squamous cell carcinoma. TM11D 3.4.21.-/S01.047 TMPRSS11D (HAT) 418 Expressed at highest levels in the vagina and TMPRSS11D, airway trypsin-like protease esophagus. Expressed also in the cells of the (HAT) submucosal serous glands of the bronchi and trachea. TM11E 3.4.21.-/S01.021 TMPRSS11E (DESC1) 423 Expressed at highest levels in the esophagus and TMPRSS11E, differentially-expressed in squa- vagina. Also expressed in the bladder. Upre- mous cell carcinoma gene 1 (DESC1) gulated in tumors of different origin. TM11 F 3.4.21.-/S01.321 TMPRSS11F (HATL4) 438 Expressed at highest levels in the esophagus and TMPRSS11 F, airway trypsin-like protease 4 vagina. Also expressed in the skin. Unique (HATL4) function in epidermal barrier formation. TMPSC 3.4.21.-/S01.291 TMPRSS12 348 Type I transmembrane protein expressed almost TMPRSS12 exclusively in the testis. Expressed in colo- rectal cancer. TMPSD 3.4.21.-/S01.087 TMPRSS13 (MSP, TMPRSS11) 586 Predominantly expressed in the skin. Also TMPRSS13, membrane-type mosaic serine expressed in the esophagus and vagina, among protease (MSP, MSPL, MSPS) other organs. Cell-surface expression regu- lated by phosphorylation of its intracellular peptide. ST14 3.4.21.109/ ST14 (PRSS14, SNC19, 855 Ubiquitously expressed, at highest levels in the TMPRSS14, matriptase, membrane-type serine S01.302 TADG15) colon and small intestine, but also in the skin, protease 1 (MT-SP1), tumor-associated lung, kidneys, breast, and vagina. Proteolytic activity essential for epithelial integrity. 4 J. Biol. Chem. (2021) 296 100135 JBC REVIEWS: On the mechanism of SARS-CoV-2 spike protein activation Table 1—Continued Length Expression pattern and relevance for Recommended MASP namea/Other names EC/MEROPSb Gene name (residues) pathology differentially expressed gene 15 protein Implicated in the development and progres- (TADG-15), epithin, SNC19 sion of several cancers. Proposed to play a role in breast cancer invasion and metastasis. Forms reciprocal zymogen activation complex with prostasin. Enteropeptidase 3.4.21.9/S01.156 TMPRSS15 (ENTK, PRSS7) 1019 Expression restricted to the small intestine. TMPRSS15, enterokinase, serine protease 7 Starts a zymogen cascade leading to digestive (PRSS7) enzyme activation by converting trypsinogen to trypsin. Prostasin 3.4.21.-/S01.159 PRSS8 343c Type I transmembrane protein with a preference PRSS8, channel-activating protease-1 (CAP1), for polybasic substrates.d Ubiquitously serine protease 8 (PRSS8) expressed, at highest levels in the colon and small intestine, but also in the liver, salivary gland, kidney (renal proximal tubular cells), lung, skin. Forms reciprocal zymogen activa- tion complex with matriptase. Testisin 3.4.21.-/S01.011 PRSS21 (ESP1, TEST1) 314e GPI-anchored protein. Expressed only in the PRSS21, eosinophil serine protease 1 (ESP1), testis but lost in testicular tumors. Also TESP5 expressed in the minor salivary gland and lungs. Important for sperm cell maturation and fertilizing ability. PRSS41 3.4.21.-/S01.417 TESSP1 318f Testis-specific GPI-anchored protein. Also Testis serine protease 1 (TESSP-1) expressed in the minor salivary gland and lungs. Required for the progression of meiosis during spermatogenesis. PRSS55 3.4.21.-/S01.299 PRSS55 (TSP1) 352g Type I transmembrane protein.h Expressed at Testis serine protease 1 (T-SP1) highest levels in the testis, but also found in the brain. Expressed in prostate and ovarian cancer. Plays a crucial role in sperm migration and sperm–egg interaction. Information has been gathered from UniProt (www.uniprot.org) and MEROPS databases (https://www.ebi.ac.uk/merops/) or from cited references. a As deposited in the UniProt database. b Accession numbers according to the Enzyme Commission (EC) and MEROPS databases. c Refers to the full-length protein; residues Met1-Gly29 form the signal peptide and are removed, as well as the Ala30-Gly32 activation peptide. The C-terminal residues Pro323- His343 are reported to be removed during maturation. d According to some authors, the protein is GPI-anchored instead. e Refers to the full-length protein; the signal peptide, Met1-Arg19 is removed. The C-terminal propeptide, Gly289-Val314, is also cleaved during maturation. f Refers to the full-length protein; the signal peptide, Met1-Gly19 is removed. The C-terminal propeptide, Thr300-Pro318, is also cleaved during maturation. g Refers to the full-length protein; the signal peptide, Met1-Leu18, is removed, and the C-terminal residues Gly326-Tyr352 are also removed during maturation. h Controversial. According to some authors, the protein is GPI-anchored instead. which the term “viral sepsis” has been recently coined (29). In more poorly by hepsin (36) has also been reported (recently this regard, it is noteworthy that ACE2 and TMPRSS2 are not reviewed in ref. (37)). Further, TMPRSS3, TMPRSS4, matrip- coexpressed in the majority of tested cells (ref. (26); see also tase, and prostasin have all been reported as ENaC activators Fig. 4A). Such results suggest that other MASPs might either (38–42). Several MASPs cleave and activate pro-uPA potentiate priming of the S protein in ACE2+/TMPRSS2+ cells (24, 32, 43–46), presumably bound to its cell surface recep- (for instance, by simultaneously targeting two or all three S2’ tor, uPAR, which is additionally targeted by TMPRSS11D (47). sites in a trimeric S protein, thus exposing multiple fusion Both hepsin and matriptase have been identified as efficient, peptides at once) or even replace TMPRSS2 altogether in cells physiologically relevant activators of pro-HGF (43, 48–50) of organs/tissues that do not express significant levels of this and of the matrix metalloproteinases, proMMP-1 and -3 serine proteinase. (36, 51, 52). Finally, TMPRSS13 has also been reported as a pro-HGF activator (53). Noncatalytic domains do not influence the position of MASP On the other hand, regardless of the architecture of their active sites relative to the cell membrane noncatalytic chains, all well-characterized MASPs are inhibited At first sight, it could be thought that members of the MASP by either one or both transmembrane inhibitors, hepatocyte subfamily with longer, more complex noncatalytic chains than growth factor activator inhibitor-1/serine proteinase inhibitor, TMPRSS2 would be less likely to function as activators of Kunitz type 1 (HAI-1/SPINT1) or the related HAI-2/SPINT2 SARS-CoV-2 spike protein or to do so less efficiently (compare (see Fig. 4B for their domain organization). Kinetic analysis Fig. 2). However, several lines of evidence indicate that the has been reported so far for hepsin inhibition by the first actual MASP domain architecture is essentially irrelevant in Kunitz domain of HAI-1 (54) as well as for the matriptase- this regard. HAI-1 pair (55, 56). In addition, HAI-1 and/or HAI-2 have First, it is striking that diverse MASPs activate trans- been identified as physiologically relevant inhibitors of hepsin membrane or membrane-proximal substrates by cleaving the (50), TMPRSS2 (57–59), TMPRSS3 (57), TMPRSS4 (57, 60), same peptide bond. Although matriptase is considered the matriptase-2 (61, 62), HATL1/TMPRSS11A (57), HAT/ most potent PAR2 activator (30, 31), activation of the GPCR TMPRSS11D (63), DESC1/TMPRSS11E (64), TMPRSS13 by TMPRSS2, TMPRSS11D, and testisin (32–35) as well as (53, 57, 65), matriptase (58, 60, 66–70), enteropeptidase/ J. Biol. Chem. (2021) 296 100135 5 JBC REVIEWS: On the mechanism of SARS-CoV-2 spike protein activation Figure 3. Expression patterns and structure of membrane-associated serine proteinases. A, organs with highest expression levels of specific MASPs. Data for some organs are indicated only for males or females, although there are no known sex-associated differences in MASP expression in these organs. Data from the GTEx Portal (gtexportal.org) (see also Fig. 4A). B–C, 3D crystal structure of human hepsin/TMPRSS1 ectodomain, represented as a cartoon highlighting major secondary structure elements; loops that shape the active-site cleft are noted. (After PDB entry 1P57: N-terminal SRCR module (deep-teal cyan); serine proteinase domain (deep salmon-red)). A small-molecule inhibitor bound in the S1 specificity pocket of the serine proteinase domain (2-{5- [amino(iminio)methyl]-1H-benzimidazol-2-YL}benzenolate) is shown as color-coded spheres (carbon, pink; oxygen, red; nitrogen, blue; and hydrogen, gray). B, side view, highlighting the proximity of the N-terminal residue of the SRCR domain, Pro50, to the transmembrane helix, Gly24-Leu44. This locates also the rigidly attached catalytic domain of the proteinase essentially flat against the cell membrane (74). A similar localization should be expected for the catalytic domains of other MASPs, which implies that the Arg815-Ser816 bond in the S protein would be cleaved close to the cell membrane, facilitating rapid interaction with the exposed viral fusion peptide and escape from immune surveillance. C, view of the proteinase in the “standard orientation”, e.g., with active-site residues (given with all their nonhydrogen atoms, color-coded) facing the viewer and substrates running from left to right. The N and C termini of the catalytic chain are noted (Ile16 and Thr253, respectively) as well as Asp189 at the bottom of the S1 pocket, which is largely responsible for the recognition and cleavage of substrates after a basic Arg/Lys residue. TMPRSS15 (57), and prostasin (68, 71–73). In fact, HAI-2 has the high degree of structural plasticity of the spike protein, been recently proposed as a broad-spectrum antiviral agent exemplified by the transition between “up” (ACE2 accessible) (58). and “down” (ACE2 inaccessible) conformations, and finally (3) The relative positions of SRCR and serine proteinase mod- the flexibility provided to ACE2 and MASPs by some of their ules in the crystal structure of hepsin strongly suggest that the interdomain linkers, which would support both large rotations catalytic domain of the proteinase lies essentially flat against and displacements of protein modules relative to each other the plasma membrane (ref. (74); see also Fig. 3B). In the light of and to the cell membrane. The combined result would be the the shared substrate and inhibitor profiles of several MASPs rapid (re)positioning of the S2’ sites of the spike protein in the discussed above, a similar localization should be expected for catalytic cleft of the activating proteinase, in a conformation the catalytic domains of all other related, membrane-bound compatible with proteolysis of the Arg815-Ser816 bond. proteinases. Thus, the combined structural and functional evidence strongly suggests that the active sites of all MASPs The catalytic domains of most MASPs could efficiently cleave would be located close to the cell membrane. An important the Arg815-Ser816 peptide bond in SARS-CoV-2 spike protein consequence for SARS-CoV-2 activation is that, upon cleavage Another issue arises when assessing the capability of MASPs of the Arg815-Ser816 bond in the S protein, the exposed viral other than TMPRSS2 to activate viral S proteins: could unique fusion peptide would be able to rapidly interact with the host- features of this serine proteinase favor recognition and cell phospholipid membrane, thus allowing escape from proteolysis of the Arg815-Ser816 peptide bond? Despite highly immune surveillance. divergent domain architectures (Fig. 2), all MASPs share Three major features would facilitate MASP access to the well-conserved, trypsin-like catalytic domains. (Mechanism S2’ sites of an ACE2-bound, trimeric viral S protein: (1) the and specificity of serine proteases are reviewed in ref. (75)). fluidity of phospholipid membranes, which allows lateral dis- Crystal structures have been reported for the catalytic domains placements of the viral protein and of its cellular receptors, (2) of hepsin (48, 74, 76, 77) (see also Fig. 3, B–C), DESC1 (78), 6 J. Biol. Chem. (2021) 296 100135 JBC REVIEWS: On the mechanism of SARS-CoV-2 spike protein activation Figure 4. Expression patterns of human ACE2, MASPs and their endogenous inhibitors. A, comparison of the expression levels of human ACE2 and a selection of those MASPs with restricted expression patterns. Spinesin/TMPRSS5 is predominantly expressed in the brain and the tibial nerve, corin in the heart, and TMPRSS12, PRSS21, PRSS41 as well as PRSS51 in the testis. Further, matriptase-2 and hepsin are expressed at highest levels in the liver or in liver and kidney, respectively. The members of the airway tract subgroup, TMPRSS11A–11F are predominantly expressed in the mucosa of the esophagus and in the vagina. B, schematic representation of domain organization in the endogenous Kunitz-type inhibitors of membrane-associated serine proteinases, HAI-1/SPINT1 and HAI-2/SPINT2. C–D, expression patterns of human HAI-1 (C) and HAI-2 (D). Note the overall complementarity of expression profiles in different organs (e.g., HAI-2 but not its paralog is expressed in the brain and arteries), although both inhibitors are similarly expressed in several organs, including kidneys, prostate, and vagina. The extremely low expression of both Kunitz inhibitors in the heart is also noteworthy, which suggests that the proteolytic activity of the heart-specific MASP, corin/TMPRSS10, would only be controlled by circulating serpins. The expression values are given as Transcripts per Million (TPM), as reported in the GTex Portal (gtexportal.org). HAI, hepatocyte growth factor activator inhibitor; MANEC, motif at N-terminus with eight-cysteines; SPINT, serine protease inhibitor Kunitz type. J. Biol. Chem. (2021) 296 100135 7 JBC REVIEWS: On the mechanism of SARS-CoV-2 spike protein activation matriptase (79–88), enterokinase (89), and prostasin (90–92), protein even more efficiently than TMPRSS2, at least in vitro either free (unliganded) or, more commonly, bound to natural (97). This is in line with previous reports on the activator or synthetic inhibitors. This wealth of structural information activity of these MASPs: TMPRSS11A had been shown to allows generation of high-quality 3D models for the remaining activate MERS spike protein as well as hemagglutinin (64), members of the subfamily, including TMPRSS2. TMPRSS11D primes SARS-CoV S protein for membrane A detailed analysis of the substrate and inhibitor specificity fusion and also cleaves ACE2 (3, 98), while TMPRSS11E had of all MASPs in the light of these structures/models has been reported as an activator of SARS and MERS coronavi- important implications for the mechanism of viral S protein ruses (95). cleavage. In particular, it can be predicted that most MASPs In addition to these reports on the activation of SARS-CoV- could target the Arg815(P1)-Ser816(P1’) bond of SARS-CoV-2 1/-2 and MERS-CoV by multiple MASPs, a large body of spike protein with similar efficiency as the canonical activator, experimental evidence indicates that hepsin, TMPRSS4, TMPRSS2.† Prominent exceptions would be enterokinase and TMPRSS11D, TMPRSS12, TMPRSS13, matriptase, and pros- TMPRSS11B, which appear to have quite restricted substrate tasin activate certain influenza strains among other respiratory specificities: acidic residues at the P4–P2 positions and side- viruses, although significantly less efficiently than TMPRSS2 in chain-less glycines at P1’ and P2’, respectively. most cases (99–110). Considering the experimentally verified Thus, furin-mediated cleavage at the S1/S2 site would involvement of certain MASPs in SARS-CoV-1/-2 priming in enhance infectivity of SARS-CoV-2 because S protein model systems, activation of the fusion process in other monomers would not need to be repositioned in the active viruses, expression levels, and similarity of their catalytic site cleft of the activating MASP, but most importantly so machineries to that of TMPRSS2, a putative ranking of MASPs because several MASPs would be able to attack the Arg815- expected ability to prime SARS-CoV-2 spike protein can be Ser816 bond at a similar rate as TMPRSS2, which would not postulated: TMPRSS4, TMPRSS13 > TMPRSS11D, 11E, 11F be the case if they had to target the S1/S2 site first. (Because > TMPRSS11A > matriptase/prostasin > matriptase-2, bulky and basic arginine residues are present at positions P3 hepsin, TMPRSS5 > testisin, matriptase-3, corin > and P4 of this site (Fig. 1A), the Arg685-Ser686 peptide bond TMPRSS9, TMPRSS12, PRSS41 > PRSS55, TMPRSS3 > is much less likely to be efficiently targeted by multiple TMPRSS1B > enterokinase. MASPs. Matriptase, for instance, prefers substrates with basic Arg/Lys residues at positions P3 or P4, but not both, and also TMPRSS11B is unlikely to be an efficient activator of SARS- TMPRSS9 has been shown to be a poor activator of sub- CoV-2 S protein but might be involved in an alternative strates with polybasic sequences). Of note, the presence of a pathway of viral cell entry furin-cleavage site in the influenza fusion protein, hemag- Analysis of MASP active site machineries suggests that only glutinin, sets apart low from high pathogenicity avian influ- side-chain-less glycine residues would be well tolerated at the enza viruses (93). positions following a basic Arg/Lys-P1 residue in TMPRSS11B substrates. The only bona fide substrate of this MASP reported Several MASPs are able to activate viral fusion machineries to date is the transmembrane protein, basigin (CD147/ In addition to TMPRSS2, other MASPs have been reported EMMPRIN) (111). The presence of a K↓GG site suggests that as activators of diverse viral fusion proteins, at least in vitro.‡ cleavage after Lys57 in the common basigin isoform would It was recently shown that TMPRSS2 and TMPRSS4 syner- shed (“solubilize”) the receptor from the cell surface, as re- gize to promote infection of small intestinal enterocytes by ported by Updegraff and coworkers. SARS-CoV-2 (94). TMPRSS13 had been previously shown to Basigin has been proposed as an alternative receptor for prime both SARS- and MERS-CoV S proteins (95), and a SARS-CoV-2 S protein (112), although other authors have recent investigation suggests that it is even a more efficient failed to detect interactions between the viral protein and activator of SARS-CoV-2 spike protein than TMPRSS2 (96). human basigin (113). By contrast to ACE2, basigin is ubiqui- These authors also demonstrate significant priming potential tously expressed at high levels, most notably in the heart. Of of three members of the HAT/DESC subgroup of membrane note, the receptor could also be targeted by TMPRSS11D serine proteinases, TMPRSS11D, TMPRSS11E, and (114), which reminds of the reported cleavage of ACE2 by TMPRSS11F, but only limited activity for TMPRSS11A and TMPRSS2 and other MASPs. Along these lines, basigin had TMPRSS11B. By contrast, another recently presented work been previously identified as a receptor essential for erythro- suggests that TMPRSS11A might process SARS-CoV-2 spike cyte invasion by the malaria parasite, Plasmodium falciparum (115), as well as for vascular colonization by Neisseria men- † According to the Schechter and Berger nomenclature, substrate residues ingitidis (116), and has also been linked to cytomegalovirus are denoted as Pm, …, P1, P1’, …, Pn’, from N to C terminus; P1-P1’ is the infection of both endothelial and epithelial cells (117). Thus, scissile peptide bond to be cleaved by the proteinase. The cognate the pathological relevance of a cell entry pathway involving binding sites for these residues in the proteolytic enzyme are termed Sm, …, S1, S1’, …, Sn’; not to be confused with the viral S1/S2/S2’ subunits. basigin—and perhaps also its closest relatives within the ‡ Several trypsin-like serine proteinases cleave the SARS-CoV S proteins and immunoglobulin superfamily, neuroplastin and embigin— thus function as entry cofactors in vitro, including trypsin itself, as well as deserves careful examination, as emergence of a second high- the procoagulant factor, FXa, and the major fibrinolytic enzyme, plasmin. However, these proteinases are unlikely to act as pathologically relevant affinity receptor for the spike protein in SARS-CoV-2 mutants activators of S proteins in vivo. might have catastrophic consequences. 8 J. Biol. Chem. (2021) 296 100135 JBC REVIEWS: On the mechanism of SARS-CoV-2 spike protein activation “Proteolytic surveillance”: endogenous MASP inhibitors are members of the subfamily. Altogether, the fusion activation likely to regulate SARS-CoV-2 infection in vivo potential of TMPRSS2 and other MASPs in vivo would be The physiological activities of all MASPs are tightly critically codetermined by the concentrations of cognate controlled by endogenous proteinaceous inhibitors that belong inhibitors of the Kunitz and serpin classes and their relative to either the Kunitz or the serpin families (I1 and I4 in the affinities for the priming proteinase. It is likely that imbalance MEROPS classification, respectively). The transmembrane in the expression levels of MASPs, on the one side, and members of the Kunitz family, HAI-1/SPINT1 and HAI-2/ HAI-1/-2 and serpins, on the other, would determine the SPINT2, have been experimentally shown to inhibit the cata- likelihood of SARS-CoV-2 infection of a given cell and thus lytic activity of every MASP tested so far. (See above and ultimately of COVID-19 progression, similar to the well- Fig. 4B for a schematic representation of HAI-1/-2 domain documented situation in cancer (128, 129). I propose the organization). Indeed, as expected from the high degree of term “proteolytic surveillance” to refer to this up to now sequence conservation at and around the active site of these unappreciated level of control of viral infection. proteinases, current structural information supports formation of stable MASP⋅HAI-1/HAI-2 complexes (80, 118). Overall, Signaling, endocytosis, and shedding the two Kunitz inhibitors show a complementary pattern of I finally mention two only poorly understood features of expression in human organs/tissues, which would allow inhi- MASPs, but that potentially impact the life cycle of these bition of MASP activity throughout the body, with the notable proteinases, and therefore their capacity to prime S proteins exception of the heart (Fig. 4, C–D). Thus, HAI-1 and HAI-2, and contribute to viral infection. It is noteworthy that the as master regulators of all MASPs, would play an important cytoplasmic domains of several members of the subfamily role in SARS-CoV-2 propagation by regulating the availability possess consensus phosphorylation sites for protein kinase C of serine proteinase active sites on host cell membranes. and/or casein kinase II (reviewed in ref. (15)). This observation Indeed, it has been recently shown that HAI-2 reduces suggests that phosphorylation and perhaps also other post- cleavage/activation and growth of several influenza viruses by translational modifications of the cytoplasmic tails of MASPs inhibiting TMPRSS2 or matriptase (58). could promote endocytosis. This is in addition to the presence On the other hand, recently published results demonstrate of specific, short linear peptide motifs in the cytoplasmic that TMPRSS2 is targeted by the serpin, α1-antitrypsin (AAT/ domains of MASPs important for proteinase trafficking, as SERPINA1), which inhibits SARS-CoV-2 infection in vitro recently reported for matriptase (130). (119, 120). Notably, other reports show that AAT inhibits The role of the endocytic pathway in the replication process protease-mediated cell entry by SARS-CoV-2 at concentra- of coronaviruses, and in particular of SARS-CoV-1/-2, remains tions much lower than those found in serum and bron- disputed, with strong evidence indicating that major auto- choalveolar tissues, which suggests that the serpin is a phagy factors of the host cell are not required for viral infec- physiologically relevant inhibitor of viral infection (121). AAT, tion (reviewed in ref. (112)). It is currently unknown whether however, is more specific for elastase-like serine proteinases, as engagement of ACE2 and TMPRSS2 or other MASPs by viral indicated by the presence of a hydrophobic methionine as P1 proteins elicits intracellular signaling pathways that might residue. Noteworthy, several additional, trypsin-like MASPs enhance cellular uptake of virions. Overall, it is believed that (hepsin, all three matriptases, and DEC1) are potently inhibi- the MASP-mediated proteolytic cleavage and concomitant ted in vitro by the most important circulating serpin, anti- direct cell–cell fusion (the so-called “early” pathway) are the thrombin (AT/SERPINC1) (122–124). A similar inhibitory pathologically relevant events in SARS-CoV-1 infection of capacity has been reported for other abundant serpins with respiratory epithelia in vivo, while endocytic trafficking (the broad specificities such as plasminogen activator inhibitor 1 “late” pathway) plays a secondary role in this regard (131). (PAI-1/SERPINE1), α2-antiplasmin (α2-AP/SERPINF2), and Similar considerations would likely apply for SARS-CoV-2 and protein C inhibitor (PCI/SERPINA5). Prostasin activity is are probably at the heart of the poor results of autophagy regulated in vivo by the closest phylogenetic relative of PAI-1, inhibitors, chloroquine and its derivative, hydroxychloroquine, protease nexin-1/SERPINE2 (125), which also potently inhibits both for prophylaxis and treatment of COVID-19. matriptase, at least in vitro (126). Finally, the ubiquitously Another issue that deserves consideration is the frequent expressed SERPINB12 has been reported as a slow-binding detection of soluble MASP forms (reviewed in ref. (15)). inhibitor of hepsin (127). AT and other serpins interact with Although alternative splicing could explain the presence of proteoglycans and glycosaminoglycans present in large some circulating variants of these proteinases, active shedding amounts on cell membranes and in the extracellular matrix. from the cell membrane upon proteolytic cleavage seems to be This interaction allows serpin trapping near the cell membrane the predominant mechanism. At least in the cases of hepsin, and therefore increases the probability of productive complex TMPRSS2, and matriptase, the same proteinases are respon- formation with active MASPs. sible for ectodomain shedding, following cleavage in trans Similar to the transmembrane Kunitz-type inhibitors, the (132). However, the identity of proteinases that shed other high degree of conservation of residues that shape the active- MASPs has not been established; matrix metalloproteinases site clefts of these serine proteinases strongly suggests that (ADAMs and MT-MMPs) are also likely to play a role in this major circulating serpins would also target most, if not all, regard, as demonstrated for HAI-1. These “sheddases” might J. Biol. Chem. (2021) 296 100135 9 JBC REVIEWS: On the mechanism of SARS-CoV-2 spike protein activation Table 2 Hypothesized correlations between expression patterns of MASPs other than TMPRSS2 and pathological observations in COVID-19 patients Likely relevance for SARS-CoV-2 infection and Putative priming MASP Relevant expression organ/tissue COVID-19 pathology TMPRSS12, PRSS21 (testisin), PRSS41 (TESSP-1), and Male reproductive organs (prostate, testis) Possible viral reservoir. More severe complications of PRSS55 (T-SP1)a + TMPRSS6 (matriptase-2) + TMPRSS7 COVID-19 in male patients, leading to significantly (matriptase-3) higher mortality rates. Airway trypsin-like proteases TMPRSS11A (HATL1), At highest levels in the esophagus and minor Alternative entry portals for SARS-CoV-2. Might TMPRSS11D (HAT), TMPRSS11E (DESC1), and salivary gland, but also expressed in the contribute to and/or worsen lung infection/ TMPRSS11F (HATL4) + TMPRSS4 + TMPRSS7 (matrip- bronchi, trachea, and lungs pneumonia. tase-3) + TMPRSS14 (matriptase) + PRSS8 (prostasin) TMPRSS5 (spinesin) Brain/tibial nerve Might explain the neurological complications re- + TMPRSS7 (matriptase-3) ported for some patients, as well as impaired motor functions. TMPRSS6 (matriptase-2) Liver Might contribute to liver damage. + TMPRSS1 (hepsin) TMPRSS10 (corin) Cardiomyocytes Might be responsible for heart damage. Might indirectly contribute to thrombotic complications through dysregulation of blood pressure. TMPRSS1 (hepsin) + TMPRSS4 + TMPRSS14 Kidney Might contribute to acute kidney injury (AKI). (matriptase) + PRSS8 (prostasin) TMPRSS14 (matriptase) + PRSS8 (prostasin) Ubiquitously expressed in epithelial cells. Involved in u-PA activation. Might be linked to thrombotic complications. Might also be linked to intestinal infection. a Note that both PRSS41 and PRSS55 are termed “testis serine protease 1”. represent yet another control element of viral uptake (e.g., the a SARS complication, and the testes of patients who died of higher the sheddase activity, the lower the density of MASPs SARS-CoV-1 infection displayed widespread germ cell on cell membranes, and thus the lower the probability that a destruction with few or no spermatozoa (138). Persistent given cell would be infected). infection of this immune-privileged site by other unrelated, single-stranded RNA viruses has also been reported. Sertoli Implications for the cell tropism of SARS-CoV-2 and cells serve as reservoir for the Zika virus (139), and the testes clinical manifestations of COVID-19 are the preferred replication site of this flavivirus (140). Long- The presence of several MASPs capable of priming the spike term testicular persistence of Ebola and Marburg viruses has proteins of SARS-CoV-2 virions, in synergy with or in place of been well documented and is responsible for sexual trans- TMPRSS2, offers straightforward explanations for clinical data mission of these filoviruses (141, 142). on COVID-19 that remain unresolved to date (Table 2). The presence of multiple priming MASPs exposed on Noteworthy, some family members are exclusively or pre- different human organs might account for the establishment of dominantly expressed in male reproductive organs, alone four putative SARS-CoV-2 reservoirs. Identification of these res- of them in the testis (TMPRSS12, PRSS21/testisin, PRSS41/ ervoirs is an urgent need in the light of the growing number of TESSP-1 and PRSS55/T-SP1; Figs. 3A and 4A). Along these patients with persistent COVID-19 symptoms (chronic or long lines, ACE2 is expressed at relatively high levels in the testis, COVID), as well as first reports of placental infection with only second to the small intestine (Fig. 4A). These features, SARS-CoV-2 (143–145) and vertical (transplacental) trans- together with the upregulation of TMPRSS2 expression by mission of the virus to the fetus (146). In addition to the testes, androgens (23, 133), might explain the more severe compli- other immune-privileged organs such as the eyes and brain cations of COVID-19 in male compared with age-matched might serve as sanctuary for the coronavirus. female patients, including a much higher death rate (2.8 The preferential expression of specific MASPs in organs/ versus 1.7%; see e.g., refs. (134, 135)). The relative contribution tissues that are well-known targets of SARS-CoV-2 infection is of MASPs other than TMPRSS2 to this sex bias is highlighted also striking (Fig. 3A) and may have important pathological by the fact that the outcome of influenza infection is generally implications. Most notably, members of the HAT/DESC sub- worse for women instead (136). (Of note, almost all MASPs group, TMPRSS11A and 11D to 11F, together with other reported to activate specific influenza strains—hepsin, MASPs expressed in the airway tract (TMPRSS4, 7, 13, and TMPRSS4, TMPRSS13, and in particular, TMPRSS11D; see 14), could contribute to the severe pneumonia observed in an above—are predominantly expressed in the female, rather than important subset of COVID-19 patients. Further, engagement in the male reproductive organs. Even the matriptase– of corin/TMPRSS10 by SARS-CoV-2 in cardiomyocytes may prostasin pair is ubiquitously expressed and at similar levels explain the frequent myocardial damage reported in COVID- in both male and female organs). 19 patients (147, 148). Myocardial infection by SARS-CoV-2 These observations raise the hypothesis that male repro- might be facilitated by the extremely low expression levels of ductive organs could be infected with SARS-CoV-2 and HAI-1 and HAI-2 in the heart (Fig. 4, B−C), which suggests perhaps serve as viral reservoirs. Indeed, the testis is one of the that the proteolytic activity of corin would be less tightly organs in which more interactions between viral and host controlled than other members of the subfamily. Indeed, first proteins were detected in a systematic study of the viral autopsy findings reveal relatively high viral RNA titers in the interactome, only second to the lungs (137). Of note, orchitis is heart, liver, brain, kidneys, blood, and/or gastrointestinal tract 10 J. Biol. Chem. (2021) 296 100135 JBC REVIEWS: On the mechanism of SARS-CoV-2 spike protein activation of several patients who died of confirmed SARS-CoV-2 realize that S protein priming is followed by dramatic struc- infection (149–151). Also along these lines, SARS-CoV-2 tural rearrangements, which lead to an unstable conformation RNA was detected in the heart and brain of infected mice in of the S1 “crown” with all three RBDs up and its concomitant a recently presented model of the disease (152). The situation shedding. Thus, released S1 subunits might function as decoy is also reminiscent of the systemic character of the influenza antigens for circulating antibodies. This feature needs to be (H5N1) infection in cats, in which the virus could be detected considered in the light of current efforts to develop vaccines most often in the heart, brain, liver, and kidney, in addition to based on immunization with the viral S protein or its RBD, as respiratory tissues (153). it might compromise the effectiveness of this approach. In The matriptase–prostasin axis might also play an important addition to immune surveillance, the fusion machinery is role as viral activator, as these proteinases have the broadest under proteolytic surveillance by both proximal, trans- expression patterns of all MASPs and are essential for the membrane Kunitz inhibitors (HAI-1/HAI-2) andcirculating maintenance of epithelial barrier integrity. Indeed, infection of serpins. Finally, pericellular proteolysis of host proteins could epithelial cells by SARS-CoV-2 in severe COVID-19 patients either enhance virion uptake, as in the case of TMPRSS2- has been recently reported (154). The matriptase–prostasin mediated cleavage of ACE2, or abort the whole membrane pair could in particular contributes to intestinal infection. fusion process, upon MASP shedding. It is also noteworthy that several MASPs are directly An important element of the mechanism, revealed upon involved in plasminogen activation and fibrinolysis via pro- inspection of the 3D structures of SARS-CoV-2 S protein in its uPA cleavage (see ref. (24) and above). Matriptase, in addi- S1/S2-cleaved, prefusion conformation (13, 158), is the fact tion, is engaged by blood coagulation factors upon activation that the S2’ activation cleavage site is not accessible to pro- of the tissue-factor-triggered clotting cascade (25). This unique teolytic attack. Most notably, the side chain of the specificity- location at the crossroads of epithelial signaling and the blood determining P1 residue, Arg815, occupies a cleft between two coagulation and fibrinolytic cascades could be particularly prominent α-helices comprising the N-terminal residues of the relevant in the light of reported thrombotic complications in fusion peptide (Phe817-Asn824) and the following Asp867- COVID-19 patients (155). In addition to direct cardiomyocyte Ile882 stretch, with its guanidinium group compensated by damage, these complications might be at least partly related to the carboxylates of neighboring acidic residues (Fig. 1, C−D). the “hijacking” of matriptase and other proteinases by SARS- The side chain of this basic arginine residue stacks on the CoV-2 and/or to their reduced expression/enhanced endocy- aromatic ring of Phe823, one of the critical residues for tosis, as part of negative feedback mechanisms. Finally, MASP membrane fusion. The P1’ residue, Ser816, is even less acces- engagement in other organs might explain a number of addi- sible to bulk solvent, as it points toward the core of the subunit tional manifestations of COVID-19 reported so far, among (Fig. 1D). In this manner, Arg815 and surrounding residues them neurological complications including cognitive stabilize the prefusion conformation and avoid untimely dysfunction (156) as well as acute kidney injury (151, 157). exposure of the fusion machinery. It follows that an important degree of flexibility (“breath- An integrated mechanism of SARS-CoV-2 spike protein ing”) of the S2 subunit, in particular of residues surrounding cleavage and activation: an explanation for enhanced the S2’ activation cleavage site, is required for formation of viral infectivity productive MASP-S protein complexes and proteolysis. In summary, current structural and functional information Although the positions of Arg815 and neighboring residues are suggests that several related, transmembrane trypsin-like essentially conserved in the spike proteins of SARS-CoV-1 and serine proteinases decisively contribute to prime SARS-CoV- MERS-CoV, there are also important sequence and structural 2 S protein either in synergy with or in place of TMPRSS2 differences, which might have a bearing on their relative and therefore promote infection of ACE2-coexpressing cells infectivities (Fig. 1, E–F). In particular, the Arg815-pocket in in vivo. A mechanism of viral protein activation that integrates SARS-CoV-1 and MERS-CoV is more acidic and overall better current structural and functional information is schematically shaped to accommodate the Arg815 side chain. Therefore, I summarized in Figure 5. hypothesize that the S2’ activation cleavage site of SARS- The viral infection process could be viewed as a race, in CoV-2 spike protein would be more frequently exposed to which successful membrane fusion and infection depend on activating proteinases than those of the related coronaviruses, the balance between multiple competing protein–protein in- which would contribute to its enhanced infectivity. teractions. On the one hand, the viral spike protein requires simultaneous or highly synchronized binding to ACE2 and to Summary and perspectives the catalytic cleft of the priming MASP, in a conformation Unique adaptations of SARS-CoV-2 for human infection compatible with proteolysis of the S2’ cleavage site. Formation (e.g., higher affinity of the S protein for human ACE2, its of these complexes and cleavage/activation of membrane presence in a preactivated state in the virions following furin fusion could be counteracted by circulating immunoglobulins cleavage at the S1/S2 site, and its higher capacity for mem- directed against e.g., the RBD in its exposed, “up” conforma- brane fusion) are at the heart of the current COVID-19 tion, or against the fusion peptide exposed upon cleavage of pandemic. In addition, MASPs other than TMPRSS2 appear the Arg815-Ser816 bond. In this regard, it is important to to prime SARS-CoV-2 S protein in vivo, which is likely to play J. Biol. Chem. (2021) 296 100135 11 JBC REVIEWS: On the mechanism of SARS-CoV-2 spike protein activation Figure 5. Mechanism of S protein activation and viral–host cell membrane fusion. The SARS-CoV-2 spike protein is found in the virions in a metastable prefusion state, in which the S1/S2 site has been cleaved by furin within the secretory pathway. (1) The RBD within the N-terminal S1 subunit of the S protein is exposed in the so-called “up” conformation and engages the host cell receptor, ACE2. (In the more stable “down” conformation, the RBD is not accessible to ACE2, which probably helps to evade immune surveillance, ref. (7)). A single ACE2 molecule is shown bound to the RBD of an S monomer, but two trimeric S proteins could simultaneously engage an ACE2 dimer in vivo (12). Also omitted for simplicity is the B0AT1 molecule that binds to the long C-terminal helix of ACE2 (after PDB 6M17). (2) RBD–ACE2 complex formation stabilizes a metastable conformation of the spike protein, stimulating “breathing” of the S2’ site and eventual exposure of the Arg815-Ser816 peptide bond to proteolytic cleavage by TMPRSS2 or another MASP. (3) MASP-mediated cleavage and activation of the S protein are controlled by endogenous inhibitors, most notably HAI-1 and/or HAI-2, but also by circulating serpins. Here, the HAI-1 ectodomain is shown, according to the reported crystal structure (PDB 5H7V; ref. (162)); the side chain of the P1 residue, Arg260, is highlighted. Other cellular processes that coregulate membrane fusion and viral uptake are (4) shedding of ACE2, MASPs and/or HAI-1/HAI-2 ectodomains, either by MASPs in trans or by membrane-bound metalloproteinases, and (5) endocytosis, which could be modulated by phosphorylation of cytosolic peptides in the human factors, or by palmitoylation of the Cys-rich endodomain of SARS-CoV-2. The scheme focuses on the “early”, endocytosis- independent pathway of virus cell entry. Endocytic trafficking (the “late” pathway) seems to play a minor role. an important role in determining the viral cell tropism as well instance, are higher relative concentrations of inhibitors as disease progression. Among the critical questions that predictors of a less virulent infection or even protect remain unanswered are: against infection?). - Preliminary investigations provide direct evidence of SARS- - Which MASPs serve as viral activating factors? The substrate CoV-2 infection in e.g., the heart, liver, kidneys, and testes of and inhibitor profiles of these proteinases need to be COVID-19 patients (149–151). How frequently are these carefully characterized, including structural investigations organs infected by the coronavirus, and does this frequency and rigorous quantum mechanical/molecular dynamics correlate with measured viral titers and the relative expres- (QM/MD) simulations of their mechanism of S protein sion levels of specific MASPs? Which are the histopatho- recognition and proteolysis. logical and ultrastructural consequences of viral infection in - Which MASPs colocalize with human ACE2 in cells from these organs? different organs and tissues? Are there sex- and age-related - The correlation between COVID-19 and other comorbid- differences that could contribute to explain current clinical ities, in particular cancer, needs to be better understood. observations, such as the higher mortality rate in male TMPRSS2 is an androgen-induced prostate cancer-specific patients? Animal models might be useful to study the marker, thus its overexpression is straightforwardly linked contribution of each MASP to SARS-CoV-2 infection of to a higher risk of infection in prostate cancer patients. relevant cell types. (e.g., by crossing transgenic mice over- Indeed, patients receiving androgen-depravation therapy expressing human ACE2 (152) with conditional knockouts of showed a significantly lower risk of SARS-CoV-2 infection individual MASPs). compared with those not treated with antiandrogens (159). - Are there correlations between the levels of circulating forms Since most other MASPs are overexpressed in several cancer of specific MASPs and their endogenous inhibitors (HAI-1/ types (Table 1), also patients diagnosed with colorectal, skin, HAI-2 and serpins) and clinical parameters of hospitalized breast, or ovarian cancers are more likely to be infected and/ COVID-19 patients? In particular, do relative expression or have more complications than sex- and age-matched in- levels of these factors are related to differences in disease dividuals. Indeed, substantial rates of hospitalization and evolution such as mild versus severe respiratory distress more severe outcomes have been reported in cancer patients and infection of organs/tissues other than the lungs? Are diagnosed with COVID-19 (160). These patients deserve these levels predictive markers of disease evolution? (For particularly close follow-up, and they should be advised to 12 J. Biol. Chem. (2021) 296 100135 JBC REVIEWS: On the mechanism of SARS-CoV-2 spike protein activation adopt the most restrictive measures suggested within their 5. Yuan, Y., Cao, D., Zhang, Y., Ma, J., Qi, J., Wang, Q., Lu, G., Wu, Y., respective communities to avoid viral infection and Yan, J., Shi, Y., Zhang, X., and Gao, G. F. (2017) Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic re- transmission. ceptor binding domains. Nat. Commun. 8, 15092 The answers to these questions would help diagnosis and 6. 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