BioMed Research International

BioMed Research International / 2021 / Article

Review Article | Open Access

Volume 2021 |Article ID 8856018 | https://doi.org/10.1155/2021/8856018

Saeid Najafi Fard, Linda Petrone, Elisa Petruccioli, Tonino Alonzi, Giulia Matusali, Francesca Colavita, Concetta Castilletti, Maria Rosaria Capobianchi, Delia Goletti, "In Vitro Models for Studying Entry, Tissue Tropism, and Therapeutic Approaches of Highly Pathogenic Coronaviruses", BioMed Research International, vol. 2021, Article ID 8856018, 21 pages, 2021. https://doi.org/10.1155/2021/8856018

In Vitro Models for Studying Entry, Tissue Tropism, and Therapeutic Approaches of Highly Pathogenic Coronaviruses

Academic Editor: San-Ji Gao
Received15 Sep 2020
Revised27 Apr 2021
Accepted05 Jun 2021
Published22 Jun 2021

Abstract

Coronaviruses (CoVs) are enveloped nonsegmented positive-sense RNA viruses belonging to the family Coronaviridae that contain the largest genome among RNA viruses. Their genome encodes 4 major structural proteins, and among them, the Spike (S) protein plays a crucial role in determining the viral tropism. It mediates viral attachment to the host cell, fusion to the membranes, and cell entry using cellular proteases as activators. Several in vitro models have been developed to study the CoVs entry, pathogenesis, and possible therapeutic approaches. This article is aimed at summarizing the current knowledge about the use of relevant methodologies and cell lines permissive for CoV life cycle studies. The synthesis of this information can be useful for setting up specific experimental procedures. We also discuss different strategies for inhibiting the binding of the S protein to the cell receptors and the fusion process which may offer opportunities for therapeutic intervention.

1. Introduction

Coronaviruses (CoVs) are enveloped nonsegmented positive-sense RNA viruses belonging to the family Coronaviridae, order Nidovirales, broadly distributed in humans, and other mammals [1]. They can cause varieties of diseases, including respiratory, enteric, renal, and neurological syndromes [2].

With 80–160 nm in diameter, CoVs contain the largest viral genome (27–32 kilobase pairs) among the RNA viruses, sharing similar genome organization [3, 4]. They encode for 14-16 nonstructural proteins and 4 structural proteins, the spike (S) glycoprotein, small envelope protein (E), integral membrane protein (M), and genome-associated nucleocapsid protein (N) [3, 5, 6]. In addition, genes encoding accessory proteins have also been identified in the three regions between S–E–M–N [3, 4]. The proteins E, M, and N are mainly responsible for virions assembly while the S protein mediates viral attachment, membrane fusion, and entry, thus, determining tissue and cell tropism as well as host range [5, 7].

CoVs are classified into four genera, including alpha-, beta-, gamma-, and delta-CoVs [8]. So far, there have been three zoonotic outbreaks of beta-CoVs. In 2002–2003, severe acute respiratory syndrome coronavirus (SARS-CoV), a lineage B beta-CoV (subgenus sarbecovirus), emerged and infected over 8,000 people causing about 800 deaths [8, 9].

In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV), a lineage C beta-CoV (subgenus Merbecovirus), was discovered as the etiologic agent of a severe respiratory syndrome in Saudi Arabia, that caused 2,494 confirmed cases and 858 deaths [10]. Nowadays, sporadic cases are registered, remaining endemic in Middle East [11]. In December 2019, cases of pneumonia of unknown cause were reported in Wuhan, Hubei Province, China, that were later confirmed to be caused by a novel coronavirus, named SARS-CoV-2 due to its close phylogenetic relation to bat SARS-like CoVs. The clinical condition caused by this novel coronavirus is called COronaVIrus Disease-19 (COVID-19). SARS-CoV-2, a lineage B beta-CoV, is the third known highly pathogenic human coronavirus infection in the last two decades after SARS-CoV and MERS-CoV [2, 8, 12].

In addition to the highly pathogenic zoonotic pathogens, four coronaviruses with low-pathogenicity are endemic in humans. Among them, human CoV 229E (HCoV-229E) and HCoV-NL63 belong to alpha-CoVs, while HCoV-OC43 and HCoV-HKU1 are members of beta-CoVs [4, 13, 14].

Several in vitro models have been used to study the biology of pathogenic coronaviruses, so far. SARS-CoV-2 was first isolated using human airway epithelial cells [2]. SARS-CoV, SARS-CoV-2, and MERS-CoV can be isolated and grow readily in Vero cells [9, 12, 1517]. The critical first step for CoVs infection is the entry into the susceptible host cells by binding to a specific receptor. SARS-CoV and SARS-CoV-2 recognize angiotensin-converting enzyme 2 (ACE2), whereas MERS-CoV recognizes dipeptidyl peptidase 4 (DPP4, also referred as cluster of differentiation 26, CD26) as its receptor (Figure 1) [1820].

In addition to the requirement of receptors for cellular entry, several cellular proteases have been reported to be key factors for CoVs infection, which determine the entry route into host cells through cleavage of the S protein and activation of its membrane fusion activity [2125].

Studies have shown that both human and animal cell lines can express ACE2 and CD26 receptors and can be used to study the mechanism of viral entry and host species restriction of different CoVs, although the expression levels and regulation of these receptors are not yet completely elucidated [8, 2633]. Different receptors are used for cell entry, and several models have been applied for in vitro studies on CoVs; however, based on our current knowledge, a comprehensive review of in vitro studies and models on CoV pathogenesis is missing. For instance, data related to the expression levels of ACE2 and CD26/DPP4 in human and animal cell lines are highly controversial, probably due to the method of detection and reagents used. Here, we aim to review in vitro studies on highly pathogenic CoVs focusing on ACE2 and CD26/DPP4 receptor expression, cell tropism, and models useful for better understanding the viral life cycles, which could potentially offer opportunities for therapeutic intervention.

2. Methods

2.1. Search Strategy

Medline, EMBASE, Scopus, and Web of Science databases were used to perform the review. Studies were identified by crossing the name coronavirus or COVID-19 with the following key terms: SARS-CoV, MERS-CoV, SARS-CoV-2, Viral entry, ACE2 and/or CD26/DPP4 receptors, cellular proteases, and cell lines. We included data from English language articles. Meeting abstracts not yet published as full article, and case reports were excluded. The literature review was extended to April 15, 2021.

3. Results

3.1. Cellular Tropism of Highly Pathogenic Human Coronaviruses

The cell and tissue tropism of viruses is defined as the capacity of a virus to infect specific cells and tissues and is determined by both viral and cellular factors. Indeed, it depends on virus attachment protein(s) (antireceptors) and the presence of the cognate cellular receptor(s). The ability of CoVs to infect a wide range of mammals and birds is primarily determined by the ability of the S protein to bind a cell surface receptor [1]. Moreover, their zoonotic potential is due to the adaptability of S proteins to receptors of other species [18]. The receptor binding is the first critical step for the virus tropism. Although both SARS-CoV and SARS-CoV-2 employ ACE2 as a receptor for cellular entry [19], the SARS-CoV-2 S protein binds ACE2 with higher affinity than SARS-CoV S, which may explain the greater transmissibility of SARS-CoV-2 [20].

Of note, both SARS-CoV and SARS-CoV-2 have a marked tropism for ciliated cells widely distributed along the upper and lower respiratory tract [19, 29]. Key factors for CoVs entry into cells are host cellular proteases such as type II transmembrane serine protease (TMPRSS2), furin, and members of the cathepsin family (e.g., B and L) [2123, 25, 3436]. Indeed, the S protein is cleaved by cellular proteases in the priming step. As the priming event is essential for virus entry, the efficacy and extent of this activation step should regulate cellular tropism and viral pathogenesis of the different CoVs [37].

3.2. Structure and Function of Coronavirus S Proteins

The S proteins are a class I viral glycoproteins, which form homotrimers protruding from the viral surface that undergo structural modifications during host receptor binding and virus-host cell membrane fusion steps. They are divided into an N-terminal S1-ectodomain that recognizes a cognate cell surface receptor and a C-terminal S2-membrane-anchored protein responsible for virus–host membrane fusion and viral entry [4, 3740].

The S1 region contains two major domains, the N-terminal domain (NTD) and C-terminal domain (CTD) that can both function as receptor-binding domain (RBD). RBDs are divided into a core subdomain and a receptor-binding motif (RBM). Among CoVs, the core subdomain is structurally similar while the RBMs differ significantly, thus, explaining the peculiar receptor binding. Upon S1 binding to the host receptor, a conformational change in S2 mediates fusion between the viral envelope and the cell membrane. The viral genome is then released into the cytoplasm of the host cell through the fusion core [4, 14].

3.2.1. The SARS-CoV S Protein

The S of SARS-CoV is a homotrimer that belongs to a group of class I viral fusion glycoproteins that includes HIV glycoprotein 160 (Env), influenza haemagglutinin (HA), paramyxovirus F, and Ebola virus Gp2 [41]. The SARS-CoV S gene encodes a surface glycoprotein precursor of 1,255 amino acids in length. The amino terminus and most of the protein are predicted to be on the outside of the virus particle surface [42]. The S protein can be cleaved into the S1 and S2 subunits by proteases, and the fusion process is similar to that mediated by class I viral fusion proteins of other viruses (e.g., HIV-1 and murine hepatitis virus (MHV)), but may occur in the acidic environment of the endosomes, rather than on the cell surface [43, 44]. S2 contains heptad repeat 1 (HR1) and HR2 domains, which play an important part in virion fusion with target cells [45]. The S protein of SARS-CoV binds to its cellular receptor, ACE2. A defined RBD on S mediates this interaction. The atomic details at the interface between the two proteins clarify the importance of residue changes that facilitate efficient cross-species infection and human-to-human transmission [40].

3.2.2. The MERS-CoV S Protein

The S protein of MERS-CoV, similarly to SARS-CoV, is a type I transmembrane glycoprotein in a trimer state, which is located at the viral envelope surface. It contains 1,353 amino acids that can be cleaved into a receptor-binding subunit S1 and a membrane-fusion subunit S2 [29, 46]. S1 is located in the N-terminal 14–751 amino acids, containing the RBD region, which binds CD26/DPP4 [47, 48]. Although the RBDs of MERS-CoV and SARS-CoV share a high degree of structural similarity in their core subdomains, their RBMs are quite different, which can explain the different receptors utilized by these viruses [49, 50]. After binding to cellular receptor, MERS-CoV S protein undergoes significant conformational changes, exposing HR1 and HR2 regions in S2 subunit, further forming a 6-helix bundle fusion core, which leads to membrane fusion [51, 52].

3.2.3. The SARS-CoV-2 S Protein

The S protein of SARS-CoV-2 shares about 76% of amino acid identities with SARS-CoV, and, in particular, the sequence of potential RBD of SARS-CoV-2 is only about 74% homologous to that of SARS CoV [53]. Studies have reported that SARS-CoV-2 uses ACE2 as the cellular receptor for entry [8]. Successful conformational changes of S protein that lead to membrane fusion not only require receptor binding but also need appropriate protease activation. The cell-surface serine protease TMPRSS2 makes S protein fusogenic [54]. Moreover, there is a furin site between S1 and S2 (amino acids 682-685, motif of Arg-Arg-Ala-Arg (RRAR)) subunits in SARS-CoV-2 S protein, but it is not clear whether this furin site has any effect on viral pathogenesis and virus spreading among humans [8, 55].

4. Viral Attachment and Entry

4.1. Receptors for Highly Pathogenic Coronaviruses

Several types of receptors have been identified for alpha and beta-CoVs to enter cells [40]. hCoV-229E uses Aminopeptidase N (APN, CD13) as its receptor and OC43 and HKU1 attach to O-acetyl-sialosides found on glycoproteins and glycolipids at the host cell surface to enable entry into susceptible cells [4, 14, 38]. As mentioned in previous sections, ACE2 has been identified as the specific receptor for SARS-CoV and SARS-CoV-2 [20], while MERS-CoV utilizes CD26/DPP4 as its cellular receptor [49].

ACE2, a type I membrane protein, has a single metalloprotease active site and a transmembrane domain. It cleavages angiotensin (Ang) I to produce Ang-(1-9) and provides a direct binding site for the S proteins of some CoVs [56]. Study of the expression of ACE2 protein, the tissue tropism, and cellular distributions of SARS-CoV and SARS-CoV-2 is crucial to understand the mechanisms of pathogenesis of these viruses. ACE2 is widely expressed in different body sites including lung, heart, kidney, testis, intestine epithelia, arterial and venous endothelial cells, and maternal-fetal interface [19, 57, 58]. In the respiratory tract, ACE2 is expressed on the epithelial cells of alveoli, trachea, and bronchi, bronchial serous glands, and alveolar monocytes and macrophages [59]. ACE2 without ectopeptidase activity is also an efficient SARS-CoV receptor, and several proteases can operate as SARS-CoV entry cofactors, including cathepsin L, elastase, trypsin, factor Xa, thermolysin, and plasmin [23]. Moreover, a truncated form of the cytoplasmic domain of ACE2 does not affect the efficiency of SARS CoV infection in a pseudotype assay, suggesting that this domain is not essential for ACE2-mediated entry of the virus [60].

Previous studies have shown that certain ACE2-expressing endothelial cells and human intestinal cell lines failed to be infected by SARS-CoV [61, 62]. The SARS-CoV cannot replicate in differentiated respiratory epithelial cells or in primary undifferentiated bronchial and alveolar epithelial cells, even when ACE2 is abundantly expressed [63]. On the other hand, type II pneumocytes and bronchial epithelial cells differentiated in vitro could be infected with SARS-CoV [64]. In contrast, some cells without a detectable expression level of ACE2 such as hepatocytes could be infected by SARS-CoV, suggesting that ACE2 presence alone is not sufficient for sustaining viral infection [65]. Altogether, these observations indicate that different receptors or coreceptors may be utilized by SARS-CoV for infecting different tissues.

Indeed, SARS-CoV has been reported to also interact with alternative receptors, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin as (DC-SIGN) and liver/lymph node SIGN (L-SIGN, e.g., CD209L) [6668]. DC-SIGNR is expressed in alveolar type II cells, the major cell type infected in fatal cases of SARS infection [69]. However, these proteins appear to be much less efficient than ACE2 in promoting SARS-CoV entry and do not support infection effectively in absence of ACE2 [45, 70].

Vimentin, an intermediate filament, has been identified as abundantly present in the SARS-CoV S protein-ACE2 complexes Vimentin may directly bind to the SARS-CoV S protein, suggesting that it may also serve as a coreceptor for SARS-CoV [68]. Interestingly, it has been preliminary reported that antibodies directed against Vimentin are able to prevent SARS-CoV-2 pseudovirus infection in vitro [71].

Regarding SARS-CoV-2, two studies published in Science identified the protein Neuropilin-1 (NRP1) as an additional cellular factor which may facilitate viral entry and infectivity [72, 73]. Moreover, a number of alternative SARS-CoV-2 entry factors, which may function as coreceptors for viral attachment, have been investigated including heparan-sulfates, Sialic Acid, L-SIGN/DC-SIGN CD147, glucose-regulated protein 78 (GRP78), angiotensin II receptor type 2 (AGTR2), and the receptor for advanced glycation end products (RAGE) [74].

Unlike SARS-CoV and SARS-CoV-2, MERS-CoV utilizes CD26/DPP4 as its main cellular receptor [49]. CD26/DPP4 is a 110 kDa type II transmembrane glycoprotein homodimer present on the cell surface, which can regulate hormone and chemokine activity by cleaving dipeptides at the N-terminus after a proline or alanine residue in the penultimate position [75]. The use of peptidases by CoVs may be related to their abundant presence on epithelial and endothelial tissues, the primary tissues of CoV infection [29, 48, 76]. CD26/DPP4 is also widely expressed on epithelial cells in kidney, small intestine, liver and prostate, and on activated leukocytes. In the human respiratory tract, it is mainly expressed on nonciliated epithelial in alveoli rather than the nasal cavity or conducting airways [29, 77]. Since CD26/DPP4 is a costimulatory factor for T cell activation, it is tempting to speculate a viral-mediated mechanism of the host immune system evasion [26, 59]. CD26/DPP4 is conserved among different mammals (e.g., bats, dromedaries, and humans), and the broad species tropism of MERS-CoV may partly be the result of this conservation [28, 78]. It has been also found that the susceptibility or resistance to the infection of different cell lines directly correlates with the presence or absence of CD26/DPP4 expression [27]. It has been shown that MERS-CoV pseudoviruses had a relatively higher infectivity in certain cells (e.g., in HT-1080, Hep-2, MT-2, and NBL-7) with undetectable CD26/DPP4 expression [79]. Moreover, HEK cells with undetectable CD26/DPP4 had relatively high MERS-CoV infectivity [80]. On the other hand, MERS-CoV pseudovirus showed a lower infectivity in CD26/DPP4 expressing A549 and 293T cells [81]. Altogether, these findings suggest that the infectivity of MERS-CoV might not be completely associated with the expression of CD26/DPP4. Thus, similar to SARS-CoV, MERS-CoV might also use alternative receptors that have not been identified yet.

Recently, Vankadari and Wilce modelled a homo-trimer structure of SARS-COV-2 S protein. They predicted that the S1 domain of SARS-COV-2 S protein potentially interacts with the human CD26/DPP4 and suggested that SARS-COV-2 may share infection modes with that of SARS-CoV and MERS-CoV [82]. However, the potential role of CD26/DPP4 in the pathogenesis of new emerged SARS-CoV-2 is yet to be delineated.

CoVs in addition to the protein-based receptors (i.e., ACE2 and CD26/DPP4) [20, 49] use low-affinity interactions with carbohydrates such as sialylated glycans or sialic acids for cell attachment [14, 38]. The N-terminal S1 subunit comprises four β-rich domains, designated A, B, C, and D, with domain A or B acting as receptor-binding domains in different coronaviruses. While the protein receptors are generally bound via S1 domain B, the sialoglycan-based receptors are mediated by the S1 domain A of S protein [38, 83]. For instance, MERS-CoV S protein selectively binds to sialic acid (Sia), and glycan array analysis revealed a preference for α2,3-linked Sia over α2,6-linked Sia. Interestingly, the α2,3-linked Sias are predominantly distributed in the upper and lower respiratory tracts of camels and humans, sites of MERS-CoV replication, respectively [83]. Furthermore, although HCoV-OC43 and HCoV-HKU1 receptors have not been fully identified, they bind the 9-O-acetylated-sialic acid modifications on glycoprotein-based receptors [13, 14].

Seyran and colleagues hypothesised that the sialic acid-binding domain of S protein allows a viral “surfing” on the epithelium for receptor scanning by SARS-CoV-2 [84]. However, it has been recently shown that the Sia cell surface depletion by neuraminidase treatment increased cell attachment by both SARS-CoV and SARS-CoV-2, because Sias present on ACE2 receptors do not allow a perfect binding between spike and ACE2 [85]. On the contrary, the binding of MERS-CoV was hampered by either Sia modifications, such as 5-N-glycolylation and (7,) 9-O-acetylation, or Sia cell surface depletion by neuraminidase treatment of Calu-3 cells [83, 85], thus, revealing a different role for Sias during CoV infections. In fact, Chu and colleagues showed that, likely, SARS-CoV-2 utilizes heparan sulfate as an attachment factor for both Calu-3 cells and lung explant infection [85].

The fundamental role of the glycosylation in the interaction between SARS-CoV-2 S proteins and ACE2 receptor has been recently investigated using glycomic-informed glycoproteomics and molecular dynamic simulations. Zhao and colleagues identified several glycosylated residues in both S proteins (i.e., N0074, N0149, N0234, and N0801) and ACE2 receptors (i.e., N053, N090, N103, N322, and N432), thus, providing a valuable tool to study the receptor binding step of the viral life cycle [86]. Work by others confirmed the complexicity and the importance of glycosylation in both S proteins [87] and ACE2 receptor [88]. Moreover, these authors modelled a possible role for glycans in antigenic shielding, highlighting the importance of glycosylation in the strategy of immunogen design for vaccination and of therapeutic strategies for viral inhibition [86].

4.1.1. In Vitro Evaluation of Receptor Expression

Identification of relevant human cell line models of CoVs infection facilitates further laboratory investigations to have a better understanding of the viral entry, pathogenesis, and various therapeutic approaches. Studies have shown that both human and nonhuman cell lines can express CD26 and ACE2 and can be used to study the mechanism of viral entry, pathogenesis, and host species restriction of different CoVs; however, the expression levels of these receptors need to be well elucidated [8, 2633]. Several studies have evaluated the expression of ACE2 and CD26/DPP4 in human and animal tissues and cell lines; however, data on the expression levels of these receptors are highly discrepant (Table 1). For instance, Hamming and colleagues reported high levels of ACE2 expression in A549 cells [58], but others observed low levels of it [70, 89] and found that exogenous overexpression of ACE2 was required to render A549 cells permissive to viral infection and suggested that this difference could be due to different lineages of cell lines that may have evolved during the in vitro cell culture.


Cell lineOriginExpression markerLevelMethod of evaluationVirusReference

Nonhuman cells:
Vero, Vero E6African green monkey kidneyCD26/DPP4N/RImmunofluorescenceMERS-CoV[90]
CD26/DPP4HighFlow cytometry (FACS), immunofluorescenceMERS-CoV[27]
CD26/DPP4HighFlow cytometry (FACS), western blotMERS-CoV[28]
CD26/DPP4HighFlow cytometry (FACS), protein electrophoresisMERS-CoV[29]
CD26/DPP4LowReal-time RT-PCR, western blotMERS-CoV[25]
CD26/DPP4Int.Western blotMERS-CoV[79]
CD26/DPP4HighFlow cytometry (FACS)MERS-CoV[83]
CD26/DPP4Int.Quantitative RT-PCRMERS-CoV[91]
CD26/DPP4Int.Quantitative RT-PCR, flow cytometry (FACS)MERS-CoV[92]
CD26/DPP4HighImmunofluorescenceMERS -CoV[93]
ACE2HighReal-time RT-PCRSARS-CoV[17]
ACE2HighRT-PCRSARS-CoV[60]
ACE2Int.ImmunofluorescenceSARS-CoV[93]
ACE2HighWestern blotSARS-CoV[94]
ACE2Int.Flow cytometry (FACS)SARS-CoV[95]
ACE2HighImmunofluorescenceSARS-CoV[96]

LLC-MK2Rhesus monkey kidneyACE2HighFlow cytometry (FACS), immunofluorescenceSARS-CoV[97]
ACE2HighImmunostainingHCoV-NL63[98]

Human cells:
MRC-5Lung cells Medical Research Council-5CD26/DPP4HighQuantitative RT-PCRMERS -CoV[99]

A549Human lung adenocarcinomaCD26/DPP4LowEnzymatic activities, immunohistochemistry (IHC)-[100]
CD26/DPP4N/RFlow cytometry (FACS)-[101]
CD26/DPP4N/RImmunofluorescenceMERS -CoV[90]
CD26/DPP4LowWestern blotMERS-CoV[79]
ACE2N/RWestern blot-[102]
ACE2HighImmunostainingSARS-CoV[58]
ACE2LowImmunoblotSARS-CoV[70]

Calu-3Human lung cancer cellsCD26/DPP4HighImmunofluorescenceMERS-CoV[30]
CD26/DPP4HighReal-time RT-PCR, western blotMERS-CoV[25]
CD26/DPP4Int.Real-time RT-PCRMERS-CoV[103]
CD26/DPP4HighFlow cytometry (FACS)MERS-CoV[82]
ACE2HighFlow cytometry (FACS), immunofluorescenceSARS-CoV[104]
ACE2HighReal-time RT-PCRSARS-CoV[105]
ACE2HighFlow cytometry (FACS), western blot-[106]
ACE2LowImmunofluorescenceSARS-CoV[96]

HEp-2Human respiratory tractACE2HighRT-PCRSARS-CoV[60]
CD26/DPP4Neg.Western blotMERS-CoV[79]

Huh-7Hepatocellular carcinomaCD26/DPP4HighFlow cytometry (FACS), immunofluorescenceMERS-CoV[27]
CD26/DPP4HighFlow cytometry (FACS), western blotMERS-CoV[28]
CD26/DPP4HighFlow cytometry (FACS), protein electrophoresisMERS-CoV[29]
CD26/DPP4N/RFlow cytometry (FACS)MERS-CoV[107]
CD26/DPP4HighReal-time RT-PCRMERS-CoV[103]
CD26/DPP4HighWestern blotMERS-CoV[79]
CD26/DPP4Int.Quantitative RT-PCRMERS-CoV[99]
ACE2HighReal-time RT-PCRSARS-CoV[17]
ACE2HighRT-PCRSARS-CoV[60]
ACE2Neg.Flow cytometry (FACS), western blot-[106]

LoVoHuman colorectal adenocarcinomaACE2Int.Real-time RT-PCRSARS-CoV[61]
CD26/DPP4HighReal-time RT-PCRMERS-CoV[103]

Caco-2Human intestinal tractCD26/DPP4Int.Western blotMERS-CoV[79]
CD26/DPP4HighQuantitative RT-PCRMERS-CoV[91]
CD26/DPP4HighQuantitative RT-PCR, flow cytometry (FACS)MERS-CoV[92]
ACE2LowFlow cytometry (FACS), western blot-[106]
ACE2HighImmunofluorescenceSARS-CoV[96]
ACE2HighRT-PCR-[108]

Abbreviations: ACE2: angiotensin-converting enzyme 2; DPP4: dipeptidyl peptidase 4; CD26: cluster of differentiation 26; SARS-CoV: severe acute respiratory syndrome coronavirus; MERS-COV: Middle East respiratory syndrome coronavirus; Int.: intermediate; N/R: not reported.

Transfection of human ACE2 or CD26/DPP4 in nonsusceptible cells of different lineages or species permitted infection with SARS-CoV, SARS-CoV-2, or MERS-CoV, respectively (Table 2).


Cell linesOriginVirusMain finding related to cell entry and tissue tropismReference

Nonhuman cells:
VeroAfrican green monkey kidneySARS-CoVTMPRSS2 is involved in virus entry.[22]
SARS-CoVCathepsin L inhibitor blocked SARS-CoV infection.[124]
SARS-CoVSmall-molecule compounds could perturb the infectivity of the virus.[125]
SARS-CoVSensitivity of S-mediated entry to protease inhibitors.[21]
SARS-CoVACE2 as SARS-CoV S pseudovirion receptor for entry.[8]
SARS-CoVReceptor blocked by polyclonal goat anti-hACE2.[97]
SARS-CoV-2ACE2 as SARS-CoV-2 S pseudovirion receptor for entry.[8]
MERS-CoVFurin inhibitor significantly decreased S-mediated entry.[99]
MERS-CoVDPP4 as the receptor for viral entry.[28, 29]
MERS-CoVDPP4 is expressed on permissive cell.[90]
MERS-CoVHR2P significantly inhibited virus replication.[46]
MERS-CoVSimultaneous treatment with inhibitors of cathepsin L and TMPRSS2 completely blocked virus entry.[25]

VeroE6African green monkey kidneySARS-CoVRabbit antisera effectively blocked binding of S1 to ACE2.[126]
SARS-CoVACE2 as the receptor for viral entry.[127]
SARS-CoVAnti-ACE2 blocked viral entry.[31]
SARS-CoVProtease Inhibitors (leupeptin, Z-lll-FMK) blocked SARS-CoV S-mediated entry.[21]
SARS-CoVProteases enhanced virus entry.[128]
SARS-CoVVimentin as a coreceptor involved in the virus entry.[68]
SARS-CoVDifferent host cell proteases activate SARS-S for virus–cell and cell–cell fusion.[129]
SARS-CoVCholesterol extraction by MβCD treatment could reduce the expression level of cell surface ACE2 at a dose-dependent manner.[95]
SARS-CoV-2SARS-CoV S polyclonal Abs inhibited SARS-CoV-2 spike mediated entry.[39]

LLC-MK2Rhesus monkey kidneySARS-CoVACE2 as the receptor for viral entry.[97]

COS-7Monkey kidneyMERS-CoVExpression of human and bat DPP4 allowed MERS-CoV S1–Fc cell surface binding, and viral entry.[29]

BHKBaby hamster kidneySARS-CoVACE2 as the receptor for viral entry.[97]
SARS-CoVProteolytic cleavage within S2 exposes a novel internal fusion peptide for SARS-CoV S.[130]
SARS-CoV, SARS-CoV-2ACE2 as the receptor for viral entry.[131]
SARS-CoV-2ACE2 and host proteases requirement for viral entry.[132]
MERS-CoVhDPP4 transfected cells became permissive.[133]
MERS-CoVVirus could infect human and bat cells expressing DPP4.[134]
MERS-CoVSpecific binding between CD26 and MERS-CoV RBD.[48]

C6Rat glioma-derivedSARS-CoVNo apparent cytopathic effects (CPE) by infection but produced virus with infectivity of 102–5 per ml. No expression of ACE2[117]

Human cells:
A549Lung adenocarcinomaMERS-CoVDPP4 is expressed on permissive cells.[90]

OLOligodendroglioma-derivedSARS-CoVNo apparent cytopathic effects (CPE) by infection but produced virus with infectivity of 102–5 per ml. No expression of ACE2[117]

Calu-3Airway epitheliumSARS-CoVhACE2 was required for cell entry.[118]
SARS-CoVAnti-ACE2 Ab blocked the cell entry in a dose-dependent manner.[104]
SARS-CoVACE2, the SARS-CoV S pseudovirion receptor for entry.[8]
SARS-CoVSimultaneous treatment of the cells with camostat and EST efficiently prevented both cell entry and the multistep growth of the virus in the cells.[105]
SARS-CoV-2ACE2 as the SARS-CoV-2 S pseudovirion receptor for entry.[8]
MERS-CoVDPP4 is expressed on permissive cells.[30]
MERS-CoVTMPRSS2 inhibitor (camostat) blocked virus entry.[25]

Huh-7Hepatocellular carcinomaSARS-CoVS-mediated entry of pseudotypes requires low pH, S is a target for neutralizing antibodies.[135]
SARS-CoVACE2 as the SARS-CoV S pseudovirion receptor for entry.[8]
SARS-CoV-2ACE2 as the SARS-CoV-2 S pseudovirion receptor for entry.[8]
MERS-CoVAnti-CD26 mAbs (2F9) inhibited viral entry.[78]
MERS-CoVpAbs to the MERS-CoV S1 efficiently neutralize virus infection.[136]
MERS-CoVFurin inhibitor significantly decreased S-mediated entry.[99]
MERS-CoVDPP4 as the receptor for viral entry.[28, 29]
MERS-CoVNanobodies significantly blocked RBD binding to DPP4.[137]
MERS-CoVAntihuman CD26/DPP4 antibody inhibited MERS-CoV infection.[27]
MERS-CoVS protein-mediated cell–cell fusion and syncytium formation.[46]
MERS-CoVInteraction between recombinant RBDs and DPP4.[107]

HEK293TEmbryonic kidneysSARS-CoVCathepsin L inhibitor blocked S mediated pseudovirus entry in ACE2+ cells.[124]
SARS-CoVLactoferrin blocked the binding of S protein to ACE2 transfected cells.[138]
SARS-CoV-2Binding of polyclonal rabbit anti-SARS S1 antibodies to SARS-CoV-2.[8]
MERS-CoVhDPP4 transfected cells became permissive.[133]
MERS-CoVHigh levels of hDPP4 and furin enhanced viral entry. Knocked down furin expression with siRNA significantly reduced the pseudovirus entry.[10]

293TEmbryonic kidney epithelialSARS-CoVS Protein efficiently binds ACE2.[139]
SARS-CoVNeutralization of SARS pseudovirus infection by mouse antisera.[140]
SARS-CoVTMPRSS2 protease-dependent viral entry.[141]
SARS-CoVProtease inhibitors (leupeptin, E64c) blocked SARS-CoV S-mediated entry.[21]
SARS-CoVCytoplasmic domain was not essential for ACE2-mediated viral entry; soluble ACE2 inhibited S-bearing pseudotype entry.[60]
SARS-CoVRabbit antisera effectively blocked binding of S1 to ACE2.[126]
SARS-CoVACE2 was required for entry.[31]
SARS-CoVSARS-CoV RBD protein inhibited virus entry.[18]
SARS-CoVACE2 as the SARS-CoV S pseudovirion receptor for entry.[8]
SARS-CoVMAbs inhibited RBD-Fc binding to ACE2.[142, 143]
SARS-CoVA compound (designated VE607) inhibited pseudovirus entry.[125]
SARS-CoVACE2 as the SARS-CoV S pseudovirion receptor for entry.[81]
SARS-CoVTMPRSS2-mediated proteolysis of both S and ACE2 enhanced viral entry.[23]
SARS-CoVS-mediated entry of lentiviral-based vectors.[144]
SARS-CoVProteases activated SARS-S-driven virus-cell fusion.[145]
SARS-CoVHost cell proteases activated SARS-S for virus–cell and cell–cell fusion.[129]
SARS-CoV-2ACE2 as the SARS-CoV-2 S pseudovirion receptor for entry.[8]
SARS-CoV-2Protease was required for S-driven entry.[131]
SARS-CoV-2SARS-CoV-2 RBD protein inhibited both SARS- and SARS-CoV-2 entry.[18]
MERS-CoVMERS-CoV RBD inhibited MERS-CoV pseudovirus entry.[18]

HeLaCervical adenocarcinomaSARS-CoVTMPRSS2 enhanced pseudotyped SARS-S and authentic SARS-CoV entry, and camostat blocked it.[105]
SARS-CoV-2ACE2 was required for viral entry.[146]

JKT-hCD26Human T cell leukemiaMERS-CoVAnti-CD26 mAbs (2F9) inhibited viral entry.[78]

LoVoColorectal adenocarcinomaSARS-CoVACE2 is expressed on permissive cell.[61]
MERS-CoVDPP4 is expressed on permissive cell.[30]

Caco-2Colorectal adenocarcinomaSARS-CoV-2Protease was required for S-driven entry.[131]
MERS-CoVProtease (TMPRSS2 and cathepsin B/L) could activate EMC-S for entry.[81]

Primary culture
HBEpCPrimary bronchial epithelial cellSARS-CoV, MERS-CoVSpecific receptors were needed for cell entry.[93]

HREpCPrimary renal epithelial cellsSARS-CoV, MERS-CoVSpecific receptors were needed for cell entry.[93]

HAEAirway epitheliumSARS-CoVhACE2 as the primary receptor for entry.[118]
SARS-CoVS protein-pseudotyped FIV infected differentiated cells abundantly express ACE2 from the apical surface.[70]
MERS-CoVBoth type I and type III IFN efficiently reduced MERS-CoV replication.[147]
MERS-CoVTMPRSS2 inhibitor (camostat) blocked virus entry.[25]

NHBENormal human bronchial epithelialMERS-CoVFurin inhibitor significantly decreased S-mediated entry.[99]

3D human organoidsInduced pluripotent stem cells- (iPSCs-) derivedSARS-CoV-2The impact of SARS-CoV-2 as a neurotropic virus and emphasize that brain organoids could model CNS pathologies of COVID-19[110]
SARS-CoV-2Neuronal infection can be prevented either by blocking ACE2 with antibodies or by administering cerebrospinal fluid from a COVID-19 patient.[113]
SARS-CoV-2Using hPSCs to generate multiple different cell and organoid derivatives to study the viral tropism and cellular responses to infection.[112]
SARS-CoV-2SARS-CoV-2 can infect neural cells. detected the expression of the ACE2 receptor, but not TMPRSS2, in the model.[111]
SARS-CoV-2ACE2 expresses in cultured human pluripotent stem cell- (PSC-) derived mixed neurons.[114]

Some human (e.g., 293T, HEK293T, and HeLa) and nonhuman (e.g., COS-7, BHK) cell lines expressing hACE2 or hCD26/DPP4 are commonly used for experimentation because of their high transfectability. For example, hACE-2 expression in 293T cells resulted in the SARS-CoV and SARS-CoV-2 S pseudovirion binding and entry [8]. Also, expression of human and bat CD26/DPP4 in COS-7 cells allowed MERS-CoV S1–Fc cell surface binding and entry [29].

Nevertheless, the receptor alone may not be sufficient to explain the infectivity of CoVs in different cells, and other cellular factors may play a crucial role in completing the full life cycle of CoVs. In this regard, Chan and coauthors examined seven established human intestinal cell lines, DLD-1, HCT-116, HT-29, LoVo, LS-180, SW-480, and SW-620, for their susceptibility to SARS-CoV infection and found that only LoVo cells, which express intermediate levels of ACE2, were permissive. On the other hand, ACE2 was also found to be highly expressed in other cell lines that were not permissive to SARS-CoV infection [61]. The host factors that determine the susceptibility of the different cell types to CoVs infection are yet to be fully identified and, therefore, need of further investigations.

In studies of the expression levels of CoVs receptors, the method of detection and types of the antibodies (Abs) used are particularly important for receptor detection. For instance, while some studies have reported high levels of ACE2 expression in Calu-3, Caco-2, and Huh-7 cell lines by using different polyclonal Abs [8, 104], Liao and colleagues, by using a monoclonal anti-ACE2 Ab and total cell lysates, found that Calu-3 had elevated ACE2 levels compared to Caco-2 cells, while ACE2 was not detected in Huh-7. Moreover, by flow cytometry, ACE2 was detected on Calu-3 cells but not on Caco-2 or Huh-7 cells [106]. Using indirect immunofluorescence, in another study, Ren and coauthors found a higher number of Caco-2 cells with surface expression of ACE2 compared with Calu-3 cells. In addition, a faint band was visible when Calu-3 cells were analyzed by western blot [96].

ACE2 expression level in the CNS, and particularly in neurons, has not been well defined yet. Previous reports indicated very little expression of ACE2 RNA in the CNS [109] and in brain organoids [110, 111]. Accordingly, Yang and colleagues detected ACE2 protein expression at relatively low levels in human pluripotent stem cells- (hPSC-) derived cortical neurons by immunostaining [112]. However, others found widespread expression of ACE2 protein in both neurons and cells close to the lumen of the organoids [113, 114]. Furthermore, the enzymatic activity of ACE2 in human brain tissues and cerebrospinal fluid (CSF) [115, 116] has been indicated the significant level of ACE2 protein expression within the human CNS, suggesting that the mRNA level of ACE2 is not the best surrogate for ACE2 protein expression [113].

Specific models of neural cell lines have not been yet developed for the study of highly pathogenic coronaviruses [109]. Although a study showed that neural cell lines such as human oligodendroglioma-derived (OL) and rat (C6) origins can be susceptible to SARS-CoV infection, low levels of viral replication were observed compared to other susceptible cell lines such as Vero E6 or Caco-2 [117]. Recently, neural progenitor cells (NPCs), neurons, and microglia derived from human induced pluripotent stem cells (hIPSCs) have been used in in vitro, showing the potential of SARS-CoV-2 to infect CNS cells [110, 112, 113].

Taken together, using different lineages of cell lines and different methods may lead to different results; however, we should consider that while cell lines represent easy and useful tools for in vitro investigation on viral tropism and pathogenesis, these models have certain limitations, due to their inability to recapitulate complex and dynamic responses or cell-cell interactions happening in the whole organism.

Alternatively, primary culture systems such as human primary bronchiolar epithelial cells (HBEpC), human airway epithelium (HAE) culture models, human-derived organoids, or pseudostratified epithelia appear to be promising models to study the viral entry and pathogenesis of CoVs [29, 109, 118]. For example, the HAE culture model expressing relevant receptors for CoVs can recapitulate not only the morphological features of the human upper airway, which allows the visualization and the cultivation of several viruses, such as the newly emerging viruses like SARS-CoV-2, but also provide an excellent model for studying different steps of the viral life cycle and responses of the lung microenvironment to viral infections [118]. More recently, organoids that are miniaturized, simplified 3D structures of an organ (e.g., lung, kidney, intestine, liver, blood vessels, and brain) produced in vitro, offer another alternative to study tropisms and complex physiological or pathological processes of highly pathogenic coronaviruses more similarly to the in vivo condition (Table 2). They can be developed from either pluripotent stem cells or organ-specific progenitors through a self-organization process and provide very useful systems to study CoV life cycle as well as to test new antiviral compounds [109]. In particular, for the new SARS-CoV-2, several antiviral compounds have been tested such as interferon lambda, remdesivir, inhibitors of tyrosine kinases (Imatinib), inosine monphosphate dehydrogenase (the immunosuppressant mycophenolic acid, MPA), TMPRSS2 (camostat mesylate), or JAK kinases (baricitinib) [119123].

4.2. The Role of Cellular Proteases in Viral Entry

Although the primary receptors of highly pathogenic CoVs, ACE2, and CD26/DPP4 are expressed on the host cells of almost all organs, these viruses mainly cause lung diseases. Furthermore, the distribution of the receptors does not strictly correlate with viral cell tropism suggesting that other factors are required for viral entry and replication into target cells [22, 58, 105].

Previous studies have shown that after receptor binding, the key factors for CoVs entry into cells are host cellular proteases such as trypsin, furin, cathepsins (a diverse group of pH-dependent endosomal and lysosomal proteases), elastase, hypoxanthine-aminopterin-thymidine (HAT), and TMPRSS2, as activators of the viral S protein, which is a prerequisite for the fusion of viral and host cell membranes during viral entry [15, 2123, 25, 34, 35, 81, 91, 105, 124, 129, 132, 148].

As the priming event is essential for virus entry, the efficacy and extent of this activation step by the proteases of the target cells should regulate cellular tropism and viral pathogenesis [5, 37]. SARS-CoV infectivity was enhanced in culture cells by the addition of exogenous elastase, which enabled virus entry via the cell surface [128]. In addition, furin and cathepsins were showed to play a critical role in CoV spread and cytopathicity [15, 21, 91, 124, 148]. HAT could activate S protein for cell-cell fusion in the context of surrogate systems while it has been found coexpressed with the viral receptor in bronchial epithelial cells and pneumocytes in vivo [145]. Moreover, TMPRSS2 can cleave and activate S protein on the cell surface following receptor binding for cell-cell and virus-cell fusion, thereby allowing cathepsin-independent host cell entry [105, 131, 141, 145].

Highly pathogenic CoVs are able to use two cellular proteolytic pathways, either an endosomal or a nonendosomal pathway, to ensure the adequate processing of the S protein. In the absence of proteases at the cell surface, they can enter cells by an endosomal pathway, and the S protein is activated by cathepsin B/L in the endosome [141, 145, 149]. In contrast, in the presence of proteases at the cell surface such as trypsin, elastase, TMPRSS2, and HAT, which induce envelope-plasma membrane fusion, viral S proteins attached to the receptor are activated and the virus directly enters into the cell from the surface [23, 25, 105, 132, 141]. However, S protein primed by TMPRSS2, but not cathepsin B/L, appears essential for viral entry [22, 25, 105], while viral replication in the presence of proteases has been shown to be 100 times higher than via the endosomal pathway in Vero E6 cells [128, 150].

The role of host cell proteases in CoVs infection appears not limited to cleavage of the S protein. It has been suggested that ACE2 is also processed by host cell proteases playing an important role in SARS-CoV entry and pathogenesis [141, 151]. SARS-CoV S protein binding to ACE2 triggers ACE2 processing by a disintegrin and metallopeptidase domain 17 (ADAM17)/tumor necrosis factor-converting enzyme (TACE), which promotes the uptake of SARS-CoV by Vero E6 cells [141, 152, 153]. In addition, ACE2 may also be processed by TMPRSS2 and HAT, suggesting that ACE2 cleavage increases S protein-mediated entry [23]. However, the mechanism underlying augmentation of infection and the role of ACE2 proteolysis in protease-dependent S protein activation have not been well defined yet [151].

The distribution of proteases appears to be correlated with CoV infection in the lung, and results obtained with surrogate cell culture systems suggest that proteases may play a significant role in CoV spread in the human respiratory tract [22, 105, 141, 154]. Indeed, Matsuyama and colleagues showed that the localization of TMPRSS2-expressing cells in normal lung tissues, rather than ACE2-expressing cells, correlated to SARS-CoV infection in mild lesions, suggesting that TMPRSS2 may determine viral tropism at an early stage of SARS-CoV infection [22]. Altogether, these studies suggest that the higher infectivity of pathogenic CoVs in the lungs could be due to an enhancement of direct viral cell entry mediated by a diverse range of cellular proteases [22, 25, 81, 91, 105, 131].

5. In Vitro Models of Targeting Viral Entry as Therapeutics

The cell entry process of highly pathogenic CoVs is critical for pathogenesis of these viruses. There are four main strategies developed to inhibit the binding of the S protein to the cellular receptors and the fusion process: (i) using antireceptors antibodies, (ii) using soluble receptors, (iii) targeting of S protein via anti-S antibodies (anti S1 or S2), and (iv) targeting the cellular proteases involved in the priming of the S protein. All these strategies have been evaluated for the development of therapeutics against highly pathogenic CoVs.

5.1. Targeting the Cellular Receptor and the S Protein

The development of inhibitors targeting the interaction between the S1 domain, particularly RBD, and its receptor, or manipulation of the receptor levels may offer several opportunities for therapeutic intervention, as well as for vaccine-induced neutralizing antibodies. Previous studies have shown that several antibodies, peptides, or small compounds that bind ACE2 may be useful for the treatment of SARS-CoV or SARS-CoV-2 infections, either by blocking the S-protein-binding site or by inducing an ACE2 conformational change unfavorable for viral binding or membranes fusion [31, 155]. Anti-ACE2 antibodies or soluble forms of ACE2 blocked virus entry and replication in Vero E6 cells, thus, confirming that ACE2 is a functional receptor for SARS-CoV [45, 97].

In particular, a soluble form of ACE2 was shown to block the association of the S1 domain to the cells and an anti-ACE2 antibody inhibited SARS-CoV replication with an EC50 of 1.5 mg/mL [31]. Moreover, the catalytically inactive form of ACE2 conjugated to the Fc domain of human IgG1 potently inhibited SARS-CoV infection in Vero cells, with an EC50 of 2 nM [156]. In addition, smaller portions of the ACE2 ectodomain exhibited inhibitory activity. The soluble ectodomain of ACE2 was shown to inhibit S-bearing pseudotype entry [60].

Several studies indicate that CD26/DPP4-targeting therapeutic agents, including anti-CD26/DPP4 antibodies and CD26/DPP4 antagonist, may prevent the interaction between MERS-CoV RBD and CD26/DPP4, inhibiting MERS-CoV infection [49]. Indeed, anti-CD26/DPP4 polyclonal antibodies inhibited in vitro MERS-CoV infection of primary human bronchial epithelial cells and Huh-7 cells [29]. Anti-CD26/DPP4 monoclonal antibodies (2F9, 1F7, and YS110) blocked the interaction between the S protein and CD26/DPP4, thereby neutralizing MERS-CoV infectivity in Huh-7 and JKT/CD26 (human T cell leukemia expressing human DPP4) cells [78]. Human anti-CD26/DPP4 antibodies inhibited infection of susceptible bat (RoNi/7.1) cells and Huh-7 cells, in a dose-dependent manner [27].

As mentioned above, the S protein RBD is a major target for anti-CoV therapeutics, and most of neutralizing antibodies target this domain. Notably, RBD-specific mAbs have more potent neutralizing activity than those targeting the S1 region outside RBD or the S2 region, suggesting that CoV RBD could serve as a main target for developing antibody-based therapeutics [5, 49]. The RBD binding antibodies inhibited the association of S1 to ACE2 and neutralized the infection of SARS-CoV of Vero E6 cells [126]. Moreover, Mou and colleagues demonstrated that polyclonal Abs directed against the RBD region efficiently neutralize MERS-CoV infection in Huh-7 cells [136].

Different assay systems have shown that neutralizing antibodies can block binding of S1, which contains RBD, to its receptor and fully protect cells from CoV infection [126], demonstrating that the S is a target for neutralizing antibodies and that such antibodies are generated in SARS-CoV-infected patients [135, 140, 142].

Recent studies have demonstrated that SARS-CoV RBD specific monoclonal and polyclonal antibodies may cross-react with SARS-CoV-2 RBD protein and inhibit SARS-CoV-2 entry into hACE2-expressing 293T cells [39, 131]. In addition, SARS-CoV RBD-specific antibodies could cross-neutralize SARS-CoV-2 pseudovirus infection, suggesting the potential to develop SARS-CoV RBD-based vaccine for prevention of infection by SARS-CoV-2 and SARS-CoV [18, 39, 69, 131].

Recently, other attempts have been made to block the SARS-CoV-2 cell entry. For instance, S pseudovirions into 293/hACE2 cells were significantly prevented by preincubation of soluble hACE2 at both 10 μg/ml and 50 μg/ml [8]. Case and colleagues used either human mAbs or hACE2-Fc Receptor Decoy Proteins to inhibit SARS-CoV-2 infections on Vero E6 cells [157]. Importantly, soluble hACE2 is able to inhibit viral infection of engineered human blood vessel or kidney organoids [158], further supporting the notion that hACE2 is the receptor and soluble hACE2 might be used as a therapeutic inhibitor against SARS-CoV-2 infection. Recently, it has been proposed to use a chimeric bispecific molecule between soluble hACE2 and anti-CD16 in order to better recruit innate immune cells against the particles of the virus or infected cells [159].

A different approach to treat CoVs infections that appears to be an attractive alternative to traditional mAbs are the nanobodies (Nbs), the single-heavy-chain antibody variant also termed the VHH domain. Zhao and coauthors showed that Nbs significantly block RBD of MERS-CoV binding to CD26/DPP4 in HuH-7 cells [137]. Different Nbs have been recently shown to block either the SARS-CoV-2 S protein attachment to ACE2 in vitro [160] or efficiently neutralize SARS-CoV-2 infection [161, 162]. Since Nbs are small, stable, efficacious, and straightforward to produce to target the RBD of S protein, they can represent a great promise as potential therapeutics candidates.

S2 subunit of CoVs S-protein also appears to be an intriguing target for developing CoV fusion inhibitors [43, 46, 163, 164]. Liu and colleagues evaluated peptides derived from the membrane-proximal (HR2) and membrane-distal (HR1) heptad repeat region of the S protein as inhibitors of SARS-CoV infection of Vero cells, founding that HR2 peptides, but not HR1, were inhibitory [43]. Moreover, Lu and coworkers designed several peptides spanning the HR1 and HR2 sequences of MERS-CoV S protein and found that one of the HR2 peptides (HR2P) potently inhibited MERS-CoV infection and its S protein-mediated cell–cell fusion in Vero and Huh-7 cells, suggesting that these peptides can be further developed as effective MERS therapeutics [46].

Interestingly, a similar membrane fusion assay has been recently utilized to identify drugs with anti-SARS-CoV-2 activity. Braga and colleagues performed a microscopy-based screening with over 3000 approved drugs to search for inhibitors of SARS-CoV-2 S protein-driven syncytia [165]. Among the 83 drugs identified, they deeply studied Niclosamide, an approved antihelminthic drug, which exert also inhibitory effects against virus replication [165]. The authors found that Niclosamide suppresses the activity of TMEM16F/Anoctamin6, a calcium-activated ion channel that mediates phosphatidylserine exposure on the cell surface [165, 166]. Interestingly, the results of a phase I trial of a formulation of niclosamide as a potent anti-SARS-CoV-2 agent have been recently published [167].

5.2. Targeting Cellular Proteases

Since the S protein priming of CoVs by cellular proteases is necessary for receptor binding and membrane fusion, these proteases can serve as targets (Table 2) for developing inhibitors of S protein-mediated viral entry into the target cells [49].

Coronaviruses enter cells via two distinct pathways, one mediated by TMPRSS2 at the cell surface and the other mediated by cathepsins in the endosome [22, 23, 25]. Since endosomal cathepsins (B/L) and TMPRSS2 can activate CoV S-mediated virus-cell entry and uptake, treatment of cells with either cathepsin (B/L) inhibitors (i.e., MDL28170 or teicoplanin) or TMPRSS2 inhibitors (i.e., camostat mesylate or peptidic inhibitors) can block virus entry into target cells [25, 49, 81, 145, 168]. SARS-CoV infection was blocked by specific inhibitors of the pH-sensitive endosomal protease cathepsin L. S-mediated entry into Vero E6 and 293T/hACE2 cells was blocked by leupeptin, Z-lll-FMK (an inhibitor of both CTSB and CTSL), and E64c (an inhibitor of cysteine proteases) [21].

Further studies showed that inhibitors of cathepsin L blocked infection by both SARS-CoV and pseudotype expressing the SARS-CoV S protein, while not affecting the infection by either HCoV-NL63 or a retrovirus pseudotyped with the HCoV-NL63 S protein in HEK293T cells expressing Cathepsins and ACE2. In addition, expression of exogenous cathepsin L substantially enhanced infection mediated by the SARS-CoV S protein and by filovirus GP proteins but not by the HCoV-NL63 S protein or the vesicular stomatitis virus G protein [124].

The role of cellular proteases in virus entry into cells was observed also for MERS-CoV, as the protease inhibitors camostat and MDL28170 efficiently inhibited entry of pseudotyped MERS-CoV into the human colorectal cell line Caco-2 cells [81]. In fact, simultaneous treatment with inhibitors of cathepsin L and TMPRSS2 completely blocked virus entry into Vero-TMPRSS2 cells, indicating that MERS-CoV employs both the cell surface and the endosomal pathway to infect TMPRSS2-expressing Vero cells. In contrast, a single camostat treatment suppressed MERS-CoV entry into human bronchial submucosal gland-derived Calu-3 cells by 10-fold and virus growth by 270-fold, although treatment with both camostat and (23,25)-trans-epoxysuccinyl-L-leucylamindo-3-methylbutane ethyl ester (EST), a cathepsin inhibitor, or treatment with leupeptin, an inhibitor of cysteine, serine, and threonine peptidases, was no more efficacious than treatment with camostat alone [25].

Recently, Hoffmann et al. showed that SARS-CoV-2-S-driven entry was fully inhibited when both camostat mesylate and E-64d, an inhibitor of cathepsin B/L, were added to Caco-2, TMPRSS2-expressing Vero, and 293T (transiently expressing ACE2) cell cultures, indicating that SARS-CoV-2 S protein can use both cathepsin B/L as well as TMPRSS2 for priming in these cell lines. Further, camostat mesylate significantly reduced MERS-CoV-S-, SARS-CoV-S-, and SARS-CoV-2-S-driven and authentic SARS-CoV-2 entry into the Calu-3 cells. Similarly, camostat mesylate treatment inhibited SARS-S- and SARS-2-S- but not VSV-G-driven entry into primary human lung cells, suggesting that SARS-CoV-2 can use TMPRSS2 for S protein priming and camostat mesylate blocks SARS-CoV-2 infection of lung cells [131].

In another study, camostat partially blocked infection by SARS-CoV and HCoV-NL63 in HeLa cells expressing ACE2 and TMPRSS2. Moreover, simultaneous treatment with camostat and EST efficiently prevented both cell entry and the multistep growth of SARS-CoV in human Calu-3 cells [105]. These observations suggest that inhibition of both proteases is required for viral entry inhibition [22, 25, 105, 169].

It has also been shown by Bergeron and colleagues that furin, a ubiquitously expressed protease, plays a key role in protease-activated CoV S-based fusion. The convertase inhibitor, membrane-permeable peptide decanoyl-RVKR-chloromethylketone (dec-RVKR-cmk) significantly reduced pro-S cleavage, viral entry, and infection. In addition, inhibition of processing by dec-RVKR-cmk completely abrogated the virus-induced cellular cytopathicity [148]. Moreover, siRNA silencing of furin activity decreased MERS-CoV S-mediated entry, and blockage of furin cleavage at the S cleavage sites significantly reduces virus infection [99]. However, this protease appears to be indispensable, and blockade of it does not affect S protein-driven cell-cell and virus-cell fusion of MERS-CoV, although it can reduce the processing of S protein in infected cells.

Finally, furin inhibition by decanoyl-RVKR-chloromethylketone (CMK) and naphthofluorescein has been found to inhibit syncytia formation and decrease SARS-CoV-2-induced cytopathic effects and viral production [170]. In particular, it has been reported that CMK blocks virus entry while naphthofluorescein suppresses mainly viral RNA transcription, thus, further suggesting that furin inhibitors may be promising antiviral agents for SARS-CoV-2 infection.

6. Conclusions

CoVs entry into cells is based on (1) binding of viral S protein to a specific receptor and (2) priming it with host cellular proteases such as TMPRSS2, furin, and cathepsin L. Different strategies are under investigation for inhibiting the binding of the S protein to the cellular receptors or the fusion process using antireceptor antibodies, soluble receptors, anti-S1, or -S2 antibodies or targeting cellular proteases. These strategies may offer opportunities to identify new antiviral drugs and are of particular relevance for vaccine development against highly pathogenic CoVs and particularly new variants of SARS-CoV-2. Inhibitors of viral entry appear to be a promising therapeutic strategy due to their ability to block the very first step of the viral life cycle and less toxic potentials because the membrane permeability may not be necessary. In this regard, identification of relevant in vitro models and cell lines permissive for CoVs facilitates further laboratory investigations; however, these cellular models have limitations, due to their inability to recapitulate complex and dynamic responses or cell-cell interactions that naturally occur in the organism.

Primary culture systems such as HBEpC, HAE culture models, human-derived organoids, or pseudostratified epithelia may be promising models to study CoVs. In particular, the HAE culture model expressing relevant receptors for CoVs can recapitulate not only the morphological features of the human upper airway, allowing visualization of the high levels of replication and destructive nature of viruses, but also provides an excellent model for studying viral entry, and comparing the responses of the lung to newly emerged viruses, including SARS-CoV-2 infections. Finally, organoids which are miniaturized, simplified, and three-dimensional versions of an organ produced in vitro can be a valid model to study tropisms and complex physiological or pathological processes of highly pathogenic CoVs, similarly to the in vivo situation.

Conflicts of Interest

The authors declare that there is no conflict of interest regarding the publication of this article.

Authors’ Contributions

SNF organized and wrote the manuscript; LP contributed to writing the manuscript and prepared the figure; TA, GM, FC, and CC contributed to writing the manuscript; MRC and DG designed the review and supervised the preparation of the work and writing of the manuscript. All authors approved the manuscript.

Acknowledgments

This research received funding from Ricerca Corrente of the Italian Ministry of Health, Linea 1 and 4; European funding: SC1-PHE-CORONAVIRUS-2020 of the project EXSCALATE4CoV, grant agreement ID: 101003551.

References

  1. P. C. Woo, S. K. Lau, Y. Huang, and K. Y. Yuen, “Coronavirus diversity, phylogeny and interspecies jumping,” Experimental Biology and Medicine (Maywood, N.J.), vol. 234, no. 10, pp. 1117–1127, 2009. View at: Publisher Site | Google Scholar
  2. N. Zhu, D. Zhang, W. Wang et al., “A novel coronavirus from patients with pneumonia in China, 2019,” The New England Journal of Medicine, vol. 382, no. 8, pp. 727–733, 2020. View at: Publisher Site | Google Scholar
  3. A. R. Fehr and S. Perlman, “Coronaviruses: an overview of their replication and pathogenesis,” Methods in molecular biology (Clifton, N.J.), vol. 1282, pp. 1–23, 2015. View at: Publisher Site | Google Scholar
  4. N. Wang, J. Shang, S. Jiang, and L. Du, “Subunit vaccines against emerging pathogenic human coronaviruses,” Frontiers in Microbiology, vol. 11, p. 298, 2020. View at: Publisher Site | Google Scholar
  5. Q. Wang, G. Wong, G. Lu, J. Yan, and G. F. Gao, “MERS-CoV spike protein: targets for vaccines and therapeutics,” Antiviral Research, vol. 133, pp. 165–177, 2016. View at: Publisher Site | Google Scholar
  6. Y. Chen, Q. Liu, and D. Guo, “Emerging coronaviruses: genome structure, replication, and pathogenesis,” Journal of Medical Virology, vol. 92, no. 4, pp. 418–423, 2020. View at: Publisher Site | Google Scholar
  7. G. Lu, Q. Wang, and G. F. Gao, “Bat-to-human: spike features determining 'host jump' of coronaviruses SARS- CoV, MERS-CoV, and beyond,” Trends in Microbiology, vol. 23, no. 8, pp. 468–478, 2015. View at: Publisher Site | Google Scholar
  8. X. Ou, Y. Liu, X. Lei et al., “Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV,” Nature Communications, vol. 11, no. 1, pp. 1–12, 2020. View at: Publisher Site | Google Scholar
  9. T. G. Ksiazek, D. Erdman, C. S. Goldsmith et al., “A novel coronavirus associated with severe acute respiratory syndrome,” The New England Journal of Medicine, vol. 348, no. 20, pp. 1953–1966, 2003. View at: Publisher Site | Google Scholar
  10. A. M. Zaki, S. van Boheemen, T. M. Bestebroer, A. D. Osterhaus, and R. A. Fouchier, “Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia,” The New England Journal of Medicine, vol. 367, no. 19, pp. 1814–1820, 2012. View at: Publisher Site | Google Scholar
  11. A. A. Rabaan, S. H. Al-Ahmed, R. Sah et al., “MERS-CoV: epidemiology, molecular dynamics, therapeutics, and future challenges,” Annals of Clinical Microbiology and Antimicrobials, vol. 20, no. 8, pp. 1–14, 2021. View at: Publisher Site | Google Scholar
  12. W. B. Park, N. J. Kwon, S. J. Choi et al., “Virus isolation from the first patient with SARS-CoV-2 in Korea,” Journal of Korean Medical Science, vol. 35, no. 7, p. e84, 2020. View at: Publisher Site | Google Scholar
  13. X. Huang, W. Dong, A. Milewska et al., “Human coronavirus HKU1 spike protein uses O-acetylated sialic acid as an attachment receptor determinant and employs hemagglutinin-esterase protein as a receptor-destroying enzyme,” Journal of Virology, vol. 89, no. 14, pp. 7202–7213, 2015. View at: Publisher Site | Google Scholar
  14. R. J. G. Hulswit, Y. Lang, M. J. G. Bakkers et al., “Human coronaviruses OC43 and HKU1 bind to 9-O-acetylated sialic acids via a conserved receptor-binding site in spike protein domain A,” Proceedings of the National Academy of Sciences of the United States of America, vol. 116, no. 7, pp. 2681–2690, 2019. View at: Publisher Site | Google Scholar
  15. W. B. Park, N. J. Kwon, P. G. Choe et al., “Isolation of Middle East respiratory syndrome coronavirus from a patient of the 2015 Korean outbreak,” Journal of Korean Medical Science, vol. 31, no. 2, pp. 315–320, 2016. View at: Publisher Site | Google Scholar
  16. J. F. Chan, S. K. Lau, K. K. To, V. C. C. Cheng, P. C. Y. Woo, and K. Y. Yuen, “Middle East respiratory syndrome coronavirus: another zoonotic betacoronavirus causing SARS-like disease,” Clinical Microbiology Reviews, vol. 28, no. 2, pp. 465–522, 2015. View at: Publisher Site | Google Scholar
  17. K. Hattermann, M. A. Müller, A. Nitsche, S. Wendt, O. Donoso Mantke, and M. Niedrig, “Susceptibility of different eukaryotic cell lines to SARS-coronavirus,” Archives of Virology, vol. 150, no. 5, pp. 1023–1031, 2005. View at: Publisher Site | Google Scholar
  18. W. Tai, L. He, X. Zhang et al., “Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine,” Cellular & Molecular Immunology, vol. 17, no. 6, pp. 613–620, 2020. View at: Publisher Site | Google Scholar
  19. HCA Lung Biological Network, W. Sungnak, N. Huang et al., “SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes,” Nature Medicine, vol. 26, no. 5, pp. 681–687, 2020. View at: Publisher Site | Google Scholar
  20. G. Zhou and Q. Zhao, “Perspectives on therapeutic neutralizing antibodies against the novel coronavirus SARS-CoV-2,” International Journal of Biological Sciences, vol. 16, no. 10, pp. 1718–1723, 2020. View at: Publisher Site | Google Scholar
  21. G. Simmons, D. N. Gosalia, A. J. Rennekamp, J. D. Reeves, S. L. Diamond, and P. Bates, “Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 33, pp. 11876–11881, 2005. View at: Publisher Site | Google Scholar
  22. S. Matsuyama, N. Nagata, K. Shirato, M. Kawase, M. Takeda, and F. Taguchi, “Efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2,” Journal of Virology, vol. 84, no. 24, pp. 12658–12664, 2010. View at: Publisher Site | Google Scholar
  23. A. Shulla, T. Heald-Sargent, G. Subramanya, J. Zhao, S. Perlman, and T. Gallagher, “A transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry,” Journal of Virology, vol. 85, no. 2, pp. 873–882, 2011. View at: Publisher Site | Google Scholar
  24. S. R. Weiss and J. L. Leibowitz, “Coronavirus pathogenesis,” Advances in Virus Research, vol. 81, pp. 85–164, 2011. View at: Publisher Site | Google Scholar
  25. K. Shirato, M. Kawase, and S. Matsuyama, “Middle East respiratory syndrome coronavirus infection mediated by the transmembrane serine protease TMPRSS2,” Journal of Virology, vol. 87, no. 23, pp. 12552–12561, 2013. View at: Publisher Site | Google Scholar
  26. J.-H. Chang, C.-W. Cheng, Y.-C. Yang et al., “Downregulating CD26/DPPIV by apigenin modulates the interplay between Akt and Snail/Slug signaling to restrain metastasis of lung cancer with multiple EGFR statuses,” Journal of experimental & clinical cancer research: CR, vol. 37, no. 1, pp. 1–16, 2018. View at: Publisher Site | Google Scholar
  27. Y. Caì, S. Q. Yú, E. N. Postnikova et al., “CD26/DPP4 cell-surface expression in bat cells correlates with bat cell susceptibility to Middle East respiratory syndrome coronavirus (MERS-CoV) infection and evolution of persistent infection,” PLoS One, vol. 9, no. 11, article e112060, 2014. View at: Publisher Site | Google Scholar
  28. N. van Doremalen, K. L. Miazgowicz, S. Milne-Price et al., “Host species restriction of Middle East respiratory syndrome coronavirus through its receptor, dipeptidyl peptidase 4,” Journal of Virology, vol. 88, no. 16, pp. 9220–9232, 2014. View at: Publisher Site | Google Scholar
  29. V. S. Raj, H. Mou, S. L. Smits et al., “Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC,” Nature, vol. 495, no. 7440, pp. 251–254, 2013. View at: Publisher Site | Google Scholar
  30. X. Tao, T. E. Hill, C. Morimoto, C. J. Peters, T. G. Ksiazek, and C. T. K. Tseng, “Bilateral entry and release of Middle East respiratory syndrome coronavirus induces profound apoptosis of human bronchial epithelial cells,” Journal of Virology, vol. 87, no. 17, pp. 9953–9958, 2013. View at: Publisher Site | Google Scholar
  31. W. Li, M. J. Moore, N. Vasilieva et al., “Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus,” Nature, vol. 426, no. 6965, pp. 450–454, 2003. View at: Publisher Site | Google Scholar
  32. X. Y. Ge, J. L. Li, X. L. Yang et al., “Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor,” Nature, vol. 503, no. 7477, pp. 535–538, 2013. View at: Publisher Site | Google Scholar
  33. A. Barlan, J. Zhao, M. K. Sarkar et al., “Receptor variation and susceptibility to Middle East respiratory syndrome coronavirus infection,” Journal of Virology, vol. 88, no. 9, pp. 4953–4961, 2014. View at: Publisher Site | Google Scholar
  34. C. G. K. Ziegler, S. J. Allon, S. K. Nyquist et al., “SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues,” Cell, vol. 181, no. 5, pp. 1016–1035.e19, 2020. View at: Publisher Site | Google Scholar
  35. I. Glowacka, S. Bertram, M. A. Muller et al., “Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response,” Journal of Virology, vol. 85, no. 9, pp. 4122–4134, 2011. View at: Publisher Site | Google Scholar
  36. K. E. Follis, J. York, and J. H. Nunberg, “Furin cleavage of the SARS coronavirus spike glycoprotein enhances cell-cell fusion but does not affect virion entry,” Virology, vol. 350, no. 2, pp. 358–369, 2006. View at: Publisher Site | Google Scholar
  37. B. Coutard, C. Valle, X. de Lamballerie, B. Canard, N. G. Seidah, and E. Decroly, “The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade,” Antiviral Research, vol. 176, p. 104742, 2020. View at: Publisher Site | Google Scholar
  38. M. A. Tortorici, A. C. Walls, Y. Lang et al., “Structural basis for human coronavirus attachment to sialic acid receptors,” Nature Structural & Molecular Biology, vol. 26, no. 6, pp. 481–489, 2019. View at: Publisher Site | Google Scholar
  39. A. C. Walls, Y. J. Park, M. A. Tortorici, A. Wall, A. T. McGuire, and D. Veesler, “Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein,” Cell, vol. 181, no. 2, pp. 281–292.e6, 2020. View at: Publisher Site | Google Scholar
  40. F. Li, W. Li, M. Farzan, and S. C. Harrison, “Structure of SARS coronavirus spike receptor-binding domain complexed with receptor,” Science (New York, N.Y.), vol. 309, no. 5742, pp. 1864–1868, 2005. View at: Publisher Site | Google Scholar
  41. W. Weissenhorn, A. Dessen, L. J. Calder, S. C. Harrison, J. J. Skehel, and D. C. Wiley, “Structural basis for membrane fusion by enveloped viruses,” Molecular Membrane Biology, vol. 16, no. 1, pp. 3–9, 1999. View at: Publisher Site | Google Scholar
  42. M. A. Marra, S. J. Jones, C. R. Astell et al., “The genome sequence of the SARS-associated coronavirus,” Science (New York, N.Y.), vol. 300, no. 5624, pp. 1399–1404, 2003. View at: Publisher Site | Google Scholar
  43. S. Liu, G. Xiao, Y. Chen et al., “Interaction between heptad repeat 1 and 2 regions in spike protein of SARS- associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors,” Lancet (London, England), vol. 363, no. 9413, pp. 938–947, 2004. View at: Publisher Site | Google Scholar
  44. B. Tripet, M. W. Howard, M. Jobling, R. K. Holmes, K. V. Holmes, and R. S. Hodges, “Structural Characterization of the SARS-Coronavirus Spike S Fusion Protein Core*,” The Journal of Biological Chemistry, vol. 279, no. 20, pp. 20836–20849, 2004. View at: Publisher Site | Google Scholar
  45. L. du, Y. He, Y. Zhou, S. Liu, B. J. Zheng, and S. Jiang, “The spike protein of SARS-CoV -- a target for vaccine and therapeutic development,” Nature Reviews. Microbiology, vol. 7, no. 3, pp. 226–236, 2009. View at: Publisher Site | Google Scholar
  46. L. Lu, Q. Liu, Y. Zhu et al., “Structure-based discovery of Middle East respiratory syndrome coronavirus fusion inhibitor,” Nature Communications, vol. 5, no. 1, p. 3067, 2014. View at: Publisher Site | Google Scholar
  47. S. Xia, Q. Liu, Q. Wang et al., “Middle East respiratory syndrome coronavirus (MERS-CoV) entry inhibitors targeting spike protein,” Virus Research, vol. 194, pp. 200–210, 2014. View at: Publisher Site | Google Scholar
  48. G. Lu, Y. Hu, Q. Wang et al., “Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26,” Nature, vol. 500, no. 7461, pp. 227–231, 2013. View at: Publisher Site | Google Scholar
  49. L. du, Y. Yang, Y. Zhou, L. Lu, F. Li, and S. Jiang, “MERS-CoV spike protein: a key target for antivirals,” Expert Opinion on Therapeutic Targets, vol. 21, no. 2, pp. 131–143, 2017. View at: Publisher Site | Google Scholar
  50. Y. Chen, K. R. Rajashankar, Y. Yang et al., “Crystal structure of the receptor-binding domain from newly emerged Middle East respiratory syndrome coronavirus,” Journal of Virology, vol. 87, no. 19, pp. 10777–10783, 2013. View at: Publisher Site | Google Scholar
  51. Y. Zhou, S. Jiang, and L. Du, “Prospects for a MERS-CoV spike vaccine,” Expert Review of Vaccines, vol. 17, no. 8, pp. 677–686, 2018. View at: Publisher Site | Google Scholar
  52. H. A. Mohd, J. A. Al-Tawfiq, and Z. A. Memish, “Middle East respiratory syndrome coronavirus (MERS-CoV) origin and animal reservoir,” Virology Journal, vol. 13, no. 1, p. 87, 2016. View at: Publisher Site | Google Scholar
  53. J. A. Jaimes, N. M. André, J. S. Chappie, J. K. Millet, and G. R. Whittaker, “Phylogenetic analysis and structural modeling of SARS-CoV-2 spike protein reveals an evolutionary distinct and proteolytically sensitive activation loop,” Journal of Molecular Biology, vol. 432, no. 10, pp. 3309–3325, 2020. View at: Publisher Site | Google Scholar
  54. J. Buchrieser, J. Dufloo, M. Hubert et al., “Syncytia formation by SARS-CoV-2-infected cells,” The EMBO Journal, vol. 40, no. 3, article e106267, 2020. View at: Publisher Site | Google Scholar
  55. G. Papa, D. L. Mallery, A. Albecka et al., “Furin cleavage of SARS-CoV-2 spike promotes but is not essential for infection and cell-cell fusion,” PLoS Pathogen, vol. 17, no. 1, article e1009246, 2021. View at: Publisher Site | Google Scholar
  56. M. Donoghue, F. Hsieh, E. Baronas et al., “A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9,” Circulation Research, vol. 87, no. 5, pp. E1–E9, 2000. View at: Publisher Site | Google Scholar
  57. M. Li, L. Chen, J. Zhang, C. Xiong, and X. Li, “The SARS-CoV-2 receptor ACE2 expression of maternal-fetal interface and fetal organs by single-cell transcriptome study,” PLoS One, vol. 15, no. 4, article e0230295, 2020. View at: Publisher Site | Google Scholar
  58. I. Hamming, W. Timens, M. L. Bulthuis, A. T. Lely, G. J. Navis, and H. van Goor, “Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis,” The Journal of Pathology, vol. 203, no. 2, pp. 631–637, 2004. View at: Publisher Site | Google Scholar
  59. Y. Yin and R. G. Wunderink, “MERS, SARS and other coronaviruses as causes of pneumonia,” Respirology (Carlton, Vic.), vol. 23, no. 2, pp. 130–137, 2018. View at: Publisher Site | Google Scholar
  60. H. Hofmann, M. Geier, A. Marzi et al., “Susceptibility to SARS coronavirus S protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor,” Biochemical and Biophysical Research Communications, vol. 319, no. 4, pp. 1216–1221, 2004. View at: Publisher Site | Google Scholar
  61. P. K. Chan, K. F. To, A. W. Lo et al., “Persistent infection of SARS coronavirus in colonic cells in vitro,” Journal of Medical Virology, vol. 74, no. 1, pp. 1–7, 2004. View at: Publisher Site | Google Scholar
  62. Y. Ding, H. Wang, H. Shen et al., “The clinical pathology of severe acute respiratory syndrome (SARS): a report from China,” The Journal of Pathology, vol. 200, no. 3, pp. 282–289, 2003. View at: Publisher Site | Google Scholar
  63. A. C. Sims, R. S. Baric, B. Yount, S. E. Burkett, P. L. Collins, and R. J. Pickles, “Severe acute respiratory syndrome coronavirus infection of human ciliated airway epithelia: role of ciliated cells in viral spread in the conducting airways of the lungs,” Journal of Virology, vol. 79, no. 24, pp. 15511–15524, 2005. View at: Publisher Site | Google Scholar
  64. M. C. Chan, R. W. Chan, G. S. Tsao, and J. S. Peiris, “Pathogenesis of SARS coronavirus infection using human lung epithelial cells: an in vitro model,” Hong Kong medical journal = Xianggang yi xue za zhi, vol. 17, no. 6, pp. 31–35, 2011. View at: Google Scholar
  65. K. F. To and A. W. Lo, “Exploring the pathogenesis of severe acute respiratory syndrome (SARS): the tissue distribution of the coronavirus (SARS-CoV) and its putative receptor, angiotensin-converting enzyme 2 (ACE2),” The Journal of Pathology, vol. 203, no. 3, pp. 740–743, 2004. View at: Publisher Site | Google Scholar
  66. S. A. Jeffers, S. M. Tusell, L. Gillim-Ross et al., “CD209L (L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 44, pp. 15748–15753, 2004. View at: Publisher Site | Google Scholar
  67. Z. Y. Yang, H. C. Werner, W. P. Kong et al., “Evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 3, pp. 797–801, 2005. View at: Publisher Site | Google Scholar
  68. Y. T. C. Yu, S. C. Chien, I. Y. Chen et al., “Surface vimentin is critical for the cell entry of SARS-CoV,” Journal of Biomedical Science, vol. 23, pp. 1–10, 2016. View at: Publisher Site | Google Scholar
  69. X. Tian, C. Li, A. Huang et al., “Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody,” Emerging microbes & infections, vol. 9, no. 1, pp. 382–385, 2020. View at: Publisher Site | Google Scholar
  70. H. P. Jia, D. C. Look, L. Shi et al., “ACE2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia,” Journal of Virology, vol. 79, no. 23, pp. 14614–14621, 2005. View at: Publisher Site | Google Scholar
  71. L. Suprewicz, M. Swoger, S. Gupta et al., “Vimentin binds to SARS-CoV-2 spike protein and antibodies targeting extracellular vimentin block in vitro uptake of SARS-CoV-2 virus-like particles,” 2021, bioRxiv. View at: Publisher Site | Google Scholar
  72. J. L. Daly, B. Simonetti, K. Klein et al., “Neuropilin-1 is a host factor for SARS-CoV-2 infection,” Science, vol. 370, no. 6518, pp. 861–865, 2020. View at: Publisher Site | Google Scholar
  73. L. Cantuti-Castelvetri, R. Ojha, L. D. Pedro et al., “Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity,” Science, vol. 370, no. 6518, pp. 856–860, 2020. View at: Publisher Site | Google Scholar
  74. N. Zamorano Cuervo and N. Grandvaux, “ACE2: evidence of role as entry receptor for SARS-CoV-2 and implications in comorbidities,” eLife, vol. 9, article e61390, 2020. View at: Publisher Site | Google Scholar
  75. N. Enz, G. Vliegen, I. De Meester, and W. Jungraithmayr, “CD26/DPP4 - a potential biomarker and target for cancer therapy,” Pharmacology & Therapeutics, vol. 198, pp. 135–159, 2019. View at: Publisher Site | Google Scholar
  76. T. Stange, U. Kettmann, H. J. Holzhausen, and D. Riemann, “Expression patterns of the ectopeptidases aminopeptidase N/CD13 and dipeptidyl peptidase IVICD26: immunoultrastructural topographic localization on different types of cultured cells,” Acta Histochemica, vol. 100, no. 2, pp. 157–169, 1998. View at: Publisher Site | Google Scholar
  77. D. K. Meyerholz, A. M. Lambertz, and P. B. McCray Jr., “Dipeptidyl peptidase 4 distribution in the human respiratory tract: implications for the Middle East respiratory syndrome,” The American Journal of Pathology, vol. 186, no. 1, pp. 78–86, 2016. View at: Publisher Site | Google Scholar
  78. K. Ohnuma, B. L. Haagmans, R. Hatano et al., “Inhibition of Middle East respiratory syndrome coronavirus infection by anti-CD26 monoclonal antibody,” Journal of Virology, vol. 87, no. 24, pp. 13892–13899, 2013. View at: Publisher Site | Google Scholar
  79. G. Zhao, L. Du, C. Ma et al., “A safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus MERS-CoV,” Virology Journal, vol. 10, no. 1, p. 266, 2013. View at: Publisher Site | Google Scholar
  80. J. F. Chan, K. H. Chan, G. K. Choi et al., “Differential cell line susceptibility to the emerging novel human betacoronavirus 2c EMC/2012: implications for disease pathogenesis and clinical manifestation,” The Journal of Infectious Diseases, vol. 207, no. 11, pp. 1743–1752, 2013. View at: Publisher Site | Google Scholar
  81. S. Gierer, S. Bertram, F. Kaup et al., “The spike protein of the emerging betacoronavirus EMC uses a novel coronavirus receptor for entry, can be activated by TMPRSS2, and is targeted by neutralizing antibodies,” Journal of Virology, vol. 87, no. 10, pp. 5502–5511, 2013. View at: Publisher Site | Google Scholar
  82. N. Vankadari and J. A. Wilce, “Emerging COVID-19 coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human CD26,” Emerging microbes & infections, vol. 9, no. 1, pp. 601–604, 2020. View at: Publisher Site | Google Scholar
  83. W. Li, R. J. G. Hulswit, I. Widjaja et al., “Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein,” Proceedings of the National Academy of Sciences of the United States of America, vol. 114, no. 40, pp. E8508–E8517, 2017. View at: Publisher Site | Google Scholar
  84. M. Seyran, K. Takayama, V. N. Uversky et al., “The structural basis of accelerated host cell entry by SARS‐CoV‐2†,” The FEBS Journal, 2020, Epub ahead of print. View at: Publisher Site | Google Scholar
  85. H. Chu, B. Hu, X. Huang et al., “Host and viral determinants for efficient SARS-CoV-2 infection of the human lung,” Nature Communications, vol. 12, no. 1, p. 134, 2021. View at: Publisher Site | Google Scholar
  86. P. Zhao, J. L. Praissman, O. C. Grant et al., “Virus-receptor interactions of glycosylated SARS-CoV-2 spike and human ACE2 receptor,” Cell Host & Microbe, vol. 28, no. 4, pp. 586–601.e6, 2020. View at: Publisher Site | Google Scholar
  87. A. Antonopoulos, S. Broome, V. Sharov et al., “Site-specific characterization of SARS-CoV-2 spike glycoprotein receptor-binding domain,” Glycobiology, vol. 31, no. 3, pp. 181–187, 2021. View at: Publisher Site | Google Scholar
  88. A. Shajahan, S. Archer-Hartmann, N. T. Supekar, A. S. Gleinich, C. Heiss, and P. Azadi, “Comprehensive characterization of N- and O- glycosylation of SARS-CoV-2 human receptor angiotensin converting enzyme 2,” Glycobiology, vol. 31, no. 4, pp. 410–424, 2021, Epub ahead of print. View at: Publisher Site | Google Scholar
  89. E. C. Mossel, C. Huang, K. Narayanan, S. Makino, R. B. Tesh, and C. J. Peters, “Exogenous ACE2 expression allows refractory cell lines to support severe acute respiratory syndrome coronavirus replication,” Journal of Virology, vol. 79, no. 6, pp. 3846–3850, 2005. View at: Publisher Site | Google Scholar
  90. I. Eckerle, V. M. Corman, M. A. Müller, M. Lenk, R. G. Ulrich, and C. Drosten, “Replicative capacity of MERS coronavirus in livestock cell lines,” Emerging Infectious Diseases, vol. 20, no. 2, pp. 276–279, 2014. View at: Publisher Site | Google Scholar
  91. H. Kleine-Weber, M. T. Elzayat, M. Hoffmann, and S. Pöhlmann, “Functional analysis of potential cleavage sites in the MERS-coronavirus spike protein,” Scientific Reports, vol. 8, no. 1, p. 16597, 2018. View at: Publisher Site | Google Scholar
  92. H. Kleine-Weber, M. T. Elzayat, L. Wang et al., “Mutations in the spike protein of Middle East respiratory syndrome coronavirus transmitted in Korea increase resistance to antibody-mediated neutralization,” Journal of Virology, vol. 93, no. 2, article e01381-18, 2019. View at: Publisher Site | Google Scholar
  93. I. Eckerle, M. A. Müller, S. Kallies, D. N. Gotthardt, and C. Drosten, “In-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during Middle East respiratory syndrome (MERS) coronavirus infection,” Virology Journal, vol. 10, no. 1, p. 359, 2013. View at: Publisher Site | Google Scholar
  94. J. Glende, C. Schwegmann-Wessels, M. al-Falah et al., “Importance of cholesterol-rich membrane microdomains in the interaction of the S protein of SARS-coronavirus with the cellular receptor angiotensin- converting enzyme 2,” Virology, vol. 381, no. 2, pp. 215–221, 2008. View at: Publisher Site | Google Scholar
  95. G. M. Li, Y. G. Li, M. Yamate, S. M. Li, and K. Ikuta, “Lipid rafts play an important role in the early stage of severe acute respiratory syndrome-coronavirus life cycle,” Microbes and Infection, vol. 9, no. 1, pp. 96–102, 2007. View at: Publisher Site | Google Scholar
  96. X. Ren, J. Glende, M. al-Falah et al., “Analysis of ACE2 in polarized epithelial cells: surface expression and function as receptor for severe acute respiratory syndrome-associated coronavirus,” The Journal of General Virology, vol. 87, no. 6, pp. 1691–1695, 2006. View at: Publisher Site | Google Scholar
  97. M. A. Müller, V. S. Raj, D. Muth et al., “Human coronavirus EMC does not require the SARS-coronavirus receptor and maintains broad replicative capability in mammalian cell lines,” MBio, vol. 3, no. 6, article e00515-12, 2012. View at: Publisher Site | Google Scholar
  98. A. Milewska, P. Nowak, K. Owczarek et al., “Entry of human coronavirus NL63 into the cell,” Journal of Virology, vol. 92, no. 3, article e01933-17, 2018. View at: Publisher Site | Google Scholar
  99. J. K. Millet and G. R. Whittaker, “Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein,” Proceedings of the National Academy of Sciences of the United States of America, vol. 111, no. 42, pp. 15214–15219, 2014. View at: Publisher Site | Google Scholar
  100. L. Juillerat-Jeanneret, J. D. Aubert, and P. Leuenberger, “Peptidases in human bronchoalveolar lining fluid, macrophages, and epithelial cells: Dipeptidyl (amino)peptidase IV, aminopeptidase N, and dipeptidyl (carboxy)peptidase (angiotensin-converting enzyme),” The Journal of Laboratory and Clinical Medicine, vol. 130, no. 6, pp. 603–614, 1997. View at: Publisher Site | Google Scholar
  101. P. K. Sharma, E. B. Wong, R. J. Napier et al., “High expression of CD26 accurately identifies human bacteria-reactive MR1-restricted MAIT cells,” Immunology, vol. 145, no. 3, pp. 443–453, 2015. View at: Publisher Site | Google Scholar
  102. M. Shao, Z. B. Wen, H. H. Yang et al., “Exogenous angiotensin (1-7) directly inhibits epithelial-mesenchymal transformation induced by transforming growth factor-β1 in alveolar epithelial cells,” Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, vol. 117, article 109193, 2019. View at: Publisher Site | Google Scholar
  103. S. Matsuyama, K. Shirato, M. Kawase et al., “Middle East respiratory syndrome coronavirus spike protein is not activated directly by cellular furin during viral entry into target cells,” Journal of Virology, vol. 92, no. 19, article e00683-18, 2018. View at: Publisher Site | Google Scholar
  104. C. T. Tseng, J. Tseng, L. Perrone, M. Worthy, V. Popov, and C. J. Peters, “Apical entry and release of severe acute respiratory syndrome-associated coronavirus in polarized Calu-3 lung epithelial cells,” Journal of Virology, vol. 79, no. 15, pp. 9470–9479, 2005. View at: Publisher Site | Google Scholar
  105. M. Kawase, K. Shirato, L. van der Hoek, F. Taguchi, and S. Matsuyama, “Simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry,” Journal of Virology, vol. 86, no. 12, pp. 6537–6545, 2012. View at: Publisher Site | Google Scholar
  106. K. Liao, D. Sikkema, C. Wang, and T. N. Lee, “Development of an enzymatic assay for the detection of neutralizing antibodies against therapeutic angiotensin-converting enzyme 2 (ACE2),” Journal of Immunological Methods, vol. 389, no. 1-2, pp. 52–60, 2013. View at: Publisher Site | Google Scholar
  107. L. du, W. Tai, Y. Yang et al., “Introduction of neutralizing immunogenicity index to the rational design of MERS coronavirus subunit vaccines,” Nature Communications, vol. 7, no. 1, p. 13473, 2016. View at: Publisher Site | Google Scholar
  108. F. J. Warner, R. A. Lew, A. I. Smith, D. W. Lambert, N. M. Hooper, and A. J. Turner, “Angiotensin-converting Enzyme 2 (ACE2), But Not ACE, Is Preferentially Localized to the Apical Surface of Polarized Kidney Cells*,” The Journal of Biological Chemistry, vol. 280, no. 47, pp. 39353–39362, 2005. View at: Publisher Site | Google Scholar
  109. I. Sanclemente-Alaman, L. Moreno-Jiménez, M. S. Benito-Martín et al., “Experimental models for the study of central nervous system infection by SARS-CoV-2,” Frontiers in Immunology, vol. 11, p. 2163, 2020. View at: Publisher Site | Google Scholar
  110. A. Ramani, L. Müller, P. N. Ostermann et al., “SARS-CoV-2 targets cortical neurons of 3D human brain organoids and shows neurodegeneration-like effects,” 2020, bioRxiv [Preprint]. View at: Publisher Site | Google Scholar
  111. C. K. Bullen, H. T. Hogberg, A. Bahadirli-Talbott et al., “Infectability of human BrainSphere neurons suggests neurotropism of SARS-CoV-2,” ALTEX, vol. 37, no. 4, pp. 665–671, 2020. View at: Publisher Site | Google Scholar
  112. L. Yang, Y. Han, B. E. Nilsson-Payant et al., “A human pluripotent stem cell-based platform to study SARS-CoV-2 tropism and model virus infection in human cells and organoids,” Cell Stem Cell, vol. 27, no. 1, pp. 125–36.e7, 2020. View at: Publisher Site | Google Scholar
  113. E. Song, C. Zhang, B. Israelow et al., “Neuroinvasive potential of SARS-CoV-2 revealed in a human brain organoid model,” 2020, bioRxiv, [Preprint]. View at: Publisher Site | Google Scholar
  114. J. Xu and E. Lazartigues, “Expression of ACE2 in human neurons supports the neuro-invasive potential of COVID-19 virus,” Cellular and Molecular Neurobiology, vol. 4, pp. 1–5, 2020. View at: Google Scholar
  115. P. G. Kehoe, S. Wong, N. Al Mulhim, L. E. Palmer, and J. S. Miners, “Angiotensin-converting enzyme 2 is reduced in Alzheimer's disease in association with increasing amyloid-β and tau pathology,” Alzheimer's Research & Therapy, vol. 8, no. 1, p. 50, 2016. View at: Publisher Site | Google Scholar
  116. J. Xu, S. Sriramula, H. Xia et al., “Clinical relevance and role of neuronal AT1Receptors in ADAM17-mediated ACE2 shedding in neurogenic hypertension,” Circulation Research, vol. 121, no. 1, pp. 43–55, 2017. View at: Publisher Site | Google Scholar
  117. M. Yamashita, M. Yamate, G. M. Li, and K. Ikuta, “Susceptibility of human and rat neural cell lines to infection by SARS- coronavirus,” Biochemical and Biophysical Research Communications, vol. 334, no. 1, pp. 79–85, 2005. View at: Publisher Site | Google Scholar
  118. A. C. Sims, S. E. Burkett, B. Yount, and R. J. Pickles, “SARS-CoV replication and pathogenesis in an _in vitro_ model of the human conducting airway epithelium,” Virus Research, vol. 133, no. 1, pp. 33–44, 2008. View at: Publisher Site | Google Scholar
  119. J. Huang, A. J. Hume, K. M. Abo et al., “SARS-CoV-2 infection of pluripotent stem cell-derived human lung alveolar type 2 cells elicits a rapid epithelial-intrinsic inflammatory response,” Cell Stem Cell, vol. 27, no. 6, pp. 962–973.e7, 2020. View at: Publisher Site | Google Scholar
  120. M. M. Lamers, J. van der Vaart, K. Knoops et al., “An organoid-derived bronchioalveolar model for SARS-CoV-2 infection of human alveolar type II-like cells,” The EMBO Journal, vol. 40, no. 5, article e105912, 2021. View at: Publisher Site | Google Scholar
  121. Y. Han, X. Duan, L. Yang et al., “Identification of SARS-CoV-2 inhibitors using lung and colonic organoids,” Nature, vol. 589, no. 7841, pp. 270–275, 2021. View at: Publisher Site | Google Scholar
  122. J. Stebbing, G. Sánchez Nievas, M. Falcone et al., “JAK inhibition reduces SARS-CoV-2 liver infectivity and modulates inflammatory responses to reduce morbidity and mortality,” Science Advances, vol. 7, no. 1, article eabe4724, 2021. View at: Publisher Site | Google Scholar
  123. S. K. Tiwari, S. Wang, D. Smith, A. F. Carlin, and T. M. Rana, “Revealing tissue-specific SARS-CoV-2 infection and host responses using human stem cell-derived lung and cerebral organoids,” Stem Cell Reports, vol. 16, no. 3, pp. 437–445, 2021. View at: Publisher Site | Google Scholar
  124. I. C. Huang, B. J. Bosch, F. Li et al., “SARS Coronavirus, but Not Human Coronavirus NL63, Utilizes Cathepsin L to Infect ACE2-expressing Cells,” The Journal of Biological Chemistry, vol. 281, no. 6, pp. 3198–3203, 2006. View at: Publisher Site | Google Scholar
  125. R. Y. Kao, W. H. Tsui, T. S. Lee et al., “Identification of novel small-molecule inhibitors of severe acute respiratory syndrome-associated coronavirus by chemical genetics,” Chemistry & Biology, vol. 11, no. 9, pp. 1293–1299, 2004. View at: Publisher Site | Google Scholar
  126. Y. He, Y. Zhou, S. Liu et al., “Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine,” Biochemical and Biophysical Research Communications, vol. 324, no. 2, pp. 773–781, 2004. View at: Publisher Site | Google Scholar
  127. X. Xiao, S. Chakraborti, A. S. Dimitrov, K. Gramatikoff, and D. S. Dimitrov, “The SARS-CoV S glycoprotein: expression and functional characterization,” Biochemical and Biophysical Research Communications, vol. 312, no. 4, pp. 1159–1164, 2003. View at: Publisher Site | Google Scholar
  128. S. Matsuyama, M. Ujike, S. Morikawa, M. Tashiro, and F. Taguchi, “Protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 35, pp. 12543–12547, 2005. View at: Publisher Site | Google Scholar
  129. G. Simmons, S. Bertram, I. Glowacka et al., “Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion,” Virology, vol. 413, no. 2, pp. 265–274, 2011. View at: Publisher Site | Google Scholar
  130. I. G. Madu, S. L. Roth, S. Belouzard, and G. R. Whittaker, “Characterization of a highly conserved domain within the severe acute respiratory syndrome coronavirus spike protein S2 domain with characteristics of a viral fusion peptide,” Journal of Virology, vol. 83, no. 15, pp. 7411–7421, 2009. View at: Publisher Site | Google Scholar
  131. M. Hoffmann, H. Kleine-Weber, S. Schroeder et al., “SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor,” Cell, vol. 181, no. 2, pp. 271–280.e8, 2020. View at: Publisher Site | Google Scholar
  132. M. Letko, A. Marzi, and V. Munster, “Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses,” Nature Microbiology, vol. 5, no. 4, pp. 562–569, 2020. View at: Publisher Site | Google Scholar
  133. A. S. Cockrell, K. M. Peck, B. L. Yount et al., “Mouse dipeptidyl peptidase 4 is not a functional receptor for Middle East respiratory syndrome coronavirus infection,” Journal of Virology, vol. 88, no. 9, pp. 5195–5199, 2014. View at: Publisher Site | Google Scholar
  134. M. Letko, K. Miazgowicz, R. McMinn et al., “Adaptive evolution of MERS-CoV to species variation in DPP4,” Cell Reports, vol. 24, no. 7, pp. 1730–1737, 2018. View at: Publisher Site | Google Scholar
  135. H. Hofmann, K. Hattermann, A. Marzi et al., “S protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients,” Journal of Virology, vol. 78, no. 12, pp. 6134–6142, 2004. View at: Publisher Site | Google Scholar
  136. H. Mou, V. S. Raj, F. J. van Kuppeveld, P. J. Rottier, B. L. Haagmans, and B. J. Bosch, “The receptor binding domain of the new Middle East respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies,” Journal of Virology, vol. 87, no. 16, pp. 9379–9383, 2013. View at: Publisher Site | Google Scholar
  137. G. Zhao, L. He, S. Sun et al., “A novel nanobody targeting Middle East respiratory syndrome coronavirus (MERS-CoV) receptor-binding domain has potent cross-neutralizing activity and protective efficacy against MERS-CoV,” Journal of Virology, vol. 92, no. 18, article e00837-18, 2018. View at: Publisher Site | Google Scholar
  138. J. Lang, N. Yang, J. Deng et al., “Inhibition of SARS pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans,” PLoS One, vol. 6, no. 8, article e23710, 2011. View at: Publisher Site | Google Scholar
  139. S. K. Wong, W. Li, M. J. Moore, H. Choe, and M. Farzan, “A 193-Amino Acid Fragment of the SARS Coronavirus S Protein Efficiently Binds Angiotensin-converting Enzyme 2*,” The Journal of Biological Chemistry, vol. 279, no. 5, pp. 3197–3201, 2004. View at: Publisher Site | Google Scholar
  140. Y. He, H. Lu, P. Siddiqui, Y. Zhou, and S. Jiang, “Receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies,” Journal of Immunology (Baltimore, Md. : 1950), vol. 174, no. 8, pp. 4908–4915, 2005. View at: Google Scholar
  141. A. Heurich, H. Hofmann-Winkler, S. Gierer, T. Liepold, O. Jahn, and S. Pohlmann, “TMPRSS2 and ADAM17 cleave ACE2 differentially and only proteolysis by TMPRSS2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein,” Journal of Virology, vol. 88, no. 2, pp. 1293–1307, 2014. View at: Publisher Site | Google Scholar
  142. Y. He, J. Li, S. Heck, S. Lustigman, and S. Jiang, “Antigenic and immunogenic characterization of recombinant baculovirus-expressed severe acute respiratory syndrome coronavirus spike protein: implication for vaccine design,” Journal of Virology, vol. 80, no. 12, pp. 5757–5767, 2006. View at: Publisher Site | Google Scholar
  143. Y. He, J. Li, L. du et al., “Identification and characterization of novel neutralizing epitopes in the receptor-binding domain of SARS-CoV spike protein: revealing the critical antigenic determinants in inactivated SARS-CoV vaccine,” Vaccine, vol. 24, no. 26, pp. 5498–5508, 2006. View at: Publisher Site | Google Scholar
  144. G. Simmons, J. D. Reeves, A. J. Rennekamp, S. M. Amberg, A. J. Piefer, and P. Bates, “Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 12, pp. 4240–4245, 2004. View at: Publisher Site | Google Scholar
  145. S. Bertram, I. Glowacka, M. A. Muller et al., “Cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease,” Journal of Virology, vol. 85, no. 24, pp. 13363–13372, 2011. View at: Publisher Site | Google Scholar
  146. P. Zhou, X. L. Yang, X. G. Wang et al., “A pneumonia outbreak associated with a new coronavirus of probable bat origin,” Nature, vol. 579, no. 7798, pp. 270–273, 2020. View at: Publisher Site | Google Scholar
  147. E. Kindler, H. R. Jónsdóttir, D. Muth et al., “Efficient replication of the novel human betacoronavirus EMC on primary human epithelium highlights its zoonotic potential,” MBio, vol. 4, no. 1, pp. e00611–e00612, 2013. View at: Publisher Site | Google Scholar
  148. E. Bergeron, M. J. Vincent, L. Wickham et al., “Implication of proprotein convertases in the processing and spread of severe acute respiratory syndrome coronavirus,” Biochemical and Biophysical Research Communications, vol. 326, no. 3, pp. 554–563, 2005. View at: Publisher Site | Google Scholar
  149. Y. W. Kam, Y. Okumura, H. Kido, L. F. P. Ng, R. Bruzzone, and R. Altmeyer, “Cleavage of the SARS coronavirus spike glycoprotein by airway proteases enhances virus entry into human bronchial epithelial cells in vitro,” PLoS One, vol. 4, no. 11, article e7870, 2009. View at: Publisher Site | Google Scholar
  150. S. Matsuyama, N. Nao, K. Shirato et al., “Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells,” Proceedings of the National Academy of Sciences of the United States of America, vol. 117, no. 13, pp. 7001–7003, 2020. View at: Publisher Site | Google Scholar
  151. L. Samavati and B. D. Uhal, “ACE2, much more than just a receptor for SARS-COV-2,” Frontiers in Cellular and Infection Microbiology, vol. 10, no. 317, 2020. View at: Publisher Site | Google Scholar
  152. S. Haga, N. Yamamoto, C. Nakai-Murakami et al., “Modulation of TNF-alpha-converting enzyme by the spike protein of SARS-CoV and ACE2 induces TNF-alpha production and facilitates viral entry,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 22, pp. 7809–7814, 2008. View at: Publisher Site | Google Scholar
  153. S. Haga, N. Nagata, T. Okamura et al., “TACE antagonists blocking ACE2 shedding caused by the spike protein of SARS- CoV are candidate antiviral compounds,” Antiviral Research, vol. 85, no. 3, pp. 551–555, 2010. View at: Publisher Site | Google Scholar
  154. S. Bertram, A. Heurich, H. Lavender et al., “Influenza and SARS-coronavirus activating proteases TMPRSS2 and HAT are expressed at multiple sites in human respiratory and gastrointestinal tracts,” PLoS One, vol. 7, no. 4, article e35876, 2012. View at: Publisher Site | Google Scholar
  155. L. Huang, D. J. Sexton, K. Skogerson et al., “Novel Peptide Inhibitors of Angiotensin-converting Enzyme 2*,” The Journal of Biological Chemistry, vol. 278, no. 18, pp. 15532–15540, 2003. View at: Publisher Site | Google Scholar
  156. M. J. Moore, T. Dorfman, W. Li et al., “Retroviruses pseudotyped with the severe acute respiratory syndrome coronavirus spike protein efficiently infect cells expressing angiotensin-converting enzyme 2,” Journal of Virology, vol. 78, no. 19, pp. 10628–10635, 2004. View at: Publisher Site | Google Scholar
  157. J. B. Case, P. W. Rothlauf, R. E. Chen et al., “Neutralizing antibody and soluble ACE2 inhibition of a replication-competent VSV-SARS-CoV-2 and a clinical isolate of SARS-CoV-2,” Cell Host & Microbe, vol. 28, no. 3, pp. 475–485.e5, 2020. View at: Publisher Site | Google Scholar
  158. V. Monteil, H. Kwon, P. Prado et al., “Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2,” Cell, vol. 181, no. 4, pp. 905–913.e7, 2020. View at: Publisher Site | Google Scholar
  159. A. Sheikhi and M. Hojjat-Farsangi, “An immunotherapeutic method for COVID-19 patients: a soluble ACE2-anti-CD16 VHH to block SARS-CoV-2 spike protein,” Human Vaccines & Immunotherapeutics, vol. 17, no. 1, pp. 92–97, 2021. View at: Publisher Site | Google Scholar
  160. J. Huo, A. le Bas, R. R. Ruza et al., “Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2,” Nature Sructral & Molecular Biology, vol. 27, no. 9, pp. 846–854, 2020. View at: Publisher Site | Google Scholar
  161. Y. Xiang, S. Nambulli, Z. Xiao et al., “Versatile and multivalent nanobodies efficiently neutralize SARS-CoV-2,” Science, vol. 370, no. 6523, pp. 1479–1484, 2020. View at: Publisher Site | Google Scholar
  162. M. Schoof, B. Faust, R. A. Saunders et al., “An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive spike,” Science, vol. 370, no. 6523, pp. 1473–1479, 2020. View at: Publisher Site | Google Scholar
  163. B. J. Bosch, R. van der Zee, C. A. de Haan, and P. J. Rottier, “The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex,” Journal of Virology, vol. 77, no. 16, pp. 8801–8811, 2003. View at: Publisher Site | Google Scholar
  164. B. J. Bosch, B. E. Martina, R. van der Zee et al., “Severe acute respiratory syndrome coronavirus (SARS-CoV) infection inhibition using spike protein heptad repeat-derived peptides,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 22, pp. 8455–8460, 2004. View at: Publisher Site | Google Scholar
  165. L. Braga, H. Ali, I. Secco et al., “Drugs that inhibit TMEM16 proteins block SARS-CoV-2 spike-induced syncytia,” Nature, vol. 594, no. 7861, pp. 88–93, 2021, Online ahead of print. View at: Publisher Site | Google Scholar
  166. S. K. S. S. Pindiprolu and S. H. Pindiprolu, “Plausible mechanisms of Niclosamide as an antiviral agent against COVID-19,” Medical Hypotheses, vol. 140, article 109765, 2020. View at: Publisher Site | Google Scholar
  167. V. Backer, U. Sjöbring, J. Sonne et al., “A randomized, double-blind, placebo-controlled phase 1 trial of inhaled and intranasal niclosamide: a broad spectrum antiviral candidate for treatment of COVID-19,” The Lancet Regional Health–Europe, vol. 4, article 100084, 2021. View at: Publisher Site | Google Scholar
  168. N. Zhou, T. Pan, J. Zhang et al., “Glycopeptide Antibiotics Potently Inhibit Cathepsin L in the Late Endosome/Lysosome and Block the Entry of Ebola Virus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)*,” The Journal of Biological Chemistry, vol. 291, no. 17, pp. 9218–9232, 2016. View at: Publisher Site | Google Scholar
  169. Y. Zhou, P. Vedantham, K. Lu et al., “Protease inhibitors targeting coronavirus and filovirus entry,” Antiviral Research, vol. 116, pp. 76–84, 2015. View at: Publisher Site | Google Scholar
  170. Y. W. Cheng, T. L. Chao, C. L. Li et al., “Furin inhibitors block SARS-CoV-2 spike protein cleavage to suppress virus production and cytopathic effects,” Cell Reports, vol. 33, no. 2, article 108254, 2020. View at: Publisher Site | Google Scholar

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