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High Temperature Increases Fluidity
Cells function best at normal physiological temperature, which is 98.6 degrees Fahrenheit in warm-blooded animals like humans. If body temperature increases, for example during a high fever, the cell membrane can become more fluid. This happens when the fatty acid tails of the phospholipids become less rigid and allow more movement of proteins and other molecules in and through the membrane. This can change the permeability of the cell, possibly allowing some potentially harmful molecules to enter. Both integral and peripheral proteins in the membrane can also be damaged by high temperatures and, if extremely high, heat might cause these proteins to break down, or denature.
Low Temperature Stiffens the Membrane
A decrease in temperature can also have a negative effect on cell membranes and cells. At low temperature, the fatty acid tails of the phospholipids move less and become more rigid. This decreases the overall fluidity of the membrane, also decreasing its permeability and potentially restricting entry of important molecules such as oxygen and glucose into the cell. Low temperature can also slow cell growth by prevent the cell's increase in size. In extreme situations, such as prolonged exposure to sub-freezing temperatures, liquid in the cell can begin to freeze, forming crystals that pierce the membrane and might ultimately kill the cell.
How does temperature affect the cell membrane?
1 Answer
Denaturation and Phase Changes
Explanation:
The cell-membrane is often depicted as a fluid mosaic model made up of a phospholipid bilayer with embedded protiens.
Phospholipids belong to a class of organic compounds called lipids (which also contains waxes, oils, and fats). As what you can observe from animal fats like butter, when heated they turn to liquid form. The same is true for phospholipids. Upon heating, they become more "runny" and fluid than normal, destroying their semi-permeability and even allowing the entrance of normally restricted substances. When chilled, the phospholipids become more rigid, restricting cell movement and even stopping the transfer of materials through the cell membrane.
Proteins are a bit more complex, but in simpler terms, they get "destroyed" or denatured from temperature fluctuations. Denaturation happens when a protein loses its original secondary, tertiary, or quaternary structure. In simpler terms, by destroying a protein's structure, you also destroy its efficiency and function. This is detrimental to the cell membrane, as its embedded proteins not only function as support but also as carriers of cell materials. Denaturation of proteins often happens due to pH and temperature changes.
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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160177/
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- a salt or ester of oleic acid.
- a thermodynamic quantity equivalent to the total heat content of a system. It is equal to the internal energy of the system plus the product of pressure and volume.
- the change in enthalpy associated with a particular chemical process."most compounds have negative enthalpies of formation"
- 1.CHEMISTRY(of a reaction or process) accompanied by or requiring the absorption of heat.
- 2.ZOOLOGY(of an animal) dependent on or capable of the internal generation of heat.
Enthalpy-driven apolipoprotein A-I and lipid bilayer interaction indicating protein penetration upon lipid binding
- PMID: 15379564
- DOI: 10.1021/bi036118k
Abstract
The interaction of lipid-free apolipoprotein A-I (apoA-I) with small unilamellar vesicles (SUVs) of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) with and without free cholesterol (FC) was studied by isothermal titration calorimetry and circular dichroism spectroscopy. Parameters reported are the affinity constant (K(a)), the number of protein molecules bound per vesicle (n), enthalpy change (DeltaH degrees), entropy change (DeltaS degrees ), and the heat capacity change (DeltaC(p) degrees). The binding process of apoA-I to SUVs of POPC plus 0-20% (mole) FC was exothermic between 15 and 37 degrees C studied, accompanied by a small negative entropy change, making enthalpy the main driving force of the interaction. The presence of cholesterol in the vesicles increased the binding affinity and the alpha-helix content of apoA-I but lowered the number of apoA-I bound per vesicle and the enthalpy and entropy changes per bound apoA-I. Binding affinity and stoichiometry were essentially invariant of temperature for binding to SUVs of POPC/FC at a molar ratio of 6/1 at (2.8-4) x 10(6) M(-1) and 2.4 apoA-I molecules bound per vesicle or 1.4 x 10(2) phospholipids per bound apoA-I. A plot of DeltaH degrees against temperature displayed a linear behavior, from which the DeltaC(p) degrees per mole of bound apoA-I was calculated to be -2.73 kcal/(mol x K). These results suggested that binding of apoA-I to POPC vesicles is characterized by nonclassical hydrophobic interactions, with alpha-helix formation as the main driving force for the binding to cholesterol-containing vesicles. In addition, comparison to literature data on peptides suggested a cooperativity of the helices in apoA-I in lipid interaction.
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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352545/
SARS-CoV-2 Evolutionary Adaptation toward Host Entry and Recognition of Receptor O-Acetyl Sialylation in Virus–Host Interaction
1. Introduction
The recent coronavirus pandemic crisis is due to viral infection of severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2), causing uncontrolled inflammatory conditions in the human lung. Soon after the first transmission emergence of SARS-CoV from animals to humans in China in 2003 [1], a genetically evolved beta-coronavirus genus similar to human viruses was discovered in Chinese horseshoe bats (Rhinolophus sinicus) [2]. To date, pneumonia is epidemiologically caused by diverse viruses. For example, adenovirus, influenza virus, Middle East respiratory syndrome virus (MERS-V), parainfluenza virus, respiratory syncytial virus (RSV), SARS-CoV and enteric enveloped CoV can cause pneumonia in human hosts.
The World Health Organization (WHO) reported the official terminology of the 2019-Novel Coronavirus (2019-nCoV) on 13 January 2020, and on February 11th, WHO edited the name of the disease caused by 2019-nCoV to Coronavirus Disease-2019 (COVID-19). In academia, the International Committee on Taxonomy of Viruses (ICTV) provided official nomenclature to the virus as SARS-CoV-2 due to the similarity between the novel coronavirus and SARS-CoV [3]. SARS-CoV-2 is spreading and causing a global health-threatening emergency [4]. Researchers have been in a race to develop anti-viral drugs against SARS-CoV-2 even before the WHO declared a worldwide pandemic threatening human lives. Preventive and therapeutic drugs for patients infected with SARS-CoV-2 are yet to be discovered.
The CoVs as enveloped forms can also infect the gastrointestinal track (GIT), although most other enteric viruses are naked in morphology [5,6]. CoVs can also rarely infect neural cells [7]. There is, unfortunately, no solid information on how the coronaviruses infect humans and animals with reciprocal infectivity and cause a zoonotic viral outbreak. This is in contrast to influenza viruses, which are known to selectively utilize sialic acid (SA) linkages [8]. Currently, only limited information is available on β-CoVs, such as SARS-CoV and its receptor usage and infectible cell types from different species. Host cell surface O-acetylated SAs are recognized by the lectin-like spike proteins of SARS CoV-2 for the first step of attachment to host cells. Infectious virus interaction with the host cell surface is mediated by sialoglycans as the most important phenomenon in eukaryote-parasite co-evolution. O-GlcNAc, a minor glycan source, is mainly found in the nucleus and cytosol (Figure 1). Apart from the general roles of glycans, CoVs recognize host cells and attach to host cell surface molecules to enter the host cells. For example, activity of the hemagglutinin-esterase (HE) enzyme relies on the typical carbohydrate-binding lectin and receptor-destroying enzyme (RDE) domains. Most β-CoVs target 9-O-acetylated SAs, but certain species have switched to recognizing 4-O-acetyl SA instead [8,9]. Crystallographic data for the molecular structure of type II HE provides an explanation for the switching mechanism to acquire 4-O acetyl SA binding. This event follows the orthodox ligand–receptor interaction (LRI), lectin–carbohydrate interaction (LCI), lectin–glycan interaction (LGI), lectin–sphingolipid interaction (LSI), protein–glycan interaction (PGI), protein–carbohydrate interaction (PCI), and also protein–protein interaction (PPI). Recently, 332 protein candidates were suggested to be SARS-CoV-2-human protein interacting proteins through PPIs. Among these, 66 human proteins, as druggable host factors, were further characterized as possible FDA-approvable drugs [10]. If PPI is involved, however, carbohydrates or glycans may serve as co-receptors or co-determinants. Previous reports suggest that carbohydrates act as receptor determinants in most cases. The general principles of PCI stereochemistry potentiate the SA–ligand switch by way of simple conformational shifts for the lectin and esterase domain. This indicates that our examination of natural adaptation should be directed to how carbohydrate-binding proteins measure and observe carbohydrates, leading to virus evolution toward transitional host tropism.
Cellular glycans are recognized by infectious agents including viruses and bacteria through protein–carbohydrate interaction (PCI) or lectin–carbohydrate interaction (LCI). The carbohydrates are uses as cellular adhesion sites in eukaryotic cells. Host cell surfaced and cytosolic glycans include glycoproteins, glycolipids and proteoglycans with minor glycan species of O-GlcNAc present in nucleus and cytosols.
The 9-carbon SAs are mainly animal-specific with anionic sugars attached to terminal sugars. SAs exist in two forms, NeuGc and NeuAc. NeuGc is a differentially modified form of the parental SA form, Neu5Ac. SAs are structurally diverse. For example, several modified SA forms are known for their structures including neuraminic acid (NeuC), N-acetyl neuraminic acid (NeuAc), N-glycolyl neuraminic acid (NeuGc), N,O-diacetyl neuraminic acid (occurs in horses), N,O-diacetyl neuraminic acid (occurs in bovines) and N-acetyl O-diacetyl neuraminic acid (occurs in bovines) (Figure 2). Sialyltransferases (STs) biosynthesize different SA linkages. SA linkage diversity occurs at the α2-3, α2-6, α2-8 or α2-9 to the SA or Gal residues (Figure 3). For example, in formation of α2,3 SA or α2,6 SA structures, α2,3-ST and α2,6-ST utilize substrates such as Galβ-1,4-GlcNAc (Figure 4). The most frequent modification of SAs is O-acetylation at positions of C4, C7, C8 and C9 of SA (Figure 5).
Diverse structures of sialic acids (SA). (A) Neuraminic acid; (NeuC); (B) N-acetyl neuraminic acid (NeuAc); (C) N-glycolyl neuraminic acid (NeuGc); (D) N, O-diacetyl neuraminic acid (occurs in horse); (E) N, O-diacetyl neuraminic acid (occurs in bovine); (F) N-acetyl O-diacetyl neuraminic acid (occurs in bovine).
2. Classification of CoVs
Mammal- and avian-infectible CoVs consist of the broad-ranged subfamily of Coronavirinae. The ICTV recommended classification is as follows: Riboviria (Realm)—Nidovirales (Order)— Cornidovirineae (Suborder)—coronavirida (Family)—orthocoronavirinae—Coronavirus genus. The four CoV genera [11] are the α-CoV, β-CoV, γ-CoV and δ-CoV. The 2019-nCoV or SARS-CoV-2 belong to the β-CoV genus and are zoonotic and cause mammalian infection, causing respiratory disease in the human lung. The α-CoV and β-CoV genus target mammal hosts while the δ-CoV and γ-CoV genus target avians and certain mammals. The β-CoV genus has A, B, C and D lineages. Among these, lineage B includes SARS-CoV and SARS-CoV-2. Lineage C includes MERS-CoV. The B lineage SARS-CoV and C lineage MERS-CoV, which are classified as β-CoVs, exhibit lethal rates of 10% and 35% in humans, respectively.
CoVs in humans are associated with respiratory infections such as colds with clinical importance, as experienced for the previous outbreak in 2003 of SARS-human CoV (HCoV), HCoV-HKU1 and HCoV-NL63 [12,13]. Human infectious HCoV includes seven species, including α-CoV (HCoV-NL63 and HCoV-229E) and β-CoV (SARS-CoV, SARS-CoV-2, HCoV-OC43, HCoV-HKU1 and MERS-CoV). CoV RNA sequences mutate at a high frequency. Among the known RNA viruses, CoVs bear the longest genome sizes of 26 to 32 kb length RNA. Nucleotide sequences of CoV ssRNA genomes isolated from COVID-19 patients in Wuhan show a high homology of 89% with the nucleotide sequence of the previously known bat SARS-like CoV-ZXC-21 strain and 89% with the previous SARS-CoV. The initial Wuhan CoV isolates belong to the β-CoV genus and were therefore termed SARS-CoV-2 or 2019-nCoV [14]. SARS-CoV-2 infects human respiratory tracts and causes outbreaks of pneumonia. SARS-CoV-2 is a novel CoV and originates from the Wuhan district in China. The genome sequence of SARS-CoV-2 exhibits 79% sequence homology with the SARS-CoV RNA sequence and 50% with the MERS-CoV sequence [15].
3. Structure, Components and Life Cycle of CoVs
CoVs are 60–140 nm in size and are enveloped (+) ssRNA viruses, which feature an RNA genome, directly available to function as mRNA and thus result in rapid infection. CoVs exhibit RNA genomes of 28–32 kb, comprised of two large overlapping open reading frames (ORFs), which encode the virus replicase (transcriptase) and structural proteins. The SARS-CoV-2 genome is 29,891 bp in size, which encodes 9860 amino acids. The ssRNA are capped and tailed with a 5′-capping structure and 3′-poly A tail at the termini. The genome is the same sense as virus mRNA indicating that the viral RNA is translated through its own (+) RNA to synthesize RNA dependent RNA polymerase (RdRp; PDB: 6M71).
Generally, viral families are determined by the genome structure and virion morphologies of an envelope or naked capsid. A virus with a naked capsid has a coat of nucleocapsid protein (N) coating the viral genome. Viruses with an envelope have lipid envelopes further surrounding the outmost protein layer. The 2019-nCoV (SARS-CoV-2) contains a spike (S) glycoprotein, E, dimeric HE enzyme, a membrane matrix glycoprotein (M), N and RNA [16]. The structural proteins are the S, N, M and E proteins, while the non-structural proteins are proteases such as Nsp3 and Nsp5 and RdRp such as Nsp12. Among the N, M and S glycoproteins, the S glycoprotein is a fusion protein that recognizes the host receptor and enters the host cells [17]. The S, M and E proteins anchored into the endoplasmic reticulum (ER) membrane are trafficked to the endoplasmic reticulum–Golgi intermediate compartment (ERGIC). The RNA genome linked with nucleoprotein buds into the ERGIC to form virus particles. Assembled virions transported to the vesicular surface are released to the extracellular milieu via exocytosis.
The RNA generates the replicase as two polyproteins, pp1a and pp1ab. The replicase-encoded viral proteases generate up to 16 nonstructural proteins (Nsps) in the cytosol to produce replicase enzyme and the replicase–transcriptase complex (RTC). These enzymes including RTC synthesize RNAs for replication and transcription to generate viral RNA genome. CoV genomes bear two or three protease genes and the coding enzymes cleave the replicases. Together with the replicases, nonstructural proteins, termed Nsps, assemble into the RTC complex. Nsp1 to Nsp16 are known to have multiple enzyme regions. For example, Nsp1 degrades cellular mRNAs and, consequently blocks protein translation in host cells and innate immune responses. Nsp2 recognizes the specific protein called prohibitin. Nsp3 is a multi-domain transmembrane (TM) protein with diverse activities. Ubiquitin-like 1 and acidic domains bind to N protein and ADP-ribose-1′-phosphatase (ADRP) activity induces cytokine expression. The papain-like protease (PLpro)(PDB:6WX4)/ deubiquitinase domain cleaves virus-produced polyprotein. Nsp4 is a TM scaffold protein for double-membrane vesicle structure. Nsp5 has a main protease domain which also cleaves virus-produced polyprotein and Nsp6 acts as a TM scaffold protein. The Nsp7 and Nsp8 proteins form the Nsp7-Nsp8 hexadecameric complex, which functions as an RNA polymerase-specific clamp and a primase enzyme. Nsp9 is an RNA-binding protein that activates ExoN and 2-O-methyltrnasferase (MTase) enzyme activity. Nsp10 binds to Nsp16 and Nsp14 to form a heterodimeric complex. Nsp12 is the RdRp and Nsps13 is the RNA helicase and 5′ triphosphatase. Nsps14 is a N7 MTase and 3′-5′ exoribonuclease. ExoN of Nsap14 acts as an N7 MTase and attaches the 5′ cap to viral RNAs. Viral exoribonuclease enzyme proofreads the viral RNA genome, where Nsp15 is a viral endoribonuclease (PDB: 6VWW). Nsp16 has 2-O-MTase enzyme activity, which shields viral RNA from melanoma differentiation associated protein-5 recognition.
3.1. Spike (S) Transmembrane Glycoprotein
In RNA viruses, the S glycoprotein (PDB: 6VSB) is the biggest protein, heavily glycosylated and its N-terminal domain (NTD) sequence binds to the host receptor to enter the ER of host cells. SARS-CoV-2 S-glycoprotein bears 22 N-glycan sequons in each protomer. Therefore, the trimeric S glycoprotein surface is dominated by 66 N-glycans. The S glycoprotein mediates direct and indirect interaction of virus with host cells in the infection cycle. All CoVs exhibit a surface S glycoprotein, which bears the receptor-binding domain (RBD). The S glycoprotein has a distinct spike structure. When S glycoprotein binds to its host receptor, a host furin-like protease cleaves the S glycoprotein, which liberates the spike fusion peptides, allowing entry of the virus into the host cell [18]. The furin-like protease-generated S1 and S2 exist as a S1/S2 complex, where S1 in a homotrimeric form interacts with the host cell membrane and S2 penetrates the cytosolic area. For SARS-CoV and MERS-CoV, the S1 C-terminal domains (CTDs) have a dual role in virus entry via attachment and fusion. The S1 NTD binds to carbohydrate receptors because the S1 domains act as the RBD. The CTD of S1 recognizes protein receptors via RBDs.
3.2. Nucleocapsid (N) Protein
In RNA viruses, the N protein recognizes the viral RNA genome. The N protein (PDB: 6M3M) binds to the RNA genome via the NTD and CTD. The N protein tethers to the viral RNA and replicase–transcriptase complex (RTC). NSP3 (PDB: 6VXS) of CoV blocks the innate immune responses of hosts. After entrance into the host cells, for CoV transcription and particle release, RNA chaperones such as nonspecific nucleic acid binding proteins potentiate ssRNA conformation shifts. Representatively, the N protein is known as the RNA chaperone protein. For example, glycogen synthase kinase 3 (GSK3) phosphorylates the SARS-CoV N-protein and thus, GSK3 inhibition contributes to reduced replication activity of SARS-CoV [19]. In addition, heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) regulates the preformed mRNA splicing in the nucleus and continuous translation. hnRNPA1 interacts with SARS-CoV N protein to form a replication and transcription complex during ssRNA genome biosynthesis [20].
3.3. Envelope (E) Protein
E protein is the most abundant structural protein needed to assemble virus particles in the cytosol. As a TM protein, E protein is the smallest structural protein with a MW of 12 kDa. The E protein has an NTD in the extracellular region and a CTD in the cytosolic region. The E protein bears an ectodomain in the NTD and an endodomain in the CTD. It has also an ion channel domain. E protein is present in the cytosolic region of infected cells and only a limited amount is incorporated into the envelope of virions [21]. Most E proteins assemble and bud in new virus particles.
3.4. Membrane Glycoprotein (M)
The M protein contains three TM domains and is an abundant structural protein with a MW of 30 kDa. It consists of a small glycosylated NTD in the extracellular region and CTD in the cytoplasmic region. The M protein forms a scaffold for virus assembly in the cytosol via binding to S glycoprotein and N protein [22]. For example, E protein and N protein are co-expressed with M protein to form virus-like particles (VLPs) that are released from the cells, as the M and E protein are involved in CoV assembly. Then, CoVs bud into the ERGIC, trafficking by membrane vesicles and transported via the exocytosis-secretory pathway [23,24]. The dimeric M protein binds to the nucleocapsid.-M protein binds to S glycoprotein for S glycoprotein retention in the ERGIC/Golgi complex. The M-N protein complex keeps the N protein–RNA complex stable, for nucleocapsid and the viral assembly.-M and E proteins constitute the virus envelope for successful release of virus-like particles.
3.5. Hemagglutinin-Esterase (HE) Dimeric Protein
HE hemagglutinates and destroys receptors. As RNA viruses, CoVs bear RDEs, which are used in effective attachment to hosts and also reversely in detachment from the hosts. For example, enveloped RNA viruses evade the hosts via their RDEs. Currently, RDE-related functional enzymes such as neuraminidase (NA) and SA-O-acetyl-esterase are known. SA-O-acetyl-esterase was originally identified in influenza C virus and in nidoviruses (CoV and torovirus) as well as in salmon anemia virus (teleost orthomyxovirus). The origin and evolution of CoV SA-O-acetylesterases are correlated to other viruses. The fusion event of S glycoprotein and HE is specific for HCoV attachment to SA-associated receptors in the host [25]. The HE has acetylesterase activity [26]. In early SA-related biology, influenza A/B viruses were found to recognize chicken erythrocytes in 1942 [27]. They caused hemagglutination through clumping by virus-borne hemagglutinin. These phenomena were widely found in influenza viruses, paramyxoviruses, Newcastle disease (NDV) and mumps virus.
4. Relationship between C4-O- and C9-O-Acetyl SA Preferences of CoVs in Host Cell Recognition
4.1. General O-Acetylation of SA
SAs have various derivatives of more than 50 chemically different structures formed from the basic N-acetylneuraminic acid (Neu5Ac) on the main ring of pyranose and the glycerol side chain. SA are modified by acetyl-, lactyl-, methyl- and sulfo-groups individually or in multiple combinations [28]. Multiple enzymes are involved in the modifications [29]. Historically, the first discovered SA was crystallized by Gunner Blix via a hot mild acid extraction of bovine submaxillary mucin in 1936. It consisted of two acetyl groups. Among these, only one acetyl group was attached to nitrogen [30]. Blix isolated a 9-O-acetyl SA of the common SA N-acetylneuraminic acid (Neu5Ac), chemically described as 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2). Neu5,9Ac2, Neu5Ac and Neu5Gc are naturally occurring SA species in mammals. A common modification is O-acetylation. In fact, O-acetylation of SAs is common in organisms.
The O-acetyl modification occurs in single positions of C-4, C-7, C-8 and C-9 of SA as well as in combined C-positions to yield Neu4,5Ac2, Neu5,9Ac2 and Neu5,7,9Ac3 SAs. Neu5Ac9NAc is a chemical and biologic mimic of Neu5,9Ac2 in the SA-glycans. The C-7 and/or C-9 O-acetylations are catalyzed by the SA O-acetyltransferase enzyme, Cas1 domain containing 1 (CasD1) (Figure 6). CasD1 catalyzes the addition of acetyl groups to the SA C-7 at the late Golgi apparatus compartment [31]. Thereafter, an enzyme termed “migrase” transfers the additional acetyl-group from C-7 to C-9, although this enzyme has not been identified [29]. CasD1 uses acetyl-coenzyme A as a donor substrate and CMP-Neu5Ac as an acceptor substrate, but with weak activity on CMP-Neu5Gc.
CasD1, SA O-acetyltransferase, transfers acetyl groups to C7 position of SA (Neu5Ac), from which it migrates to the C9 position (Neu5,9Ac2). The additional acetyl group is added to C7 of SA (Neu5,7,9Ac3) by the same CasD1. The SA O-acetylesterase cleaves of the acetyl groups.
Biologically, the SA O-acetylation event confers merits to hosts such as protection from pathogenic invasion and maintenance of systemic self-homeostasis. The O-acetylation event of SAs protects the SA-containing glycans from neuraminidase (NA)/sialidase action, because O-acetyl-groups inhibit microbial NA activity. The chemical structure of the O-acetyl group is quite unstable and susceptible to esterase enzymes. Sialic acid cleavage of the di-acetylated Neu5,7,9Ac3 by bacterial NAs decreases two-fold, when compared to mono-O-acetylated Neu5Ac. The O-acetylated glycan modification invites interaction with viruses, antibodies and mammalian lectins [32]. Therefore, the SA O-acetylation modification confers specific functions to organisms.
4.2. Evolutionary Acquisition of C4-O-Acetyl and C9-O-Acetyl SA Recognition by HE Enzymes
4.2.1. C4-O-Acetyl Modification
For example, in the horse, C4-O-acetyl modification of Neu5Ac (SA) occupies more than 50% of the total SA content. The C4-O-acetylated Neu5Ac, Neu4,5Ac2, inhibits the influenza A2 virus HA. De-acetylation reagents such as NaOH or NaIO4 treatment completely hemagglutinate Neu4,5Ac2 by elimination of the C4-O-acetyl group [33]. The C4-O-acetyl Neu5Ac species are found in various sources such as equine erythrocyte GM3, starfish A. rubens and fish [34,35,36,37,38]. C4-O-acetylated Neu5Ac facilitates the initial attachment of viruses to target cells. Like the influenza C virus, infectious salmon anemia virus (ISAV), a member of the Orthomyxoviridae family, contains HE and HEF proteins to mediate virus entry and exit. C4-O-Ac Neu5Ac is the major receptor determinant of ISAV in receptor binding and destruction [38], while the influenza C virus recognizes C9-O-Ac Neu5Ac. The acetylesterase RDE of ISAV cleaves C4-O-Ac via 4-SA-O-acetylesterase with a short turnover time, whereas C9-O-Ac Neu5Ac is cleaved by 9-SA-O-acetylesterase with a long turnover time [34].
The position of SA O-acetylation is linked to functions including substrate differentiation of enzymes such as NAs and esterase by C4 O-Ac. Previous development of O-Ac site-selective NA inhibitors were based on the conceptual consideration of different O-Ac positions. The O-Ac of SAs is site-specific, as C4 of Neu5Ac is considered to be a potential position for modification. Historically, inhibitors of influenza A and B viruses-sialidases were designed by Von-Itzstein in 1993 [39]. The Ac group-based C4 substitution interacts with amino acid Glu-119 present in the active site of sialidase. Guanidine-attached C4 of C2–C3 unsaturated SA (Neu5Ac2en) inhibits activity of sialidases isolated from influenza A virus (Singapore/1/57) and B virus (Victoria/102/85). The same scenario was applied for sialidase inhibition of the human parainfluenza virus type 3, which has HN and fusion proteins [40]. The C4 of Neu5Ac2en was substituted by alkyl groups such as the O-ethyl group. For example, Zanamivir has a substitution with a 4-guanidino group with an IC50 of 25 μM. Thus, sialidase inhibition is important for C4 modification of Neu5Ac2en. Later, oseltamivir with the tradename Tamiflu (Basel, Switzerland) and zanamivir with the tradename Relenza (London, UK) were established [41]. These drugs exhibit some adverse side effects that restrict clinical use.
4.2.2. SA C9-O-Acetyl Modification
The SA 9-O-acetylation in hosts allows hosts to evade influenza A virus hemagglutinin (HA) recognition and some lectins of factor H (FH), CD22/Siglec-2 and sialoadhesin/Siglec-1. Instead, the influenza C virus HA recognizes the hosts. β-elimination and permethylation eliminate the 9-O-acetyl group from SAs. Chemical modification of the C-9 position of Neu5,9Ac2 generates a 9-N-acetyl analog, 9-acetamido-9-deoxy-N-acetylneuraminic acid (Neu5Ac9NAc), a mimic of Neu5,9Ac2 with influenza C virus-binding capacity, which is not cleaved by the HE [42]. SA O-acetylesterase regulates the presence of 7,9-O-Ac and 9-O-Ac. SA O-acetylation and deacetylation are involved in development, cancer and immunology. SA O-acetylation alters host lectin bindings such as siglecs [29]. The presence of 9-O-Ac can also reduce the activity of NAs [43]. SA modifications regulate pathogen binding or pathogen NAs. Influenza A/B/C/D viruses use SA as their entry receptors. Influenza A and B subtypes bind to SAs via HA and NA to allow endocytosis of the virus and fusion of the viral envelope with endosomes. In contrast, influenza C and D subtypes bear only one coated glycoprotein, termed the HE fusion protein (HEF). The HEF acts as the HA and NA. HEF recognizes 9-O-acetyl SA for entry into cells, while the esterase domain removes 9-O-acetyl-groups and liberates the virus from mucus and mis-assembled virus aggregates after budding. The 9-O-Ac on cells prevents the NA activity and HA binding of the influenza A type virus [44].
5. HE of CoVs
5.1. Evolutionary Origin and Classification of the CoV HE
Certain viruses use glycoproteins such as HA, HE, S and HEF for host receptor binding or destruction. Coronaviridae, Orthomyxoviridae, Paramyxoviridae and Adenoviridae utilize SAs as binding molecules for attachment and entry. However, only limited human pathogens recognize O-Ac SA.
5.1.1. Influenza Virus A and B Spike Proteins of HA and NA
Influenza A and B viruses bear two spikes of receptor-binding HA and NA [45].
5.1.2. Influenza C virus HA-HEF
HEF is indeed an ancient type of SA-O-acetylesterase. In contrast to A/B, the influenza C virus bears one spike with triple functions of HEF as a homotrimer [46]. Each HEF subunit bears two Neu5,9Ac2-binding sites and binds to the 9-O-acetyl group. In parallel, another modification of O-acetylation is found. Indeed, influenza C virus bears SA-O-acetylesterase [47], which converts 5-N-acetyl-9-O-NeuAc (Neu5,9Ac2) to 5-Neu5Ac. The 9-O-acetyl SA is a unique determinant for the influenza C virus receptor and Neu5,9Ac2 is crucial for receptor activity, but not Neu5Gc or Neu5Ac [48]. Neu5,9Ac2 is an essential determinant for influenza virus C type-specific host cell tropism. NAs cleave the α-ketosidic linkages to the D-Gal or GalNAc. SA-O-acetylesterases cleave different O-acetyl linkages (Figure 5). The OH-group of Tyr224 and the guanidino group of Arg236 interact with the CH3CO-carbonyl oxygen [49]. HEF SA-O-acetylesterase is found in several enveloped (+) ssRNA viruses of influenza C virus and also in certain CoVs and toroviruses [47]. The CoVs are different from the orthomyxoviruses, which hold a segmented (−) ssRNA genome and are instead evolutionary linked to the family Coronaviridae, order Nidovirales [45].
5.1.3. CoV SA-O-Acetylesterase HE
CoVs and toroviruses of the Coronaviridae family are specific for the O-Ac SA receptors. Their S and HE glycoproteins are similar to influenza C virus HEF. CoVs and all toroviruses bear HE gene form class I envelope membrane proteins of about 400 amino acid residues which bear 7 to 12 N-glycosylation sites [50]. HE multimer forms enter virions. Bovine CoV (BCoV) and HCoV-OC43, similar to influenza C virus, recognize Neu5,9Ac2 and bear SA-9-O-acetylesterase [8]. CoV HEs are all O-acetylesterases. The HE enzymes found in torovirus, CoV and influenza C virus are evolutionarily interspecies-mutated with about 30% homology by heterologous RNA recombination [51] and horizontal gene transfer. Therefore, viral HEs are diverse and widespread over evolution.
5.2. Substrate Diversity of the CoV HEs
HEs as envelope proteins are found in CoVs, orthomyxoviruses and toroviruses. Coronaviral HEs are involved in virus attachment to SA species. HE protein in β-CoVs binds to Neu5,9Ac2 form SA and agglutinates the red blood cells (RBCs) of rodents [52]. As with SA-O-acetylesterase, HE potentiates viral entry with the S protein and spreading via the mucosal glycans. It contains a carbohydrate-recognizing domain (CRD) known in lectin. The HE glycan-binding domain (GBD) mediates virus attachment to SAs on host cells. HE is the only HA. This indicates that compared to the S glycoprotein, HE is only minor a HA and the S glycoprotein mainly attaches to the cell surface. The HE protein of murine hepatitis virus (MHV), an enveloped CoV, binds to SA-4-acetylester or SA-9-O-acetylester of the carcinoembryonic antigen cell adhesion molecule 1a (CEACAM; known as CD66a) as the key receptor [53]. Murine CoVs HEs acquired by horizontal gene transfer, bind to C9-O-Ac Neu5Ac. However, some murine CoV HEs cannot bind to C4-O-Ac Neu5Ac. The original mouse MHV HE binds to C9-O-Ac Neu5Ac, while the MHV S-strain HE evolutionarily acquired the ability to bind to C4-O-Ac Neu5Ac [12,53,54]. In terms of structure, the C5 N- and C9 O-Ac Neu5Ac-accomodating hydrophobic pocket was shifted to a C5 N- and C4-O-Ac Neu5Ac-accomodating pocket [55].
Type I HE is specific for the 9-O-acetylated SAs (9-O-Ac-SAs). Type II HE is specific for 4-O-Ac-SAs. The SA-binding shift indicates quasi-synchronous adaptations of the SA-recognition sites of the lectin and esterase domains. Type I HE monomers of β-CoV lineage A have a bimodular enzyme–lectin domain similar to cellular glycan/carbohydrate-modifying proteins. Originally, HE homologs are found in various viruses including toroviruses and orthomyxoviruses such as the influenza virus C/D and isavirus, as well as the exceptional case of β-CoV lineage A among CoVs. The HE gene was transmitted to a β-CoV lineage A progenitor via horizontal gene transfer from a 9-O-Ac-Sia–recognizing HEF, as shown in influenza virus C/D. HE acquisition and expansion occurred by cross-species transmission over HE evolution and this phenomenon reflects viral evolutionary adaptation to host SA-containing glycans. Therefore, CoV HE receptor switching precedes virus evolution driven by SA-containing glycan diversity of hosts. For instance, the BcoV HE prefers 7,9-di-O-Ac-SAs, which is also a target of the bovine torovirus HE. For a more outstanding case, such a switching event occurred in the murine CoVs for the β-CoV lineage A type switch toward O-Ac-SA recognition. In the HE specificity of murine CoVs, two different murine CoV subtypes of virus group exist with one subtype possessing the typical 9-O-Ac-SA (type I) attachment factor and the other exclusively 4-O-Ac-SA (type II) attachment virus group [56].
The first coronaviral HE proteins identified were from the porcine hemagglutinating encephalomyelitis virus (PHEV), BCoV and HCoV-OC43, which bear SA-9-O-acetylesterases similar to HEF [8]. Rat CoV (RCoV) has SA-4-O-acetylesterases, converting Neu4,5Ac2 to Neu5Ac [53,57,58]. Some murine CoVs prefer 4-O-Ac-SAs and others 9-O-Ac-SAs. HCoV-OC43 and BCoV prefer α2-6-SA 9-O-acetylation by their SA-O-acetyleseterases. The S glycoproteins of BCoV and HCoV-OC43 are Neu5,9Ac2-recognizing lectins and agglutinate murine, rat and chicken erythrocytes due to the enriched 9-O-Ac-SA species [52]. BCoV and HCoV-OC43 adapted to SA receptor determinants of 9-O-Ac-SA receptors [59]. For a second receptor, the binding of S glycoprotein to Neu5,9Ac2 receptor is essential for entry into cells. BCoV-infection is prevented by prior treatment of cells with NA enzyme or with viral SA-O-acetylesterases, blocking the roles of HE and S glycoprotein in SA-dependent entry to host cells.
6. CoVs Infection of Human Hosts
6.1. CoVs Utilize SAs and SA Linkages as Attachment and Entry Sites to Human Host Cells
Several β-CoV genera such as BCoV bind to O-acetylated SAs and bear an acetylesterase enzyme to act as a host cell RDE. Certain α-CoV and γ-CoV are deficient for the comparable acetylesterase enzyme but have a preference to NeuAc or NeuGc type SA species. Infectious bronchitis virus (IBV) and transmissible gastroenteritis virus are such examples. Additionally, both α-CoV and γ-CoV also include sub-members deficient of any SA-recognizing activity. During evolution, some subtypes of SARS-CoV and HCoV-229E acquired SA-binding capacity. The SA-binding activities of BCoV, transmissible gastroenteritis coronavirus (TGEV) and IBV are well known [60].
6.1.1. α-Coronavirus
In α-CoVs such as TGEV, HA-activity is attributed to the SA-recognizing activity to α2,3-NeuGc [61,62]. The SA-binding site is present on the N-terminal region of the S-glycoprotein of TGEV. TGEV has two types with enteric and respiratory tropism. The respiratory TGEV has the porcine aminopeptidase N (pAPN)-binding domain and SA-binding domain. Nucleotide 655 of the S gene is essential for enteric tropism and the S219A mutation of the S glycoprotein confers the enteric to respiratory tropism shift. In addition, a 6-nucleotide insertional mutation at nucleotide 1124, which yields the Y374-T375insND shift of the S glycoprotein, causes enhanced enteric tract tropism. TGEV interacts with SA species on mucin-like glycoprotein (MGP), a highly glycosylated protein, in an SA-dependent manner, on mucin-secreting goblet cells [6]. MGP SA-binding allows virus entry via the mucus layer to the intestinal enterocytes. Different from TGEV, the S glycoprotein of porcine CoV has no hemagglutination activity due to deletion of the SA-binding site of the S glycoprotein [61]. The loss of SA-binding activity is correlated to the non-enteropathogenicity. SAs function as HA-mediated entry determinants for TGEV, causing the enteropathogenic outcome of the virus, and SA-recognition activity is also responsible for virus amplification in cells. SA-binding activity-deficient TGEV can propagate in cells through pAPN, known as CD13, as a receptor [62,63]. The SA-binding activity potentiates infection and is crucial for intestinal infection.
6.1.2. β-Coronavirus
In β-CoV, HE mediates viral attachment to O-Ac-SAs and its function relies on the combined CBD and RDE domains. Most β-CoVs target 9-O-Ac-SAs (type I), but certain strains switched to alternatively targeting 4-O-Ac-SAs (type II). For example, the SA-acetylesterase enzyme in BCoVs and HCoV-OC43 is known to have hemagglutinizing activities as a type of SA-9-O-acetylesterase [8]. The SA-acetylesterase is the HE surface glycoprotein in BCoV. The three-dimensional structure of BCoV HE is similar to other viral esterases [9]. The HE gene is found only in the β-CoV genus. The acetylesterase of murine CoVs differs in its substrate binding specificity from that of BCoV and HCoV-OC43, which is specific for O-acetyl residue release from SA C-9. Murine CoVs prefer to esterize 4-O-acetyl-NeuAc [64]. The β-CoV acetylesterase destroys the receptors and this specificity is similar to that of influenza viruses. Acetylesterase activity can be inhibited by diisopropyl fluorophosphate and this agent decreases viral infection levels [65]. As deduced from the SA acetylesterase of HCoV-OC43 [8], the 9-O-Ac-SA species is a receptor binding determinant for erythrocytes and entry into cells [59]. The BCoV HE protein has dual activity of acetylesterase and HA [9]. BCoV widely agglutinates erythrocytes and purified HE only agglutinates Neu5,9Ac2-enriched erythrocytes of rats and mice. BCoV and HCoV-OC43 can agglutinate chicken erythrocytes, while purified HE cannot. In contrast to the HE protein, purified S glycoprotein can agglutinate chicken erythrocytes [52], indicating that the major HA is the S protein which acts as the major SA-binding protein. However, the role of O-Ac-SAs is not certain to be essential in receptors, and SA-binding activity may be essential only to the HE protein, but not to the S glycoprotein [54].
6.1.3. γ-Coronavirus
In γ-CoVs, IBV strains, known as poultry respiratory infectious pathogens, can agglutinate erythrocytes. IBV prefers to recognize α2,3-NeuAc and the SA functions as a host entry receptor for infection [66]. Glycosylation of IBV M41 S1 protein RBD is crucial for interaction with chicken trachea tissue and RBD N-glycosylation confers receptor specificity and enables virus replication. The heavy glycosylated M41 RBD has 10 glycosylation sites. N-glycosylation of IBV determines receptor specificity. However, the host receptor has not yet been found. NA treatment reduces the binding of soluble S to kidney and tracheal epithelial cells. The IBV S protein recognizes epithelial cells in a SA-dependent manner. The SA-binding ability of IBV is necessary for infection of tracheal epithelial cells and lung respiratory epithelial cells [67]. The SA-binding site is located on S1 of the IBV S protein, although the IBV-specific protein receptor is not known. In contrast to BCoV or HCoV-OC43, IBV lacks an RDE. SA binding of IBV is likely more essential than in other viruses such as TGEV.
6.1.4. Torovirus
In torovirus, which belongs to the family Coronaviridae, the toroviruses are grouped into the Torovirinae subfamily and the Torovirus genus. The known toroviruses can infect four species of hosts, constituting bovine, equine, porcine and human toroviruses. They mildly infect swine and cattle through the HE protein, which is similar to the β-CoV HE protein [68]. The HE protein is a class I membrane glycoprotein which forms homodimers with a MW of 65 kDa. The RDE protein HE reversibly binds to glycans [15] through binding to SAs. The acetyl-esterase activity disrupts SA binding. HE hemagglutinates mouse erythrocytes and cleaves the acetyl-ester linkage of glycans and acetylated synthetic substrate p-nitrophenyl acetate (pNPA) [69]. Similar to CoV, torovirus HE is an acetylesterase type, which cleaves the O-acetyl group from the SA C-9 position using Neu5,9Ac2 and N-acetyl-7(8),9-O-NeuAc [64]. However, torovirus HE exhibits a restricted specificity for the Neu5,9Ac2 substrate, but not for the Neu5,7(8),9Ac3 substrate, with a unique SA-binding site generated by a single amino acid difference in porcine Thr73 and bovine Ser64 for each HE [70].
6.2. SARS-CoV-2 Recognizes 9-O-Acetyl-SAs and MERS-CoV Recognizes α2,3-SAs as Attachment Receptors
The S glycoprotein SARS-CoV-2 initiates infection of the host cells. The molecular basis of CoV attachment to sugar/glycan receptors is an important issue, as demonstrated by recent cryo-EM defining the structure of the CoV-OC43 S glycoprotein trimer complexed with a 9-O-acetylated SA [56]. Cryo-EM structures of the trimeric ectodomain of S glycoprotein were observed using forms complexed with Neu5Ac, Neu5Gc, sialyl–LewisX (SLeX), α2,3-sialyl-N-acetyl-lactosamine (α2,3-SLacNAc) and α2,6-SLacNAc, respectively. The receptor-binding site is commonly conserved in all CoV S glycoproteins, which attach to 9-O-Ac-SA species with similar ligand-binding pockets to the CoV HEs and influenza virus C/D HEF glycoproteins, indicating conserved recognizing structures [25]. The S glycoprotein-9-O-acetyl-SA interaction resembles the ligand-binding pockets of CoV HEs and influenza virus C/D HE fusion glycoproteins. HCoV-OC43 and BCoV recognize 9-O-Ac-SA. S glycoproteins engage 9-O-acetyl-SAs. The 9-O-acetyl SAs are the binding site for HCoV-OC43 S glycoprotein and related β-1 CoV S glycoproteins, however SA-binding sites on the 9-O-acetyl sialyl receptors of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are different [71]. Thus, CoVs use two different entry and attachment receptors. Therefore, S glycoproteins of CoVs are distinct from influenza virus A HAs, which bind to the Neu5Ac species by conserved binding sites. The ligand-binding sites of BCoV HE enzyme, influenza HEF enzyme and CoV S glycoprotein have evolved 9-O-Ac-SA binding through hydrogen bonding with the 9-O-acetyl carbonyl group and hydrophobic pocket formation with the 9-O-acetyl methyl group [71,72]. However, influenza HA cannot bind to 9-O-acetyl-SAs but can bind to NeuGcs [73]. The HCoV-OC43 S glycoprotein, HCoV-HKU1 S glycoprotein, BCoV S glycoprotein and PHEV S glycoprotein, therefore, share the ligand-binding specificity of influenza C/D HEF enzyme, although they are functionally more similar to influenza virus A/B HA, whereas CoV HE or influenza virus A/B NA have RDE activities
CoV HEs are functionally similar to influenza virus C/D HEF glycoproteins. In CoV, the S glycoprotein recognizes the 9-O-Ac-SA sugar, while the HE acts as the RDE enzyme with SA-O-acetyl-esterase activity to release virions from infected host cells. For example, HCoV-OC43 also has a similar HE as an RDE [71]. In influenza C and D viruses, HEF glycoproteins act similarly to the CoV HE [74]. In influenza A virus, RDE NA releases virions from host cells. However, MERS-CoV does not have a similar enzyme and thus MER-CoV binding to SA receptors is mediated by energetically reversible interactions of the lipid rafts with increased SA receptors [75], thus enhancing dipeptidyl peptidase 4 (DPP4) or carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) recognition power and viral entry [76] and membrane-associated 78-kDa glucose-regulated protein (GRP78) [77].
MERS-CoV S glycoprotein can hemagglutinate human erythrocytes and mediates virus entry into human respiratory epithelial cells. MERS-CoV S glycoprotein attachment is not observed for 9-O-acetylated or 5-N-glycolyl SAs, but is observed for α2,3-SA linkage over α2,6-SA linkages. SA-binding sites of MERS-CoV S glycoprotein and HCoV-OC43 S glycoprotein are not conserved [78], although they engage α2,3-SAs on the avian host cell surface [79]. MERS-CoV recognizes α2,3-SA and to a lesser extent the α2,6-SAs and sulfated SLeX for binding preference. Thus, S glycoproteins may have independently evolved SA recognition. The acquisition of SA-binding ability of MERS-CoV S seems to be an evolutionarily recent event, because HKU4 S1 and HKU5 S1 cannot hemagglutinate human erythrocytes [75], indicating flexible evolutionary exchange allowing cross-species transmission towards host cell tropism of CoVs. In conclusion, CoV recognition of 9-O-Ac-SAs for infection is based on a conserved sequence for engagement of SA-related carbohydrate ligands across CoVs and orthomyxoviruses.
6.3. Host Receptors of CoVs
CoV S spikes recognize diverse surface molecules as the attachment or entry site. Animal and human coronaviruses evolve to acquire the same host receptors and attachment factors and overcome the interspecies barrier from animals to human. Specifically, S glycoprotein interaction with its binding receptor determines host tropism, pathogenicity and therapeutic clues [80]. CoVs recognize multiple host receptors via distinct S domains. The host receptors for β-CoV SARS-CoV includes angiotensin-converting enzyme 2 (ACE2). As a lineage C β-CoV, the MERS-CoV S glycoprotein binds to DPP4 [81,82,83]. MERS-CoV S glycoprotein recognizes α2,3-SA over α2,6-SA-bearing receptors. The N-terminal subunits of the S1/S1A/S1B/S1D complex of MERS-CoV recognize DPP4. MERS-CoV recognizes CEACAM5 as the attachment factor for entry [78]. Among the six HCoVs, the α-CoV HCoV-229E S protein recognizes human APN (hAPN) [84]. α-CoV HCoV-NL63 and the lineage B β-CoV SARS-CoV S glycoproteins bind to ACE2. Meanwhile the protein receptors specific for lineage A β-CoVs such as HCoV-HKU1 and HCoV-OC43 are not known yet.
BCoV, HCoV-OC43, HCoV-HKU1 and TGEV recognize O-acetyl-SAs as attachment molecules. In addition to O-acetyl-SA, HCoV-HKU1 spikes additionally bind to major histocompatibility complex class I (MHC-I) C as attachment sites [85]. SARS-CoV uses dendritic cell (DC)-specific intercellular adhesion molecule (ICAM)-3–grabbing nonintegrin (DC-SIGN) for attachment [86]. For glycan interaction, HCoV-NL63 and mouse hepatitis virus utilize heparan sulfate (HS) proteoglycans as attachment enhancers [87]. In general, ACE2, APN, heat shock protein A5 (HSPA5), furin, heparan sulfate proteoglycans (HSPGs) and O-acetyl-SA are CoVs-recognizing candidates.
6.3.1. Angiotensin-Converting Enzyme 2 (ACE2) as the SARS-CoV Host Receptor
Structure and Role of the Host SARS-CoV Receptor ACE2
SARS-CoV-2 needs ACE2 for entry. Host proteases such as human ACE2 help viral entry through removement of a barrier to enter human cells through unknown receptors. Human ACE2 is known for its role as the SARS-CoV-2 entry receptor and the SARS-CoV receptor. The enzyme ACE-2 in the renin-angiotensin system (RAS) is associated with CoV entry into lungs. ACE2 mediates SARS-2002 entry into host cells via S glycoprotein interaction with the ACE2 receptor. The ACE2 levels on the plasma membrane correlate with virus infectivity. ACE2 expression is present in most tissues such as the lung epithelium. It is highly expressed by respiratory epithelial cells and type I/II lung alveolar epithelial cells [88]. The host receptor is not linked to the classification of CoVs. MERS-CoV, a β-CoV, does not recognize the ACE2 receptor. In contrast, the α-CoV HCoV-NL63 recognizes the ACE2 receptor. ACE2 is a membrane-anchored carboxypeptidase with 805 amino acid residues and is captopril-insensitive. It contains 17 amino acid residues as a signal peptide in the N-terminal region, a type I membrane-anchored domain in the C-terminal region, an extracellular N-terminal domain with heavy N-glycans, a N-terminal SARS-CoV-binding and carboxypeptidase site and a short C-terminal cytoplasmic tail. The ACE2 gene is located on chromosome Xp22. Two ACE2 forms are known, a membrane-bound form and a soluble form.
ACE cleaves angiotensin I (Ang I) substrate to Ang II. Ang II recognizes the Ang II receptor type 1 (AT1R), contributing to systemic and local vasoconstriction, fibrosis and salt retention in vascular organs. ACE2 has the opposite function of ACE. ACE2 is a close homolog to human ACE. ACE2 activity on Ang II is about 400-fold higher than that on Ang I. Ang-1 to Ang-7 recognize the G protein-coupled receptor (GPCR) Mas to activate vasorelaxation, cardioprotection, antioxidative action, antiinflammation and anti-Ang II-signaling. Therefore, the ACE2-Ang-1 to Ang-7 axis is a target candidate for cardiovascular diseases. ACE2 shows similar binding structures between nCoV and SARS-CoV. The three proteins of ACE, Ang II and AT1R contribute to progression of lung injury in humans. ACE2 removes a single amino acid residue from Ang II to yield the vasodilator, named Ang 1-Ang 7. ACE2 cleaves Ang-I to Ang 1–Ang 9 and Ang II to Ang-1 to Ang-7. The biggest difference between ACE2 and ACE is that ACE2 has a non-inhibitory property by ACE inhibitors.
Pulmonary ACE2 is potentially a candidate target in CoV-involved inflammatory pathogenesis. If ACE inhibitors and Ang II-AT1 blockers are dosed, ACE2 expression is increased. However, currently we have no conclusive evidence that the inhibitors help SARS-CoV or SARS-CoV-2 entry. Rather, SARS-CoV infection reduces ACE2 expression. Therefore, SARS-CoV-2 host tropism is not related to ACE2 expression. ACE2 levels and ANG II/ANG 1–7 levels regulate the pathogenic progression. ACE2 expression is upregulated by gene polymorphisms and ACE inhibitors or Angiotensin II receptor blockers such as sartans.
Host Cell ADAM17 and TMPRSS2 Competitively Cleave ACE2
A disintegrin and metallopeptidase domain (ADAM) family of Zn-metalloproteinases belongs to membrane proteins. The well-known ADAM17 is a TNF-α-converting enzyme (TACE), called the sheddase for TNF-α. Other ADAM sheddase family members include ADAM9, ADAM10 and ADAM12. ADAM17 mediates ACE2 shedding. SARS-CoV S glycoprotein activates cellular TACE and consequently facilitates virus entry. Soluble ACE2 as the N-terminal carboxypeptidase domain form is derived from the original ACE2 form by an ADAM17 metalloprotease in the membrane [89]. ADAM17 is indeed an enzyme that can convert membrane type pro-TNF-α to soluble TNF-α, a functional proinflammatory cytokine. Therefore, ADAM17 inhibition indicates an anti-inflammatory response and ADAM17 inhibitors are promising candidates for TNF-α-induced inflammatory diseases. The short C-terminal domain of ACE2 is removed by ADAM17 and TMPRSS2. However, TMPRSS2 cleaves ACE2 competitively with the ADAM17 metalloprotease. SARS-S protein-ACE2 binding leads to ADAM17/TNF-α-converting enzyme (TACE)-cleavage of ACE2, facilitating extracellular ACE2 shedding and consequent SARS-CoV entry into host cells [90,91]. Only TMPRSS2 cleavage allows SARS-CoV entry into host cells through endocytosis and fusion. Soluble ACE2 also recognizes the virus and prevents SARS-CoV-2 infection. SARS-CoV-2 infection requires membrane ACE2 and TMPRSS2. The ACE2–B0AT1 complex binds to the S glycoprotein of SARS-CoV-2. Intestinal membrane ACE2 and lung TMPRSS2-shedded ACE2 can act as alternative entry sites for SARS-CoV-2. SARS-CoV-2 infects the lungs and intestine via TMPRSS2-cleaved ACE2. If TMPRSS2 is engaged in SARS-CoV-2 entry and ACE2 downregulation, TMPRSS2 inhibition would lead to COVID-19 prevention. Although ACE2 is expressed both in type I and type II lung alveolar epithelial cells, SARS-CoV and SARS-CoV-2 target only type II epithelial cells due to the ACE2–TMPRSS2 interaction. Therefore, supplementation of ACE2 (soluble ACE2) or Ang-1 to Ang-7 should be a way to reduce SARS-CoV-2-related symptoms.
TMPRSS2-cleaved ACE2 is involved in SARS-CoV and MERS-CoV infections. SARS-CoV-2 uses ACE2 for cell entry through TMPRSS2 priming of the S glycoprotein (Figure 7). Infection of the H7N9 influenza and H1N1 influenza A subtype viruses are also mediated by TMPRSS2-cleaved ACE2. This implies that TMPRSS2 can be targeted as a strategic antiviral therapy [92]. Transmembrane protease serine 2, termed TMPRSS2, a type II TM Ser protease (TTSP), also cleaves ACE2. The human TMPRSS2 gene, located on chromosome 21, comprises androgen receptor elements (AREs) in the upstream 5′-flanking region [93]. TMPRSS2 expression is regulated in an androgen-dependent manner. The TMPRSS2 gene encodes 492 amino acids. The original form is cleaved into the major membrane form and the minor soluble form. TMPRSS2 activates protease activated receptor 2 (PAR-2) and activated PAR-2 upregulates matrix metalloproteinase-2 (MMP-2) and MMP-9. TMPRSS2-activated hepatocyte growth factor (HGF) induces c-Met receptor signaling. TMPRSS2 activates SARS-CoV and MERS-CoV. The SARS-CoV S glycoprotein is cleaved by host-borne TMPRSS2, human airway trypsin-like protease (HAT), TM protease, serine 13 (MSPL), serine protease DESC1 (DESC1), furin, factor Xa and endosomal cathepsin L/B. SARS-CoV can enter cells upon cleavage by protease TMPRSS2 or endosomal cathepsin L/B [90]. Virus S protein precursor is cleaved by host proteases. The spikes are cleaved by endosomal cathepsin and by Golgi or plasma membrane TMPRSS2 in the step of assembly or attachment and release. The serine protease inhibitor camostat effectively blocks lethal SARS-CoV infection to mice. However, serine protease and cathepsin inhibitors are not effective. Thus, TMPRSS2 is suggested to be an acting protease for SARS-CoV entry into host cells, but not by cathepsin. Cis-cleavage liberates SARS-CoV S glycoprotein fragments into the extracellular supernatant. Trans-cleavage activates the SARS-CoV S glycoprotein on the target cells, potentiating efficient SARS-CoV S glycoprotein-driven viral fusion. TMPRSS2-activated SARS-CoV facilitates enveloped virus entry into cells. TMPRSS2 is important for SARS-CoV entry and infection [81,94,95,96].
SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) for cell entry through TMPRSS2 priming of S glycoprotein.
The fact that SARS- and MERS-CoV infections are potentiated by TMPRSS2 indicates that TMPRSS2 is a promising target for therapeutic agents. For example, several Ser protease inhibitors such as camostat mesylate inhibit TMPRSS2–ACE2-involved SARS-CoV-2 entry. camostat, a serine protease inhibitor, reduces influenza virus titers in cell culture. camostat-treated TMPRSS2 inhibition in Calu-3 cells greatly reduces SARS-CoV viral titers and improves survival rate in SARS-CoV infected mice. A treatment of 10-μM camostat blocks MERS-CoV entry to African green monkey kidney (Vero)-TMPRSS2 cells and blocks viral RNA synthesis in Calu-3 cells upon MERS-CoV infection. Aprotinin is a polypeptide with 58 amino acid residues that was isolated from bovine lungs. Another serine protease inhibitor, nafamostat, inhibits MERS-CoV entry and infection by TMPRSS2 inhibition [93]. nafamostat mesylate blocks the TMPRSS2–ACE2-involved SARS-CoV-2 envelope–PM fusion and prevents SARS-CoV-2 entry [95]. nafamostat mesylate inhibits viral entry and thrombosis in COVID-19 patients. Similarly, an FDA-approved mucolytic cough suppressant, Bromhexine hydrochloride (BHH), inhibits TMPRSS2 (IC50 0.75 μM) and hence blocks infection of CoV and influenza virus. MPRSS2 as a host factor plays a pivotal role in SARS-CoV and MERS-CoV infections. FDA-approved TMPRSS2 inhibitors are yet under development. Because TMPRSS2 mediates efficient viral entry and replication, it should be a promising target for new therapeutics against CoV infection.
6.3.2. Dipeptidyl peptidase-4 (DPP4) as MERS-CoV Receptor
The Ser exopeptidase DPP-4/human CD26 (PDB: 4L72), a type II TM ectopeptidase, functions as a host cell receptor for MERS-CoV. The RBD structure was characterized by crystallography approaches of the MERS-CoV S glycoprotein–DPP4 complex. DPP4 is a single type II TM glycoprotein with a small cytoplasmic tail in the N-terminal region and is present as a homodimeric form. DPP4 cleaves X-proline dipeptides from the N-terminal region. S glycoprotein recognizes SA species and DPP44 as the attachment and entry receptors, respectively. The MERS-CoV S1 N-terminal domain attaches to DPP4 as the host receptor [81]. The S2 C-terminal domain of MERS-CoV anchors to cellular PM to enter. MERS-CoV S glycoprotein is cleaved at a sequence between the S1 and S2 domains [96]. Another cleavage site S2′ is present in the S2 domain. MERS CoV S glycoprotein sialyl receptors are expressed in the camel nasal respiratory epithelial cells and the human lung alveolar epithelial cells, which express DPP4. Binding capacities are hindered by the SA 9-O-acetyl group or SA 5-N-glycolyl group [75].
6.3.3. CEACAM Receptor
Entry of host cells needs binding of S glycoproteins to the CEACAM receptor, forming S-protein-mediated membrane fusion. The trimeric S glycoprotein bears three S1 receptor heads. The three S1 heads of the virus bind to three receptor molecules on the host cell. Cholesterol is indirectly involved in membrane fusion through CEACAM engagement into “lipid raft” microdomains, increasing multiple S protein interaction with the receptors and triggering membrane fusion [97]. The enveloped CoV, MHV, binds to CEACAMs on cholesterol-depleted cells in BHK cell cultures. The NTD of S1 recognizes CEACAM1. For MERS-CoV, another CEACAM5 isoform is the attachment factor for virus entry [75]. The CoV S1 NTD has a similar tertiary structure to human galactose-recognizing galectins. MHV S1 NTD binds murine CEACAM1a and BCoV S1 NTD binds sugar [98,99,100]. CEACAM1a is a cell adhesion protein (CAM) and its mRNA is alternatively spliced. The cryo-EM structure of MHV S complexed with CEACAM1a was elucidated [101]. Thus, HCoVs evolutionarily combined the galectin gene of hosts into their S1 glycoprotein gene, while BCoV S1 protein is present without such gene recombination but contains the sugar-recognizing lectin capacity. MHV S1 protein also evolutionarily acquired murine CEACAM1a-recognizing activity [102]. Therefore, CoVs are under evolution to adapt their host receptor interaction to infect cross-species hosts [80,103]. On the host side, to escape the lethal pressure from CoV infections, hosts have also evolved to acquire SA-binding proteins such as siglecs to inhibit or activate the innate immune cells.
Both raft and non-raft CEACAMs are involved in the virus–cell membrane fusion event. Formation of CEACAM-associated MHV particles or CEACAM-induced MHV fusion is possible by GPI-anchored CEACAMs through the binding between CEACAM and S proteins. However, MHV can bind to both GPI- and TM-anchored CEACAMs. In addition, soluble CEACAMs also mediate S glycoprotein-driven fusion [104]. This implies that membrane anchors are not intrinsically necessary. In fact, CEACAMs are present in different tissue-specific isoforms [105]. Nevertheless, GPI-anchored CEACAMs are more effective for MHV infection than TM-anchored CEACAMs. Soluble CEACAM receptors can bind to viral S glycoproteins and induce conformational shifts to acceptable S glycoprotein-involved membrane fusions [106]. For example, soluble CEACAM forms interacts with S1 fragments [107] and alters the S1–S2 association stability [108] and S1 oxidation confirmation [109]. S proteins are structurally shifted prior to membrane fusion. For the cross-linking of viruses and cells, integral hydrophobic peptides of the S2 chain are embedded into membranes via membrane hydrophobic cholesterols.
6.3.4. Membrane-Associated 78-kDa Glucose-Regulated Protein (GRP78) or HSPA5
MERS-CoV S glycoprotein also recognizes a 78-kDa glucose–regulated protein (GRP78) or heat shock 70 kDa protein 5 (HSPA5), known as binding immunoglobulin protein (BiP) or Byun1, which is encoded by the HSPA5 gene in humans. HSP5A is a ER-resident unfolded protein response (UPR) protein. Stressed cell status such as viral infection increase expression and translocation of HSPA5 to the PM to form a membrane protein complex. GRP78 modulates MERS-CoV entry in the presence of the DPP4 as a host cell receptor. Additionally, lineage D β-CoV and bat CoV HKU9 (bCoV-HKU9) also bind to GRP78 [76]. A cell surface receptor, GRP78, was predicted to be another COVID-19 receptor as an S glycoprotein binding site [110]. The prediction was made using the combined technology of molecular modeling docking with structural bioinformatics. GRP78 or BiP is a chaperone protein located in the ER lumen [111]. Known ER-bound enzymes include activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1) and protein kinase RNA (PKR)-like ER kinase (PERK) [112]. Depending on threshold of unfolded protein accumulation, GRP78 releases IRE1, ATF6 and PERK, and is activated, resulting in translation inhibition and refolding. Stress-overexpressed GRP78 can avoid ER retention and is translocated to the membrane. GRP78 translocated to the cell PM can recognize viruses by its substrate-binding domain (SBD) for virus entry into the cell (Figure 8). In sequence and structural alignments and protein–protein docking, RBD of the CoV spike protein recognizes the GRP78 SBDβ as the host cell receptor. The predicted region III (C391–C525) and region IV (C480–C488) of the S glycoprotein and GRP78 are highly potential binding sites. Region IV is the GRP78 binding-driving force. These nine amino acid residues are being molecularly targeted for the designation and simulation of COVID-19-specific drugs. This process is the mechanism underlying the cell surface HSPA5 (GRP78) exposure and this is exploited to be used for pathogen entry. Such pathogenic entry into host cells has been observed in multiple infections including pathogenic human viruses such as human papillomavirus, Ebola virus, Zika virus and HcoVs—as well as fungal Rhizopus oryzae [113,114,115,116]. Therefore, natural products can inhibit cell-surface HSPA5 recognition of the viral S glycoprotein.
6.3.5. Aminopeptidase N (APN) is a Receptor of α-CoV HCoV-229E
Among the six HCoVs, the α-CoV HCoV-229E S protein recognizes hAPN known as CD13 or membrane alanyl aminopeptidase (EC 3.4.11.2). Porcine epidemic diarrhea coronavirus virus (PEDV) binds to protein receptor APN of human- and pig NeuAc species as its co-receptor. Apart from hAPN, TGEV and PEDV bind to SA species [117], although SA recognition by TGEV is not essential in the first step of entry cycle. HCoV-229E recognizes hAPN known as CD13 for its entry receptor. hAPN (PDB: 4FYQ) or CD13 (EC 3.4.11.2), which is a Zn-dependent metalloprotease, has a MW 150 kDa with 967 amino acids. CD13 is a type II TM protein with a short cytoplasmic domain in the N-terminal region and long extracellular region in the CTD. The CTD has a pentapeptide sequence specific for the Zinc–MMPs. The APN binding domain is located on the CTD of PEDV S1 (amino acid 477–629 residues), while the SA-binding domain is found in the N-terminal region of PEDV S1 (amino acid 1–320 residues) [118]. CD13 is also a receptor for HCoV-229E, human cytomegalovirus, porcine CoV TGEV, feline infectious peritonitis virus (FIPV), feline enteric virus (FeCV) and canine-infectious CoVs [119,120,121,122]. Homodimeric CD13 digests luminal peptides. The hAPN-encoding ANPEP gene is a dominant component in proximal tubular epithelial cells, small intestinal cells, macrophages, granulocytes and synaptic membranes. If this gene is defective, leukemia or lymphoma are transformed [123]. Porcine and human APN exhibit about 80% protein identity. FIPV and FeCV are in the same group as HCoV-229E and TGEV. Thus, porcine APN is also an attachment site for pig TGEV with an additional second receptor. HCoV-229E first binds to CD13 and consequently clusters CD13 in caveolae-associated lipid rafts [120].
6.3.6. Heparan Sulfate (HS) is the HCoV-NL63 Attachment Site
For glycan interaction, HCoV-NL63 and MHV utilize heparan sulfate proteoglycans (HSPGs) as attachment enhancers [87,124]. Viruses recognize HSPGs as attachment molecules. In the spike (S) protein-deficient virions, the M protein recognizes HSPG. The S proteins generally bind to the viral cellular receptor. However, the M protein also acts as a receptor in the early step of HCoV-NL63 infection. The M membrane protein of HCoV-NL63 recognizes the attachment site of HSPGs. HCoV-NL63 M protein binds to HSPG for the initial attachment of virus to host cells and thereafter, the M and S proteins cooperate for virus entrance into the host cells [125]. HSPGs are glycosaminoglycan (GAG)-carrying proteins frequently used as a secondary receptor for viral entry. HSPGs are composed of covalent-bonded HS chains as a GAG form. The HS GAG linkage structure of tetrasaccharide exhibits GluAβ1,3GlcNAcα1,4Galβ1,3Galβ1,4Xylβ-O-serine. Glycosyltransferases involved in HS GAG synthesis include GlcAT-II (glucuronosyltransferase) and GlcNAcT-II (N-acetylglucosaminyltransferase II) for heparan sulfate synthesis (Figure 9). GAG is used as docking sites for virus interaction with the host cell surface. GAGs contain negatively charged N- and O-sulfated sugars [126]. The biosynthetic pathway and biologic roles in early embryogenic morphogenesis and vulval morphogenesis of HS and chondroitin sulfate GAG have been elucidated in Caenorhabditis elegans [127]. The negative charges mediate the interaction of GAGs and their ligands through electrostatic forces. Interaction of HSPG with ligands potentiates many virus infectious cycles. For examples, adeno-associated virus, human T cell lymphotropic virus type 1, human papilloma virus 16, herpes viruses, hepatitis B and C viruses, Kaposi’s sarcoma-associated herpesvirus, human papilloma viruses and Merkel cell polyoma virus recognize the HSPGs [128,129]. HSPGs increase virulence upon interaction with viral factors required for viral attachment and replication.
Structure of glycosaminoglycan (GAG)-linkage tetrasaccharide, GluAβ1,3Galβ1,3Galβ1,4Xylβ-O-serine and HS; (A) HS structure; (B) Sulfation of sugar residues; (C) Synthesis and localization of HS in host cells. Glycosyltransferases involved in GAG synthesis include (i) GlcAT-II (glucuronosyltransferase) and (ii) GlcNAcT-II (N-acetylglucosaminyltransferase II) for heparan sulfate synthesis. β-D-glucuronic acid (GlcA), α-L-iduronic acid (IdoA) and 2-O-sulfo-α-L-iduronic acid (IdoA(2S) are composed [124].
6.3.7. Major Histocompatibility Complex Class I (MHC-I) C is an Attachment Site for HCoV-HKU1
Although HCoV-HKU1 utilizes O-acetyl-SAs as attachment sites, the HCoV-HKU1 S protein also interacts with MHC-I C (HLA-C) as an additional attachment molecule [85].
6.3.8. DC-SIGN (CD209) is a Binding Candidate for SARS-CoV Entry
SARS-CoV uses the C-type lectins of DC-SIGN and DC-L-SIGN as additional or secondary receptors. Glycans on the S glycoprotein are recognized by DC/L-SIGN for virus attachment and entry. Seven glycosylation sites of the S glycoprotein have been found to be essential for DC/L-SIGN-driven virus entry [86,130].
6.3.9. Tetraspanin CD9 is a Surface factor for MERS-CoV Entry Via Scaffold Cell Receptors and Proteases
Tetraspanin CD9, but not tetraspanin CD81, associates with DPP4 and the type II TM serine protease (TTSP) member TMPRSS2, a CoV-activating protease, to form a cell surface complex [131]. This CD9–DPP4–TMPRSS2 complex permits MERS-CoV pseudovirus entrance into the host cells. The tetraspanins have four TM spanning regions linked by one large and one small loop in the extracellular region. Tetraspanins form virus entry baselines and open CoV entry routes. To help viral entry into host cells, MERS-CoV S interacts with DPP4 receptors via the RBD. Receptor involvement causes cleavage using proteases such as the previously described TMPRSS2. Association of tetraspanin CD9 with the DPP4–TMPRSS2 complex triggers the S glycoprotein. MERS-CoVs enter the cells via endocytosis and cathepsins cleave the S proteins [132].
6.4. Effects of Receptor and Ligand S Glycosylation on Virus–Host Interaction
SAs are predominant surface determinants for pathogen attachment, adherence and entry to host cells. Eleven representative vertebrate virus families utilize SAs as initial entry receptors or as attachment factors. Interaction of virus with SA-containing glycans is complex because virus SA-binding lectins are inherently of very low affinity. Viruses acquire enzymes to catalyze virion elution by regional depletion of binding receptors [56]. TM S glycoprotein recognizes oligosaccharide receptors. Using cryo-EM technology and observed structures of S glycoprotein trimers of CoV OC43 complexed with 9-O-acetylated SA, S glycoprotein was demonstrated to mediate virus adhesion and entry to host cells. All CoV S proteins show conservation in binding to 9-O-acetyl-SAs. MERS-CoV also recognizes 9-carbon sugar SA species. MERS-CoV S-1A binds to SA species. For example, SAα2,3- over SAα2,6-linkages expressed in human erythrocytes and mucins are preferentially targeted by MERS-CoV S-1A. Binding is hence blocked by SA modification to 5-N-NeuGc and 7, 9-O-NeuAc species [73]. For example, impairment of ACE2 receptor glycosylation does not influence S-glycoprotein-ACE2 interaction, however, SARS-CoV-2 virus entry into respiratory epithelial host cells was downregulated [133]. Changes in ACE2 N-glycans do not apparently influence interaction with the SARS-CoV S glycoprotein, but instead, impair viral S glycoprotein-mediated membrane fusion. The receptor glycan structures decide the entry of some human viruses. Changes in ACE2 receptor sialylation influences interaction affinity between virus ligands and host receptor. Inter-species or individual genetic variations such as drift and mutation may occur in SARS-CoVs. This explains currently emerging differences in CoV responses within the same population such as humans.
On the other hand, from the aspect of virus ligand, the S glycoprotein decorates viral surfaces and is, therefore, the target for vaccination design. Virus internalization requires potential glycosylation of viral glycoproteins. Among the three viral envelope components, S and M are the major glycoproteins and E is nascent and not glycosylated. The M glycoprotein consists of a short glycosylated ectodomain in the N-terminal region. The S glycoprotein expressed in hemagglutinating encephalomyelitis virus is an HA that recognizes N-acetyl-9-O-NeuAc as a binding receptor expressed on erythrocyte surfaces [134]. For example, BCoVs attach to the surface receptor of N-acetyl-9-O-NeuAc (9-O-acetylated SAs) on host cells. TGEV and PEDV are currently known as a similar class of such CoVs. PEDV infects multiple hosts including bat, pig, human and monkey, where bats are considered to be the evolutionary origin for PEDV. The S glycoprotein of SARS-CoV-2 utilizes different glycosylation patterns to recognize its receptors. The glycosylation sites in minimal RBD exhibits similar sites to other CoVs. The trimeric SARS-CoV-2 S glycoprotein is also highly glycosylated with 66 N-glycans, but a few O-glycans [135]. Glycosylation of S glycoproteins leads to immune evasion. In the MERS-CoV and the bat-specific CoV-HKU4, glycosylation is linked to zoonotic infection for fusion-based entry [136].
7. Pharmacology of Glycan-Related Anti-SARS-CoV-2 Agents
The emerging CoV-pandemic requires therapeutic agents to block the recognition, binding, replication, amplification and propagation of the CoV in humans. Protease inhibitors, RNA synthase inhibitors and S2 inhibitors are potential targets, and several agents are currently being evaluated. Efficient therapeutic drugs are the most reliable option for patients. The first attachment step of the viral amplification cycle is initiated on the respiratory cell surfaces, driven by the viral S protein. This is a potential therapeutic target. Soon after the SARS-CoV-2 outbreak initiated, the CoV S glycoprotein was demonstrated to recognize ACE2 as a binding receptor on human cells. Human TMPRSS2 enzyme influences the CoV S glycoprotein activation, to facilitate virus infection. ACE2 binding and TMPRSS2 activation facilitate the CoVs to attach to human host cells. Mouse, nonhuman primate and human cells have been analyzed using single-cell RNA new generation sequencing (NGS). For example, for human infection, CoVs can enter nasal goblet secretory cells, because these cells express the proteins required for SARS-CoV-2 infection. In the lungs, the proteins are stored in the alveoli like air sacs of type II pneumocytes. In the intestine, the two proteins are expressed in entero-epithelial cells. ACE2 gene expression correlates with the IFN-related genes [137]. ACE2 helps lung cells to tolerate cellular damage. Therefore, CoVs may evolutionally take advantage of the defense mechanisms of host cells, hijacking such host-borne proteins.
In SARS-CoV-2, the ACE2 receptor is an attachment, entry and infection receptor into the cell, when the S glycoprotein is cleaved by a specific serine protease. SARS-CoV-2 infection is regulated by glycosylated SARS-CoV-2 viral particles and glycosylated ACE2 in the lung epithelial cells. RBD of the CoV S glycoprotein recognizes ACE2 [82]. Amino acid residues 442, 472, 479, 480 and 487 located on the receptor-binding motif (RBM) of the S glycoprotein RBD recognize human ACE2 [138]. Trimeric viral S glycoprotein is glycosylated and cleaved by a protease, furin, into two subunits, S1 and S2. Subunit S1 is further cleaved into the SA and SB domains and the SB domain recognizes human ACE2. The N-glycosylated S2 subunit is involved in virus-ACE2 complex formation [139]. Therefore, the glycosylated ACE2 receptor is a key molecule for virus binding and fusion.
Plasma sera prepared from infected patients is an alternative medication. The WHO has suggested this trial since the 2014 Ebola epidemic and 2015 MERS-CoV outbreak. In addition, Mab therapy is another option. For example, LCA50 Mab mimics produced by modification of plasma antibodies isolated from MERS-CoV patients was valuable [140]. Low molecular molecules are being examined for anti-virus activities from alkaloids, glycan derivatives and terpenoids. Recently, anti-CoV drugs are being approached using molecular modeling, docking and simulation methods. Computation-assisted drugs via molecular modeling and docking toward drug targets are applied as anti-viral compounds against CoVs. They target human ACE2, PLpro (PDB: 3e9s), the CoV main proteinase (PDB: 6Y84), 3-chymotrypsin-like (3C-like protease; 3CLpro), RdRp, helicase, N7 methyltransferase, human DDP4, RBD, protease cathepsin L, type II TM Ser protease or TMPRSS2. CoV 3CLpro (PDB: 6WX4) and the PLpro cleave the polyproteins to assemble virus proteins. For newborn RNA genomes, RdRp is used as a replicase for the complementary RNA strand synthesis, which uses the virus RNA template.
7.1. N-Glycosylation Inhibition by Chloroquine (CLQ) and Hydroxychloroquine (CLQ-OH)
CLQ and CLQ-OH are under investigation worldwide to treat COVID-19 (Figure 10). CLQ and its derivative CLQ-OH block CoV replication, amplification and spread in in vitro culture via inhibition of ACE2 receptor glycosylation. In HCoVs, interaction of the S glycoprotein with gangliosides initially occur as the first entry step during the replication cycle of the virus. CLQ and CLQ-OH have been alternative drugs for RA and several autoimmune diseases for 70 years, although they are anti-malaria prophylaxis drugs. CLQ-OH is an aminoquinoline with less toxicity than CLQ. CLQ-OH bears an N-hydroxyethyl side chain, which increases its solubility compared to CLQ [141]. CLQ-OH modulates activated immune cells via downregulation of TLR signaling and IL-6 production [142]. Clinical trials are also under consideration for the efficacy and safety of these drugs. Regarding the action mechanism(s), CLQ and CLQ-OH-mediated inhibition of ACE2 terminal glycosylation was considered. In in vitro Vero E6 cells, CLQ significantly inhibits SARS-CoV spread by interfering with ACE2 function, acting at the entry and post-entry steps of SARS-CoV-2 replication and infection. The binding affinity of ACE2 to S glycoprotein is simulated to be lowered by treatment with CLQ-OH or CLQ. CLQ may modify the binding affinity between ACE2 and S glycoprotein by alterations in ACE2 glycosylation or modification. CLQ-OH (EC50 0.72 μM) and CLQ (EC50, 5.47 μM) inhibit SARS-CoV-2 [143].
Using computer simulation techniques, CLQ and CLQ-OH have been suggested to recognize the enzymatic active site of the UDP-GlcNAc 2-epimerase, known as an essential enzyme in SA biosynthesis [144], blocking the sialylation of host cells. The mechanism underlying the glycosylation inhibition may support the antiviral properties of CLQ and CLQ-OH through interactions of CLQ or CLQ-OH with NDP-saccharide mutases or glycosyltransferases [145]. CLQ was reported to inhibit quinone reductase 2 [146], known as a catalytic mimetic or structural neighbor of UDP-GlcNAc 2-epimerases [147,148]. If CLQ or CLQ-OH inhibits SA synthesis, the inhibitory properties may support the antiviral activity of CLQ or CLQ-OH against SARS-CoVs because the SARS-CoV receptor ACE2 contains SA species. In fact, CLQ exhibits in vitro anti-SARS-CoV-1 activity via defective glycosylation of viral ACE2 in Vero cells [149]. In addition. the interference of CLQ or CLQ-OH with SA synthesis may broadly be applicable as an antiviral because the HcoVs or other orthomyxoviruses also utilize SAs as entry molecules [150]. However, the detailed mechanisms should be further elucidated. The CLQ treatment efficacy in Covid-19 patients has, however, not been conclusively determined.
7.2. Interaction of Membrane Gangliosides in Lipid Rafts with CLQ and CLQ-OH
Lipid rafts are also viral attachment sites. Viruses such as IBV, dengue virus, Ebola virus, hepatitis C virus, HIV, human herpes virus 6, measles virus, Newcastle disease virus, poliovirus, West Nile virus, foot-and-mouth disease virus, simian virus 40, rotavirus, influenza virus and Marburg virus also use lipid rafts for virus entry [151,152,153,154,155,156]. In avian CoV IBV, structural proteins of the IBV virus are co-localized with PM lipid rafts embedded with the ganglioside GM1. HCoV-229E entry is prevented by cholesterol depleted conditions because HCoV-229E clusters in caveolae-associated lipid rafts [157]. Caveolae of caveolin-1, -2 and -3 are cross-linked [158] and control the molecular distribution between rafts and caveolae in a regulatory mechanism. S protein-CD13 cross-linking occurs via CD13-caveolin-1 sequestering. HCoV-229E particles similarly exhibit a longitudinal distribution property. HCoV-229E-colocalized caveolin-1 undergoes the next step of virus infection. Caveolin-1 knockdown inhibited HCoV-229E endocytosis and entry and thus caveolin-1 is essential for HCoV-229E infection. TGEV also endocytoses by a clathrin-mediated mechanism in MDCK cells [159]. Other viruses including HCoV-OC43 also use an entry receptor sequestered to cross-linked caveolae [160]. In SARS-CoV, the first entry step to host cells needs ACE2 in intact lipid rafts by the S glycoprotein [151]. ACE2 is associated with caveolin-1 and GM1 in membrane rafts depending on its cell-type specific localization [161]. Raft integrity with cholesterol and ACE2 is necessary for SARS-CoV pseudovirus entry into Vero E6 cells and for SARS-CoV-microdomain-based entry. C-type lectin, CD209 L (L-SIGN), can also form lipid rafts and acts as a SARS-CoV receptor [162]. Information of the CoV entry pathways is important for therapeutic designation of SARS-CoV-targeting drugs, for example, if agents disrupt lipid-raft localization of the ACE2 receptor.
CLQ binds the SAs and gangliosides in lipid rafts with a high affinity. Therefore, CLQ or CLQ-OH prevents the S glycoprotein–ganglioside binding. CLQ (or CLQ-OH) binding to SA consequently prevents S glycoprotein binding to host receptors. The N-terminal region of SARS-CoV-2 S glycoprotein interacts with gangliosides. A ganglioside-binding site (GBS) or ganglioside-binding domain (GBD) is present in the NTD of the S glycoprotein of SARS-CoV-2. Using molecular modeling and simulation technology, CLQ has been suggested to recognize the SAs and gangliosides. Human type Neu5Ac binds to CLQ and CLQ-OH. Thus, SAs are binding targets of CLQ and CLQ-OH. CLQ and CLQ-OH have two specific recognition sites in the polar sugar residues of ganglioside GM1. The first site is found at the tip of the sugar residues of GM1 with an interaction energy of −47 kJ/mol. The CLQ rings face the GalNAc residue of GM1, while the second site is in a large region of the sugar-ceramide junction and the sugar residues. Several amino acid residues of the S protein NTD, which are Phe-135, Asn-137 and Arg-158, recognize the ganglioside GM1. The S glycoprotein NTD-GM1 complex is suggested to form a trimolecular complex with two molecules of ganglioside GM1 anchored to the NTD of S protein [163]. The ACE2-binding RBD is suggested to be a potential GBS located on a differential site of the S glycoprotein NTD. The protein sequence interfacing surface of the NTD is the consensus GBDs [164]. The amino acids Gly, Pro and/or Ser residues found in GBD motifs are in the same 111–158 amino acids of the NTD as the ganglioside-attachment interface. The GBD is conserved throughout viral isolates from worldwide COVID-19 patients. The GBD potentially increases viral attachment ability to PM lipid rafts and contact between host ACE-2 and S protein [165]. The interaction between CLQ-OH and 9-O-acetyl-NeuAc is also similar to the 9-O-acetyl-NeuAc-CLQ interaction. The CLQ-OH OH group enhances the interaction of CLQ with SA via a hydrogen bond [163]. In conditions with CLQ or CLQ-OH derivative treatment, the S glycoprotein cannot bind to gangliosides in in silico studies, which are used to uncover the action mechanism. CLQ and CLQ-OH prevent the binding of S glycoprotein to gangliosides. The CLQ-SA complex is formed in a mixed surface and balls by the positioning of the negative charged COOH group of Neu5Ac and one of the two cationic charges of CLQ [163]. CoVs preferentially bind to 9-O-acetyl-NeuAc [60], differentiating with other viral properties.
As CLQ interacts with the GM1 sugar part, the N-terminal domain of the S protein loses viral attachment capacity to the cell receptors [166]. The S protein NTD and the CLQ/CLQ-OH maintain the same position during GM1 binding, consequently preventing GM1 binding to the S protein and the drug at the same time, because the NTD and the CLQ/CLQ-OH simultaneously recognize GM1. Asn-167 forms a hydrogen bond with the GalNAc residue, whereas an aromatic Phe-135 stacks to the Glc residue of GM1. Therefore, the antiviral activities of CLQ and CLQ-OH is to block the interaction between the SARS-CoV-2 S glycoprotein and gangliosides on host cell surfaces. The lipid composition of host cell PM can also be a potential target for preventive and therapeutic drugs against such viruses.
8. Conclusions
The SARS-CoV-2-caused COVID-19 pandemic is a global public health issue in the 21st century. In order to coordinate efforts against and mitigate the public health consequences of the spreading and outbreak of the disease, the international community has been exchanging independent information and knowledge. The scientists and epidemiologists exchange COVID-19 information to highlight interdisciplinary approaches. The pandemic outbreak issue has raised interest in the pathology and epidemiology of the disease. The current COVID-19 pandemic resulted in establishment of the COVID Action Platform of the World Economic Forum (WEF) to perform evidence-based cutting-edge research and analyze the fast-evolving pandemic. The Virus Outbreak Data Network (VODAN) of the GoFair Data Alliance also pursues to apply the best remedy against the pandemic infection. In the aspect of basic biology, most β-CoVs recognize 9-O-acetyl SAs, but certain viruses have switched to binding 4-O-acetyl SA. Originally, HE is found in other viruses such as toroviruses and orthomyxoviruses (influenza C/D and isavirus). The exceptional β-CoV lineage A is the only one to bear HE among the CoVs. Virus entry and replication inhibitors need to be urgently developed. Computation-fused artificial intelligence accelerates therapeutic agent development. The SARS-CoV-2 genome shows 80% similarity to the previous SARS-CoV and SARS-targeting agents can be commonly treated to the related SARS-CoV-2 patients. For better understanding of the entry pathway of SARS-CoV-2, the importance of the carbohydrates including SAs on cell and virus surfaces is again emphasized.
Acknowledgments
This study was in part supported by a grant (NRF-2018R1D1A1A09081927 to CHK) of the Basic Science Research Program through National Research Foundation (NRF).
Abbreviations
| HE | hemagglutinin-esterase |
| COVID-19 | coronavirus disease 2019 |
| SA | sialic acid |
| SARS-CoV | severe acute respiratory syndrome-related coronavirus |
| MERS-V | Middle East respiratory syndrome virus |
| RSV | respiratory syncytial virus |
| WHO | World Health Organization |
| 2019-nCoV | 2019-Novel Coronavirus |
| GIT | gastrointestinal track |
| RDE | receptor-destroying enzyme |
| LRI | ligand–receptor interaction |
| LCI | lectin–carbohydrate interaction |
| LGI | lectin–glycan interaction |
| LSI | lectin–sphingolipid interaction |
| PGI | protein–glycan interaction |
| PCI | protein–carbohydrate interaction |
| PPI | protein–protein interaction |
| NeuC | neuraminic acid |
| NeuAc | N-acetyl neuraminic acid |
| NeuGc | N-glycolyl neuraminic acid |
| ST | sialyltransferase |
| ICTV | International Committee on Taxonomy of Viruses |
| HCoV | human CoV |
| ORF | open reading frame |
| RdRp | RNA-dependent RNA polymerase |
| E | envelope |
| N | nucleocapsid |
| S | spike glycoprotein |
| M | membrane matrix glycoprotein |
| ER | endoplasmic reticulum |
| ERGIC | endoplasmic reticulum-Golgi intermediate compartment |
| Nsp | nonstructural protein |
| RTC | replicase–transcriptase complex |
| TM | transmembrane |
| ADRP | ADP-ribose-1′-phosphatase |
| PLpro | papain-like protease |
| MTase | methyltransferase |
| NTD | N-terminal domain |
| CTD | C-terminal domain |
| RBD | receptor-binding domain |
| ssRNA | single stranded RNA |
| GSK3 | glycogen synthase kinase 3 |
| hnRNP A1 | heterogeneous nuclear ribonucleoprotein A1 |
| VLP | virus-like particle |
| NA | neuraminidase |
| NDV | Newcastle disease |
| Neu5Ac | N-acetylneuraminic acid |
| Neu5:9Ac2 | 9-O-acetyl-N-acetylneuraminic acid |
| CasD1 | Cas1 domain containing 1 |
| ISAV | infectious salmon anemia virus |
| FH | factor H |
| Neu5Ac9NAc | 9-acetamido-9-deoxy-N-acetylneuraminic acid |
| HEF | HE fusion protein |
| Neu5:9Ac2 | 5-N-acetyl-9-O-NeuAc |
| BCoV | bovine CoV |
| RBC | red blood cell |
| CRD | carbohydrate-recognizing domain |
| GBD | glycan-binding domain |
| MHV | murine hepatitis virus |
| CEACAM | carcinoembryonic antigen cell adhesion molecule 1a |
| 9-O–AC-SA | 9-O-acetylated SA |
| PHEV | porcine hemagglutinating encephalomyelitis virus |
| RCoV | rat CoV |
| TGEV | transmissible gastroenteritis coronavirus |
| IBV | infectious bronchitis virus |
| pAPN | porcine aminopeptidase N |
| MGP | mucin-like glycoprotein |
| pNPA | p-nitrophenyl acetate |
| SLeX | sialyl-LewisX |
| α2:3-SlacNAc | α2,3-sialyl-N-acetyl-lactosamine |
| DPP4 | dipeptidyl peptidase 4 |
| GRP78 | membrane-associated 78-kDa glucose-regulated protein |
| ACE2 | angiotensin-converting enzyme 2 |
| hAPN | human APN |
| MHC-I | major histocompatibility complex class I |
| DC-SIGN | dendritic cells-specific intercellular adhesion molecule-3-grabbing nonintegrin |
| HSPG | heparan sulfate proteoglycan |
| HSPA5 | heat shock protein A5 |
| RAS | renin-angiotensin system |
| Ang I | angiotensin I |
| AT1R | Ang II receptor type 1 |
| GPCR | G protein-coupled receptor |
| ADAM | A disintegrin and metallopeptidase domain |
| TACE | TNF-α-converting enzyme |
| TMPRSS | transmembrane protease serine |
| TTSP | type II TM Ser protease |
| PAR-2 | protease activated receptor 2 |
| MMP | matrix metalloproteinase |
| HGF | hepatocyte growth factor |
| HAT | human airway trypsin-like protease |
| MSPL | serine 13 |
| BHH | bromhexine hydrochloride |
| DPP4 | dipeptidyl peptidase-4 |
| BiP | immunoglobulin protein |
| UPR | unfolded protein response |
| ATF6 | activating transcription factor 6 |
| IRE1 | inositol-requiring enzyme 1 |
| PERK | protein kinase RNA (PKR)-like ER kinase |
| SBD | substrate-binding domain |
| PEDV | porcine epidemic diarrhea coronavirus virus |
| FIPV | feline infectious peritonitis virus |
| FeCV | feline enteric virus |
| GAG | glycosaminoglycan |
| GlcAT-II | glucuronosyltransferase |
| GlcNAcT-II | N-acetylglucosaminyltransferase II |
| NGS | new generation sequencing |
| RBM | receptor-binding motif |
| 3CLpro | 3-chymotrypsin-like protease |
| CLQ | chloroquine |
| CLQ-OH | hydroxychloroquine |
| GBS | ganglioside-binding site |
| GBD | ganglioside-binding domain |
| WEF | World Economic Forum |
| VODAN | Virus Outbreak Data Network |
Author Contributions
Conceptualization, C.-H.K.; writing—original draft preparation, C.-H.K.; writing, review and editing, C.-H.K. All authors have read and agreed to the published version of the manuscript.
References
Articles from International Journal of Molecular Sciences are provided here courtesy of Multidisciplinary Digital Publishing Institute (MDPI)
https://www.pnas.org/content/105/26/9065
Edited by Ralph S. Baric, University of North Carolina, Chapel Hill, NC, and accepted by the Editorial Board April 11, 2008
↵ †Q.Z. and M.A.L. contributed equally to this work. (received for review January 18, 2008)
Abstract
The hemagglutinin-esterases (HEs) are a family of viral envelope glycoproteins that mediate reversible attachment to O-acetylated sialic acids by acting both as lectins and as receptor-destroying enzymes (RDEs). Related HEs occur in influenza C, toro-, and coronaviruses, apparently as a result of relatively recent lateral gene transfer events. Here, we report the crystal structure of a coronavirus (CoV) HE in complex with its receptor. We show that CoV HE arose from an influenza C-like HE fusion protein (HEF). In the process, HE was transformed from a trimer into a dimer, whereas remnants of the fusion domain were adapted to establish novel monomer–monomer contacts. Whereas the structural design of the RDE-acetylesterase domain remained unaltered, the HE receptor-binding domain underwent remodeling to such extent that the ligand is now bound in opposite orientation. This is surprising, because the architecture of the HEF site was preserved in influenza A HA over a much larger evolutionary distance, a switch in receptor specificity and extensive antigenic variation notwithstanding. Apparently, HA and HEF are under more stringent selective constraints than HE, limiting their exploration of alternative binding-site topologies. We attribute the plasticity of the CoV HE receptor-binding site to evolutionary flexibility conferred by functional redundancy between HE and its companion spike protein S. Our findings offer unique insights into the structural and functional consequences of independent protein evolution after interviral gene exchange and open potential avenues to broad-spectrum antiviral drug design.
Many different respiratory and enteric viruses initiate infection by binding to sialic acids. These nine-carbon amino sugars are common terminal residues of glycoconjugates and occur in a wide variety, which arises from differential modification of the parental molecule, 5-N-acetyl neuraminic acid (Neu5Ac), and from differences in glycosidic linkage (1). Although viruses may attain host and cell specificity by being selective for particular sialic acid subtypes (1–4), even these receptors may still occur abundantly on off-target host cells (including the infected cell), on viral progeny, and on soluble and insoluble molecules in the extracellular environment. To prevent irreversible binding to “decoy receptors” and consequent loss of infectivity, orthomyxo-, paramyxo-, and coronaviruses encode virion-associated receptor-destroying enzymes (RDEs) (5–7). In turn, RDEs make prime targets for antiviral intervention, as exemplified by the influenza A virus neuraminidase (8–10).
The coronaviruses are a group of enveloped (+)strand RNA viruses that comprises several important pathogens of human clinical and veterinary importance, including severe acute respiratory syndrome (SARS)-coronavirus, human respiratory coronavirus (HCoV) OC43 and its proposed zoonotic ancestor (11), bovine coronavirus (BCoV). HCoV-OC43 and BCoV use 9-O-acetylated sialic acids as receptor and correspondingly possess sialate-9-O-acetylesterases as RDE (12, 13). Enzymatic activity is associated with the hemagglutinin-esterase (HE), a homodimeric class I membrane protein (for a review, see ref. 7). Although among coronaviruses, HE is exclusive to members of phylogenetic subgroup 2a (7), closely related proteins do occur in toroviruses (14–16) and in orthomyxoviruses of mammals (17) and fish (18). Best studied is the influenza C virus HE fusion protein (HEF), a homotrimeric multifunctional spike protein that mediates virion attachment, receptor destruction, and membrane fusion (19–22).
The presence of homologous HE genes in the genomes of viruses from three distinct taxa is generally ascribed to RNA recombination and would have required two lateral gene transfer events at the minimum (7, 14, 23). The source of the HE module and the direction of gene flow remain to be established, however (7). Moreover, the influenza, toro-, and coronavirus HEs followed independent evolutionary trajectories, the functional and structural consequences of which are poorly understood. Unlike HEF, CoV HEs lack membrane fusion activity and, in the virion, they are accessory to the spike protein S, a receptor-binding fusion protein. Furthermore, although CoV HEs do bind to sialic acid, their role in virion attachment is subject to debate (7). Reportedly, CoV HEs serve primarily as RDE (24), but this view is at variance with observations that esterase-deficient HE promotes viral spread in vivo (25). Intriguingly, there is no obvious sequence homology between the HEF receptor-binding site and corresponding regions in HE, raising the question of whether CoV HEs possess a separate receptor-binding site or mediate sialic acid-binding and catalysis through a single site in the esterase pocket (7).
To further our understanding of the function and evolution of coronavirus HEs, we expressed BCoV HE and solved its structure, free and in complex with its receptor.
Results and Discussion
Expression, Purification, and Biochemical Analysis of CoV HE.
We produced the ectodomain of BCoV HE (residues 19–388) in enzymatically active (HE) and inactive form (HE0, containing an active site Ser40-to-Ala substitution) as Fc-fusion proteins by transiently transfecting N-acetylglucosaminyltransferase-deficient HEK293S cells with expression plasmids encoding the respective molecules. Soluble CoV HE was purified by protein A-affinity chromatography of the Fc chimeras followed by on-the-bead thrombin digestion. The proteins were tested in a series of assays to confirm that biological functions had been retained also as an indication for proper folding. Indeed, in cleavage assays with a synthetic di-O-acetylated Sia (5-N-acetyl-4,9-di-O-acetylneuraminic acid α-methylglycoside, αNeu4,5,9Ac32Me) as substrate, HE-Fc displayed sialate-9-O-acetylesterase activity (Fig. 1 A). In addition, HE-Fc, but not its esterase-deficient derivative HE0-Fc, specifically destroyed BCoV receptors on rat erythrocytes and on Madin–Darby bovine kidney (MDBK) cells (Fig. 1 B and C). Moreover, HE-Fc and HE0-Fc retained lectin activity as demonstrated by hemagglutination assay (Figs. 1 C and 4 E) and by Sia-specific immunofluorescence staining of tissue culture cells (Fig. 1 D).
BCoV HE-Fc chimeras retain esterase and lectin activity. (A) HE-Fc displays sialate-9-O-acetylesterase activity. Purified HE-Fc was assayed for substrate specificity as described (16) with the synthetic di-O-acetylated sialic acid analogue αNeu4,5,9Ac32Me as substrate. Graphs show total ion current gas chromatograms. Sialic acids were identified by mass spectrometry: αNeu4,5,9Ac32Me (peak 1), αNeu4,5Ac22Me (peak 2); peaks 1′ and 3 represent nonsialic acid compounds. (B) HE-Fc, but not esterase-deficient HE0-Fc, destroys BCoV receptors on the surface of tissue culture cells. MDBK cell monolayers were mock-treated, treated with neuraminidase (NA), or with the HE-Fc chimeras and subsequently infected with BCoV. Infected cells were visualized by an in situ acetylesterase staining. (C) HE-Fc destroys BCoV receptors on rat erythrocytes. Erythrocytes were mock-treated or treated with HE-Fc and subsequently subjected to hemagglutination assay with two-fold dilutions of BCoV (+) or with PBS (−). (D) HE0-Fc lectin activity allows immunohistochemical detection of 9-O-acetylated sialic acid on monolayers of polarized MDBK cells (green). Cell contacts were visualized by staining for PAN-Cadherin (red) and nuclei by staining with HOECHST 33258 (blue).
Structure Determination and Overall Structure.
Crystals of free HE and of a complex of HE0 with αNeu4,5,9Ac32Me diffracted to 2.1 and 1.8 Å resolution, respectively. The structures were solved by molecular replacement by using influenza C HEF residues 35–410 as search model (for detailed crystallographic statistics, see Table 1).
Data collection and refinement statistics
The data reveal that the CoV HE monomer is composed of three modules, each with an equivalent in influenza C HEF subunit 1 (HEF1): a receptor-binding domain (R), an acetylesterase domain (E), and a membrane-proximal domain (MP; Fig. 2 A–C). There is no equivalent of HEF subunit 2 (HEF2), which in HEF comprises the bulk of the fusion domain (F) (Fig. 2 B and C). Bound to the R domain is a well defined ligand molecule [supporting information (SI) Fig. S1] providing first and direct evidence for the presence of a genuine receptor-binding site. This observation unambiguously defines BCoV HE as a receptor-binding protein per se.
Structure of HE and comparison to HEF. (A) Ribbon representation of the BCoV HE dimer. One HE monomer is colored gray, the other by domain: lectin domain (R, blue) with bound αNeu4,5,9Ac32Me (cyan sticks) and potassium ion (magenta sphere); esterase domain (E, green) with Ser-His-Asp active site triad (magenta sticks); membrane-proximal domain (MP, red). (B) Structure of the influenza C HEF trimer. One monomer is colored by domain with domains R and E color-coded as in a; the fusion domain F is shown in red. Remaining monomers are shown in pink or gray. (C) Linear order of the sequence segments in HE and HEF color-coded by domains as in a and b. Gray segments indicate the transmembrane domain in HE and the fusion peptide in HEF. The arrowhead indicates the HEF cleavage site. (D) Top views showing the arrangement of the lectin domains in the BCoV HE homodimer (Left) and the influenza C HEF homotrimer (Right). The four-stranded β-sheets forming a continuous eight-stranded β-sheet in the HE dimer are emphasized by darker coloring.
In the virion, CoV HE forms dimers that are disulphide-linked through Cys385 near the C-terminal membrane anchor (7). This bond, although present in HE crystals (Fig. S2), was not visible in electron density maps because of disorder of residues 377–388. The biological dimer could, however, be identified uniquely from the proximity of C termini and size of interaction surface. It consists of identical molecules, related by twofold crystallographic symmetry, with an interface comprised of two major protein contact regions (CRs). CR1, associated with 952 Å2 buried solvent-accessible surface, involves a continuous eight-stranded β-sheet bridging the two R domains (Fig. 2 D). CR2 buries 1,182 Å2 solvent-accessible surface and involves MP.
Origin and Evolution of CoV HE.
The structure of MP provides a decisive clue to the origin of CoV HE. As shown in Fig. 3, MP is strikingly similar to the F1/F2 globular region of HEF1 (22) and the F′ subdomain in the influenza A virus HA (26). In the native metastable forms of HEF and HA, these subdomains pack against a heptad-repeat loop that, as a prelude to membrane fusion, refolds into an α-helix at endosomal pH (26). HEF and HA are evolutionarily related but diverged long ago; their fusion domains are similar in structure, as are their R domains, but overall sequence identity has dwindled to only 12% (22). In contrast, HEF1 and CoV HE share 30% identity, indicative of a more recent split-off (7, 23). The presence in HE of an element that already must have been part of a fusion protein ancestral to HA and HEF (22) thus indicates that CoV HE originates from an HEF-like fusion protein and strongly argues against evolutionary models in which orthomyxo- and coronaviruses independently acquired an HE module solely comprised of R and E (7). In the simplest scenario (23), a group 2a CoV progenitor obtained through recombination the 5′-end of an HEF gene, including the coding sequence for the fusion peptide that came to serve as transmembrane domain (Fig. 2 C). Without the HEF2 helical subdomain to direct trimerization, fortuitous interactions between transmembrane and/or F2 segments might have caused the HE protein to assemble into dimers instead; of note, the face of MP involved in dimer formation is equivalent to that of HEF segment F2 contacting the heptad repeat loop (Fig. 3). A four-stranded β-sheet in the R domain that remains unpaired in HEF and HA trimers (22) may have been remodeled subsequently to create a second, membrane-distal intermonomer contact to increase dimer stability (Fig. 2 D).
MP is a remnant of a fusion domain. (A) Ribbon representations of MP in the HE dimer and of the fusion domains of the HEF and HA monomers. MP and corresponding subdomains in HEF and HA are shown in red; the remainder of the HEF and HA fusion domains are shown in gray. A conserved disulfide bond is shown in yellow stick representation. (B) Superposition of HE MP (red) and corresponding HEF (magenta) and HA subdomains (cyan). Sequence identity and rms deviations on Cα positions for HE-HEF and HE-HA are 33% and 1.9 Å, and 22% and 2.5 Å, respectively.
The Esterase Domain.
The E domain of CoV HE, harboring the RDE activity, has an SGNH-hydrolase fold (27) highly similar to that of HEF (1.4-Å rms deviation on Cα positions at 34% sequence identity). In fact, the active-site architecture of the HEF sialate-9-O-acetylesterase is fully conserved in BCoV HE with Ser40, His329, and Asp326 as catalytic triad and with the side chain of Asn104 and the NH groups of Ser40 and Gly75 forming an oxyanion hole (Fig. 4 A). HE crystals soaked with 9-O-acetylated sialic acid did not contain this substrate in the enzymatic pocket. Instead, the oxyanion hole was occupied by an acetate ion, i.e., one of the catalytic products. The HEF and HE E domains do differ in size and structure of an extended surface loop (residues 47–54) that shields the catalytic pocket and that may prevent antibodies from accessing the functionally important esterase site (Fig. 4 A). This loop is highly variable even among coronavirus HEs (7), tentatively identifying it as a site of antigenic variation.
HE and HEF display conserved enzymatic but divergent receptor-binding sites. (A) Superposition of active site residues of HE (green) and HEF (gray). An acetate ion occupying the oxyanion hole in HE is shown in stick representation (carbon, cyan; oxygen, red). Catalytic triad and oxyanion hole hydrogen bonds are indicated by dashed black lines. Also shown is an adjacent loop that is highly variable also among CoVs. (B) Comparison of the receptor-binding regions of HE, HEF, and HA. Bound ligands, αNeu4,5,9Ac32Me in HE, αNeu5,9Ac22Me in HEF, and αNeu5Ac2Me in HA are shown in stick representation (carbon, cyan; nitrogen, blue; oxygen, red). Proteins are depicted in similar orientations obtained by superposition of their conserved central cores. (C) Ligand bound to the HE receptor-binding site in stick representation. Water molecules are shown as red spheres, a potassium ion as magenta sphere. Hydrogen bonds with receptor are shown as dashed black lines. (D) Surface representation of the HE receptor-binding site revealing two pockets accommodating the 9-O- and 5-N-acetyl groups of the receptor. (E) The effect of Ala substitutions on receptor binding. Relative binding affinity of wild-type HE and its derivatives was assessed by hemagglutination assay with rat erythrocytes and twofold serial dilutions of each of the HE-Fc chimeras (1,000 to 0.5 ng of per well, arrow).
A Receptor-Binding Site in CoV HE.
The R domain mediates receptor recognition (Fig. 1 D). Although its core, an eight-stranded “Swiss roll,” is structurally well conserved and closely resembles that of HEF and HA (Fig. S3), its receptor-binding region is unique. This region is shaped by five variable loops, four grafted on the conserved core and one extending from the E domain. Differences between HE and HEF in loop size and conformation result in dramatically different binding sites that engage 9-O-acetylated sialic acid in nearly opposite orientations. Compared with the HEF–receptor complex, the receptor analogue bound to HE is rotated by 147° and shifted by 4 Å (Figs. 2 D and 4 B). Conversely, HEF and HA are similar with respect to receptor orientation and binding site topology (22) (Figs. 4 B and Fig. S3). In HE, a β-hairpin, replacing an α-helix in HEF and HA interacts with the receptor through hydrogen bonds from main chain atoms of Leu212 and Asn214 to the 5-N-acetyl- and carboxylate groups, respectively, and from the side chain of Ser213 to O-8 (Fig. 4 C). The α-2-O-methyl group of the receptor analogue points into the solvent, of relevance, because at this position, the natural ligand would be linked to the penultimate residue of the sugar chain. The 5-N-acetyl group fits into a pocket, its methyl interacting with a hydrophobic patch formed mainly by Phe211 (Fig. 4 D). The 9-O-acetyl methyl of the receptor analogue docks into an adjacent hydrophobic pocket, formed by Tyr184, Phe211, Leu266, and Leu267 (Fig. 4 C and D), with the latter two residues in a loop stabilized by a potassium ion coordinated by Asp220, Ser221, Gln222, Ser263, Glu265, Leu267, and one water (Fig. S4). Given that the 9-O-acetyl moiety is a prerequisite for receptor binding and serves as a switch for virion attachment and release, this hydrophobic pocket must be key to ligand recognition. Indeed, Ala substitution of Phe211 or of Leu266 and Leu267 in tandem abrogated receptor binding, whereas substitution of Tyr184 decreased affinity significantly (Fig. 4 E).
Plasticity of the CoV HE Receptor-Binding Site.
HEF also binds 9-O-acetylated sialic acid by contacting the 5-N-acetyl group through a hydrophobic patch and by burying the 9-O-acetyl in a nonpolar pocket (22). Therefore, despite the considerable differences in receptor-binding site architecture, HEF and HE use similar strategies to discriminate functional receptors from non-O-acetylated sialic acids. It is, however, surprising that the HE receptor-binding site has changed to this extent at all. Over a much larger evolutionary distance, the architecture of the HEF receptor-binding site has been preserved in HA, despite differences in receptor specificity (Neu5,9Ac2 vs. α2,3- or α2,6-linked Neu5Ac, respectively) and throughout antigenic variation (22, 26, 28) (Fig. 4 B). Unlike HA and HEF, however, CoV HE acts in concert with another receptor-binding protein, S, a fusion protein essential for entry, and whereas HE-mediated virion attachment may be advantageous (25), it is not vital (16, 25, 29). We propose that redundancy in function between S and HE (24) afforded CoVs leeway to explore HE-binding site topologies and even to shift in coreceptor usage from 9-O- to 4-O-acetylated sialic acids in one branch of murine coronaviruses (16, 30–32).
The plasticity of the CoV HE receptor-binding site contrasts with the strict conservation of the E domain and the esterase catalytic site. From a pragmatic point of view, this may potentially allow the development of broad-spectrum antivirals targeting both orthomyxo- and coronavirus sialate-O-acetylesterases.
Materials and Methods
Construction of Expression Plasmids.
A synthetic codon-optimized sequence for the HE ectodomain of BCoV strain Mebus (amino acid residues 19–388) was cloned in a derivative of expression plasmid S1-Ig (33). The resulting construct, pCD5-BCoVHE-T-Fc, encodes a chimeric HE protein provided with an N-terminal CD5 signal peptide and at its C terminus, separated by a thrombin cleavage site, the Fc domain of human IgG1. To allow expression of an enzymatically inactive HE-Fc chimera (HE0-Fc), we generated a derivative of pCD5-BCoVHE-T-Fc in which the codon for the esterase catalytic residue Ser40 was substituted by Ala by site directed mutagenesis using the QuikChange XL II kit (Stratagene). Ala substitutions of key residues in the HE receptor-binding site were made likewise.
Protein Expression and Purification.
HEK293S cells lacking N-acetylglucosaminyl-transferase I (GnTI) activity (34) were transiently transfected with the expression plasmids using polyethyleneimine. At 18 h after transfection, the transfection mixture was replaced by 293 SFM II expression medium (Invitrogen), supplemented with sodium bicarbonate (3.7 g/liter), glucose (2.0 g/liter), Primatone RL-UF (3.0 g/liter), penicillin (100 units/ml), streptomycin (100 μg/ml), glutaMAX (Gibco), and 1.5% DMSO. Tissue culture supernatants were harvested 5–6 days after transfection, and HE-Fc and HE0-Fc were purified by protein A-affinity chromatography. For crystallography, the HE and HE0 ectodomains were cleaved off by on-the-bead thrombin digestion, concentrated to 10–15 mg/ml, and dialyzed against crystallization buffer (Tris·HCl, pH 8.2; 50 mM NaCl).
Biological and Biochemical Characterization of HE-Fc Chimeras.
Molecular weights of the proteins were determined by SDS/PAGE (under reducing and nonreducing conditions) and by gel filtration chromatography. O-acetylesterase activity was routinely measured by pNPA assay (19). Enzymatic de-O-acetylation of αNeu4,5,9Ac32Me was analyzed by GC-MS (16, 35). To assess esterase activity of the expression products toward cell-bound 9-O-acetylated sialic acids, MDBK cell monolayers in 16-mm diameter wells were treated with 20 μg/ml HE-Fc or HE0-Fc in PBS for 2 h at 37°C and subsequently infected with BCoV strain Mebus. Monolayers, mock-treated or treated with Vibrio cholerea neuraminidase (NA; 0.6 units/ml), were included as controls. Infected cells were visualized by staining using an in situ acetylesterase assay (36). Receptor-destroying activity toward 9-O-acetylated sialic acids on rat erythrocytes was assessed by incubating the cells with 20 μg/ml HE-Fc in PBS for 2.5 h at 37°C, before hemagglutination assay with twofold serial dilutions of BCoV. In situ detection of 9-O-acetylated sialic acids in cultured cells was demonstrated by performing a standard indirect immunofluorescence assay. Briefly, monolayers of polarized MDBK cells, grown on glass coverslips, were fixed with 2% paraformaldehyde and successively incubated with ≈25 μg/ml HE0-Fc in PBS, 0.05% Tween20 and with FITC-conjugated anti-human IgG1 (Nordic, 1:100) as secondary antibody. The samples were mounted on glass slides in FluorSave (Calbiochem) and examined by confocal fluorescence microscopy (Leica TCS SP2).
Crystallization.
Crystallization conditions were screened by the sitting-drop vapor diffusion method using a Honeybee 961 (Genomic Solutions). Drops were set up with 0.1 μl of HE protein solution in 10 mM Tris·HCl, pH 8.0, and 0 l reservoir solution. Diffracting crystals were obtained from JCSG solution D12 [0.04 M KH2PO4, 16% (wt/vol) PEG 8000, 20% (wt/vol) glycerol] (JCSG Technologies) and PEGIon-lite solution 89 [0.1 M KH2PO4, 10% (wt/vol) PEG 3350] (Hampton Research) at 18°C. Crystals for diffraction experiments were grown with the hanging-drop vapor diffusion method set up by hand with 0.8 μl of reservoir solution and 0.8 μl of protein solution at 18°C. Under both crystallization conditions, crystals of wild-type HE and HE0 grew within 2 weeks with the shape of hexagonal bipyramids to a size of ≈0.25 × 0.16 × 0.16 mm. Their space group is P6522. Crystals of wild-type HE grown at 0.1 M KH2PO4, 10% (wt/vol) PEG 3350 were flash-frozen in liquid nitrogen using reservoir solution with 22.5% (wt/vol) glycerol as the cryoprotectant.
Data Collection and Structure Solution.
To determine the HE structure in complex with its receptor analog Neu4,5,9Ac32Me (16), HE0 was used to prevent substrate degradation during soaking. Crystals obtained from 0.1 M KH2PO4, 10% (wt/vol) PEG 3350 cracked during ligand soaking and the flash-freezing procedure. Therefore, we used for substrate-soaking crystals of the mutant grown from 0.04 M KH2PO4, 16% (wt/vol) PEG 8000, 20% (wt/vol) glycerol, which did not require addition of cryoprotectant. Soaking was performed by adding 2 μl of reservoir solution with 10 mM Neu4,5,9Ac32Me directly into the margin of the drop with crystals, resulting in a final substrate concentration of ≈7 mM. Crystals were flash-frozen after 5–10 min.
Diffraction data of native and ligand-soaked crystals of BCoV HE were collected at European Synchrotron Radiation Facility station ID14-3 and ID 23-1, respectively. Diffraction data were processed by using XDS (37) and scaled using SCALA from the CCP4 suite (38). Molecular replacement was performed by using PHASER with Influenza C HEF residues 35–410 as template [Protein Data Bank (PDB) ID code 1FLC]. Density modification was carried out by using RESOLVE (PHENIX package) (39) and CNS (40). Approximately 280 residues were built automatically by ARP/WARP (41); the remaining residues were built manually with Coot (42). Refinement was carried out by using REFMAC (43); water molecules were added by using ARP/WARP. Molecular graphics were generated with PYMOL (http://pymol.sourceforge.net).
The structure of HE0 in complex with Neu4,5,9Ac32Me was solved by molecular replacement by using the wild-type HE structure as template. The structure was refined as described for wild-type HE. The library file for refinement of the ligand in REFMAC was made by adding chemical and geometric descriptions of the 4- and 9-O-acetyl groups to that of sialic acid (SIA) in the monomer library of the CCP4 suite by using the program SKETCHER (CCP4 Suite).
Acknowledgments
We thank Peter Rosenthal (MRC, Cambridge, UK) for providing coordinates of the HEF-inhibitor complex. We also thank P. Gros, J. de Groot-Mijnes, B. Bosch, P. Rottier, and P. Rosenthal for advice and for reading of this manuscript; G. Gerwig and J. Kamerling for help in preparing the sialic acid analogue; and P. Reeves (University of Essex, Colchester, UK) and M. Farzan (Harvard Medical School, MA) for sharing HEK293S GnTI(−) cells and expression plasmids, respectively. We acknowledge the European Synchrotron Radiation Facility for providing beamline facilities and the beamline scientists at ID-14-EH3 and ID23-1 for help with data collection. This work was supported by “Pionier” program and ECHO grants of the Council for Chemical Sciences of The Netherlands Organization for Scientific Research (NWO-CW) and by a grant of the Mizutani Foundation for Glycoscience.
Footnotes
- §To whom correspondence may be addressed. E-mail: e.g.huizinga@uu.nl or r.j.degroot@uu.nl
Author contributions: Q.Z., M.A.L., E.G.H., and R.J.d.G. designed research; Q.Z., M.A.L., and A.L.W.v.V. performed research; Q.Z., M.A.L., E.G.H., and R.J.d.G. analyzed data; and Q.Z., M.A.L., E.G.H., and R.J.d.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. R.S.B. is a guest editor invited by the Editorial Board.
Data deposition: The data reported in this paper have been deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 3CL4 (wild-type BCoV HE) and 3CL5 (BCoV HE0 complexed with Neu4,5,9Ac32Me)].
See Commentary on page 8807.
This article contains supporting information online at www.pnas.org/cgi/content/full/0800502105/DCSupplemental.
- © 2008 by The National Academy of Sciences of the USA
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The metabolic route by which oleate is converted into cholesterol in rat hepatocytes
- PMID: 3741380
- PMCID: PMC1146642
- DOI: 10.1042/bj2350019
Abstract
The effect of (-)-hydroxycitrate on the conversion of [1-14C]oleate into cholesterol was dependent on the time of day at which the cells were prepared and on the extracellular oleate concentration. In hepatocytes prepared during the light phase of the diurnal cycle (L2-hepatocytes), (-)-hydroxycitrate inhibited the conversion of L-[U-14C]lactate (2 mM) and of 0.13 mM-[1-14C]oleate into cholesterol. However, when [1-14C]oleate was present at 1.3 mM, most of the sterol carbon was derived from this source, and under these conditions (-)-hydroxycitrate had no inhibitory effect on [14C]cholesterol formation. In these cells, non-radioactive acetoacetate blocked the conversion of 1.3 mM-[1-14C]oleate, but not of 0.13 mM-[1-14C]oleate, into cholesterol. In cells prepared during the dark phase of the diurnal cycle (D6-hepatocytes), irrespective of the concentration of [1-14C]oleate, (-)-hydroxycitrate decreased its conversion into cholesterol. In both types of cell preparation, the inhibitory effect of (-)-hydroxycitrate on the conversion of L-[U-14C]lactate into cholesterol was greater than that on the overall rate of cholesterol production from all endogenous sources. These results provide evidence for the following. (1) The major metabolic route by which oleate is converted into cholesterol is dependent on its extracellular concentration. (2) When oleate is the major source of hepatic sterol carbon, the flux of substrate through citrate into cholesterol is dependent on the nutritional state of the animal. (3) When endogenous substrates are the sole source of sterol carbon, a substantial proportion of the carbon enters the cholesterol pathway through routes not involving citrate cleavage.
Oleic acid is the most abundant fatty acid in human adipose tissue,[14] and second in abundance in human tissues overall, following palmitic acid.
ABSTRACT
Introduction of a new influenza virus in humans urges quick analysis of its virological and immunological characteristics to determine the impact on public health and to develop protective measures for the human population. At present, however, the necessity of executing pandemic influenza virus research under biosafety level 3 (BSL-3) high-containment conditions severely hampers timely characterization of such viruses. We tested heat, formalin, Triton X-100, and β-propiolactone treatments for their potencies in inactivating human influenza A(H3N2) and avian A(H7N3) viruses, as well as seasonal and pandemic A(H1N1) virus isolates, while allowing the specimens to retain their virological and immunological properties. Successful heat inactivation coincided with the loss of hemagglutinin (HA) and neuraminidase (NA) characteristics, and β-propiolactone inactivation reduced the hemagglutination titer and NA activity of the human influenza virus 10-fold or more. Although Triton X-100 treatment resulted in inconsistent HA activity, the NA activities in culture supernatants were enhanced consistently. Nonetheless, formalin treatment permitted the best retention of HA and NA properties. Triton X-100 treatment proved to be the easiest-to-use influenza virus inactivation protocol for application in combination with phenotypic NA inhibitor susceptibility assays, while formalin treatment preserved B-cell and T-cell epitope antigenicity, allowing the detection of both humoral and cellular immune responses. In conclusion, we demonstrated successful influenza virus characterization using formalin- and Triton X-100-inactivated virus samples. Application of these inactivation protocols limits work under BSL-3 conditions to virus culture, thus enabling more timely determination of public health impact and development of protective measures when a new influenza virus, e.g., pandemic A(H1N1)v virus, is introduced in humans.
- Copyright © 2010 American Society for Microbiology
11. Jonges M, Liu WM, van der Vries E, Jacobi R, Pronk I, Boog C, et al. Influenza virus inactivation for studies of antigenicity and phenotypic neuraminidase inhibitor resistance profiling. J Clin Microbiol. 2010;48(3):928–40. Epub 2010/01/22. 10.1128/JCM.02045-09 ; PubMed Central PMCID: PMCPMC2832438. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
- (of a solid) hold (molecules of a gas or liquid or solute) as a thin film on the outside surface or on internal surfaces within the material."the dye is adsorbed onto the fiber"
Coronavirus membrane fusion mechanism offers a potential target for antiviral development
Associated Data
1. Introduction
Coronavirus (CoV) cell infection begins with viral entry, in which the viral particle recognizes a host cell receptor and fuses its membrane with the host cell membrane (Belouzard et al., 2012). These two steps are mediated by the coronavirus spike (S) protein. In addition to mediating entry, the S protein is the principal antigenic determinant and the target of neutralizing antibodies (Walls et al., 2016; Yuan et al., 2017; Zhang et al., 2004). This makes the S protein a valuable target in vaccine and antiviral efforts, as summarized in (Du et al., 2017, 2009; Xia et al., 2014). As the urgency to develop effective therapeutics against coronaviruses rises, it is important to also consider broad-spectrum properties of such therapies, as there will likely be future outbreaks for which we will need to be prepared for a rapid response.
Of the two functions that the S protein mediates, there have been considerably more efforts studying therapeutics against receptor binding, as it contains major antigenic determinants (Ying et al., 2015). However, across the coronavirus family, the receptor binding domain is poorly conserved (Belouzard et al., 2012; Graham et al., 2013; Li, 2015), and so therapeutics that target the receptor binding function have low potential as a pan-coronavirus solution. In fact, it was observed that monoclonal antibodies that recognized the SARS-CoV receptor binding domain did not recognize the SARS-CoV-2 receptor binding domain, highlighting the low cross-reactivity in this region (Wrapp et al., 2020). On the other hand, the membrane fusion domain is amongst the most conserved areas in the S protein (Lai et al., 2017; Madu et al., 2009b), and so, targeting membrane fusion may have greater cross-functional success against future coronavirus outbreaks (Xia et al., 2019).
In this article, we review the S protein structure, function, and its role in mediating fusion, and notable therapeutic strategies for blocking fusion, focusing on results obtained on SARS-CoV, MERS-CoV, and recent peer-reviewed studies on SARS-CoV-2. We also provide a supplemental file containing background information on the biology, epidemiology, and clinical features of all human infecting coronaviruses, along with a phylogenetic tree of these coronaviruses.
2. S protein structure and function
As discussed, the CoV S protein has an important role in an infection as it regulates viral entry into host cells and is also the major antigenic determinant, which is usually targeted by the host antibody response (Bosch et al., 2003; Walls et al., 2016; Yuan et al., 2017). These roles emphasize the importance of this protein in both viral entry and interaction with the host's immune system. The S protein is one of 4 structural proteins encoded by the CoV single-stranded, positive-sense RNA genome. In the viral membrane, the S protein participates in two key events: binding to the cellular receptor and inducing fusion between the viral and the cellular membranes (Belouzard et al., 2012). The accomplishment of these two events will drive the release of the viral RNA genome in the host cell and the subsequent start of the viral replication cycle (Masters and Perlman, 2013).
Following viral genome release into the host cell, translation of the ORF produces 16 non-structural proteins that form the viral replicase-transcriptase complex (Fig. 1 A). This complex aids in viral genome replication and subgenomic transcription. The subgenomic RNAs encode four structural proteins, spike (S), envelope (E), membrane (M), and nucleocapsid (N), along with several accessory proteins. Upon translation, the S, E, and M structural proteins are inserted into the rough endoplasmic reticulum. From there, the proteins travel along the secretory pathway to the endoplasmic reticulum-Golgi apparatus intermediate compartment (ERGIC), the site of CoV particle assembly. Afterwards, the virus is released from the cell via exocytosis (de Haan and Rottier, 2005).
Coronavirus spike (S) protein. A. Cartoon figure of the CoV particle (top) and complete CoV viral genome (bottom). CoVs have a lipid envelope with three structural transmembrane proteins: spike (S), membrane (M), and envelope (E). The virus interior contains the viral genome encapsulated by the nucleocapsid (N) protein. The CoV single stranded genome encodes for 16 non-structural proteins, including the papain-like protease (PLpro), 3C-like protease (3CLpro), RNA-dependent RNA polymerase (RdRp), helicase (Hel), and exonuclease (ExoN). The subgenomic RNAs encode four structural proteins: spike (S; dark pink), envelope (E; dark blue), membrane (M; purple), and nucleocapsid (N; magenta) and a number of accessory proteins (Chan et al., 2020; de Wit et al., 2016). B. Cartoon figure of the CoV S protein trimer. C. The CoV S gene denoting the functional components of the protein. The CoV S protein is composed of the two subunits: S1 and S2, encompassing the major functional components: SP (signal peptide, pink); NTD (N-terminal domain; green), CTD (C-terminal domain; light blue), FP (fusion peptide; red), HR1 (heptad repeat 1; purple), HR2 (heptad repeat 2; orange), TM (transmembrane; yellow), and CP (cytoplasmic; dark blue). The S protein has two cleavage sites denoted with dark purple (S1/S2) and pink (S2’) arrows. D. Sequence alignment of S1/S2 cleavage site (dark purple arrow) and S2’ cleavage site (pink) between MERS-CoV, SARS-CoV, and SARS-CoV-2. E. Within the genome, the fusion peptide is highlighted, denoting the sequences from MERS-CoV FP and SARS-CoV FP. Red denotes the conserved residues between MERS-CoV, SARS-CoV, and SARS-CoV-2 FP sequences; blue denotes the SARS-CoV and SARS-CoV-2 FP conserved residues; green denotes the SARS-CoV and MERS-CoV FP conserved residues; purple denotes the MERS-CoV and SARS-CoV-2 conserved residues. The fusion peptide sequence of SARS-CoV-2 was determined by performing a pairwise alignment with MUSCLE through Geneious (version 2020.0.5). Amino acid sequence of the spike proteins was obtained from NCBI Genbank based on the following: SARS-CoV-2 (MN908947.3), MERS-CoV (AFS88936.1), SARS-CoV (AAP13441.1).
In the ERGIC, the S protein monomer is also extensively modified via N-glycosylation and trimerizes (Heald-Sargent and Gallagher, 2012). For some coronaviruses (i.e. MERS-CoV, but not SARS-CoV), the S protein can also be partially processed by furin and furin-like proteases during protein biosynthesis in the Golgi apparatus depending on the host cell type (Kleine-Weber et al., 2018; Millet and Whittaker, 2014; Yang et al., 2014). In some coronaviruses, the S protein that is not incorporated into the viral particle could be transferred to the cell surface (Fehr and Perlman, 2015).
Structurally, the S protein is a ~180–200 kDa type I transmembrane protein, with the N-terminus facing the extracellular space, held in the viral membrane via its transmembrane domain, with a short C-terminal segment facing the intracellular space (Bosch et al., 2003). The extracellular domain is split into two subunits or domains, S1 and S2, which mediate receptor binding and membrane fusion, respectively (Fig. 1B and C). Visually, the S protein trimers form a characteristic bulbous, crown-like halo surrounding the viral particle, for which coronaviruses were named. Structural modeling of coronavirus S protein monomers show that the S1 and S2 subunit form the bulbous head and stalk region, respectively. Aspects of the structure will be referred to throughout the article (Fig. 2 ).
MERS-CoV (A), SARS-CoV (B), and SARS-CoV-2 (C) protein models. Models were built to show the predicted structure of the S1/S2, the S2′ cleavage site and the FP, which are not solved in cryo-EM structures. Trimers and monomers were modeled using SARS-CoV (PDB# 5X58) and MERS-CoV (PDB# 6Q05) structures using the methodology described in (Jaimes et al., 2020b). Color scheme is as described for Fig. 1.
The S1 subunit contains two subdomains, the N-terminal domain (NTD) and the C-terminal domain (CTD) (Fig. 1C). One or both subdomains can serve as the receptor-binding domain (RBD) with the piece of the RBD that directly contacts the receptor termed the receptor-binding motif (RBM). A general rule of thumb is that the NTD mediates viral binding to sugar-based receptors, whereas the CTD mediates binding to protein-based receptor, though there are exceptions, for example the mouse hepatitis virus NTD recognizes proteinaceous CEACAM1 as its receptor (Peng et al., 2011).
Both SARS-CoV and SARS-CoV-2 utilize the CTD to bind angiotensin-converting enzyme 2 (ACE2) (Li et al., 2003; Zhou et al., 2020), abundantly detected on lung and small intestine cells (Hamming et al., 2004). The amino acid differences in the RBM (~50% sequence homology) across SARS-CoV and SARS-CoV-2 have been hypothesized to strengthen or weaken RBM-ACE2 associations, though it is unclear if the overall effect of all these differences results in stronger recognition of SARS-CoV-2 for ACE2 (Yan et al., 2020). It is reported that SARS-CoV-2 S does bind ACE2 with 10- to 20-fold higher affinity than SARS-CoV S binding to ACE2 (Wrapp et al., 2020). However, another study reported that purified SARS-CoV-2 and SARS-CoV RBD binds ACE2 with similar affinities (Walls et al., 2020), so further experiments should be conducted to determine the impact of residue changes on ACE2 binding.
MERS-CoV utilizes the CTD to bind proteinaceous dipeptidyl peptidase 4 (DPP4) (Raj et al., 2013), detected on lung and kidney cells (van Doremalen et al., 2014). The well-conserved nature of DPP4 across a wide variety of species (e.g. bats, dromedaries, humans) provides more insight into the zoonotic capabilities of MERS-CoV. Although MERS-CoV does not infect murine cells since MERS-CoV S does not bind to mouse DPP4, it is possible to infect mice by replacing blades 4 & 5 of mouse DPP4 with blades from animals susceptible to MERS-CoV S binding, forming chimeric DPP4s (Barlan et al., 2014). With the ability to interchange blades 4 & 5, it was determined that MERS-CoV S preferentially binds to human, horse, camel, goat, and bat DPP4, listed in decreasing order (Barlan et al., 2014). Another contributing factor to the zoonotic potential of MERS-CoV comes from reports that MERS-CoV S has the ability to bind sialic acid receptors in addition to DPP4 (Li et al., 2017). Recent work further revealed that sialic acid receptors play a role in transmissibility between species, as they reaffirmed the binding affinity reported by the Barlan group (Widagdo et al., 2019).
Significant progress has been made to understand the RBD-receptor interaction, and crystal structures of the SARS-CoV S (Li et al., 2005; Song et al., 2018), MERS-CoV S (Wang et al., 2013) and SARS-CoV-2 S (Yan et al., 2020) in complex with its receptor have been determined. Similar studies have revealed that receptor binding requires one of the trimers to be in the “up” position for SARS-CoV and SARS-CoV-2, but not for MERS-CoV (Gui et al., 2017; Song et al., 2018; Yan et al., 2020; Yuan et al., 2017).
The CoV S protein is also classified as a class I viral fusion protein, based on the structure of its fusion subunit (White et al., 2008). Within this class, the fusion subunit is largely composed of α-helical secondary structures (Fig. 2), and its function is regulated through proteolytical priming or cleavage at specific sites to induce the fusion-competent state of the S protein (White and Whittaker, 2016). The S2 or fusion subunit contains a variety of motifs (Fig. 1C), starting with the fusion peptide (FP) which is the functional fusogenic element of the S protein. The FP describes a short segment (15–25 amino acids), conserved across the viral family that is composed of mostly hydrophobic residues, such as glycine (G) or alanine (A), which inserts in the host cell membrane to trigger the fusion event (Epand, 2003). Fusion peptides tend to be sensitive to point mutations, in that a single mutation can negate fusion (Madu et al., 2009b). However, the fusion peptide is loosely defined, as these requirements are not absolute but rather serve as guidelines to identify the fusion peptide region.
For SARS-CoV, several regions have been suggested as the FP. Using a Wimley and White interfacial hydrophobicity scale to identify regions with a higher propensity to insert into membranes, the region 770–788 was identified and a peptide corresponding to this region was shown to induce fusion and membrane leakage in large unilamellar vesicles (Sainz et al., 2005). Further work identified regions 873–888 and 1185–1202 as strong membrane interacting regions and proposed that these regions, in conjunction with 770–788, work synergistically to mediate fusion (Guillén et al., 2008a, 2008b). Separately, the region 798–835 was also identified as a fusion peptide, since single point mutagenesis studies demonstrated its importance in fusion, with an extremely conserved region, SFIEDLLFNKV (798–808) (Madu et al., 2009b, 2009a). An excellent review that discusses the journey of identifying the SARS-CoV FP and key findings is (Millet and Whittaker, 2018).
Research specifically focusing on the MERS-CoV FP has been rather limited. Sequence alignment, using MUSCLE software from the Geneious bioinformatic software platform, based on the SARS-CoV FP suggested that the highly conserved SFIEDLLFNKV motif is similar within MERS-CoV and a potential region of the MERS-CoV FP was revealed (Lai et al., 2017). The sequence region 888–898, RSARSAIEDLLFDKV, was strongly suggested to comprise the MERS-CoV FP based on single point mutagenesis screening with giant unilamellar vesicles identifying the critical hydrophobic residues for syncytium-forming ability (Alsaadi et al., 2019).
Based on current understanding of the SARS-CoV FP, we suggest here a preliminary SARS-CoV-2 FP using a pairwise sequence alignment. The suggested FP has 93% sequence homology with SARS-CoV FP, displaying strong conservation across these two viruses. The current understanding of FP residues are as described in Fig. 1E.
Downstream of the FP are the heptad repeat regions (HR1 and HR2), with HR1 more N-terminal and longer than HR2. Both HR1 and HR2 are composed of repetitive heptapeptide, HPPHCPC, in which H represents hydrophobic or traditionally bulky residues, P are polar or hydrophilic residues, and C are other charged residues (Chambers et al., 1990). This allows the HR region to adopt an α-helix secondary structure with a hydrophobic interface to drive membrane fusion (White et al., 2008). Following the HR2 region is the transmembrane (TM) domain, which anchors the S protein in the viral membrane. Lastly, at the C-terminal end of the S protein is the cytoplasmic tail.
3. Proteolytic activation of CoV S
As reiterated, the CoV S drives receptor binding and membrane fusion. However, in order to catalyze the membrane fusion reaction, the S protein needs to be primed by an appropriate protease at the S1 and S2 interface (S1/S2) and triggered immediately upstream of the FP (S2’) (Fig. 1D). What is fascinating about this triggering event is that several different proteases can trigger, and it is the protease requirements that drive viral tropism. Both SARS-CoV and MERS-CoV S can be triggered to fuse at either the plasma membrane or the endosomal membrane, and the route of entry is dependent on protease availability (Fig. 3 ). The emerging SARS-CoV-2 also utilizes both pathways (Hoffmann et al., 2020). If the plasma membrane-route proteases are available, the virus can fuse via an “early pathway” at the plasma membrane, but if not, the virus can fuse via a “late pathway” at the endosomal membrane. Intriguingly, the activation of coronavirus fusion varies, depending on the protease in the local environment, highlighting the flexibility of coronavirus S proteins to respond to the particular chemical cues available. In this section, we will review both routes, as well as the proteases involved in activating S.
Model of coronavirus dual entry pathway. This model depicts the two methods of viral entry: early pathway and late pathway. As the virus binds to its receptor (1), it can achieve entry via two routes: plasma membrane or endosome. For SARS-CoV: The presence of exogeneous and membrane bound proteases, such as trypsin and TMPRSS2, triggers the early fusion pathway (2a). Otherwise, it will be endocytosed (2b, 3). For MERS-CoV: If furin cleaved the S protein at S1/S2 during biosynthesis, exogeneous and membrane bound proteases, such as trypsin and TMPRSS2, will trigger early entry (2a). Otherwise, it will be cleaved at the S1/S2 site (2b) causing the virus to be endocytosed (3). For both: Within the endosome, the low pH activates cathepsin L (4), cleaving S2′ site, triggering the fusion pathway and releasing the CoV genome. Upon viral entry, copies of the genome are made in the cytoplasm (5), where components of the spike protein are synthesized in the rough endoplasmic reticulum (ER) (6). The structural proteins are assembled in the ER-Golgi intermediate compartment (ERGIC), where the spike protein can be pre-cleaved by furin, depending on cell type (7), followed by release of the virus from the cell (8, 9). For SARS-CoV-2: Studies currently show that SARS-CoV-2 can utilize membrane bound TMPRSS2 or endosomal cathepsin L for entry and that the S protein is processed during biosynthesis. Other factors that can influence the viral entry pathway are calcium and cholesterol (not shown).
3.1. Plasma membrane route (“early pathway”)
An early indication that SARS-CoV can utilize the plasma membrane pathway came from cell-cell fusion studies that found that HEK293T cells transiently expressing the SARS-CoV S protein mediated cell-cell fusion with target Vero E6 cells when treated with a low concentration of trypsin, whereas a similar setup utilizing a low pH pulse in the absence of trypsin did not mediate any fusion (Simmons et al., 2004). Likewise, trypsin was also found to induce MERS-CoV and SARS-CoV-2 S protein mediated cell-cell fusion of Vero E6 cells and 293T cells, respectively (Ou et al., 2020; Shirato et al., 2013). Subsequent studies using retroviral pseudoparticles expressing SARS-CoV S protein (SARSpp) or MERS-CoV S protein (MERSpp) revealed that trypsin treatment of these pseudoparticles after binding to their respective receptor resulted in effective infection at the plasma membrane surface (Qian et al., 2013; Simmons et al., 2005). In fact, pretreating the S protein prior to receptor binding was found to abrogate infection, and this is hypothesized to occur because trypsin treatment causes the S protein to undergo irreversible conformational changes so that it cannot mediate fusion (Matsuyama et al., 2005; Park et al., 2016; Simmons et al., 2005). This phenomenon has yet to be investigated for SARS-CoV-2. Another comprehensive study investigating the impact of a panel of additional exogenous proteases on SARS-CoV and MERS-CoV live virus infection of Vero E6 cells revealed that thermolysin and elastase also efficiently mediate cellular entry at the plasma membrane after binding (Belouzard et al., 2010; Matsuyama et al., 2005; Shirato et al., 2013). The finding that elastase could mediate entry is clinically important, since elastase is produced by inflammatory cells in the lungs during SARS-CoV infection and could thus promote the progression of SARS-CoV infection.
Although these exogeneous proteases are capable of activating fusion, they do not provide insights into the fusion mechanism in the human respiratory tract, a major target for coronaviruses. This is because coronaviruses should only be cleaved after receptor engagement and not before, but this timing would be difficult to control if exogenous proteases were the in vivo activator. Thus, transmembrane proteases are of interest as they are localized on the plasma membrane surface where the virus encounters the receptor. The type II transmembrane serine proteases (TTSPs) are a family of such proteases anchored in the cellular membrane and have already been implicated in influenza virus infection (Choi et al., 2009). In particular, the transmembrane protease/serine subfamily member 2 (TMPRSS2) and TMPRSS4 were found to activate and enable influenza virus spread, even in the absence of extracellular trypsin (Bertram et al., 2010; Böttcher et al., 2006; Chaipan et al., 2009).
Inspired by the impact of TTSP on influenza virus spread, researchers have also sought to investigate whether similar TTSPs could impact SARS-CoV and MERS-CoV fusion. An initial study demonstrated that a soluble form of a TTSP protease, TMPRSS11a, could cleave and activate SARS-CoV S protein for fusion (Kam et al., 2009). Subsequent studies investigated whether membrane-bound TTSP proteases were capable of activating the SARS-CoV S protein also. It was determined that cell lines transiently expressing TMPRSS2 support both SARSpp and live SARS-CoV infection at the plasma membrane without exogeneous or late-pathway proteases (Glowacka et al., 2011; Matsuyama et al., 2010; Shulla et al., 2011). Similar studies with MERSpp and live MERS-CoV also identified membrane-bound TMPRSS2 as an activator of MERS-CoV infection (Gierer et al., 2013; Qian et al., 2013; Shirato et al., 2013). SARS-CoV-2 pseudoparticles (SARS2pp) and live SARS-CoV-2 also utilize membrane-bound TMPRSS2 for plasma membrane entry (Hoffmann et al., 2020; Matsuyama et al., 2020), much like SARS-CoV and MERS-CoV.
Other TTSP proteases have been found to support, in various degrees, CoV infection at the plasma membrane surface. Utilizing MERSpp, it was shown that TMPRSS11a and TMPRSS11e can also activate MERS-CoV S for viral infection (Zmora et al., 2018). However, the TTSP family is not a blanket activator of coronavirus S mediated fusion. Although TMPRSS4 was found to activate SARS-CoV and MERS-CoV S mediated cell-cell fusion, it was ineffective in promoting viral infection (Glowacka et al., 2011; Qian et al., 2013). Transient expression of TMPRSS2, 4, 11a, 11d, and 11e were shown to enhance SARS-CoV-2 S-mediated cell-cell fusion, though it is unknown if these proteases activate S for infection (Ou et al., 2020). The discrepancies in these findings could result from high S expression and/or interaction of large surface areas in cell-cell fusion assays that encourage more fusion than would be possible in viral-cell fusion (Glowacka et al., 2011). More intriguingly, TTSP activation can also differ between SARS-CoV and MERS-CoV. TMPRSS11d, also known as human airway trypsin-like protease (HAT), is unable to activate SARS-CoV S infection, but can activate MERS-CoV S infection (Bertram et al., 2011; Zmora et al., 2018). While differences between SARS-CoV and MERS-CoV activation will be further discussed in the following paragraph, it is noteworthy to mention that the coronavirus S has evolved to have flexibility in a variety of proteases that can cleave and activate it, providing a possible rationale for their zoonotic potential.
Although a high degree of sequence conservation exists between the SARS-CoV and MERS-CoV S2 membrane fusion domains, there are key differences in their fusion mechanism for plasma membrane entry. For one, MERS-CoV S contains a furin cleavage site at S1/S2, whereas SARS-CoV S does not (Fig. 1D) (Millet and Whittaker, 2014). Furin or furin-like proteases found in the trans-Golgi network (TGN) can encounter and act on such cleavage sites in coronavirus S as it is being synthesized (Millet and Whittaker, 2015). MERS-CoV has been observed to be cleaved during biosynthesis at the S1/S2 position (Millet and Whittaker, 2014), and this pre-cleavage was shown to promote subsequent MERS-CoV cleavage by TMPRSS2 at the S2′ position to activate plasma membrane fusion, whereas if there was no pre-cleavage, immediate entry via TMPRSS2 was not observed (Park et al., 2016). Furin activation of MERS-CoV at the S2′ position for entry has also been observed, based on the presence of specific basic residues (Millet and Whittaker, 2014), but the exact role is still unclear (Matsuyama et al., 2018). There are no indications that SARS-CoV or SARS-CoV-2 S can be cleaved by furin at S2’.
SARS-CoV S is not cleaved during biosynthesis and does not require a S1/S2 pre-cleavage event for plasma membrane fusion. While it is suspected that the S1/S2 cleavage event can cause conformational changes that further exposes the S2’ site for immediate plasma membrane fusion, it is noted that there can be alternative ways to cause these conformational changes, such as receptor binding (Park et al., 2016). Indeed, it is observed that SARS-CoV S binds its receptor with a 10- to 20- fold higher affinity compared to that of MERS-CoV S with its receptor, so stronger receptor binding can perhaps compensate for uncleaved S1/S2, at least in the case for SARS-CoV to support plasma membrane entry. It has been suggested that the S1/S2 site may enable these coronaviruses to spread and infect new organisms with low receptor affinity. Interestingly, TMPRSS2 overexpression was observed to allow uncleaved MERS-CoV to infect cells, bypassing the required S1/S2 cleavage event (Kleine-Weber et al., 2018), once again highlighting the adaptability of the coronavirus S protein.
Unexpectedly, SARS-CoV-2 S also possesses a potential furin cleavage site at the S1/S2 region, which is unique for SARS-like CoVs (Coutard et al., 2020; Walls et al., 2020; Wrapp et al., 2020; Jaimes et al., 2020a). Western blots have shown that S proteins in SARS2pp can be processed at the S1/S2 boundary during biosynthesis, similar to what was reported for MERS-CoV (Walls et al., 2020). However, the impact of the potential furin cleavage site and the accompanying extended structural loop at S1/S2 remains to be determined.
3.2. Endosomal route (“late pathway”)
In the absence of exogenous or membrane-bound proteases that enable entry at the plasma membrane surface, coronaviruses can be internalized via clathrin- and non-clathrin-mediated endocytosis (Inoue et al., 2007; Wang et al., 2008). A caveat is that if MERS-CoV is not pre-cleaved at the S1/S2 site during biosynthesis, then it will also be endocytosed, regardless of the presence of plasma membrane proteases (Park et al., 2016). As the virus is shuttled along the endocytic pathway towards the cell interior, the pH in the endosome decreases. For some viruses (e.g. influenza, vesicular stomatitis virus), the presence of low pH triggers fusion. As such, treating the cells with lysosomotropic agents to prevent endosomal acidification abrogates infection of these pH-dependent viruses (Ochiai et al., 1995). Initial experiments infecting Vero E6 cells with SARSpp in the presence of lysosomotropic agents suggested that SARSpp was sensitive to low pH conditions (Simmons et al., 2004). However, since a pH pulse could not mediate cell-cell fusion and SARSpp exposure to low pH prior to binding did not reduce infectivity, there were grounds to suspect other factors may be involved in CoV endosomal fusion.
The low pH environment also activates endosomal proteases, such as cathepsins, a family of cysteine proteases. Of interest are cathepsin B and cathepsin L, which become active in the early and late endosome, respectively, and are known activators of other members in the CoV family (Qiu et al., 2006; Regan et al., 2008). Indeed, subsequent studies demonstrated that the cathepsin B/L inhibitor MDL28170 drastically reduced SARSpp (Huang et al., 2006; Simmons et al., 2005) and MERS-CoV entry into MRC-5 cells (Gierer et al., 2013). Studies on SARS-CoV-2 have echoed similar themes; SARS2pp are sensitive to cathepsin B/L inhibitors (Hoffmann et al., 2020; Ou et al., 2020). Confirmation that all these coronaviruses are activated by cathepsin L for fusion in the late endosome was determined using inhibitors specific for either cathepsin B or L (Ou et al., 2020; Qian et al., 2013; Simmons et al., 2005), along with direct observation of SARS-CoV trafficking to late endosomes (Mingo et al., 2015). Thus, it is believed that SARS-CoV, MERS-CoV, and SARS-CoV-2 dependency on low pH in the endosomal route is indirect; acidic conditions are required to activate cathepsin L protease, which in turn then act on S, resulting in a virus primed to undergo subsequent fusion steps. To further confirm the independence of these coronaviruses entry from pH, it is important to reiterate that both viruses can undergo plasma membrane fusion, setting a precedence for neutral pH fusion.
3.3. SARS-CoV and MERS-CoV cleavage sites
Although both the SARS-CoV and the MERS-CoV S protein can be activated by a similar array of proteases, it is crucial to mention that these proteases act on the S protein at different sites, which may lead to slightly different activities (Table 1 ). TMPRSS2 has been reported to cleave both SARS-CoV and MERS-CoV at the S2’ site to activate plasma membrane fusion (Kleine-Weber et al., 2018; Reinke et al., 2017). However, for this activation, MERS-CoV must be pre-cleaved by furin at the S1/S2 site during S biosynthesis (Kleine-Weber et al., 2018; Millet and Whittaker, 2014; Park et al., 2016), whereas SARS-CoV does not need cleavage at the S1/S2 for activation (Reinke et al., 2017). Although likely, it is yet to be formally determined if SARS-CoV-2 also needs to be pre-cleaved at the S1/S2 site for plasma membrane fusion.
Table 1
SARS-CoV and MERS-CoV activating proteases, locations, and sites. Both SARS-CoV and MERS-CoV S protein contain two sites (S1/S2 and S2’) that can be cleaved by various proteases. For each protease, this table details its location in the cell (TGN: trans-Golgi network, plasma membrane bound, endosome, exogenously found), the fusion pathway it triggers, and its cleavage sites. Since cleavage at a particular site may not necessarily activate the S protein for fusion, this table distinguishes between sites that are just cleaved versus sites that result in S activation.
| Protease | Location | Pathway | SARS-CoV Sites | MERS-CoV Sites |
|---|---|---|---|---|
| Furin | TGN | Biosynthesis | Does not activate | Cleaves at RSVR (S1/S2) (Kleine-Weber et al., 2018; Millet and Whittaker, 2014) |
| Trypsin | Exogeneous | Plasma Membrane | Activates at R667 (S1/S2), R797 (S2′) sequentially (Belouzard et al., 2009) | Activates at unidentified sites (Shirato et al., 2013) |
| Elastase | Exogeneous | Plasma Membrane | Activates at T795 (S2′) (Belouzard et al., 2009) | Activates at unidentified sites (Shirato et al., 2013) |
| Thermolysin | Exogeneous | Plasma Membrane | Activates at unidentified sites (Matsuyama et al., 2005) | Activates at unidentified sites (Shirato et al., 2013) |
| TMPRSS2 | Membrane Bound | Plasma Membrane | Cleaves at R667 (S1/S2), activates at R797 (S2′) (Reinke et al., 2017) | Activates at RSAR (S2′) Needs prior S1/S2 cleavage (Kleine-Weber et al., 2018) |
| Cathepsin L | Late endosome | Endosomal | Cleaves S1/S2 at T678 (Bosch et al., 2008), activates at unidentified sites | Activates at RSAR (S2′) and unidentified sites (Kleine-Weber et al., 2018) |
The role of the furin cleavage site in the S1/S2 position as it relates to viral tropism and pathogenicity is of interest to the scientific community. In the case of influenza virus, low-pathogenicity influenza strains contain a single basic residue cleavage site, whereas highly pathogenic influenza strains have a polybasic furin cleavage site (Sun et al., 2010). It has been suggested that the presence of furin cleavage sites in influenza HA expands viral tropism, as furin and furin-like proteases are ubiquitously expressed in most cell lines. Thus, it has been hypothesized that the additional furin site present at S1/S2 may also allow SARS-CoV-2 to spread more efficiently than other SARS-like-CoVs (Coutard et al., 2020; Walls et al., 2020), though experimental work will need to be done to show whether this is the case. Of interest is to also determine how SARS-CoV-2 acquired this cleavage site, whether from animal-to-human or human-to-human transmission (Andersen et al., 2020), in order to determine the pathogenicity of emerging coronaviruses.
Trypsin, an exogeneous activator of plasma membrane fusion, has different cleavage requirements than TMPRSS2. For SARS-CoV, trypsin shows elements of a two-step activation process; it first cleaves arginine at the SARS-CoV S1/S2 site (R667), followed by the S2′ site (R797) for fusion (Belouzard et al., 2009), whereas TMPRSS2 does not require the S1/S2 cleavage site. This aspect highlights the flexibility of the CoV FP as it can be cleaved by different proteases resulting in slightly different activities. As further evidence of CoV FP flexibility, elastase cleaves SARS-CoV S at the S2′ site at T795 (Belouzard et al., 2010), which is two residues upstream of the trypsin cleavage site at R797, suggesting that the S protein can even accommodate cleavages at different sites. However, mutagenesis studies have demonstrated that elastase mediates maximal fusion when cleaving at the 797 site, showing that while the S protein is flexible in cleavage location, it has preferred sites. For MERS-CoV and SARS-CoV-2, the exact sites at S1/S2 and S2’ position for exogeneous protease cleavage have yet to be determined and it would be interesting to observe if small variations in the sites exist across different proteases in these cases as well.
Regarding endosomal pathway-related cleavage, it has been suggested that cathepsin L can cleave SARS-CoV at a site slightly downstream of the S1/S2 site at T678, though fusion functional studies have yet to be conducted (Bosch et al., 2008). For MERS-CoV, functional studies have shown that S1/S2 and S2′ sites are dispensable, though cathepsin L activation of MERS-CoV is slightly reduced when the S2′ is mutated (Kleine-Weber et al., 2018). This seems to indicate that cathepsin L may be using auxiliary S1/S2 and/or S2’ sites for activation. It is difficult to screen for these sites since cathepsin has a relatively indiscriminate recognition motif, though studies have shown that cathepsin L prefers aromatic residues at the P2 location (Biniossek et al., 2011). While it seems that SARS-CoV, MERS-CoV, and SARS-CoV-2 exhibit similar behavior when entering via the endocytic route, it is important to emphasize that the cathepsin L cleavage sites are unknown and it is possible that the location or the number of sites vary and could in turn, impact entry efficiency of MERS-CoV or SARS-CoV-2 endocytic route. All in all, the extensive range of proteases that activate SARS-CoV, MERS-CoV, and SARS-CoV-2 attest to their promiscuity in infecting a wide range of cell types that present these proteases and may explain the expanded tropism of these viruses.
3.4. CoV entry remarks
As described above, SARS-CoV, MERS-CoV, and SARS-CoV-2 can enter cells using an early pathway or a late pathway, depending on protease availability and cell type (Fig. 3). If membrane-bound proteases, most notably TMPRSS2, are available on the host cell, they can cleave both viruses for early fusion at the plasma membrane surface after receptor binding. As noted, MERS-CoV also requires the S1/S2 cleavage during biosynthesis for plasma membrane fusion (this has yet to be determined for SARS-CoV-2). Otherwise, the virus is endocytosed and the increasingly acidic conditions can activate cathepsin L to trigger fusion at the endosomal membrane. Table 2 summarizes cell lines susceptible to SARS-CoV and MERS-CoV infection, as well as their infection pathways. Initial studies of SARS-CoV-2-susceptible cell lines can be found in (Hoffmann et al., 2020; Ou et al., 2020). A general rule of thumb is that these coronaviruses will utilize the plasma membrane route in lung cells as TMPRSS is commonly expressed in lung cells and the endosomal route for all other cell types.
Table 2
SARS-CoV and MERS-CoV entry pathways in commonly used cell lines. For each cell line, the pathway that SARS-CoV or MERS-CoV uses to infect that cell line is described. If the pathway is unknown, it is noted if the cell line is susceptible to either SARS-CoV or MERS-CoV or if the cell line has been tested.
| Cell Type | Cell Type | SARS-CoV Infection Notes | MERS-CoV Infection Notes |
|---|---|---|---|
| 16HBE | Human bronchial epithelia | Likely using early pathway (Kam et al., 2009) | Not tested |
| A549 | Human alveolar basal epithelial carcinoma | Lacks ACE2, does not infect (Kam et al., 2009; Simmons et al., 2004) | Weak/No infection (Gierer et al., 2013; Qian et al., 2013) |
| BEAS-2B | Human bronchial epithelial | Lacks ACE2, does not infect (Kam et al., 2009) | Not tested |
| BSC-1 | African Green Monkey respiratory epithelial | Late pathway (Mingo et al., 2015) | Not tested |
| Caco-2 | Human colon epithelial adenocarcinoma | Infects (Mossel et al., 2005) | Early pathway (Gierer et al., 2013; Kleine-Weber et al., 2018) Could use both pathways (Park et al., 2016) |
| Calu-3 | Human bronchial epithelial adenocarcinoma | Early pathway (Kawase et al., 2012) | Early pathway (Park et al., 2016; Shirato et al., 2013) |
| COS-7 | African Green Monkey kidney fibroblast | Lacks ACE2, does not infect (Simmons et al., 2004) Late pathway with ACE2 transfection (Inoue et al., 2007) | Not tested |
| EA-HY | Human endothelium | Not tested | Does not infect (Gierer et al., 2013) |
| HEK293T | Human embryonic kidney | Late pathway (Simmons et al., 2004) Late pathway with ACE2 transfection (Bertram et al., 2011; Huang et al., 2006; Shulla et al., 2011; Wang et al., 2008) | Late pathway with DPP4 transfection (Gierer et al., 2013) |
| HeLa | Human cervix epithelial adenocarcinoma | Late pathway with ACE2 transfection (Kawase et al., 2012) | Lacks DPP4, does not infect (Shirato et al., 2013) |
| HepG2 | Human liver carcinoma | Late pathway (Inoue et al., 2007) | Not tested |
| HOS | Human bone, fibroblast/epithelial osteosarcoma | Does not infect (Simmons et al., 2004) | Infects (Gierer et al., 2013) |
| HT1080 | Human fibrosarcoma | Infects (Simmons et al., 2004) | Not tested |
| Huh-7 | Human liver carcinoma | Infects (Simmons et al., 2004) | Late pathway (Millet and Whittaker, 2014; Park et al., 2016) |
| Human Airway Epithelial | Primary | Not tested | Early pathway (Park et al., 2016) |
| LLCMK2 | Rhesus Macaque kidney epithelial | Not tested | Late pathway (Qian et al., 2013) |
| MRC-5 | Human lung fibroblast | Not tested | Likely late pathway (Gierer et al., 2013; Millet and Whittaker, 2014; Shirato et al., 2013) |
| NHBE | Primary normal human bronchial epithelial cells | Not tested | Late pathway (Millet and Whittaker, 2014) |
| RPE | Human retina epithelial | Not tested | Does not infect (Gierer et al., 2013) |
| U373 | Human glioblastoma | Not tested | Does not infect (Gierer et al., 2013) |
| Vero E6 | African Green Monkey kidney epithelial | Late pathway (Glowacka et al., 2011; Huang et al., 2006; Matsuyama et al., 2010; Simmons et al., 2004) | Late pathway (Kleine-Weber et al., 2018; Millet and Whittaker, 2014; Qian et al., 2013; Shirato et al., 2013) |
| WI-38 | Human lung fibroblast | Not tested | Likely late pathway (Millet and Whittaker, 2014; Shirato et al., 2013) |
Although a variety of triggers exist to dictate the pathway of coronavirus entry and fusion, it is worth considering whether these pathways are unique or redundant. Early studies seem to suggest that the plasma membrane pathway is the preferred route, as it enables the virus to directly enter the cells and disseminate. In fact, it has been shown that exogeneous trypsin treatment enhanced SARS-CoV replication in Vero cells by 100-fold when entering via the plasma membrane route as opposed to the endosomal route (Matsuyama et al., 2005). The delayed viral growth kinetics associated with the endosomal route could be attributed to the 30-minute lag time the virus requires to traffic from the cellular membrane to the endosomal membrane.
Moreover, the plasma membrane pathway has also been implicated as the clinically relevant pathway (Shulla et al., 2011). SARS-CoV, MERS-CoV, and SARS-CoV-2 replicate rapidly in the lungs during disease progression, and lung epithelial cells have been reported to have high TMPRSS2 expression (Donaldson et al., 2002), suggesting that these viruses can employ TMPRSS2 for entry. This view is further supported by observations that SARS-CoV receptor, ACE2, was found to colocalize with TMPRSS2 so that after receptor binding, SARS-CoV can immediately be processed by TMPRSS2 for entry (Shulla et al., 2011). It will be of interest to observe if TMPRSS2 colocalization with ACE2 can also immediately process SARS-CoV-2 entry, and likewise for MERS-CoV, if the MERS-CoV receptor, DPP4, can also colocalize with TMPRSS2 to support plasma membrane fusion.
Further support for the relevance of the plasma membrane pathway comes from experiments with Calu-3 cells, an immortalized human lung cell line reported to have mRNA expression levels of ACE2, DPP4, TMPRSS2, and cathepsin L similar to the human lung, so that they may serve as a good representative model for SARS-CoV, MERS-CoV, and SARS-CoV-2 susceptibility. Using Calu-3 cells, researchers have shown that both SARS-CoV and MERS-CoV infection are mitigated using a TMPRSS2 inhibitor, camostat, but not with a cathepsin inhibitor, EST (Kawase et al., 2012; Shirato et al., 2013). This implies that TMPRSS2-mediated entry is the preferred pathway, since cathepsin inhibitors have minimal impact on entry when the TMPRSS2 route is available. In fact, mice treated with a TMPRSS2 inhibitor had a higher survival rate (60%) than mice treated with a cathepsin inhibitor (10%) when challenged with a lethal dose of SARS-CoV (Zhou et al., 2015). Similar studies also allude to the importance of TMPRSS2 in SARS-CoV-2 infection as camostat significantly reduced both SARS2pp and live SARS-CoV-2 infection of Calu-3 cells (Hoffmann et al., 2020). Interestingly, treatment of Calu-3 cells with both camostat and EST drastically reduced SARS-CoV, but not MERS-CoV infection, much more so than with camostat alone (Kawase et al., 2012; Shirato et al., 2013). This suggests that SARS-CoV can more easily employ the late pathway than MERS-CoV.
These studies bring into question the importance of the late pathway, especially as cathepsin L is not as highly expressed as TMPRSS2 in respiratory cells (Park et al., 2016). Furthermore, for other human coronaviruses, it has been suggested that replication in cell culture may select for cathepsin L activation (Kleine-Weber et al., 2018). Intriguingly, usage of non-lung cell culture lines that support SARS-CoV and MERS-CoV infection, such as Vero E6 (SARS-CoV/SARS-CoV-2) or Huh-7 (MERS-CoV) have demonstrated that the cathepsin L pathway is much more prominent as these cells express minimal TMPRSS2 and infection is strongly reduced in the presence of cathepsin inhibitors (Hoffmann et al., 2020; Park et al., 2016; Simmons et al., 2005). While the TMPRSS2 route seems to hold higher clinical significance, it is important to consider that in the later stages of SARS-CoV and MERS-CoV infection, the virus may spread beyond the lungs, especially as MERS-CoV infection may result in kidney failure. Thus, studies of other cellular systems are highly valued to enable comprehensive understanding SARS-CoV and MERS-CoV entry pathways and how it depends on cell type.
4. S protein membrane fusion
Viral membrane fusion is the process by which enveloped viruses merge their membrane with the host cell membrane so that the virus can deliver its genome in the cell, resulting in the eventual production of new virions (Harrison, 2015, 2008). Coronavirus membrane fusion occurs after receptor binding, so that both its membrane and the host cell membrane are proximal. However, membrane fusion is not a spontaneous process, as there are high energy requirements to bring the membranes close together (Cohen and Melikyan, 2004; Martens and McMahon, 2008). It is the role of the viral fusion protein to serve as the catalyst by providing the energy requirement to drive this reaction.
Viral fusion proteins are grouped into three distinct classes (I, II and III) based on their structure and function (White et al., 2008; White and Whittaker, 2016). As mentioned previously, the coronavirus S protein is a class I fusion protein, due to the structural characteristics of its fusion domain, the need for protease cleavage to be fusion-competent and the presence of heptad repeats that fold into a six-helix bundle (Bosch et al., 2003). Class I fusion proteins catalyze the membrane fusion reaction though a sequence of states: (1) pre-fusion native state, (2) pre-fusion metastable state, (3) pre-hairpin intermediate state, (4) post-fusion stable state (Fig. 4 ). As the S protein is synthesized, it adopts the pre-fusion native state. Proteolytic processing at the S1/S2 will cause the S protein to adopt a pre-fusion metastable state. This priming event generates separate S1 and S2 domains, which are then non-covalently associated. (Tripet et al., 2004).
Coronavirus viral fusion pathway model based on class I fusion protein understanding. The captions above the figure describe the state of the fusion protein, the captions below describe the state of the membranes. The S protein starts in the pre-fusion native state (1) and undergoes priming of the S1 subunit by relevant proteases to achieve the pre-fusion metastable state (2). Subsequent triggering by relevant proteases will enable the FP to insert in the host membrane and allow the S protein to form the pre-hairpin intermediate (3). The pre-hairpin begins to fold back on itself due to HR1 and HR2 interactions forming the pre-bundle (4), bundle (5), and eventual post-fusion stable (6) states. During the S protein foldback, the two membranes will approach each other until the outer leaflets merge (hemifusion) and eventually the inner leaflets merge (pore formation). Adapted from (White and Whittaker, 2016).
In this metastable state, the fusion protein must overcome a kinetic barrier to transition to the next state. The energy to overcome this barrier can be provided by a trigger that will interact with the fusion protein, resulting in a series of conformational changes that will enable the fusion protein to insert its FP into the host membrane, forming a pre-hairpin intermediate state. The triggering event(s) are usually environmental cues that inform the virus about its microenvironment. As an example, the influenza virus fusion protein is triggered by low pH; as the virus is trafficked through the endosome, the increasingly acidic conditions eventually destabilize its fusion protein, so that the fusion peptide is able to insert into the endosomal membrane and commence the fusion process (Carr and Kim, 1993). Since the triggering step initiates the fusion cascade, it is very well regulated to ensure that the virus fuses in an appropriate location. This is especially important as class I viral fusion proteins are generally not reversible; they should not be triggered to fuse at a non-optimal condition (i.e. not bound to a cell). Furthermore, the triggering event also exposes the influenza fusion peptide, as it is buried within the subunit prior to trigger to protect its hydrophobic nature from surrounding aqueous environment. However, for SARS-CoV and MERS-CoV, recently determined cryo-EM structures have shown that both of their FPs are partially exposed at the surface (Yuan et al., 2017). This seems to be a feature of coronaviruses, as the murine hepatitis virus fusion peptide is also exposed in its prefusion state (Walls et al., 2016).
After insertion of the FP, the three HR1 regions assemble into a coiled-coil trimer and three HR2 regions bind to the hydrophobic grooves of the HR1 trimer in an antiparallel manner. (Guillén et al., 2005). The assembly of the HR1 and HR2 domains is known as the fusion core or six-helix bundle, and it is this conformational rearrangement that pulls the viral and host cell membrane into proximity so that they can fuse, bringing the fusion protein into a stable post-fusion state. The process of membrane fusion itself is composed of two stages: hemifusion and pore formation (Fig. 4). The hemifusion stage is defined as the merging of only the outer leaflets of the opposing membranes, with still distinct inner leaflets and no content mixing. The hemifusion stage is considered a transient intermediate that will either dissociate and form two separate vesicles or proceed to pore formation (Lentz et al., 2000). In pore formation, both outer and inner leaflets are merged, forming a connection between the interior of the virus and the host cell cytoplasm, so that the virus can transfer its genetic material through the newly formed pore. Additional reviews on the mechanics of (viral) membrane fusion are (Chernomordik and Kozlov, 2003, 2008; Harrison, 2015).
4.1. Role of cholesterol in fusion
The membrane fusion reaction is dependent on the lipid composition of the viral and/or cellular membranes (Chernomordik and Kozlov, 2008). In both of them, sphingolipids and cholesterol molecules tend to pack together and form microdomains termed “lipid rafts” floating within the "sea" of phospholipids (Simons and Ikonen, 1997). Sphingolipids possess long, saturated acyl chains that have reduced Van der Waals interactions with the unsaturated acyl chains of phospholipids in membranes, due to the inability to align the unsaturated carbon chains. Cholesterol can fill gaps between associating sphingolipids, leading to tighter packing and ordering of cholesterol and sphingolipids that gives lipids rafts detergent-resistance above their non-raft counterparts. The high lipid tail ordering in rafts also influences the distribution of protein and other lipids (Munro, 2003). As an example, GPI-anchored or palmitoylated proteins tend to be raft-associated (Levental et al., 2010) and sphingolipids like GM1 tend to also be enriched in lipid rafts.
Studies on the role of lipid rafts in viral membrane fusion have shown two major themes: (1) viral transmembrane receptors may be concentrated within rafts and serve as "hotspots" for viral entry, and (2) cholesterol in rafts promotes fusion by reducing the energy needed to form fusion intermediates (Yang et al., 2016). For human immunodeficiency virus (HIV), membrane fusion requires multiple fusion protein and receptor interactions, and rafts have been proposed as a means to enrich fusion proteins and receptors for viral entry. Biophysical studies have shown HIV fusion protein interactions with raft domain lipids (Hammache et al., 1999). In vitro studies treating HIV particles with a cholesterol sequestering drug, β-cyclodextrin, was shown to inactivate the virus by rendering it incapable of membrane fusion (Liao et al., 2001), highlighting a potential fusion inhibition strategy.
Attempts to understand CoV interactions with lipid rafts have met with limited success. For SARS-CoV, the location of its ACE2 receptor in rafts is still controversial. Earlier studies showed that ACE2 was found in non-raft fractions in CHO cells transiently expressing it and Vero E6 cells endogenously expressing it (Li et al., 2007; Warner et al., 2005). Other studies have observed that ACE2 colocalizes with established raft proteins caveolin-1, flotillin-2, and ganglioside GM1 in Vero E6 cells (Glende et al., 2008; Lu et al., 2008), with the discrepancies between these results attributed to different experimental techniques (Glende et al., 2008). For the study with transiently expressed ACE2 in CHO cells, overexpression could cause ACE2 to also partition into non-raft areas. For the studies with Vero E6 cells, endogenous expression of ACE2 and its distribution depend on the time point following cell seeding and it is possible that the different groups might have measured ACE2 location at different time points. Further studies on the influence of cholesterol on SARS-CoV infectivity have strengthened the notion that ACE2 is a raft protein (Glende et al., 2008; Lu et al., 2008). Treating Vero cells with a cholesterol-chelating drug, methyl-β-cyclodextrin (mβCD) that disrupts raft formation, has shown that SARS-CoV can still infect in the absence of lipid rafts. It was observed that SARSpp infection was inhibited 60–90% in cells following mβCD treatment. A possible explanation is that ACE2 is a raft protein. By disrupting raft formation, ACE2 is no longer concentrated in microdomains, and this reduced receptor availability lessens SARS-CoV docking and binding efficiency. It will be of interest to observe if SARS-CoV-2 infectivity is similarly inhibited by this drug treatment.
In summary, it is suggested that ACE2 is a raft protein and SARS-CoV entry requires cholesterol. However, there remains a number of questions on the mechanism by which cholesterol is influencing infection. Rather than simply organizing receptors into rafts, it is possible that cholesterol is directly influencing membrane fusion dynamics, by encouraging fusion intermediate formation, but formal evidence for this role for coronaviruses has not been obtained. With regard to MERS-CoV, its DPP4 receptor has been reported to partition into the raft fraction, in Jurkat cells, a human T cell line (Ishii et al., 2001). It would be interesting to observe if DPP4 also partitions into the raft phase in MERS-CoV susceptible cell lines and to determine the dependence of MERS-CoV entry on cholesterol.
4.2. Role of calcium in fusion
As previously discussed, the extracellular environment heavily influences CoV membrane fusion. Extracellular proteases and pH both serve direct and/or indirect roles in enabling SARS-CoV and MERS-CoV fusion. Recently, the ion content of the cellular environment has been investigated for its role in viral fusion, as ions have been shown to be crucial for other membrane fusion processes, such as synaptic vesicle fusion (Martens and McMahon, 2008). Initial studies have discovered that the rubella virus fusion machinery coordinates with calcium ions (Ca2+) for proper orientation and insertion into the host membrane (Dubé et al., 2014). This coordination/requirement was specific to Ca2+ as both fusion assays and infectivity experiments demonstrated that magnesium, manganese, and zinc cations did not enable fusion when supplemented at the same concentration as Ca2+ for rubella virus. Later it was shown by (Nathan et al., 2019) that the Ebola virus fusion machinery also coordinates with Ca2+ in entry, further corroborated by (Das et al., 2020).
Inspired by these studies, we investigated whether the infectivity of SARS-CoV and MERS-CoV was also influenced by Ca2+ and if this is also correlated to the entry steps. By employing extracellular and/or intracellular calcium chelating compounds, it is possible to infect cells in Ca2+ depleted conditions and probe resulting infectivity (Dubé et al., 2016). Infectivity assays with SARSpp and MERSpp demonstrated that SARS-CoV and MERS-CoV entry into Vero e6 and Huh7 cell lines was reduced when intracellular calcium was chelated (Lai et al., 2017; Straus et al., 2019). These results suggest that SARS-CoV and MERS-CoV entry into cells using the endocytic route has aspects of calcium dependence. Ca2+ interactions crucial for CoV entry were further explored with biophysical techniques. Using electron spin resonance (ESR), it was observed that both SARS-CoV and MERS-CoV FP induces the ordering of the lipid acyl chains upon interaction, termed membrane ordering. Increased membrane ordering aids fusion by increasing the negative curvature of the bilayer as it bends during the fusion process, which will reduce the repulsive energy between two opposing membranes (Ge and Freed, 2009). In the presence of Ca2+, the FPs are able to induce greater membrane ordering, indicating that Ca2+ may promote fusion by stabilizing a structure of the FP that organizes the lipids in a manner that facilities membrane merging.
Although both SARS-CoV and MERS-CoV FP interact with calcium, there are differences in their interactions; the SARS-CoV FP was seen to inducer greater membrane-ordering effects than the MERS-CoV FP (Straus et al., 2019). This aspect was manifested in infectivity experiments showing that SARSpp infection was reduced more compared to MERSpp infection when Ca2+ was chelated. Further evidence highlighting the differences between these CoVs comes from isothermal calorimetry titration (ITC) experiments demonstrating that MERS-CoV FP binds one Ca2+ ion, whereas the SARS-CoV FP binds two. After partitioning the SARS-CoV FP domain into two separate domains, FP1 and FP2, it was determined using ESR experiments that each of these domains induces greater membrane order in the presence of Ca2+, suggesting that SARS-CoV FP binds Ca2+ in both the FP1 and FP2 domain. In contrast, mutagenesis experiments conducted on the MERS-CoV FP suggested only one negatively-charged amino acid (E891) in the FP1 region that could bind Ca2+. Although these experiments suggest that MERS-CoV FP can only bind one Ca2+ in its FP1 region, while SARS-CoV FP can bind two, one in each of its FP1 and FP2 regions, further experiments should be conducted to complete this data set. ESR experiments should verify that only the MERS-CoV FP1, not FP2, has a membrane-ordering effect dependent on Ca2+ and mutagenesis experiments should find sites in the SARS-CoV FP1 and FP2 domain that bind Ca2+. Regardless, it is remarkable that despite exhibiting such high sequence conservation, there are subtle differences between SARS-CoV and MERS-CoV entry requirements.
5. SARS-CoV and MERS-CoV current antifusogenics
As previously noted, after insertion of the viral fusion peptide in the target cellular membrane, the more N terminal heptad repeat region (HR1) folds into a trimeric helical coiled-coil structure. Subsequently, the C-terminal heptad repeat region (HR2) dissociates into monomers and packs against the grooves of the HR1 trimer in an antiparallel manner (Peisajovich and Shai, 2003). This packing is driven by hydrophobic interactions between HR1 and HR2 and forms the viral fusion core, also known as the six-helix bundle. The formation of the fusion core brings the viral and target membranes together so membrane fusion can commence. Thus, disrupting fusion core formation would seem to be a valid anti-fusion strategy (Fig. 5 ).
Model of major antiviral inhibitor pathway. This model depicts the inhibitory mechanism of a major CoV inhibitory peptide: HR2 peptide. Exogeneous HR2 peptides present during the CoV membrane fusion can competitively bind with CoV HR1. This prevents CoV HR2 from locking with HR1 and arrests the membrane fusion reaction, subsequently preventing pore formation.
In the early 1990s, two groups discovered that peptides derived from the HIV-1 gp41 HR2 region could bind to the HR1 region in the pre-intermediate state, preventing the gp41 HR2 from binding and forming the fusion core (Jiang et al., 1993; Wild et al., 1994). By competitively binding to the HR1 region, these HR2 peptides potently inhibited HIV-1 infection at nanomolar concentrations. One of these peptides, DP-178, was tested in proof of principle clinical studies and demonstrated that patients receiving peptide treatment displayed viral load reduction. Further clinical studies established the long-term safety and efficiency of DP-178 and eventually it was approved by the US Food and Drug Administration for HIV/AIDS treatment named Fuzeon (enfuvirtide) as the first fusion inhibitor drug (Matthews et al., 2004).
As these coronaviruses utilize the fusion core to drive membrane fusion, it is worth considering whether CoV fusion could be arrested using HR2-derived peptides. Fuzeon's inhibition potential is specific to HIV-1, as studies with the related HIV-2 were not as promising (Wild et al., 1994). This is due to sequence variation in the HR regions between these two viruses and so CoV specific HR peptides are required for inhibiting CoV fusion. Shortly after the SARS outbreak in 2003, three independent groups designed peptides based on the SARS-CoV HR2 region and found that they were inhibitory (Bosch et al., 2004; Liu et al., 2004; Yuan et al., 2004). Circular-dichroism (CD), a technique that reports on the structural and disordered content of a peptide, showed that HR2 peptides bound to viral HR1 regions have more alpha-helical content than only HR2, suggesting that HR2 peptides form a stable structure when bound to HR1 (Bosch et al., 2004; Liu et al., 2004). These results demonstrate that HR2 peptides can compete with the virus fusion protein's own HR2 region to bind HR1 and prevent fusion. A similar tale can be told for MERS-CoV HR2-based peptides. Two groups investigated MERS-CoV fusion core formation and found that an analogous HR2 peptide inhibited infection (Gao et al., 2013; Lu et al., 2014). CD experiments proved that the HR2 peptide bound to HR1, suggesting that the inhibition mechanism is identical to that of SARS-CoV HR2 and HIV HR2 peptides (Lu et al., 2014).
Sequence alignment of HR1 and HR2 region between SARS-CoV and SARS-CoV-2 show 92.6% and 100% sequence homology, respectively, suggesting that HR2 peptides may also inhibit SARS-CoV-2. Preliminary studies with analogous SARS-CoV-2 HR2 peptides have displayed similar inhibitory behavior in blocking SARS2pp infection of ACE2 expressing cells. CD experiments further confirmed SARS-CoV-2 HR1 and HR2 interact, exhibiting alpha-helical content characteristic of six-helix bundle formation (Xia et al., 2020). Detailed information regarding the peptide sequence and its half maximal inhibitory concentration values (IC50) or related values can be found in Table 3 .
Table 3
SARS-CoV and MERS-CoV HR2 based inhibitory peptides data. The inhibition data describes IC or analogous values reported for the various inhibitors in blocking infection of SARS-CoV or MERS-CoV infection. As the procedures to test inhibition varied among different groups, the different viral strains and cell lines are noted. The * for P21S10 refers to the position of the residue used to form the staple.
| Virus | Peptide Name | Sequence | Inhibition Data | References |
|---|---|---|---|---|
| SARS-CoV | HR2-8 | ELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK | EC50: 17 μM (SARS-CoV Strain 5688 infection of Vero E6 cells) | Bosch et al. (2004) |
| SARS-CoV | CP-1 | GINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYE | IC50: 19 μM (SARS-CoV Strain WHU infection of Vero E6 cells) | Liu et al. (2004) |
| SARS-CoV | HR1-1 | NGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTA | EC50: 0.14 μM (HIV pseudotyped SARS-CoV S infection of VeroE6 cells) EC50: 3.68 μM (SARS-CoV Strain BJ01 infection of Vero E6 cells) | Yuan et al. (2004) |
| SARS-CoV | HR2-18 | IQKEIDRLNEVAKNLNESLIDLQELGK | EC50: 1.19 μM (HIV pseudotyped SARS-CoV S infection of VeroE6 cells) EC50: 5.22 μM (SARS-CoV Strain BJ01 infection of Vero E6 cells) | Yuan et al. (2004) |
| SARS-CoV | P6 | YQDVNCTDVSTAIHADQLTP | IC90: 113 μM (SARS-CoV Strain GZ50 infection of FRhK-4 cells) | Zheng et al. (2005) |
| SARS-CoV | P8 | QYGSFCTQLNRALSGIAAEQ | IC90: 24.9 μM (SARS-CoV Strain GZ50 infection of FRhK-4 cells) | Zheng et al. (2005) |
| SARS-CoV | P10 | IQKEIDRLNEVAKNLNESLI | IC90: 73.5 μM (SARS-CoV Strain GZ50 infection of FRhK-4 cells) | Zheng et al. (2005) |
| SARS-CoV | P6+P8+P10 | IC90: 0.9 μM (SARS-CoV Strain GZ50 infection of FRhK-4 cells | Zheng et al. (2005) | |
| SARS-CoV | SR9 | ISGINASVVNIQKEIRLNEVAKNLNESLIDLQEL | EC50: <100 nM (SARS-CoV infection of Vero E6 cells with trypsin treatment) | Ujike et al. (2008) |
| SARS-CoV | HR2P | DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL | IC50: 2.81 μM (HIV pseudotyped SARS-CoV S infection of ACE2/293T cells) | Xia et al. (2019) |
| MERS-CoV | P1 | LTQINTTLLDLTYEMLSLQQVVKALNESYIDLKEL | IC50: 3 μM (HIV pseudotyped MERS-CoV S infection of Huh7 cells) | Gao et al. (2013) |
| MERS-CoV | HR2P | SLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKEL | IC50: 0.6 μM (MERS-CoV EMC/2012 infection of Huh7 cells) IC50: 0.6 μM (MERS-CoV EMC/2012 infection of Calu-3 cells) IC50: 13.9 μM (MERS-CoV EMC/2012 infection of HFL cells) | Lu et al. (2014) |
| MERS-CoV | HR2P-M2 | SLTQINTTLLDLEYEMKKLEEVVKKLEESYIDLKEL | IC50: 0.55 μM (MERS-CoV S mediated HEK293T and Huh7 cell-cell fusion) | Lu et al. (2014) |
| MERS-CoV | P21S10 | LDLTYEMLSLQQVV K*LNE*Y | EC50: 3.03 μM (HIV pseudotyped MERS-CoV S infection of Huh7 cells) | Wang et al. (2018) |
| SARS-CoV-2 | 2019-nCoV-HR2P | DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL | IC50: 0.98 μM (Pseudotyped SARS-CoV-2 S infection of ACE2/293T cells) | Xia et al. (2020) |
| Pan-CoV | EK1 | SLDQINVTFLDLEYEMKLEEAIKLEESYIDLKEL | IC50: 0.26 μM (HIV pseudotyped MERS-CoV S infection of Huh7) IC50: 0.62 μM (MERS-CoV Strain EMC/2012 infection of Calu-3 cells) IC50: 2.23 μM (HIV pseudotyped SARS-CoV S infection of ACE2/293T cells) IC50: 2.38 μM (Pseudotyped SARS-CoV-2 S infection of ACE2/293T cells) | (Xia et al., 2020, 2019) |
Although the inhibition mechanism is identical, it is observed that MERS-CoV HR2 peptides inhibited MERS-CoV replication in Vero cells 32-fold more than their SARS-CoV counterpart (Lu et al., 2014). Crystal structures of the SARS-CoV and MERS-CoV fusion cores showed that residue differences in the HR1 and HR2 region enabled MERS-CoV to form more hydrogen bonds in the fusion core, suggesting that MERS-CoV HR2 peptides have a greater binding affinity for the HR1 region compared with SARS-CoV HR2 peptides. Another explanation focuses on the differences in the fusion pathways between the two viruses. In the cell lines used, MERS-CoV is hypothesized to infect via the plasma membrane route, whereas SARS-CoV infects via the endosomal route. Since the peptides are likely more available in the extracellular space than in an endosome, this could explain why MERS-CoV is more sensitive to HR2 inhibitors. In fact, one study observed that the SARS-CoV HR2 peptide did block SARS-CoV infection of Vero E6 cells more potently when trypsin treatment caused SARS-CoV to utilize the plasma membrane pathway, rather than the endosomal pathway (Ujike et al., 2008). To be more conclusive, it would be important to study the efficacy of these peptides in inhibiting infection of lung-derived cell lines, such as Calu-3, since they could more accurately recapitulate in vivo infection. This would also shed light on the viral entry pathway that these peptides inhibit and how efficient they block viral entry at the plasma membrane pathway versus the endosomal pathway.
A common theme amongst these studies and those of other enveloped viruses is that only the HR2 peptide can bind viral HR1 in an inhibitory manner; analogous HR1 peptides exhibit no antiviral capacity. This is because singular HR1 peptides tend to form self-associated oligomers and aggregate in solution (Lu et al., 1995). However, by noting that the major HR1 helical content resulting in oligomerization was located in the downstream sections of HR1, one group was able to report success with a SARS-CoV HR1 peptide derived from upstream sections of HR1 (Yuan et al., 2004). CD experiments confirmed that this peptide has low α-helical content, preventing self-oligomerization.
These studies also demonstrated that the SARS-CoV and MERS-CoV HR2 peptides are not cross reactive: SARS-CoV HR2 peptides do not inhibit MERS-CoV infection, and vice versa (Gao et al., 2013; Lu et al., 2014). Recently, one group challenged this notion and screened HR2-derived peptides from all human-infecting CoV (HCoV), including SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43 (OC43), and HCoV-HKU1 (Xia et al., 2019). They found that the HR2 peptide from HCoV-OC43 inhibited all these aforementioned CoVs. The unique pan inhibition property can be attributed to key amino acid differences that strengthen HR1-HR2 interactions. As an example, the crystal structures of MERS-CoV and SARS-CoV HR1 interaction with the OC43 HR2 peptide showed that the OC43 HR2 peptide has a valine that fits neatly into the hydrophobic pockets of MERS-CoV and SARS-CoV HR1. The corresponding SARS-CoV and MERS-CoV HR2 residues, alanine and threonine, fit the pocket loosely, or not at all. This study also found that the OC43 peptide inhibits SARS-like CoV, which have reservoir in bats and recognize human receptors (Ge et al., 2013; Menachery et al., 2016). This suggests that the OC43 peptide exhibits promise as a treatment during coronavirus outbreaks.
While there is great potential towards developing CoV HR2 peptides, especially the OC43 peptide as a CoV therapeutic, there remains work to improve the IC50 values, which are roughly three order of magnitude higher than the IC50 values reported for HIV HR2 peptides (Du et al., 2009). Strategies aimed at improving IC50 and pharmacokinetic values have included introducing mutations or adding conjugates. It has been previously reported that introducing negatively and positively charged amino acids Glu (E) and Lys (K) with three or four residues in between them can form E-K or K-E salt bridges that enhance the peptide's stability, solubility, and antiviral activity (Lu et al., 2014; Marqusee and Baldwin, 1987; Otaka et al., 2002). Combining these mutations with others not involved in HR1 binding on the OC43 HR2 peptide resulted in decreased IC50 and increased solubility values. This mutated peptide, named EK1, was found to be more inhibitory than the native HR2 peptide for SARS-CoV (IC50 values of 2.23 and 2.81, respectively) and MERS-CoV (IC50 values of 0.26 and 1.06, respectively) pseudovirus infection. EK1 was also evaluated for its ability to protect mice against live MERS-CoV challenge. Infection was uniformly lethal for untreated mice, while those treated either prophylactically or therapeutically had 100% and 75% survival, respectively (Xia et al., 2019). Most promisingly, EK1 also significantly inhibits SARS-CoV-2 pseudoparticle infection, though slightly less potent than its native HR2 peptide (IC50 values of 2.38 and 0.98, respectively) (Xia et al., 2020). Regardless, these results highlight fusion inhibitory compounds as a promising strategy to block coronavirus infections.
Additional efforts sought to decrease HR2 IC50 values by improving peptide localization to the virus. One group conjugated a MERS-CoV HR2 peptide, HR2P-M2, to a monoclonal antibody that recognizes the MERS-CoV S RBD region. This showed potent synergism in blocking both receptor binding and membrane fusion by inhibiting MERS-CoV S-mediated cell-cell fusion as well as MERSpp infection (Wang et al., 2019). Furthermore, since CoV receptors and proteases tend to partition in lipid-enriched domains, another group tested the possibility of directing HR2 peptides towards these lipid-enriched domains where CoV entry occurs. Conjugating palmitate, which partitions into lipid rafts, to HR2P-M2 was found to increase inhibition of SARSpp and MERSpp cell entry (Park and Gallagher, 2017). On the other hand, conjugating tocopherol, which partitions into non-raft domains, to HR2P-M2 had minimal impact on cell entry. An intriguing aspect of this work is that these palmitate-conjugated peptides also strongly inhibited CoV entry via the endosomal route, which is believed to be relatively resistant to the inhibitory action of these peptides. Indeed, immunofluorescence microscopy showed that lipid-tagged peptides were detected intracellularly, whereas non-lipid-tagged peptides were not detected on cells.
Additional efforts sought to improve the pharmacokinetic values which describe the living organism's response to a drug of HR2P-M2 and can include parameters such as drug bioavailability, metabolism, or half-life. Peptide stapling is one technique, which generates a "brace" that locks a specific conformation for the peptide, resulting in increased target affinity, cell permeability, and serum stability (Frank et al., 2014; Hojo et al., 2016). For HR2 peptides, peptide stapling can stabilize the α-helical nature of the peptide, which is critical to their potency. An all-hydrocarbon peptide stapling of HR2P-M2, named P21S10, resulted in a 27-fold increase in the area under the plasma concentration-time curve value, which measures the total systemic exposure to a drug, while maintaining a similar EC50 and in vivo half-life when compared to HR2P-M2 (Wang et al., 2018). An alternative strategy includes conjugating peptides to gold nanorods. Nanorods have emerged as a promising drug delivery system for improving pharmacokinetic values. One group conjugated HR2-based peptides to a gold nanorod with a polyethylene glycol coating (PIH-AuNR), which demonstrated a 10-fold inhibition increase of MERS-CoV S mediated cell-cell fusion and longer inhibitory activity over its non-conjugated counterpart. In vivo studies demonstrated that PIH-AuNR are biocompatible with mice; over 12 days, there were no significant differences in body weight and behavioral abnormalities between PIH-AuNR treated and control groups (Huang et al., 2019).
An alternative strategy of using peptides to target non-HR regions has also been studied, albeit to a limited degree. While screening a variety of peptide fragments spanning the SARS-CoV S protein for SARS-CoV infection inhibitory activity, researchers identified inhibitory peptides P6, P8, and P10. P10 was derived from the HR2 region, and thus its inhibitory capabilities results from blocking 6-HB formation (Zheng et al., 2005). Intriguingly, P6 and P8 were derived from regions slightly upstream of the S1/S2 and S2’ cleavage sites, which suggest that non-HR regions may be of interest as an anti-fusogenic strategy. The authors hypothesized that the antiviral activity of P6 could result from interference with S1/S2 cleavage, preventing conformational changes for fusion.
Although these studies demonstrate that anti-fusogenic peptides can inhibit pathogenic coronavirus infection, they have not been evaluated for use in humans. There are in vivo mouse trials with the HR2P-M2 peptide (Channappanavar et al., 2015; Jiang et al., 2015), though only time will tell if these peptides will become FDA-approved. An alternative strategy is to screen FDA-approved drugs for their ability to inhibit CoV membrane fusion because the time frame and monetary cost associated with repurposing approved drugs is generally less than developing novel drugs. An initial screen identified imatinib, an Abelson kinase inhibitor (Dyall et al., 2014). Abelson kinases are tyrosine kinases that regulate a variety of cellular pathways and have been previously shown to inhibit viral replication of other viruses, such as Ebola virus, coxsackie virus, and vaccinia virus, at different stages. Subsequent studies demonstrated that imatinib inhibits SARS-CoV and MERS-CoV pseudovirus fusion with BSC-1 and Huh7 endosomal membranes, respectively (Coleman et al., 2016). As Abelson kinases are involved in pathways that enable cytoskeleton rearrangements, it is hypothesized that they inhibit fusion by preventing key cytoskeleton rearrangements for fusion (Sisk et al., 2018).
6. Next steps in research
SARS-CoV, MERS-CoV, and the emerging SARS-CoV-2 cell entry is governed by the S protein. The conserved nature of the fusion mechanism, as well as the fusion subunit sequence, makes it a potentially valuable target for developing a pan-CoV therapeutic. Increased understanding of the fusion mechanism is necessary to know which critical areas to target.
While the surface route is believed to be more clinically relevant, as lung cells express TMPRSS2, this will need to be verified with in vivo experiments. TMPRSS2 knockout mice demonstrated less severe immunopathology when infected with SARS-CoV or MERS-CoV, however, at least for SARS-CoV, viral spread was still detected in the alveoli (Iwata-Yoshikawa et al., 2019). This suggest that other proteases, such as cathepsin L, could be activating the virus, and that the endosomal route may hold clinical relevance. Thus, it is important to expand research efforts to understand the role of these other proteases. As a starting point, identifying the elusive cathepsin L cleavage sites would provide greater insight into additional fusion-relevant domains within the S protein. Furthermore, both MERS-CoV and SARS-CoV-2 have predicted cleavage sites at the S1/S2 and S2’ positions (Coutard et al., 2020; Millet and Whittaker, 2014; Walls et al., 2020; Wrapp et al., 2020). Although furin has been shown to process the MERS-CoV S1/S2 site during biosynthesis, the role of furin or furin-like proteases for SARS-CoV-2 entry is currently unknown, but will likely fundamentally affect viral entry. It is also important to note that key in vitro results should be corroborated with in vivo experiments, as knowledge of clinically relevant routes, proteases, and S domains can guide therapeutic development to block viral entry. In a similar manner, studies to determine the impact of cholesterol and Ca2+ on SARS-CoV-2 would be of interest to guide novel antiviral strategies.
Current studies on anti-CoV fusogenics have identified HR2 peptides as a promising countermeasure. Studies with the EK1 peptide have demonstrated its potential effectiveness as a pan-CoV solution, as it inhibited SARS-CoV, MERS-CoV, and SARS-CoV-2 pseudoparticle infection with IC50 values around 2 μM (Xia et al., 2019, 2020). Furthermore, in vivo studies have shown that EK1 protects mice from MERS-CoV challenge when administered either prophylactically or therapeutically, with 100% or 75% survival, respectively. As the treatment group was administered EK1 peptides 30 min post-infection, it would be of interest to observe the effectiveness of EK1 on a clinical timescale, perhaps several days after infection, to assess its therapeutic potential.
To address the pandemic caused by SARS-CoV-2 and future coronaviruses rapidly, it is important to continue studies on their entry mechanism, which can guide novel therapeutic efforts. Many aspects of the viral fusion reaction (S domains, proteases, lipid compositions, ionic environments) may be valuable targets in arresting membrane fusion by a variety of agents (small molecules, antibodies, peptides). The research community is poised to see what novel therapeutics can address CoV infections.
Acknowledgements
The authors express their gratitude to members of the Daniel and Whittaker groups for their helpful discussions. We have received financial support for coronavirus-related projects from National Institutes of Health research grant R01AI35270. TT is supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE-1650441.
Footnotes
Appendix ASupplementary data to this article can be found online at https://doi.org/10.1016/j.antiviral.2020.104792.
Appendix A. Supplementary data
The following is the Supplementary data to this article:
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