1.5  Where are these important functions in SARS-CoV and SARS-CoV-2?
Even though such glycan binding domains and enzymes as neuraminidases are found in many coronaviruses, there seems to be no such enzymes in SARS-CoV and SARS-CoV-2. Viruses of the lower respiratory tract, such as influenza virus, respiratory syncytial virus, and SARS-related coronaviruses, are generally considered as having key differences that require different therapeutics [10] even though relatively little is lost in considering already approved drugs for one of such viruses against the other (e.g. Refs. [11]). Typically, the apparent absence of glycan binding and enzymic sites in SARS-CoV and SARS-CoV-2 has been dismissed as due to the fact that the virus enters on ACE2, i.e. angiotensin converting enzyme type 2 (e.g. see Ref. [5] for discussion), not on a glycoprotein. This does not, however, escape from the intuitively important need for preliminary binding, cell entry and exit through the glycan layer, and probably the decoy-related function discussed above. There appears to be growing evidence of significant lectin-binding capability. Lectins are the carbohydrate-binding proteins that are highly specific for sugar groups of other molecules. Activation of C-type lectin receptor and other similar receptors contributes to pro-inflammatory response to many coronavirus infections. There also are studies over several years that locate glycan binding and even related catalytic activities in the spike glycoprotein. It has been noted that E3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity [8], and the 3D structure of coronavirus hemagglutinin-esterase offered insight into coronavirus and influenza virus evolution, with implications for drug and antibody discovery [9].
The location of any sialic acid glycan binding region of SARS-CoV-2 is, a priori unclear, although intuitively (a) it would likely be associated with the cap or knob at the outer end of the spike protein, or (b) at least not involve exactly the same domain as is required for other important functions. Although throughout the coronaviruses various external proteins and domains can recognize either protein or sugar receptors or both, the majority of such studies like those above implicate the S1 region in their spike glycoproteins, but as discussed in the present paper, there are other potential sugar binding sites that are still within the spike protein. Overall, the SARS-CoV-2 spike glycoprotein has 1273 amino acid residues and until early 2020 understanding of structure was heavily based on SARS-CoV spike glycoprotein (1255 amino acids) with 20–27% amino acid residues similarity among non-SARS coronaviruses. Most of the spike protein appears to be involved in the specific stages of cell entry. The spike glycoprotein of SARS-CoV and SARS-CoV-2 is translated as a large polypeptide that is later cleaved to S1 and S2 sites. After binding to the main receptor that that is held to be primarily ACE2, the host proteases activate the virus by cleaving first at the S1/S2 boundary (i.e. S1/S2 site) and then within S2, i.e. at the S2’ site. The spike of similar coronavirus have long been considered as being in two main states (i) the pre-fusion form (the form of the mature virion) and (ii) post-fusion form, the form after membrane fusion has been completed). More detailed studies have split the latter into a pre-hairpin intermediate state, and post-fusion hairpin state. Somewhat like in all virus Class I fusion proteins, the S2 protein contains two heptad repeat regions (HRs) of which one (HR2) is located close to the transmembrane anchor. Membrane fusion occurs when there is a conformational change in the HRs to form a fusion core. The HRs of the protein fold into a coiled-coil structure, known as the “fusogenic state”. As virus and target cell membranes fuse, the coiled coil regions (called heptad repeats) become a trimer-of-hairpins structure. The S2’ cleavage site appears particularly important by being well conserved [[3], [4], [5]] and proteolysis by cathepsin appears sufficient to expose the fusion peptide of S2 and activate fusion within the host cell endosome. In general, S2’ is now considered as the key viral fusion peptide which is unmasked following S2 cleavage. Subsequently, S1 dissociates from S2, allowing S2 to transition to the post-fusion structure.
The following locations in the sequences of amino acid residues apply specifically to SARS-CoV-2. These vary somewhat with author, and the following are used here. The signal peptide (SP) comprises residues 1–19. On the inside of the lipid membrane, the carboxyl terminus (C-terminus) is comprised of the transmembrane region (TM) comprising residues 1214–1236 and the cytoplasmic tail (CT) residues1237-1273. The extracellular domain of the spike glycoprotein is comprised of N-terminal domain (S1-NTD) comprising residues 20–286, and is of particular interest here. The host cell receptor binding domain (RBD) comprising residues 319–541. In summary the key regions are as follows.SP 1-19
S1-NTD 20–286
RBD 319-541
S2 686-1213
TM 1214-1236
CT 1237-1273
Fig. 1 shows the external part 20–1213 of the spike glycoprotein of SARS-CoV-2 in the closed state prior to ACE2 binding, with S1-NTD domain (the “ears”, dark blue) of interest here, RBD (at tip, subdomains light blue, blue-green), S2 (subdomains, orange, green, yellow). The orange-white, green-white and yellow-white helical structures are the α-helices of the trimer that form the neck associated with S2, and the red-white helical structures are the start of the transmembrane α-helices TM.
Fig. 1 Spike protein of SARS-CoV-2 PDB entry 6VVX, showing S1-NTD domain (dark blue). See text in regard to the significance of the other colors.