Spike glycoprotein S2, coronavirus (Spike_S2_CoV)
Type: Domain

The type I glycoprotein S of Coronavirus, trimers of which constitute the typical viral spikes, is assembled into virions through noncovalent interactions with the M protein. The spike glycoprotein is translated as a large polypeptide that is subsequently cleaved to S1 (

) and S2 . The cleavage of S can occur at two distinct sites: S2 or S2' . The spike is present in two very different forms: pre-fusion (the form on mature virions) and post-fusion (the form after membrane fusion has been completed). The spike is cleaved sequentially by host proteases at two sites: first at the S1/S2 boundary (i.e. S1/S2 site) and second within S2 (i.e. S2' site). After the cleavages, S1 dissociates from S2, allowing S2 to transition to the post-fusion structure .
Both chimeric S proteins appeared to cause cell fusion when expressed individually, suggesting that they were biologically fully active . The spike is a type I membrane glycoprotein that possesses a conserved transmembrane anchor and an unusual cysteine-rich (cys) domain that bridges the putative junction of the anchor and the cytoplasmic tail .

SARS-CoV S is largely uncleaved after biosynthesis. It can be later processed by endosomal cathepsin L, trypsin, thermolysin, and elastase, which are shown to induce syncytia formation and virus entry. Other proteases that are of potential biological relevance in potentiating SARS-CoV S include TMPRSS2, TMPRSS11a, and HAT which are localized on the cell surface and are highly expressed in the human airway . The furin-like S2' cleavage site at KR/SF with P1 and P2 basic residues and a P2' hydrophobic Phe downstream of the IFP is identical between the SARS-CoV-2 and SARS-CoV. One or more furin-like enzymes would cleave the S2' site at KR/SF . Deletion of SARS-CoV-2 furin cleavage site suggests that it may not be required for viral entry but may affect replication kinetics and altered sites have been still seen proteolytically cleaved. Several substitutions within the S2' cleavage domain of SARS-COV-2 have been reported, including P812L/S/T, S813I/G, F817L, I818S/V, but further experimental study of their consequences and the replication properties of the altered viruses are required to understand the role of furin cleavage in SARS-CoV-2 infection and virulence .

The S2 subunit normally contains multiple key components, including one or more fusion peptides (FP), a second proteolytic site (S2') and two conserved heptad repeats (HRs), driving membrane penetration and virus-cell fusion. The HRs can trimerize into a coiled-coil structure built of three HR1-HR2 helical hairpins presenting as a canonical six-helix bundle and drag the virus envelope and the host cell bilayer into close proximity, preparing for fusion to occur . The fusion core is composed of HR1 and HR2 and at least three membranotropic regions that are denoted as the fusion peptide (FP), internal fusion peptide (IFP), and pretransmembrane domain (PTM). The HR regions are further flanked by the three membranotropic components. Both FP and IFP are located upstream of HR1, while PTM is distally downstream of HR2 and directly precedes the transmembrane domain of SARS-CoV S. All of these three components are able to partition into the phospholipid bilayer to disturb membrane integrity. . During the pandemic, many conservative amino acid changes in FP segment of SARS-CoV-2 have been reported (i.e., L821I, L822F, K825R, V826L, T827I, L828P, A829T, D830G/A, A831V/S/T, G832C/S, F833S, I834T), although their impact is not known as the active conformation and mode of insertion of SARS-CoV-2 fusion peptide have not been experimentally characterised. Differences in HR1 sequences between SARS-CoV and SARS-CoV-2 suggest that SARS-CoV-2 HR2 makes stronger interactions with HR1. However, the substitutions observed in the solvent accessible surface of the HR1 domain (e.g., D936Y, S943P, S939F) of SARS-CoV-2 do not seem to be involved in stabilizing interactions with HR2. Substitutions in HR2 (e.g., K1073N, V1176F) or the TM or cytoplasmic tail domains have also been observed, but further experimental work is required to determine the effects of these changes .