The spike protein (S protein) is a large type I transmembrane protein ranging from 1,160 amino acids for avian infectious bronchitis virus (IBV) and up to 1,400 amino acids for feline coronavirus (FCoV) (Figure 1). In addition, this protein is highly glycosylated as it contains 21 to 35 N-glycosylation sites. Spike proteins assemble into trimers on the virion surface to form the distinctive "corona", or crown-like appearance. The ectodomain of all CoV spike proteins share the same organization in two domains: a N-terminal domain named S1 that is responsible for receptor binding and a C-terminal S2 domain responsible for fusion (Figure 2). CoV diversity is reflected in the variable spike proteins (S proteins), which have evolved into forms differing in their receptor interactions and their response to various environmental triggers of virus-cell membrane fusion.
A notable distinction between the spike proteins of different coronaviruses is whether it is cleaved or not during assembly and exocytosis of virions. With some exceptions, in most alphacoronaviruses and the betacoronavirus SARS-CoV, the virions harbor a spike protein that is uncleaved, whereas in some beta- and all gammacoronaviruses the protein is found cleaved between the S1 and S2 domains, typically by furin, a Golgi-resident host protease. Interestingly, within the betacoronavirus mouse hepatitis virus (MHV) species, different strains, such as MHV-2 and MHV-A59 display different cleavage requirements. This has important consequences on their fusogenicity.
Figure 1. Spike protein of coronavirus
The coronavirus spike protein is a class I fusion protein. The formation of an α-helical coiled-coil structure is characteristic of this class of fusion protein, which contain in their C-terminal part regions predicted to have an α-helical secondary structure and to form coiled-coils. The S2 subunit is the most conserved region of the protein, whereas the S1 subunit diverges in sequence even among species of a single coronavirus (Figure 2). The S1 contains two subdomains, a N-terminal domain (NTD) and a C-terminal domain (CTD). Both are able to function as receptor binding domains (RBDs) and bind variety of proteins and sugars.
Coronavirus spike proteins contain two heptad repeats in their S2 domain, a feature typical of a class I viral fusion proteins. Heptad repeats comprise a repetitive heptapeptide abcdefg with a and d being hydrophobic residues characteristic of the formation of coiled-coil that participate in the fusion process. For SARS-CoV and MHV, the post-fusion structures of the HR have been solved; they form the characteristic six-helix bundle. The functional role of MHV and SARS-CoV HR was confirmed by mutating key residues and by inhibition experiments using HR2 peptides.
Figure 2. SARS-CoV spike protein schematic. The spike protein ectodomain consists of the S1 and S2 domains. The S1 domain contains the receptor binding domain and is responsible for recognition and binding to the host cell receptor. The S2 domain, responsible for fusion, contains the putative fusion peptide (blue) and the heptad repeat HR1 (orange) and HR2 (brown). The transmembrane domain is represented in purple. Cleavage sites are indicated with arrows.
The CoVs are widely distributed in nature and their zoonotic transmissions into human populations can cause epidemic disease. After entering into respiratory or gastrointestinal tracts, these viruses establish themselves by entering and infecting lumenal macrophages and epithelial cells. The cell entry programs for these viruses are orchestrated by the viral spike (S) proteins that bind cellular receptors and also mediate virus-cell membrane fusions. Take SARS-CoV for example. The spike protein (S protein) of SARS-CoV has pivotal roles in viral infection and pathogenesis. S1 recognizes and binds to host receptors, and subsequent conformational changes in S2 facilitate fusion between the viral envelope and the host cell membrane.
Models depicting the S-mediated membrane fusion event have extended from knowledge of S protein structures and functions. In part, these models are deemed reasonable because the postfusion 6-HB conformations in SARS and MHV S proteins are so strikingly similar to postfusion forms of influenza HA2, paramyxovirus F2, Ebolavirus GP2 and HIV gp41. In analogy to these more widely-studied and well-understood viral fusion proteins, the CoV S-mediated membrane fusion process is generally viewed as schematized in Figure 3.
Figure 3. Schematic of CoV spike protein mediated membrane fusion. The illustrations represent several steps of S protein conformational changes that may take place during membrane fusion. In the first step, receptor binding, pH reduction and/or S protein proteolysis induces dissociation of S1 from S2. This step is documented for some MHVs. In the second step, the fusion peptide (FP) is intercalated into the host cell membrane. This is the fusion-intermediate stage. In the third stage, the part of the S protein nearest to the virus membrane refolds onto a heptad repeat 1 (HR1) core to form the six-helix bundle (6-HB), which is the final postfusion configuration of the S2 protein.
The roles of spike protein (S protein) in receptor binding and membrane fusion indicate that vaccines based on the spike protein could induce antibodies to block virus binding and fusion or neutralize virus infection. Among all structural proteins of SARS-CoV, spike protein is the main antigenic component that is responsible for inducing host immune responses, neutralizing antibodies and/or protective immunity against virus infection. Spike protein has therefore been selected as an important target for coronavirus vaccine and anti-viral development. A comparison of these approaches is provided in Table 1.
Table 1. Spike protein-based vaccines and antiviral therapies against SARS-CoV
|Full-length S protein||Induces effective neutralizing-antibody and T-cell responses, as well as protective immunity||Might induce harmful immune responses that cause liver damage or enhanced infection|
|DNA-based||Easier to design; induces immunoglobulin G, neutralizing-antibody and T-cell responses and/or protective immunity||Might have low efficacy in humans; repeated doses may cause toxicity|
|Viral vector-based||Induces neutralizing-antibody responses, protective immunity and/or T-cell responses||Might induce ADE effect; possibly present pre-existing immunity|
|Recombinant S protein-based||Induces high neutralizing-antibody responses and protective immunity||Mainly humoral responses; need repeated doses and adjuvants|
|RBD||Induces highly potent neutralizing-antibody and T-cell responses and protective immunity||Not identified|
|DNA-based||Induces neutralizing-antibody and T-cell responses and/or protective immunity||Induces low responses; might not neutralize mutants|
|Viral vector-based||Induces neutralizing-antibody responses, protective immunity and/or T-cell responses||Possible genomic integration of foreign DNA; viral vector instability|
|Recombinant RBD protein-based||Safer and more effective than other RBD vaccines; induces neutralizing-antibody and T-cell responses, protective immunity and cross protection||Needs repeated doses and adjuvants|
|Peptides||Inhibits virus infection by preventing S protein-mediated
receptor binding and blocking viral fusion and entry
|Low antiviral potency|
|RBD–ACE2 blockers||Blocks RBD–ACE2 binding and S protein-mediated infection||Not identified|
|S cleavage inhibitors||Might interfere with S cleavage||Not identified|
|Fusion core blockers||Easy to design; inhibits virus infection with high specificity||Not identified|
|Highly potent virus inhibition and/or neutralization activity
against homologous and heterologous SARS-CoV isolates
|Might enhance SARS-CoV entry; further
|Easier to generate than human neutralizing
antibodies; neutralizes SARS-CoV in vitro and prevents virus
|Repeated use can cause HAMA response;
might not recognize mutants with key
substitutions in S protein
|Inhibits virus entry, neutralizes virus infection, induces cross
protection and reduces disease severity and viral burden; more
suitable to development as human immunotherapeutics
|Small compounds||Oral bioavailability||Low antiviral potency|
|Protease inhibitors||Blocks virus entry and/or inhibits protease (cathepsin L)
|S protein inhibitors||Specifically inhibits S protein-mediated SARS-CoV fusion and
entry into the host cell
|Small interfering RNAs||Reduces virus replication and/or silences S gene expression||Low antiviral potency; limited usefulness|
|*All candidates are at the preclinical study stage. ACE2, angiotensin-converting enzyme 2; ADE, antibody-dependent enhancement; HAMA, human–anti-mouse antibody, RBD, receptor-binding domain, SARS-CoV; severe acute respiratory syndrome-coronavirus.|