The function of the spike
glycoprotein is to mediate
viral entry into the
host cell by first interacting with molecules on the exterior cell surface and then fusing the viral and cellular
membranes. Spike glycoprotein is a
class I fusion protein that contains two regions, known as S1 and S2, responsible for these two functions. The S1 region contains the receptor-binding
domain that binds to receptors on the cell surface. Coronaviruses use a very diverse range of receptors;
SARS-CoV (which causes
SARS) and
SARS-CoV-2 (which causes
COVID-19) both interact with
angiotensin-converting enzyme 2 (ACE2). The S2 region contains the
fusion peptide and other fusion infrastructure necessary for membrane fusion with the host cell, a required step for infection and
viral replication. Spike glycoprotein determines the virus'
host range (which organisms it can infect) and
cell tropism (which cells or tissues it can infect within an organism).[4][5][7][8]
Spike glycoprotein forms
homotrimers in which three copies of the protein interact through their ectodomains.[5][7] The trimer structures have been described as club- pear-, or petal-shaped.[3] Each spike protein contains two regions known as S1 and S2, and in the assembled trimer the S1 regions at the N-terminal end form the portion of the protein furthest from the viral surface while the S2 regions form a flexible "stalk" containing most of the
protein-protein interactions that hold the trimer in place.[7]
The S1 region of the spike glycoprotein is responsible for interacting with receptor molecules on the surface of the host cell in the first step of
viral entry.[4][7] S1 contains two
domains, called the N-terminal domain (NTD) and C-terminal domain (CTD),[2][7] sometimes also known as the A and B domains.[14] Depending on the coronavirus, either or both domains may be used as receptor-binding domains (RBD). Target receptors can be very diverse, including
cell surface receptor proteins and sugars such as
sialic acids as receptors or coreceptors.[2][7] In general, the NTD binds sugar molecules while the CTD binds proteins, with the exception of
mouse hepatitis virus which uses its NTD to interact with a protein receptor called
CEACAM1.[7] The NTD has a
galectin-like
protein fold, but binds sugar molecules somewhat differently than galectins.[7] The observed binding of
N-acetylneuraminic acid by the NTD[15] and loss of that binding through mutation of the corresponding sugar binding pocket in emergent variants of concern has suggested a potential role for tranisent sugar-binding in the zoonosis of SARS-CoV-2, consistent with prior evolutionary proposals.[16]
The CTD is responsible for the interactions of
MERS-CoV with its receptor
dipeptidyl peptidase-4,[7] and those of
SARS-CoV[7] and
SARS-CoV-2[5] with their receptor
angiotensin-converting enzyme 2 (ACE2). The CTD of these viruses can be further divided into two subdomains, known as the core and the extended loop or receptor-binding motif (RBM), where most of the residues that directly contact the target receptor are located.[5][7] There are subtle differences, mainly in the RBM, between the SARS-CoV and SARS-CoV-2 spike proteins' interactions with ACE2.[5] Comparisons of spike proteins from multiple coronaviruses suggest that divergence in the RBM region can account for differences in target receptors, even when the core of the S1 CTD is structurally very similar.[7]
Within coronavirus lineages, as well as across the four major coronavirus subgroups, the S1 region is less well
conserved than S2, as befits its role in interacting with virus-specific host cell receptors.[4][5][7] Within the S1 region, the NTD is more highly conserved than the CTD.[7]
Spike proteins are activated through
proteolytic cleavage. They are cleaved by host cell
proteases at the S1-S2 boundary and later at what is known as the S2' site at the N-terminus of the fusion peptide.[4][5][7][8]
Conformational change
Like other
class I fusion proteins, the spike protein undergoes a very large
conformational change during the fusion process.[4][5][7][8] Both the pre-fusion and post-fusion states of several coronaviruses, especially
SARS-CoV-2, have been studied by
cryo-electron microscopy.[5][21][22][23] Functionally important
protein dynamics have also been observed within the pre-fusion state, in which the relative orientations of some of the S1 regions relative to S2 in a trimer can vary. In the closed state, all three S1 regions are packed closely and the region that makes contact with host cell receptors is
sterically inaccessible, while the open states have one or two S1 RBDs more accessible for receptor binding, in an open or "up" conformation.[5]
Expression and localization
Genomic information
Genomic organisation of isolate Wuhan-Hu-1, the earliest sequenced sample of SARS-CoV-2, indicating the location of the S gene
The
gene encoding the spike protein is located toward the
3' end of the virus's
positive-sense RNAgenome, along with the genes for the other three structural proteins and various virus-specific
accessory proteins.[4][5]Protein trafficking of spike proteins appears to depend on the coronavirus subgroup: when
expressed in isolation without other viral proteins, spike proteins from
betacoronaviruses are able to reach the
cell surface, while those from
alphacoronaviruses and
gammacoronaviruses are retained intracellularly. In the presence of the
M protein, spike protein trafficking is altered and instead is retained at the
ERGIC, the site at which viral assembly occurs.[20] In
SARS-CoV-2, both the M and the
E protein modulate spike protein trafficking through different mechanisms.[24]
The spike protein is not required for viral assembly or the formation of
virus-like particles;[20] however, presence of spike may influence the size of the envelope.[26] Incorporation of the spike protein into virions during assembly and budding is dependent on
protein-protein interactions with the M protein through the C-terminal tail.[20][24] Examination of virions using
cryo-electron microscopy suggests that there are approximately 25[27] to 100 spike trimers per virion.[22][26]
Function
The spike protein is responsible for
viral entry into the
host cell, a required early step in
viral replication. It is
essential for replication.[2] It performs this function in two steps, first binding to a receptor on the surface of the host cell through interactions with the S1 region, and then fusing the viral and cellular membranes through the action of the S2 region.[7][8][9] The location of fusion varies depending on the specific coronavirus, with some able to enter at the
plasma membrane and others entering from
endosomes after
endocytosis.[8]
Attachment
The interaction of the receptor-binding domain in the S1 region with its target receptor on the cell surface initiates the process of viral entry. Different coronaviruses target different cell-surface receptors, sometimes using sugar molecules such as
sialic acids, or forming
protein-protein interactions with proteins exposed on the cell surface.[7][9] Different coronaviruses vary widely in their target receptor. The presence of a target receptor that S1 can bind is a determinant of
host range and
cell tropism.[7][9][28] Human serum albumin binds to the S1 region, competing with ACE2 and therefore restricting viral entry into cells.[29]
Human coronaviruses and their cell surface receptors
Proteolytic cleavage of the spike protein, sometimes known as "priming", is required for membrane fusion. Relative to other class I fusion proteins, this process is complex and requires two cleavages at different sites, one at the S1/S2 boundary and one at the S2' site to release the
fusion peptide.[5][7][9] Coronaviruses vary in which part of the viral life cycle these cleavages occur, particularly the S1/S2 cleavage. Many coronaviruses are cleaved at S1/S2 before viral exit from the virus-producing cell, by
furin and other
proprotein convertases;[7] in
SARS-CoV-2 a polybasic furin cleavage site is present at this position.[5][9] Others may be cleaved by extracellular proteases such as
elastase, by proteases located on the cell surface after receptor binding, or by proteases found in
lysosomes after
endocytosis.[7] The specific proteases responsible for this cleavage depends on the virus, cell type, and local environment.[8] In
SARS-CoV, the
serine proteaseTMPRSS2 is important for this process, with additional contributions from
cysteine proteasescathepsin B and
cathepsin L in endosomes.[8][9][37]Trypsin and trypsin-like proteases have also been reported to contribute.[8] In
SARS-CoV-2, TMPRSS2 is the primary protease for S2' cleavage, and its presence is reported to be essential for viral infection,[5][9] with cathepsin L protease being functional, but not essential.[37]
Membrane fusion
Like other
class I fusion proteins, the spike protein in its pre-fusion conformation is in a
metastable state.[7] A dramatic
conformational change is triggered to induce the
heptad repeats in the S2 region to refold into an extended
six-helix bundle, causing the
fusion peptide to interact with the cell membrane and bringing the viral and cell membranes into close proximity.[5][7] Receptor binding and proteolytic cleavage (sometimes known as "priming") are required, but additional triggers for this conformational change vary depending on the coronavirus and local environment.[40]In vitro studies of
SARS-CoV suggest a dependence on
calcium concentration.[8] Unusually for coronaviruses,
infectious bronchitis virus, which infects birds, can be triggered by low
pH alone; for other coronaviruses, low pH is not itself a trigger but may be required for activity of proteases, which in turn are required for fusion.[8][40] The location of membrane fusion—at the
plasma membrane or in
endosomes—may vary based on the availability of these triggers for conformational change.[40] Fusion of the viral and cell membranes permits the entry of the virus'
positive-sense RNAgenome into the host cell
cytosol, after which
expression of viral proteins begins.[2][4][9]
In addition to fusion of viral and host cell membranes, some coronavirus spike proteins can initiate membrane fusion between infected cells and neighboring cells, forming
syncytia.[41] This behavior can be observed in infected cells in
cell culture.[42] Syncytia have been observed in patient tissue samples from infections with
SARS-CoV,
MERS-CoV, and
SARS-CoV-2,[42] though some reports highlight a difference in syncytia formation between the SARS-CoV and SARS-CoV-2 spikes attributed to sequence differences near the S1/S2 cleavage site.[43][44][45]
Immunogenicity
Because it is exposed on the surface of the virus, the spike protein is a major
antigen to which
neutralizing antibodies are developed.[2][9][46][47] Its extensive
glycosylation can serve as a
glycan shield that hides
epitopes from the
immune system.[9][18] Due to the outbreak of
SARS and the
COVID-19 pandemic, antibodies to
SARS-CoV and
SARS-CoV-2 spike proteins have been extensively studied.[46] Antibodies to the SARS-CoV and SARS-CoV-2 spike proteins have been identified that target epitopes on the receptor-binding domain[9][46][48] or interfere with the process of conformational change.[9] The majority of antibodies from infected individuals target the receptor-binding domain.[46][49][50] More recently antibodies targeting the S2 subunit of the spike protein have been reported with broad neutralization activities against variants.[51]
In response to the
COVID-19 pandemic, a number of
COVID-19 vaccines have been developed using a variety of technologies, including
mRNA vaccines and
viral vector vaccines. Most vaccine development has targeted the spike protein.[10][11][12] Building on techniques previously used in vaccine research aimed at
respiratory syncytial virus and
SARS-CoV, many SARS-CoV-2 vaccine development efforts have used constructs that include
mutations to stabilize the spike protein's pre-fusion conformation, facilitating development of antibodies against
epitopes exposed in this conformation.[52][53]
According to a study published in January 2023, markedly elevated levels of full-length spike protein unbound by antibodies were found in people who developed postvaccine myocarditis (vs. controls that remained healthy). However, these results do not alter the risk-benefit ratio favoring vaccination against COVID-19 to prevent severe clinical outcomes.[54][non-primary source needed]
Throughout the
COVID-19 pandemic, the
genome of SARS-CoV-2 viruses was
sequenced many times, resulting in identification of thousands of distinct
variants.[61] Many of these possess
mutations that change the
amino acidsequence of the spike protein. In a
World Health Organization analysis from July 2020, the spike (S) gene was the second most frequently mutated in the genome, after ORF1ab (which encodes most of the virus'
nonstructural proteins).[61] The
evolution rate in the spike gene is higher than that observed in the genome overall.[62] Analyses of SARS-CoV-2 genomes suggests that some sites in the spike protein sequence, particularly in the receptor-binding domain, are of evolutionary importance[63] and are undergoing
positive selection.[49][64]
Spike protein mutations raise concern because they may affect
infectivity or
transmissibility, or facilitate
immune escape.[49] The mutation
D614
G has arisen independently in multiple viral lineages and become dominant among sequenced genomes;[65][66] it may have advantages in infectivity and transmissibility[49] possibly due to increasing the density of spikes on the viral surface,[67] increasing the proportion of binding-competent conformations or improving stability,[68] but it does not affect vaccines.[69] The mutation N501Y is common to the Alpha, Beta, Gamma and Omicron
Variants of SARS-CoV-2 and has contributed to enhanced infection and transmission,[70] reduced vaccine efficacy,[71] and the ability of SARS-CoV-2 to infect new rodent species.[72] N501Y increases the affinity of Spike for human ACE2 by around 10-fold,[73] which could underlie some of fitness advantages conferred by this mutation even though the relationship between affinity and infectivity is complex.[74] The mutation P681R alters the furin cleavage site, and has been responsible for increased infectivity, transmission and global impact of the
SARS-CoV-2 Delta variant.[75][76] Mutations at position
E484, particularly
E484
K, have been associated with
immune escape and reduced
antibody binding.[49][62]
The
SARS-CoV-2 Omicron variant is notable for having an unusually high number of mutations in the spike protein.[77] The SARS CoV-2 spike gene (S gene, S-gene) mutation 69–70del (Δ69-70) causes a
TaqPathPCR test probe to not bind to its S gene target, leading to S gene target failure (SGTF) in SARS CoV-2 positive samples. This effect was used as a marker to monitor the propagation of the
Alpha variant[78][79] and the
Omicron variant.[80]
Additional Key Role in Illness
In 2021, Circulation Research and Salk had a new study that proves COVID-19 can be also a vascular disease, not only respiratory disease. The scientists created an “pseudovirus”, surrounded by SARS-CoV-2 spike proteins but without any actual virus. And pseudovirus resulted in damaging lungs and arteries of animal models. It shows SARS-CoV-2 spike protein alone can cause vascular disease and could explain some covid-19 patients who suffered from strokes, or other vascular problems in other parts of human body at the same time. The team replicated the process by removing replicating capabilities of virus and showed the same damaging effect on vascular cells again.[81][82]
During the
COVID-19 pandemic,
anti-vaccinationmisinformation about COVID-19 circulated on social media platforms related to the spike protein's role in
COVID-19 vaccines. Spike proteins were said to be dangerously "
cytotoxic" and mRNA vaccines containing them therefore in themselves dangerous. Spike proteins are not cytotoxic or dangerous.[83][84] Even though studies has found that spike proteins are causing
amyloid-disease associated blood coagulation and fibrinolytic disturbances, along with neurologic and cardiac problems.[needs copy edit][85] Spike proteins were also said to be "shed" by vaccinated people, in an erroneous allusion to the phenomenon of
vaccine-induced viral shedding, which is a rare effect of
live-virus vaccines unlike those used for COVID-19. "Shedding" of spike proteins is not possible.[86][87]
Evolution, conservation and recombination
The
class I fusion proteins, a group whose well-characterized examples include the coronavirus spike protein,
influenza virushemagglutinin, and
HIVGp41, are thought to be evolutionarily related.[7][88] The S2 region of the spike protein responsible for membrane fusion is more highly
conserved than the S1 region responsible for receptor interactions.[4][5][7] The S1 region appears to have undergone significant
diversifying selection.[89]
Within the S1 region, the N-terminal domain (NTD) is more conserved than the C-terminal domain (CTD).[7] The NTD's
galectin-like
protein fold suggests a relationship with structurally similar cellular proteins from which it may have evolved through gene capture from the host.[7] It has been suggested that the CTD may have evolved from the NTD by
gene duplication.[7] The surface-exposed position of the CTD, vulnerable to the host
immune system, may place this region under high
selective pressure.[7] Comparisons of the structures of different coronavirus CTDs suggests they may be under diversifying selection[90] and in some cases, distantly related coronaviruses that use the same cell-surface receptor may do so through
convergent evolution.[14]
^Yonker, Lael M.; Swank, Zoe; Bartsch, Yannic C.; Burns, Madeleine D.; Kane, Abigail; Boribong, Brittany P.; Davis, Jameson P.; Loiselle, Maggie; Novak, Tanya; Senussi, Yasmeen; Cheng, Chi-An; Burgess, Eleanor; Edlow, Andrea G.; Chou, Janet; Dionne, Audrey; Balaguru, Duraisamy; Lahoud-Rahme, Manuella; Arditi, Moshe; Julg, Boris; Randolph, Adrienne G.;
Alter, Galit; Fasano, Alessio; Walt, David R. (4 January 2023).
"Circulating Spike Protein Detected in Post–COVID-19 mRNA Vaccine Myocarditis". Circulation. 147 (11): 867–876.
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S2CID255475007. Extensive antibody profiling and T-cell responses in the individuals who developed postvaccine myocarditis were essentially indistinguishable from those of vaccinated control subjects, [...] A notable finding was that markedly elevated levels of full-length spike protein (33.9±22.4 pg/mL), unbound by antibodies, were detected in the plasma of individuals with postvaccine myocarditis, [...] (unpaired t test; P<0.0001).
^Methods for the detection and identification of SARS-CoV-2 variants (Technical report). Stockholm and Copenhagen: European Centre for Disease Prevention and Control/World Health Organization Regional Office for Europe. 3 March 2021. Diagnostic screening assays of known VOCs.