Coronavirus spike protein
|Coronavirus spike glycoprotein S1|
|Coronavirus spike glycoprotein S2|
|Betacoronavirus spike glycoprotein S1, receptor binding|
|Betacoronavirus-like spike glycoprotein S1, N-terminal|
Spike (S) protein (sometimes also spike glycoprotein, formerly known as E2) is the largest of the four major structural proteins found in coronaviruses. The spike protein assembles into trimers that form large structures, called spikes or peplomers, that project from the surface of the virion. The distinctive appearance of these spikes when visualized using negative stain transmission electron microscopy, "recalling the solar corona", gives the virus family its name.
The function of the spike protein 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. The spike protein 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. The spike protein determines the virus' host range (which organisms it can infect) and cell tropism (which cells or tissues it can infect within an organism).
The spike protein is highly immunogenic. Antibodies against the spike protein are found in patients recovered from SARS and COVID-19. Neutralizing antibodies target epitopes on the receptor-binding domain. Most COVID-19 vaccine development efforts in response to the COVID-19 pandemic aim to activate the immune system against the spike protein.
The spike protein is very large, often 1200-1400 amino acid residues long; it is 1273 residues in SARS-CoV-2. It is a single-pass transmembrane protein with a short C-terminal tail on the interior of the virus, a transmembrane helix, and a large N-terminal ectodomain exposed on the virus exterior.
The spike protein forms homotrimers in which three copies of the protein interact through their ectodomains. The trimer structures have been described as club- pear-, or petal-shaped. 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.
The S1 region of the spike protein is responsible for interacting with receptor molecules on the surface of the host cell in the first step of viral entry. S1 contains two domains, called the N-terminal domain (NTD) and C-terminal domain (CTD), sometimes also known as the A and B domains. 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. 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. The NTD has a galectin-like protein fold, but binds sugar molecules somewhat differently than galectins.
The CTD is responsible for the interactions of MERS-CoV with its receptor dipeptidyl peptidase-4, and those of SARS-CoV and SARS-CoV-2 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. There are subtle differences, mainly in the RBM, between the SARS-CoV and SARS-CoV-2 spike proteins' interactions with ACE2. 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.
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. Within the S1 region, the NTD is more highly conserved than the CTD.
The S2 region of the spike protein is responsible for membrane fusion between the viral envelope and the host cell, enabling entry of the virus' genome into the cell. The S2 region contains the fusion peptide, a stretch of mostly hydrophobic amino acids whose function is to enter and destabilize the host cell membrane. S2 also contains two heptad repeat subdomains known as HR1 and HR2, sometimes called the "fusion core" region. These subdomains undergo dramatic conformational changes during the fusion process to form a six-helix bundle, a characteristic feature of the class I fusion proteins. The S2 region is also considered to include the transmembrane helix and C-terminal tail located in the interior of the virion.
The spike protein is a glycoprotein and is heavily glycosylated through N-linked glycosylation. Studies of the SARS-CoV-2 spike protein have also reported O-linked glycosylation in the S1 region. The C-terminal tail, located in the interior of the virion, is enriched in cysteine residues and is palmitoylated.
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.
Like other class I fusion proteins, the spike protein undergoes a very large conformational change during the fusion process. Both the pre-fusion and post-fusion states of several coronaviruses, especially SARS-CoV-2, have been studied by cryo-electron microscopy. 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.
Expression and localization
|NCBI genome ID|
|Genome size||29,903 bases|
|Year of completion||2020|
The gene encoding the spike protein is located toward the 3' end of the virus's positive-sense RNA genome, along with the genes for the other three structural proteins and various virus-specific accessory proteins. 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. In SARS-CoV-2, both the M and the E protein modulate spike protein trafficking through different mechanisms.
The spike protein is not required for viral assembly or the formation of virus-like particles; however, presence of spike may influence the size of the envelope. 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. Examination of virions using cryo-electron microscopy suggests that there are approximately 25 to 100 spike trimers per virion.
The spike protein is responsible for viral entry into the host cell, a required early step in viral replication. It is essential for replication. 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. 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.
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. 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.
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. 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; in SARS-CoV-2 a polybasic furin cleavage site is present at this position. 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. The specific proteases responsible for this cleavage depends on the virus, cell type, and local environment. In SARS-CoV, the serine protease TMPRSS2 is important for this process, with additional contributions from cysteine proteases cathepsin B and cathepsin L in endosomes. Trypsin and trypsin-like proteases have also been reported to contribute. In SARS-CoV-2, TMPRSS2 is the primary protease for S2' cleavage, and its presence is reported to be essential for viral infection.
Like other class I fusion proteins, the spike protein in its pre-fusion conformation is in a metastable state. 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. 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. In vitro studies of SARS-CoV suggest a dependence on calcium concentration. 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. The location of membrane fusion—at the plasma membrane or in endosomes—may vary based on the availability of these triggers for conformational change. Fusion of the viral and cell membranes permits the entry of the virus' positive-sense RNA genome into the host cell cytosol, after which expression of viral proteins begins.
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. This behavior can be observed in infected cells in cell culture. Syncytia have been observed in patient tissue samples from infections with SARS-CoV, MERS-CoV, and SARS-CoV-2, 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.
Because it is exposed on the surface of the virus, the spike protein is a major antigen to which neutralizing antibodies are developed. Its extensive glycosylation can serve as a glycan shield that hides epitopes from the immune system. 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. Antibodies to the SARS-CoV and SARS-CoV-2 spike proteins have been identified that target epitopes on the receptor-binding domain or interfere with the process of conformational change. The majority of antibodies from infected individuals target the receptor-binding domain.
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. 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.
Monoclonal antibodies that target the spike protein have been developed as COVID-19 treatments. As of July 8, 2021, three monoclonal antibody products had received Emergency Use Authorization in the United States: bamlanivimab/etesevimab, casirivimab/imdevimab, and sotrovimab. Bamlanivimab/etesevimab was not recommended in the United States due to the increase in SARS-CoV-2 variants that are less susceptible to these antibodies.
Throughout the COVID-19 pandemic, the genome of SARS-CoV-2 viruses was sequenced many times, resulting in identification of thousands of distinct variants. Many of these possess mutations that change the amino acid sequence 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). The evolution rate in the spike gene is higher than that observed in the genome overall. 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 and are undergoing positive selection.
Spike protein mutations raise concern because they may affect infectivity or transmissibility, or facilitate immune escape. The mutation D614G has arisen independently in multiple viral lineages and become dominant among sequenced genomes; it may have advantages in infectivity and transmissibility possibly due to increasing the density of spikes on the viral surface, increasing the proportion of binding-competent conformations, or improving stability. Mutations at position E484, particularly E484K, have been associated with immune escape and reduced antibody binding.
During the COVID-19 pandemic, anti-vaccination misinformation 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. 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.
Evolution, conservation and recombination
The class I fusion proteins, a group whose well-characterized examples include the coronavirus spike protein, influenza virus hemagglutinin, and HIV Gp41, are thought to be evolutionarily related. The S2 region of the spike protein responsible for membrane fusion is more highly conserved than the S1 region responsible for receptor interactions. The S1 region appears to have undergone significant diversifying selection.
Within the S1 region, the N-terminal domain is more conserved than the C-terminal domain. 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. It has been suggested that the CTD may have evolved from the NTD by gene duplication. The surface-exposed position of the CTD, vulnerable to the host immune system, may place this region under high selective pressure. Comparisons of the structures of different coronavirus CTDs suggests they may be under diversifying selection and in some cases, distantly related coronaviruses that use the same cell-surface receptor may do so through convergent evolution.
The genomic region that encodes for the Spike is also a hotspot for homologous as well as non-homologous recombination. Especially for Spike recombination events among different coronavirus subgenera, the cross-over sites are not single, but they tend to be double, meaning that the Spike moves to another subgenus in a cassette-like mode. So far, there is no evidence that the Spike has moved (via recombination) from one subgenus to another, within the Beta-Coronavirus genus (that includes human SARS-CoV, SARS-CoV-2, MERS, HKU1, OC43). However, many such Spike recombination events have occurred among different subgenera in Alpha, Gamma and Delta coronavirus genera, and they are a significant cause for concern and vigilance, concerning the evolution of the COVID19 pandemic.
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