The development of vaccines and treatments against COVID-19 must focus on the S protein of SARS-CoV-2 because it is a highly dynamic and complex molecule that is essential for viral entrance into host cells and interacts with the host immune system ^[1][2][12,13].

The SARS-CoV-2 virus’s S protein, which is its most thoroughly studied component, is essential for the virus’s entry and multiplication inside the host ^[3][14]. S1, S2, and a transmembrane domain make up the three components of the S protein, a complex glycosylated protein. The angiotensin-converting enzyme 2 (ACE2) receptor on host cells is bound by the receptor-binding domain (RBD) of the S1 subunit, which facilitates viral entrance ^[3][4][14,15]. The transmembrane domain secures the protein to the viral membrane, and the S2 subunit includes the fusion peptide that helps the viral and host cell membranes fuse ^[5][16]. During viral entry, the highly dynamic S protein changes shape from a closed conformation to an open conformation, exposing the RBD for ACE2 binding ^[6][17].

In order to create novel vaccines and treatments against SARS-CoV-2 and to ensure that existing vaccines are still effective against new variants, more research into the structure and function of the S protein and its variants is required ^[7][18].

There are several SARS-CoV-2 variants with S protein mutations, and these alterations have been linked to both greater transmissibility and decreased neutralization by antibodies induced by existing vaccinations. Investigations are still ongoing into how these alterations affect viral pathogenesis and infectivity functionally ^[8][9][19,20].

Angiotensin-converting enzyme 2 (ACE2) receptor identification and binding through the SARS-CoV-2 spike (S) protein facilitates viral entry into host cells ^[10][21]. The S1 subunit of the S protein’s receptor-binding domain (RBD) preferentially binds to the ACE2 receptor on the surface of the host cell, causing conformational changes that enable the fusion of the viral and host cell membranes and result in viral entrance ^[11][22]. The affinity of the RBD for ACE2 has been demonstrated to be a crucial factor of viral infectivity and pathogenicity. The S protein is highly selective for the ACE2 receptor ^[12][23]. The S protein of the SARS-CoV-2 virus has a stronger affinity for the ACE2 receptor than the S protein of the original SARS-CoV virus, according to structural analyses ^[13][24]. This stronger affinity could be a factor in SARS-CoV-2’s higher transmissibility when compared to SARS-CoV ^[14][15][25,26]. Additionally, the S protein has a furin cleavage site that enables host proteases to cleave the S protein, facilitating viral entrance into host cells.

2. Structural Consequences of Spike Protein Mutations

2.1. Alterations in Spike Protein Conformation and Stability

Mutations in the spike protein can alter its stability and antigenicity in addition to impacting receptor binding. Approximately two-thirds of the spike protein’s surface is covered with glycans, indicating that it is extensively glycosylated ^[16][17][61,62]. The spike protein’s structural flexibility and stability may be impacted by mutations in the glycosylation pattern. This may lessen the effectiveness of vaccinations and therapeutic antibodies by interfering with the recognition and binding of neutralizing antibodies ^[18][63]. For instance, the RBD and N-terminal domain (NTD) alterations in the original South African B.1.351 variation have been linked to decreased neutralization by certain monoclonal antibodies and convalescent plasma ^[19][20][48,64]. Creating efficient COVID-19 preventive and treatment plans requires an understanding of the structural effects of spike protein mutations. This entails creating treatments and vaccines that can target the spike protein in its many conformations and patterns of glycosylation, as well as keeping an eye on the emergence and dissemination of novel variants with mutations that can alter the effectiveness of these interventions.

2.2. Effects on Spike Protein Interactions with Host Factors

The interactions of the spike protein with host components like antibodies, immune cells, and other host proteins may impact the pathogenicity and infectiousness of viruses ^[21][22][65,66]. Mutations may result in the loss of the spike protein’s epitopes, or regions of the protein that neutralizing antibodies may identify ^[23][24][67,68]. By reducing the potency of therapeutic antibodies and vaccinations that target specific epitopes, this may lead to breakthrough infections and decreased protection against reinfection ^[25][69]. For instance, the E484K mutation seen in the B.1.351 and P.1 variants has been connected to decreased neutralization by certain monoclonal antibodies and convalescent plasma ^[8][26][19,70]. Spike protein mutations can alter not only antibody recognition but also how the protein interacts with immune cells like T cells and natural killer cells ^[27][71]. Spike protein mutations may impact how antigen-presenting cells process and display viral antigens, thereby lowering T cell activation and compromising the antiviral immune response ^[28][29][72,73]. The spike protein’s interactions with the host protein are important in viral entry and replication, such as the ACE2 receptor and host proteases, which can also be impacted by mutations ^[5][30][16,74]. This may have an impact on the effectiveness of viral entrance and replication, thereby influencing viral pathogenesis and the severity of the disease. For the purpose of creating efficient COVID-19 prevention and treatment techniques, it is essential to comprehend the consequences of spike protein mutations on how it interacts with host factors. In addition to creating treatments that can specifically target the spike protein and host components involved in viral entry and replication, this involves keeping an eye on the development and dissemination of novel variations with mutations that may influence viral infectivity and pathogenicity.

2.3. Influence on Spike Protein Cleavage and Processing

The SARS-CoV-2 spike protein is a glycoprotein that goes through a lot of cleavage and processing when it enters host cells ^[31][75]. To allow for viral entrance, the spike protein has a polybasic cleavage site (RRAR) that is digested by host proteases such as furin and TMPRSS2 ^[32][76]. The effectiveness of cleavage and processing can be changed through mutations in the spike protein, which may have an impact on the pathogenicity and infectiousness of viruses ^[33][34][77,78]. The D614G mutation in the spike protein, which has been found in several SARS-CoV-2 variations and is linked to increased infectivity and transmissibility, is one of the most prominent changes in the protein ^[15][26]. It has been demonstrated that the D614G mutation, which affects the receptor-binding domain (RBD) of the spike protein, makes the spike trimer more stable and makes it easier for host proteases to digest the spike protein. These modifications could be a factor in the D614G variant’s heightened contagiousness and transmissibility ^[35][79]. It has also been demonstrated that other mutations in the spike protein alter cleavage and processing. For instance, the B.1.1.7 variant’s P681H mutation has been linked to improved furin cleavage efficiency, which may help explain why the variety is more contagious ^[36][37][80,81]. On the other hand, it has been demonstrated that alterations in the polybasic cleavage site, such as the E484K mutation present in most variants of concern, impair cleavage efficiency and impact viral entrance ^[38][82]. For the purpose of creating efficient COVID-19 therapies, it is essential to comprehend the effects of spike protein mutations on cleavage and processing. Protease inhibitors are one example of a therapeutic strategy that targets these processing steps and may be useful in inhibiting viral entrance and replication. More so, keeping an eye on the development and dissemination of novel variants with mutations that impact cleavage and processing can reveal crucial details about the pathogenesis and evolution of SARS-CoV-2.

3. Functional Consequences of Spike Protein Mutations

A SARS-CoV-2 mutation in the spike protein can have substantial functional ramifications because it is essential for viral entrance and pathogenesis ^[11][22].

3.1. Changes in Spike Protein Receptor Binding Affinity and Specificity

To enable viral entrance, the SARS-CoV-2 spike protein interacts with the angiotensin-converting enzyme 2 (ACE2) receptor on host cells ^[39][83]. Alterations to the spike protein’s binding affinity and specificity for ACE2 can have an impact on the pathogenicity and transmission of viruses ^[40][84]. For instance, the RBD of the spike protein has several changes in the B.1.1.7 variation, including N501Y, which has been demonstrated to increase the binding affinity for ACE2 and improve viral infectivity ^[41][85]. Similar to the B.1.351 variant, the B.1.351 variant features many mutations in the RBD, including E484K, which decreases the binding affinity for some monoclonal antibodies and might have an impact on how well the host immune system neutralizes the virus ^[8][42][19,86]. Mutations in the spike protein can alter ACE2 binding as well as result in the development of novel receptor binding capacities ^[43][87]. The P681R mutation in the B.1.617 variation confers improved binding to the host protease furin, which may make it easier for the virus to enter host cells that do not express a lot of ACE2 ^[44][88]. This mutation has been found in a number of variants that are cause for concern, underscoring the possibility that the development of new receptor-binding capacities may be what propels the evolution and appearance of SARS-CoV-2 variants ^[45][89]. Neutralizing antibodies and ACE2 decoys are examples of therapeutic approaches that target the interaction between the spike protein and ACE2 and may be useful in limiting viral entrance and replication ^[46][90]. Furthermore, keeping an eye on the appearance and spread of novel variations with mutations that impair receptor binding will shed light on the pathogenesis and evolution of SARS-CoV-2 ^[47][91].

3.2. Effects on Spike Protein Fusion and Membrane Fusion

SARS-CoV-2 enters host cells through the spike-protein-mediated fusion of the viral and host cell membranes, which is an important stage in the process ^[5][33][16,77]. For instance, the enhanced cleavage, which is characterized by the D614G mutation and alterations in the S1/S2 furin cleavage region of the spike protein, can impact the proteolytic processing necessary for membrane fusion ^[48][49][92,93]. Mutations in the spike protein can directly alter the conformational changes necessary for membrane fusion in addition to having an impact on proteolytic processing. Multiple SARS-CoV-2 variants have the E484K mutation, which has been found to stabilize the spike protein’s prefusion conformation and improve the effectiveness of membrane fusion ^[5][50][16,94]. Moreover, this mutation has been linked to a decreased susceptibility to neutralizing antibodies and perhaps a decreased effectiveness of vaccination ^[51][95]. Fusion inhibitors and other therapeutic approaches that focus on the fusion process may be useful in limiting viral entry and reproduction ^[52][96]. Therefore, keeping an eye on the development and dissemination of novel variations with mutations that interfere with membrane fusion can reveal vital details about the pathogenesis and evolution of SARS-CoV-2.

3.3. Modulation of Spike Protein Proteolytic Activation and Inactivation

The spike protein’s proteolytic activation and inactivation are crucial processes in SARS-CoV-2’s entry and egress from host cells ^[53][97]. Numerous protease cleavage sites found in the spike protein are crucial for viral pathogenicity and infectivity ^[54][98]. The effectiveness of proteolytic processing and the activation and inactivation of the spike protein can both be altered by mutations in these cleavage sites ^[55][99]. The D614G mutation, which has been demonstrated to improve the efficacy of furin cleavage at the S1/S2 region of the spike protein, is one illustration of such a mutation. This results in increased viral transmissibility and infectivity ^[56][100]. The S686G mutation, among others, can disrupt cleavage at the S2’ site and may have an impact on viral entrance and pathogenesis ^[57][101]. On the other hand, mutations that hinder the spike protein’s activation and proteolytic processing can affect viral pathogenicity ^[58][43]. For example, changes near the furin cleavage site, like the P681H mutation, have been linked to a reduction in furin-mediated cleavage and, possibly, a reduction in viral entrance and pathogenicity ^[59][102]. For the development of efficient therapeutic approaches against COVID-19, it is essential to comprehend the effects of spike protein mutations on proteolytic processing. It may be possible to thwart viral entrance and reproduction by using protease inhibitors that target the proteases responsible for cleaving spike proteins. Furthermore, keeping an eye on the appearance and dissemination of novel variants with mutations that alter proteolytic processing can reveal crucial details about the pathogenesis and evolution of SARS-CoV-2.