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SARS-CoV-2 belongs to the family of coronaviruses, which are characterized by spikes that sit densely on the surface of the virus. The spike protein (Spro) is responsible for the attachment of the virus to the host cell via the ACE2 receptor on the surface of the host cell. The strength of the interaction between the receptor-binding domain (RBD) of the highly glycosylated spike protein of the virus and the host cell ACE2 receptor represents the key determinant of the infectivity of the virus. The SARS-CoV-2 virus has mutated since the beginning of the outbreak, and the vast majority of mutations has been detected in the spike protein or its RBD. Since specific mutations significantly affect the ability of the virus to transmit and to evade immune response, studies of these mutations are critical. We investigate GISAID data to show how viral spike protein mutations evolved during the pandemic. We further present the interactions of the viral Spro RBD with the host ACE2 receptor. We have performed a large-scale mutagenesis study of the Spro RBD-ACE2 interface by performing point mutations in silico and identifying the ambiguous interface stabilization by the most common point mutations in the viral variants of interest (beta, gamma, delta, omicron).
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia
Radostina Alexandrova
Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Bulgaria
Sebastian Pleško
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia
Reactor Physics Department, Jozef Stefan Institute, Slovenia
Urban Bren
Faculty of Chemistry and Chemical Engineering, University of Maribor, Slovenia
Faculty of Mathematics, Natural Sciences and Information Technologies, University of Primorska, Slovenia
Marko Jukič*
Faculty of Chemistry and Chemical Engineering, University of Maribor, Slovenia
Faculty of Mathematics, Natural Sciences and Information Technologies, University of Primorska, Slovenia
*Address all correspondence to: crtomir.podlipnik@fkkt.uni-lj.si and marko.jukic@um.si
1. Introduction
The SARS-CoV-2 virus has been with us for more than 2 years now. More than half a billion people have contracted the virus in a little over 2 years, and more than 6 million people have died from the virus [1, 2]. Subsequent pandemic waves of the disease can be prevented by social distancing during local outbreaks and vaccination. In spite of current pandemic, coronaviruses have always been present and are commonly the cause of colds. The first virus from the family mentioned above, isolated in 1962, was classified as an enveloped, single-stranded (+ssRNA) RNA virus. The new virus family was named after its characteristic morphological appearance, the crown spikes (spike protein or Spro) on its surface (Figure 1) [4].
Figure 1.
The cross-section of SARS-CoV-2 with characteristic spikes at the virus’s surface (left) and the fusion of the virus with the cell membrane (right). The figure is the artwork of David Goodsell (PDB-101: Educational resources supporting molecular explorations through biology and medicine [3]).
Until the outbreak of the SARS (Severe Acute Respiratory Syndrome; causative virus named SARS-CoV) pandemic in mainland China and Hong Kong in 2003, coronaviruses did not receive so much attention from the scientific community. Timely action at that time prevented the outbreak and evolution to the pandemic. However, in 2012, the respiratory syndrome coronavirus (MERS-CoV) led to an outbreak of the Middle East respiratory syndrome (MERS) in Saudi Arabia, mainland China, the United Arab Emirates, and the Republic of Korea [2, 5, 6]. In late 2019, SARS-CoV-2, a member of the Coronaviridae family, emerged in Wuhan, China. As a result, a creeping spread among the human population began, and the WHO declared a pandemic on March 11, 2020 [7, 8]. At the time of writing, COVID-19 disease (caused by SARS-CoV-2) has spread worldwide, claiming more than 6.5 million lives. As the SARS-CoV-2 virus has become a critical health concern, scientists immediately began research on this topic. COVID-19 disease is of great concern worldwide because, while the majority of cases have mild symptoms, a variable percentage (0.2 to >5%!) of patients progresses to pneumonia and multiple organ failure, which can lead to death especially without medical assistance [9, 10].
Vaccines against SARS-CoV-2 are now available, few therapeutic options have been authorized for emergency use by FDA and only one antiviral agent has been approved for COVID-19 treatment, however novel drug research is ongoing [11, 12, 13, 14, 15, 16]. Since vaccines are the poster child in the fight against the COVID-19 pandemic, a high viral mutation rate may lead to changes in the structures of essential viral proteins, rendering available vaccines ineffective [17]. This concern is exacerbated by the fact that, with the exception of inactivated SARS-CoV-2 vaccines, all other vaccines (RNA vaccines, Adenovirus-based and protein-based vaccines) currently in clinical use are targeted at the same structure in the virus – the spike protein from the viral envelope. Its biological functions and surface location, make it a major target for the formation of neutralizing antibodies. However, this also determines the high frequency of mutations in this region, which can help the virus escape from the immune response [18].
The first identified mutations and respectively recognized subtypes / variants of the virus were announced in March and April 2020 [19, 20]. In late 2020, the first SARS-CoV-2 variant of concern (VOC) was reported—the B.1.1.7 variant (UK variant, designated alpha by WHO as of June 7, 2021; https://www.who.int/). Alpha is often designated by canonical mutations: N501Y, 69/70 deletion, P681H. This was followed by the appearance of several other variants of concern—beta, gamma, delta and omicron, as well as a number of variants of interest (VOI; for variant classification the reader is referred to a wonderful classification at CDC: https://www.cdc.gov/coronavirus/2019-ncov/variants/variant-classifications.html). The second in the list of VOC was B.1.351 variant (beta; canonical mutations: K417N, E484K, N501Y) or South African variant [21, 22]. Both variants carry an N501Y mutation in the RBD (receptor binding domain) of the Spro, which is associated with increased viral transmission [23]. In addition, the South African variant carries mutations K417N and E484K, which may be responsible for decreased binding to host antibodies [24]. P.1 (gamma, canonical mutations: K417T, E484K, and N501Y) variant has been reported in Brazil with the known N501Y, E484K and the novel K417T mutations [25]. Epsilon or B.1.427 or B.1.429 followed with canonical mutations: S13I, W152C, L452R, D614G. In early 2021, a novel SARS-CoV-2 variant B.1.617.2 (delta, canonical mutations: L452R, T478K, D614G, and P681R) nicknamed “the double mutant” or Indian variant was reported to cause infections in India and slowly spread throughout the world via global travel practices [26]. Acquired critical mutations in the Spro, particularly in the receptor-binding domain (RBD), are currently under heavy investigation (Delta Plus variant) as they may have higher infectivity and transmissibility or even escape the host immune response [27]. Last but not least is the observed omicron or B.1.1.529 variant first detected in Botswana and then in South Africa in November 2021. Omicron is described with at least 34 mutations in Spro, of which 15 are in RBD, 7 in the NTD and 3 close to the furin cleavage site.
2. The structure of the spike protein and its role
Since the first announcement of the existence of the new virus, many scientific groups around the world began an intensive search for a suitable drug. In their attempts to develop drugs to treat COVID-19, scientists are focusing on a variety of strategies, including identifying / creating agents that attack and neutralize the virus; agents that affect inflammation, prevent the formation of blood clots, etc. [28]. The structure of the Spro, as well as its biological properties and its role in the entry of SARS-CoV-2 into the cell have been the subject of extensive studies and are the key towards development of adequate preventive and curative approaches [29, 30]. The spike glycoprotein is the crucial protein that determines viral host selection and pathology and thus one of the most important targets for diagnosis and therapy. With a total length of 1273 amino acids, the Spro consists of an extracellular N-terminus, a transmembrane domain anchored in the viral membrane (TM), and a short intracellular C-terminal segment. Bound to the protein are specific polysaccharides whose function is to prevent the host immune system from recognizing the viral protein. Once the virus interacts with the host cell, the conformational changes of the Spro lead to the fusion of the virus with the host cell membrane. The protein consists of the signaling protein (1-13) and the S1 (14-685) and S2 (686-1273) subunits. In addition, the S1 domain, which is responsible for receptor binding, is divided into an N-terminal domain (NTD; 14-305) and a receptor-binding domain (RBD; 319-541). The S2 subunit, whose function is fusion, consists of the fusion peptide (FP) (788-806 residues), heptapeptide repeat sequence 1 (HR1) (912-984), HR2 (1163-1213), the TM domain (1213-1237), and the cytoplasmic domain (1237-1273). A polybasic insertion (PRRAR) characteristic of joining the S1/S2 and S′ domains of SARS-CoV-2 can be cleaved by furin, and this cleavage is essential for membrane fusion. The model of the Spro with labeled domains shown in Figure 2 and results from a detailed all-atom molecular dynamics simulation (μs trajectory timeframe) of the fully glycosylated full-length Spro in a viral membrane [32].
Figure 2.
The structure of the Spro with labeled domains. The model is based on the cryo-EM structure of SARS-CoV-2 spike glycoprotein trimer in prefusion conformation with a single receptor-binding domain (RBD) in “up” conformation (PDB-ID: 6vsb) [31].
The Spro is densely coated with polysaccharides. Each monomer of SARS-CoV-2 Spro has 22 N-linked glycans, 18 of which were conserved between SARS-CoV and SARS-CoV-2 Spro [33]. The glycan shielding has several effects on Spro folding, its processing by host cell proteases, immune evasion, and the elicitation of a humoral immune response. Extensive glycan shielding of the Spro, which blocks the surface of the protein, can thereby hide specific epitopes from neutralization by antibodies, masking them and facilitating immune evasion [33]. In addition, it has been observed that both glycosylated and de-glycosylated S ectodomains bind with almost identical affinity to ACE-2 (1.7 nM vs. 1.5 nM); therefore, it has been suggested that glycosylation of the Spro does not alter the binding affinity of the Spro to ACE-2 [18]. However, the glycan shield of the Spro of SARS-CoV-2 is thought to be less dense and less effective compared to glycoproteins of other viruses such as HIV-1, which may be advantageous for the induction of humoral immunity and vaccine development. Therefore, there is great interest in investigating the potential immunogenicity of glycan components as vaccine candidates [34]. Furthermore, the structure bound to ACE2 shows that the omicron variant spike trimer contains an unusual RBD-RBD interaction and other interactions at the ACE2-RBD interface, both of which contribute to the higher affinity of ACE2 for the omicron spike trimer, which is six to nine times higher than that of the wild type, WT. The structural analysis of the omicron spike trimer also explains why the omicron escapes the most therapeutic antibodies and reduces the efficacy of vaccinations. The interaction of omicron spike trimer with ACE2 and Fab antibody is shown in Figure 3 [35].
Figure 3.
The structure of SARS-CoV-2 (omicron variant) Spro trimer in complex with ACE2 receptor (PDB ID: 7wpa, left) and with Fab antibody (PDB ID: 7wpf, right) [35].
3. SARS-CoV-2 variants and mutations in the spike protein
Genetic sequencing studies have revealed numerous neutral or mildly deleterious mutations, mainly single nucleotide polymorphisms (SNPs) and insertions/deletions (indels). However, a small percentage of mutations can alter fitness and help the virus adapt. These substitutions or deletions can alter peptide polarity and affect the structure and functionality of viral proteins responsible for infectivity, transmissibility, and antigenicity [36]. Several databases and nomenclature systems have been established to classify genome sequences and track the epidemiology and genetic evolution of SARS-CoV-2. GISAID (The Global Initiative on Sharing All Influenza Data, https://www.epicov.org) contains millions of SARS-CoV-2 whole-genome sequences [37]. The GISAID nomenclature categorizes genomes into clades (based on marker mutations) that help understand large-scale diversity patterns and geographic dispersal. Pango nomenclature (https://cov-lineages.org/) is one of the most widely used nomenclatures that assigns newly identified genomes to a lineage based on the global phylogenetic tree. Based on the extensive sequencing data and observations available, the WHO has classified the SARS-CoV-2 variants that may pose an increased public health risk into the following three groups:
Variants of Concern (VOC): VOC is defined by increased transmissibility and virulence or a decrease in practiced public health and social interventions and available therapeutics.
Variants of Interest (VOI): VOI is defined by variants that have been observed to spread in the community and occur in multiple cases or clusters or have been detected in different countries.
Variants under Surveillance (VUM): VUM is defined as a variant with genetic alterations that are believed to affect viral properties, and that may pose some risk to public health and safety in the future. Increased surveillance and ongoing assessment are needed to gather evidence of these variants’ phenotypic or epidemiologic impact.
Summary of VOI, VOC, and VUM as published by the WHO.
RNA viruses have the highest mutation rates, 1:10,000 to 1:1,000,000 mutations per base pair, due to the lack of proofreading ability of RNA-dependent RNA polymerases (RdRp) [38]. However, coronavirus family viruses have a proofreading mechanism due to the exoribonuclease (ExoN) domain of nsp14 [39]. Although this was expected to contribute to a low mutation rate, more than 6 million viral genomes were captured within 2 years (GISAID). Furthermore, the first fitness-enhancing mutation at the spike protein was identified only a few months after SARS-CoV-2 emerged [40]. These findings could be a consequence of the sheer magnitude of infection numbers on a global scale. In addition, Gribble et al. [41] have experimentally demonstrated that nsp14-ExoN may play a critical role in RNA recombination events during viral replication that can generate genetic variants (Table 2).
No.
Mutation
Region
Virus variant
Impact in viral pathogenicity
1
D614G
RBD
several lineages
the most prevalent mutation, increase spike density.
increase ACE binding, antibody escape resistance to antibody drugs
6
Q677P/H
S1/S2
Several lineages
Increasing virus fusion with human cell
7
T478K, Q493K, Q498R
RBD
B.1.1.529
Increase ACE2-RBD binding
8
Δ69–70
NTD
B.1.1.7,B.1.1.529
Immune escape
Table 2.
Collected key mutations of SARS-CoV-2, which have a substantial impact on the viral pathogenicity.
As we see in Table 2, the most essential mutations of the SARS-CoV-2 are found on the spike protein’s receptor-binding domain (RBD), but that is not the whole story. Other important regions are critical to the success of different lineages, like mutations in the antibody binding regions found on/near the RBD, on the N-terminal domain (NTD) and the S2 domain. Such mutations mainly contribute to the lineages’ antibody escape. Some mutations stabilize binding with stabilization of the open conformation and add/remove glycosylation sites which are also important. There is also the furin cleavage site, one of the vital mutation points that was present in the delta variant. The other aspect of mutations is also the influence on testing capabilities. For example the mutation S:69/70del that caused the so-called S-drop out in the PCR testing, which was caused by the mutation in the PCR primer region. This mutation was later cleverly exploited to quickly distinguish between alpha and non-alpha infections and omicron and non-omicron. There are also mutations on the N-gene that are present mainly in the omicron lineage, which cause lower sensitivity and even failure of detection using lateral flow tests (so-called quick antigen tests). Lastly, there are also mutations in other proteins that are significant for novel drug design. The phylogenetic analysis of SARS-CoV-2 is presented in Figure 4 (The reader is referred to an excellent resource: https://covariants.org/).
Figure 4.
On the chart we can see the molecular clock, i.e. the number of mutations in a particular sequence dependent on the time of sampling. The colors represent variants (clades). The thick line represents the average mutation rate. We can see that 21 L (BA.2) omicron, as well as 22A (BA.4), 22B (BA.5), 22C (BA.2.12.1), clearly deviate from the average. Nextstrain; 2022-05-31 [42, 43].
The omicron variant mutations potentially attenuate the efficacy of therapeutic antibodies and enhance the binding of ACE2. Of even more significant concern, omicron infections have been reported in individuals vaccinated in South Africa and Hong Kong [44]. The recent study by Yin et al. reported the biochemical and structural characterization of the spike protein trimer of SARS-CoV-2 omicron variant and its binding to ACE2. Data show that omicron variant RBD is less stable and more dynamic than WT RBD. [35] Omicron differs significantly from all previous versions of SARS-CoV-2. These mutations in turn have led to important consequences in the behavior of the virus, including significant epidemiological characteristics [42, 43]. Namely, mutations altered the area recognized by neutralizing antibodies and reduced the effectiveness of the immune response elicited by previous variants and vaccines. Of particular interest are the neutralizing antibodies that still recognize the virus, because knowing them will help us to improve prophylactic and therapeutic strategies and to respond adequately to future variants. There is excellent research done on antibody escape by J.D. Bloom et al. [45]. Bloom’s lab plotted the antibody escape in dependence on the spike mutation site for Moderna’s vaccine serum and convalescent serum. From the plots presented in Figure 5 it’s nicely seen that the mutation on 484 or 456 sites would cause an antigen to escape from any previously acquired immunity. The mutation on 484th residue, present in beta and gamma lineages as E484K and in omicron as E484A, confirmed Bloom’s work. The mutation 456 on the other hand, is not present so far in any of the WHO’s variants of interests.
Figure 5.
Bloom plots (https://jbloomlab.github.io/SARS2_RBD_Ab_escape_maps/) top: Moderna’s vaccine serum and bottom: Convalescent serum; on 2022-05-31 [45].
Omicron variant mutations thus contributed to the successful transmission of the virus from person to person and its faster spread. Surprisingly, the virus with so many mutations continues to effectively bind to the ACE2 receptor. Omicron is thought to combine various mutations (we know some of them from other variants), some of which simultaneously help it escape the immune response and bind to ACE2 [43]. The mutations have affected the mechanism of viral entry into the cell. Unlike other variants, which use the mechanism of fusion with the cytoplasmic membrane of the host cell, omicron uses another mechanism of entry, namely the uptake by the endosome. According to one hypothesis, this may at least partially explain omicron’s preference for the upper respiratory tract (nose and throat) [42]. The mutations have also affected the spectrum of hosts—it is known that SARS-CoV-2 can infect a wide range of domestic and wild animals, including cats, dogs, ferrets, hamsters, leopards, minks, deer, etc., but not mice and rats. However, unlike “conventional” SARS-Cov-2 variants, omicron can bind to the ACE2 receptor in turkeys, chickens and mice, as well as rats (linked to N501Y and Q498R mutations) [1, 45].
All in all, omicron consists of several genome sublines / subvariants (BA.1, BA.2, BA.3, BA.4, BA.5), some of which (BA.1, BA.2) have already been established worldwide, while the growth of the others (BA.4, BA.5) is increasing at the moment. BA4 and BA5 appeared in late December 2021 and early January 2022, they are better transmissible than earlier versions of omicron (BA2 and especially BA1) and may partially escape the immune protection provided by infection with previous variants or vaccination. At the beginning of May 2022, infections with BA.4 and BA.5 were 60–75% of the cases in South Africa, and have been registered in a number of other countries in Europe and North America [42, 46]. According to the European Centre for Disease Prevention and Control (ECDC; https://www.ecdc.europa.eu/en/news-events/epidemiological-update-sars-cov-2-omicron-sub-lineages-ba4-and-ba5), as of May 13, 2022, BA.5 represents already 37% of the cases in Portugal, and the expectation is to become dominant by May 22, 2022. Omicron has a large number of mutations (50 as compared to the original variant of SARS-CoV-2 isolated in Wuhan in the end of 2019). Its origin is not yet fully established. Clarifying it is extremely important not only from a theoretical point of view, but also because it will help us to be better prepared to manage with future variants [42]. Among the more recognized hypotheses, we can distinguish four: First, accumulation of mutations during its transmission from person to person. It is known that, unlike other RNA viruses, coronaviruses, including SARS-CoV-2, carry an editing enzyme system that helps it to correct errors occurring during in RNA molecule synthesis [47, 48]; Second, the appearance of such large number of mutations can be facilitated by infecting immunosuppressed individuals in whose body the virus persists for a long time, which creates conditions for its continuous reproduction and selection of mutations that avoid the immune response; Third, the emergence can be related to its circulation (and consequent accumulation of mutations) in animal organisms. This shows that we need to monitor the fate of the virus also in the animal kingdom [42]; and Fourth, the changes in coronaviruses can be induced through a process of recombination—in this case, the formation of the next vital generation may be the result of combining genetic information. It has been reported that this process takes place not only in bats, but in humans as well and can lead to the emergence of new variants and strains [49, 50].
We can postulate new viral variants along with key (canonical) mutations, especially at the receptor binding domain (RBD) of the Spike protein (Spro), improve the ability of the virus to recognize relevant host receptor (ACE2) via steric adaptation and new interactions with the binding partner. In order to inspect all possible mutations at the Spro RBD we performed a comprehensive in silico mutagenesis study using FoldX [46]. 3D complexes of Spro wild type along with Spro FoldX mutants were iteratively used for ΔΔG prediction. All possible mutations of RBD binding domain of SARS-CoV-2 S protein (PDB ID: 6M0J) with sequence from K417 towards Y505 (length of 89; KIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY) for a total of 1780 point mutations were calculated and the resulting heatmap is presented in Figure 6 [49]. Individual point mutation calculations were repeated once and mutations with no structural change left for validation purposes where all no-change mutation produced ΔΔG energies below 0.1 kcal/mol.
Figure 6.
We conducted a full RBD 417–505 mutagenesis study using FoldX in order to assess the key mutations and their effects on the stability of the system; in total 1780 point mutations.
Upon structural inspection and superimposition (PDB ID: 6M0J, 7DK3), the Spro RBD-ACE2 interface was identified as: 417 LYS, 445 VAL, 446 GLY, 449 TYR, 453 TYR, 455 LEU, 456 PHE, 473 TYR, 475 ALA, 476 GLY, 484 GLU, 486 PHE, 487 ASN, 489 TYR, 493 GLN, 496 GLY, 498 GLN, 500 THR, 501 ASN, 502 GLY, 503 VAL, 505 TYR. Reference key mutations were placed on this interface such as E484K, Q493N, Q493Y, and N501Y with ample experimental data for validation. Namely, N501Y confers increased binding affinity to human ACE2 while N501T shows reduced host ACE2-binding affinity in vitro all according to P0DTC2 Uniprot reference [47, 48]. We observed FoldX total energies of 0.37, 0.62, and −0.95 kcal/mol upon point mutations E484K, Q493N, and Q493Y respectively in accordance with literature [50]. Furthermore, canonical delta L452R, and E484Q displayed insignificant FoldX force field Δ energies of 0.04, 0.09 kcal/mol, respectively [51]. If we focus on omicron variant, it possesses the following mutations at the Spro RBD not in contact with ACE2 binding partner: G339D, S371L, S373P, and S375F. In the ACE2 PPI, however the following mutations are present with calculated FoldX Δ energies in kcal/mol: K417N: −0.34, G446S: 2.99, N440K: −0.61, S477N: 0.14, T478K: −0.18, E484A: 1.31, Q493K: −1.20, G496S: −0.06, Q498R: −0.93, N501Y: 6.18 and Y505H: 1.62. The results confirm the observed ensemble of mutations substantially modify the resulting PPI in accordance with the literature [52, 53, 54, 55, 56, 57, 58, 59, 60]. It should be stressed, that FoldX evaluations are single-point only and detailed binding energetics should be further studied by experiment supported MD. We postulate however such in silico interaction profiling approaches could be further developed to quickly assess key mutations or mutation ensembles for further study in the future.
One of the most interesting questions is undoubtedly in which direction the evolution of the SARS-CoV-2 will continue. Viruses strive for a successful dissemination and more intensive replication, which means that they evade the immune response and bypass the effects of antiviral agents. The omicron variant (and especially its subvariants) represents one of the fastest spreading viruses known. In addition, it is more successful at evading the immune response triggered by previous variants and/or available vaccines. What would be the profile of the next variant(s) to replace omicron? It is clear that the new variant must outperform the old one in order to prevail—this is achieved by bypassing the immune response and spreading efficiently, as evidenced by the evolution of new (sub)variants. From a public health perspective, the most important question is how severe the disease pattern of the next variant(s) will be and how effectively we can study the evolution of viral variants along with their impact on the drug/vaccine development in the future, with the final goal of preventing such future pandemics.
This study is supported by the National Science Fund, Bulgarian Ministry of Education and Science—Grant No. КП-06-КОСТ/16 from 16.12.2020 (AR) as well as by the Slovenian Research Agency (ARRS) through the research programs P1-0201 (CP), P1-0403, P2-0046 and the research project J1-2471 (UB, MJ).
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Written By
Črtomir Podlipnik, Radostina Alexandrova, Sebastian Pleško, Urban Bren and Marko Jukič
Submitted: 03 June 2022Reviewed: 17 June 2022Published: 22 July 2022
Open access peer-reviewed chapter
