Structural proteins of SARS-CoV-2

Introduction

Similar to the appearance of the influenza virus A (H1N1) (so-called “Spanish grippe, Spanish flu”), which caused a pandemic in the 20th century and claimed more than 20 million lives, the new etiological agent SARS-CoV-2 has become the cause of the pandemic of the 21st century, which covered all the continents. The high contagiousness of the pathogen, its similarity to SARS-CoV, as well as rapid spread in many countries of the world, explain the increased attention of researchers to it. The representatives of the Coronaviridae family have a wide range of features that ensure their pathogenicity, and the study of the interaction of the virus with the macroorganism inevitably involves the search and investigation of the factors and mechanisms underlying their high pathogenetic potential in the viral agent. In recent years, the biological function of structural proteins in many coronaviruses has been clarified, and their role in pathogenesis has been established. The new coronavirus is a unique structure determined primarily by its proteins, which have antigenic and immunogenic properties, participate in the recognition of host cells and interaction with them, determine the tropism of the virus, and trigger pathological processes in the body. It is not by chance that the structural proteins of SARS-CoV-2 are in the focus of attention of scientists around the world, a detailed study of which will help lead to the solution of many strategically priority tasks facing the World Health Organization, Rospotrebnadzor, and the Ministry of Health of the Russian Federation related to the creation of modern domestic vaccines and drugs for the diagnosis, treatment, and prevention of coronavirus.

SARS-CoV-2 structural S protein

The new virus exhibits a greater degree of plasticity in relation to the ways it penetrates into the host cells due to structural (S, N, M, E) proteins. Surface spike transmembrane glycoprotein S (peplomer protein) with a molecular weight of 142 kDa and an isoelectric point of 6.24, encoded by the ORF2 gene, is a class I trimmer protein. The interest of scientists around the world in this unique protein is currently not accidental and is primarily due to the fact that the spike is the main “tool” of the virus, triggering a cascade of events leading to its high contagiousness, pathogenicity, and survival, affecting the course and outcome of viral infection. Both foreign colleagues and Russian specialists have conducted fundamental research in a short period of time focused on studying the structure, properties, and functions of this protein using various modern methodological approaches [1–7].

The S protein consists of several functional domains (ectodomain, transmembrane and short cytoplasmic), and the ectodomain, in turn, consists of two functionally different subunits (receptor-binding subunit S1, responsible for binding the virus to the receptor of the host cell, and membrane-bound subunit S2, involved in the fusion of viral and cell membranes). In the native state, protein S exists as an inactive precursor. In a viral infection, target cell proteases activate protein S by changing its conformational state and splitting it into S1 and S2 subunits, and transmembrane serine protease type II-TMPRSS2 is used as a primer for the spike protein when it binds to the receptor. It has been rightly noted [8] that the spike protein SARS-CoV-2 plays an essential role in the pathogenesis of viral infection compared to SARS-CoV, using different cellular receptors and proteases for its rapid spread. In addition to the TMPRSS spike, SARS-CoV-2 can be proteolytically activated by a variety of other cellular proteases, including cathepsin B and L (endosomal cysteine proteases), as well as furin, elastase, factor X and trypsin, capable of similar “priming” proteolysis, triggering the process of virus entry into host cells. It has been shown that SARS-CoV-2, like SARS-CoV, uses the ACE2 receptor and, in addition, contains a furin cleavage site (Arg-Arg-Ala-Arg) between S1 and S2 subunits of a spike (not found in SARS-CoV), which actually expands the possibilities of SARS-CoV-2 transmission, considering the fact that furin protease is actively expressed in the respiratory tract. Proteolysis is the “key” to the penetration of coronavirus, and therefore one of the approaches in the strategy to combat SARS-CoV-2 may be the search and creation of its inhibitors, and this vector requires further study. The specific interaction between S1 and the receptor causes a conformational change in S2, leading to the fusion of the viral envelope with the cell membrane and the penetration of the nucleocapsid into the cytoplasm of the host cell. Interaction with the receptor is a determining factor in the tissue tropism of the coronavirus to human target cells. Previously, it has been revealed that some coronaviruses use cell membrane enzymes as receptors. For example, HCoV-229E uses aminopeptidase N, MERS-CoV uses dipeptidyl-peptidase 4, HCoV-OC43, HCoV-HKU1 take 9-O-acetylated sialic acid as a receptor, and HCoV viruses (such as NL63, SARS-CoV and SARS-COV-2) use ACE2.

The receptor-binding subunit of the SARS-COV-2 virus contains RBD (receptor-binding domain), an extremely important domain for the virus, providing infection, recognizing as an ACE2 receptor, an enzyme that is a carboxypeptidase and encoded by the ACE2 gene located on chromosome 22, similar to the SARS-CoV virus, affecting the same cells [9]: epithelial cells of the respiratory tract, alveoli, vascular endothelial cells, causing serious lung damage. It is interesting to note that the free energy of RBD in SARS-COV-2 is lower than in SARS-COV, as is its solvation energy, as a result of which the interaction of SARS-COV-2 with ACE2 is much easier and may be a consequence of the evolution of the virus or adaptation to the host organism. In addition, the RBD of the new coronavirus is more flexible in contrast to the similar SARS-COV domain, in order to communicate with ACE2, it must overcome a large entropy, on the basis of which the RBD SARS-COV-2 complex becomes unstable, which suggests the likelihood of a decrease in the growth rate of the pandemic with an increase in ambient temperature [10]. SARS-COV-2 can interact in addition to ACE2 with CD26, cyclophilins, and ezrin [6].

Each protomer of S-glycoprotein contains 22 sequons with N-glycans. Therefore, 66 N-glycans [11] involved in its conformational changes were exposed on the surface of the trimmer glycoprotein. Viral glycosylation plays an essential role in viral pathobiology, indirectly affecting the structure and functions of proteins, as well as the formation of viral tropism. Glycosylation sites are under selective pressure because they contribute to evading the human immune system due to molecular mimicry of glycans, protecting certain epitopes from neutralization by antibodies. The role of glycans for the new virus is obvious, although for other coronaviruses, their participation in the occlusion (masking) of epitopes of immunogenic proteins has been demonstrated. Several unique glycosylation sites were found in SARS-COV-2 compared to SARS-COV glycoprotein, which suggests differences in the screening glycan masking of spike proteins from the host immune system protection [6], contributing to the virus penetration into cells. The previously found features in the structure of neuraminidase (antigen and pathogenicity factor) of the WSN RNA-containing influenza virus (expressed in the absence of a glycosylation site at position 146 and the presence of lysine at the C-end of the molecule, according to Goto and Kawaoka (1998) and Webster (1999)), were one of the reasons for the unusual pathogenicity pandemic influenza virus (“Spanish flu”).

Oligosaccharides can also influence priming and modulation of access to host proteases [1][8] and antibody recognition. Glycans also play an active role in the penetration of the virus into cells, stabilizing the configuration of the RBD SARS-COV-2 complex.

The amino acid sequence of the SARS-CoV-2 S protein turned out to be 76% identical to the amino acid sequence of the SARS-CoV protein and 93% and 97% — to BatCoV RaTG13 and Pangolin-CoV, respectively, which suggested the possibility of spike protein interacting with similar target proteins. However, despite the high identity of amino acid sequences, the antigenicity and epitopes of spike glycoproteins of coronaviruses are different, even if they use the same receptor protein.

A comparative analysis of the amino acid composition of the structural proteins of the coronaviruses SARS-CoV-2 and SARS-CoV, conducted in 2020 by Kharchenko [7], revealed several large inserts in the S1 subunit of the spike protein of the new virus, significant changes in the amino acid composition with a predominance of positively charged amino acids, which is characteristic of the surface proteins of viruses with high contagiousness (influenza virus, measles, rubella, hepatitis A, E, etc.). This allowed considering the positive polarity of the surface proteins of viruses as a molecular marker of their high contagiousness due to the fact that the proportion of basic amino acids, such as arginine, lysine, and histidine, increased in the S protein with a decrease in the number of dicarboxylic amino acids. The revealed higher content of lysine and arginine, characteristic of the S protein of the new coronavirus, in comparison with the similar SARS-CoV protein, additionally creates sites for the action of cellular trypsin proteases necessary for its activation and most common in organs and tissues, expanding the tropicity and, consequently, facilitating and accelerating the transmissivity of the virus, which causes its high contagiousness. Conservativeness was shown for the S2 subunit, and the quantitative ratio of basic and acidic amino acids turned out to be close to the S2 subunit of SARS-CoV.

The results of computer analysis [12] revealed a large number of sequences in the S protein SARS-COV-2, homologous to different human proteins, which can cause “mosaic” symptoms of the disease with often prolonged course, systemic damage to the body, and cytokine storm. It was also found that all structural proteins of SARS-COV-2 were distinguished by a high content of sequences homologous to hemostasis proteins and their release by proteolysis can be considered, not without reason, as a trigger of increased thrombosis in COVID-19. These data once again emphasize the uniqueness of the new coronavirus, and, of course, they should be taken into account when creating modern vaccines and predicting possible risks associated with their use.

Recently, an increase in the number of variants of SARS-CoV-2 with mutations has been noted in different countries. Increased attention to this problem is due to fears of increased contagiousness of the virus, as well as a decrease in the effectiveness of vaccines against variants with mutations in the RBD of the spike protein. In total, more than 12 thousand mutations were detected in the SARS-CoV-2 genome, most of which are single-nucleotide polymorphisms. Sequencing data showed that SARS-CoV-2 accumulated two single nucleotide mutations per month in its genome [13]. Back in May 2020, Indian scientists discovered a point mutation in the RBD at position 407. In this site, arginine (a positively charged amino acid) was replaced by isoleucine (a hydrophobic, branched amino acid). This mutation, by changing the secondary structure of the protein, can potentially affect the binding of the virus to the receptor [14]. In addition, SARS-COV-2 was circulating in the USA with the D614G mutation in the S-protein, which is assumed to have led to greater contagiousness of the virus and influenced the nature of the pandemic in this country [15]. The D614G mutation (23403A>G), located in the proximal junction S1-S2, leads to conformational changes in the structure of the S-protein. Negatively charged amino acids form ionic and hydrogen bonds through their side chains and stabilize proteins, so replacing negatively charged aspartate with nonpolar glycine can cause the unfolding of the S-protein loop, making the furin cleavage site (664-RRAR-667) more flexible, thereby enhancing virus penetration [16-17]. Currently, the D614G mutation is found in almost every sequence worldwide [18].

In December 2020, new variants of SARS-CoV-2 were identified in the UK (line B.1.1.7) and South Africa (line B.1.351), and in February 2021, the Brazilian variant (line B. 1.1.248). A new B. 1.1.7 option contains 17 mutations in the genome, many of which have already been detected in other strains of the virus around the world. Variant-specific non-synonymous mutations and deletions were found in the spike protein, including deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H. The N501Y mutation leads to the substitution of the asparagine amino acid for tyrosine in the 501^st amino acid position of the spike protein, which is located inside RBD and can increase the affinity for ACE2. The N501Y mutation was found to be associated with increased virulence in a mouse model. Researchers also associate the P681H mutation located inside the RBD with increased virulence, and deletion of the spike protein at position 69-70 is associated with evasion of the immune response [19].

The new 501.V2 virus strain in South Africa carries three mutations in RBD: N501Y, K417N, E484K – the last two reduce the activity of binding antibodies to the virus. The Brazilian line has 12 mutations, including N501Y, E484K and K417N [20]. The mutations of E484K and K417N also lead to conformational changes in the RBD of the S-protein and, as a consequence, to a decrease in the activity of binding antibodies to the virus and a change in affinity for ACE2 [21]. Since the spring of 2021, there has been an intensive spread of B.1.617.1 and B.1.617.2 lines (first identified in India) carrying the L452R mutation. The appearance of the L452R mutation with the replacement of the hydrophobic leucine residue at position 452 with a polar, highly hydrophilic arginine residue alarms many specialists.

In recent years, numerous variants of SARS-COV-2 (alpha, beta, delta and gamma, etc.) have been discovered, each of which contained a characteristic set of mutations. The Omicron variant (B.1.1.529), first discovered in southern Africa in November 2021, has spread rapidly in more than 60 countries, including Russia. At least 34 mutations were found in its spike protein, including at least 15 in the RBD [22–23]. Mutations were detected in RBD domains G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y and Y505H. The significance of some of them has now been determined: mutations such as K417N, S447N, E484A and Q493R are associated with the possibility of evading the immune response, and the N501Y mutation in the RBD contributes to a high rate of transmission of SARS-CoV-2 [24] and occurs in several variants, such as B.1.1.7, B.1.351 and P.1. However, other mutations in the Omicron variant in the RBD and other domains require additional research. Increased contagiousness and/or the ability to avoid an immune response in virus strains associated with mutations in the spike protein requires in-depth research.

SARS-CoV-2 structural N protein

The nucleocapsid protein N encoded by the ORF9 gene is one of the most important structural proteins of SARS-CoV-2, consisting of 419 amino acid residues with a molecular weight of about 46 kDa and a pI of 10.09.

The amino acid sequence of the protein N SARS-CoV-2 turned out to be 90.5% identical to the amino acid sequence of the protein SARS-CoV. Independent studies conducted by several laboratories in different countries in the study of this protein allowed asserting that it is a key regulatory protein-chaperone of a new virus. It is a capsid protein; its main function is to package (assemble) genomic RNA. Previously, the ability of N protein, as well as proteins ORF3 and ORF6, to suppress the production of type I interferons and modulate the host cell apparatus in vitro, affecting the life cycle of the host cell, was revealed in SARS-CoV [25]. In the “arms race with the human immune system”, SARS-CoV-2 uses a more sophisticated strategy of evading the response of the immune system, affecting the transmission of signals for the production of interferon (IFN-I) during a viral infection. In addition, increased expression of protein N may enhance viral replication [26]. It was found earlier that the SARS-CoV nucleocapsid protein underwent various posttranslational modifications due to acetylation and phosphorylation, being a substrate for a variety of cellular kinases. In addition, it has been proven that it can be phosphorylated by mitogen-activated protein kinase (MAP kinase), cyclin-dependent kinase (CDK), glycogen-synthetase kinase 3 (GSK3) and casein kinase 2 (CK2), and is also able to inhibit the activity of the cyclin-cyclin-dependent kinase complex (cyclin-CDK). It has been reported that the SARS-CoV N protein can interact with numerous host cell proteins, such as phosphoprotein B23, chemokine Shc16, pyruvate kinase, etc. [27]. It is currently relevant to compare various interactions of host cell proteins with coronavirus protein N, which can provide valuable information about the specificity and evolution of interactions between them, which will help form an idea about the development of antiviral drugs against SARS-CoV-2 coronavirus aimed at the interaction between host cell proteins and coronavirus protein N. Protein N, also like E and M proteins, is characterized by conservativeness. The conservativeness of the primary structures of these proteins does not exclude a change in their conformations set by the secondary and tertiary structures, since each of these proteins is characterized by many synonymous substitutions in their mRNA. The variations of synonymous substitutions in codons can change the cotranslational folding of proteins in the cell, and in viruses, accordingly, change the properties of virions. Nucleoproteins, as well as surface proteins of virions, are crucial in preserving the virus in the environment and maintaining their contagiousness. It was found that noticeable changes occurred in the N-protein SARS-CoV-2. It has become shorter by 3 amino acids compared to the similar SARS-CoV protein. The quantitative ratios between hydrophobic amino acids have changed. Despite the fact that the quantitative ratios of dicarboxylic amino acids changed, this did not affect the total number of negative groups in the N protein. With the preservation of the positions and the total number of positively charged amino acids (arginine, lysine and histidine), changes in their ratio led to a decrease in the positive charge in the N protein, which may result in a weakening of its connection with RNA and acceleration of viral replication processes. It is assumed that this shift can be considered as a possible marker of increased contagiousness and pathogenicity of SARS-CoV-2 at a lower ambient temperature, which is also observed in the nucleoprotein (NP) H3N2 subtype of influenza A virus, characterized by higher variability and pathogenicity and often prevailing in epidemic seasons, causing high mortality. The shift in the ratio and quantitative content of the main amino acids in NP influenza viruses is associated with their adaptation to a certain type of organism. According to a comparison of the amino acid composition of the nucleoproteins of influenza viruses and coronaviruses conducted in Russia in 2020 [7], in the N protein of coronaviruses SARS-CoV-2, SARS-CoV, MERS-CoV and Bat Cov HKU3, in contrast to NP influenza viruses (H3N2 and H1N1), the content of arginine and lysine practically coincide. The NP protein of influenza viruses revealed a higher content of positively and negatively charged amino acids than the N protein of coronaviruses. Taking into account the greater difference between the number of positively and negatively charged amino acids in the N protein of SARS-CoV-2 compared to NP influenza viruses, a decrease in the spread of the new coronavirus can be expected later than the decline in the flu epidemic (in late spring or with the onset of summer). With an increase in ambient temperature, the N-protein-RNA bond in virions is destabilized, which can lead to changes affecting the exposure of the polar side groups of some S-protein amino acids. This should ultimately prevent the survival of SARS-CoV-2 in the external environment (according to Kharchenko).

A unique feature of the SARS-CoV-2 N protein in comparison with representatives of Coronaviridae was the absence of cysteine (C), which is associated with the peculiarities of genomic RNA stacking in the virion.

One of the most important steps to limit the outbreak of any viral disease is the ability to diagnose some markers of the pathogen SARS-CoV-2 as soon as possible, which may include the N protein. It can be considered a possible diagnostic tool for detecting SARS-CoV-2, since it is known that, for example, with SARS-CoV, this protein is detected already on the first day of infection and it is considered an ideal target for detecting viral antigens with SARS-CoV-2 [28]. It is also advisable to consider it a potential candidate for the design of modern vaccines, given its stability.

Taking into account the importance of the N protein for the vital processes of SARS-CoV-2, its participation in the realization of the pathogenic properties of virions, it seems relevant to conduct a further in-depth study of this protein, and the information obtained about its structure and properties will help expand understanding of the biology of SARS-CoV-2 and influence the development of more effective methods for its diagnosis and treatment. Therefore, the SARS-CoV-2 N protein is a multifunctional structural protein, which is advisable to consider a biomarker of increased contagiousness and pathogenicity of a new coronavirus involved in the formation of its adaptive potential and supporting its life cycle.

SARS-CoV-2 structural E protein

Integral membrane protein E is one of the structural proteins of SARS-CoV-2, consisting of 75 amino acids, pI 8.59, encoded by the QRF4(E) gene, whose molecular weight is about 5 kDa. Protein E was detected only in representatives of the Coronaviridae. It has been shown that protein E, being a pronounced hydrophobic protein-viroporin, plays an important role in the replication cycle of the virus, its assembly, release, penetration, as well as in pathogenesis [29–30]. In acute respiratory viral infections and MERS E, the protein is considered one of the determinants of pathogenicity [31]. Recombinant variants of the MHV virus with deletion of the E gene had low replication activity. Protein E, participating in the interaction of the SARS-CoV virus with host cells and its proteins, is able to induce apoptosis through a caspase-dependent mechanism, cause lymphopenia and inhibit the stress response of host cells by participating in the overexpression of cytokines [32–33]. Proteins E SARS-CoV and SARS-CoV-2 are 94.7% identical [29]. Despite the fact that protein E from SARS-CoV-2 differs from SARS-CoV by only three substitutions and one deletion, experts have suggested that such changes may affect the conformation and properties of the protein [30]. The structure of SARS-CoV-2 protein E lacks amino acids such as histidine (H), glycine (Q) and tryptophan (W). The presence of conserved cysteine residues and palmitoylation are important for the stability of proteins and the functioning of virions [34]. It is noted that this protein can use the ion channel and peptide-binding motif in order to trigger a cytokine storm that activates inflammasomes, leading to increased pulmonary edema and ultimately to acute respiratory distress syndrome, one of the main causes of death from SARS-CoV and SARS-CoV-2. Despite the fact that the E protein contains only a few copies per virion, it is an important factor of pathogenicity and is rightly considered the “most mysterious” protein, whose functions have not yet been fully studied. In addition, these functions may be different for different viruses. Literature data suggest that this protein may be an attractive molecular target for drug development with a clear calculation of their docking. Meanwhile, it is obvious that structural protein E, being a structural multifunctional protein, can claim to be a biomarker of the pathogenicity of the new SARS-CoV-2 virus.

SARS-CoV-2 structural M protein

The integral structural membrane glycoprotein M SARS-CoV-2, encoded by the ORF5 gene, consists of 222 amino acid residues with a pH of 9.51, the molecular weight of which is about 25 kDa. The amino acid sequence of the protein M of SARS-CoV-2 turned out to be 90.5% identical to the amino acid sequence of the protein of SARS-CoV. Like other structural proteins of the coronavirus, it plays a central role in its assembly, turning cell membranes into “workshops” for the assembly of new viral particles [35], morphogenesis [36] budding [37], pathogenesis [38], interacting with other major structural proteins of the coronavirus S, N, and E [33]. Moreover, in alpha-coronaviruses, this protein interacts with the spike protein at the initial stages of interaction and introduction into the cell. The data from Siu back in 2008 demonstrated that structural proteins M, N, and E are key molecules in the assembly of SARS-CoV, and proteins E and N must be co-expressed with protein M for efficient assembly, transport, and release of viral particles. It has been shown that the SARS-CoV coronavirus M protein can cause apoptosis, and in SARS-CoV-2, it can affect the human immune system by inhibiting interferon production [39]. Currently, research is underway aimed at creating inhibitors that act at the virion assembly stage, reducing the accuracy of virus assembly processes and changing the architecture of viral particles. Structural multifunctional protein M can be considered a biomarker of the pathogenicity of the new SARS-CoV-2 virus.

Conclusion

The solution of issues related to the diagnosis, treatment, prevention, and pathogenesis of the new coronavirus lies in the knowledge of the structure and function of structural and non-structural viral proteins. Currently, the structure, properties, functions, and role of SARS-CoV-2 structural proteins are being actively studied, which is an extremely relevant research vector making it possible to find out their true biological potential of action and, in the future, evaluate possible ways of forming its new variants due to gradual changes in the properties of its surface antigens (“drift”) or a complete change in one or two surface proteins (“shift”), and develop a strategy and tactics of emergency response when new strains appear.

Taking into account the above data, it is possible to conclude that the structural proteins (antigens) of the new coronavirus, being multifunctional proteins, can serve as biomarkers of high contagiousness and pathogenicity of SARS-CoV-2, forming its pathogenetic and adaptive potentials. The polyfunctionality of the structural proteins S, N, M, and E of the new coronavirus allows it to respond quickly to fluctuations in the conditions of existence through the use of alternative approaches to implement the mechanisms underlying its pathogenetic potential. The direction of research in the field of studying SARS-CoV-2 proteins is developing rapidly, which allows hoping that in the near future, it will be possible to identify structural and functional connections in the proteins of the new coronavirus, get closer to understanding the mechanisms underlying its high contagiousness, pathogenicity, adaptation. At last, the results obtained can be used to solve strategically priority tasks of the present time aimed at creating modern domestic vaccines and developing effective methods of diagnosis, treatment, and prevention of COVID-19, given that viral proteins can be the main molecular targets for this.

Authors’ contribution:

The contribution of the authors in writing the work is equivalent.

Conflict of interest. Authors declares no conflict of interest.