IN SILICO IDENTIFICATION OF A VIRAL SURFACE GLYCOPROTE IN SITE SUITABLE FOR THE DEVELOPMENT OF LOW MOLECULAR MASS INHIBITORS FOR VARIOUS VARIANTS OF SARS-COV-2

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is a new coronavirus today has an extremely significant impact on both the global economy and society as a whole, due to its pandemic status and risk of complica-tions. Therefore, understanding the molecular features of the interaction of receptor binding domain (RBD), which determines most of the dangerous properties of this pathogen, with human angiotensin-converting enzyme 2 (hACE2) is an important step in the process of developing a successful strategy to combat SARS-CoV-2. In addition, given the significant rate of accumulation of mutations in RBD, it makes sense to consider its different variants. Goal. Identification of a pocket potentially suitable for the search for low molecular mass inhibitors of the interaction of different variants of SARS-CoV-2 RBD and hACE2. Methods. The initial structure of different variants of the RBD/hACE2 complex was obtained from Protein Data Bank (PDB). Separate RBD variants were isolated from the same data. To obtain the Y453F mutant, variant P.1 was mutagenized in PyMol 1.8. The construction of the system, which included the resulting associ-ate or individual protein, solvent, and physiological concentration of sodium chloride, was performed using CHARMM-GUI (graphical user interface for CHARMM) tools according to the standard protocol for glycoproteins. The actual simulation and balancing of the system were performed in GROMACS (GROningen MAchine for Chemical Simulation) version 2019.6 for 50 ns. Results. The interface of the RBD/hACE2 interaction is formed by amino acids Q24, D30, H34, E35, E37, Y41, Y83, K353, D355, and R393 for hACE2 and K417, Y453, F486, N487, Y489, Q493, Q498, T500, N501, and Y505 — for RBD. However, it is heterogeneous and can be divided into two subinterfaces, and each either of them includes its own pool of interactions: hACE2 Q24/Y83 + RBD N487, hACE2 H34 + RBD Y453, hACE2 E35 + + RBD Q493, and hACE2 D30 + RBD K417 for N-terminal relative to H1 hACE2 subinterface and hACE2 E37/R393 + + RBD Y505, hACE2 K353 + RBD Q498/G502, and hACE2 D355 + RBD T500 — for C-terminal. According to the considered N501Y mutation, changes are observed in the mentioned interaction patterns — hydrogen bonds of hACE2 Q42 + RBD Q498, hACE2 K31 + RBD Q493, and hACE2 K31 + RBD F490 are formed, and hACE2 H34 + RBD Y453 is lost. Similar aberrations, except for the hydrogen bond with F490, are observed in the case of the N501Y + Y453F vari-ant. Despite significant changes in the pool of interactions, the gross number of hydrogen bonds for the complexes of all three variants is relatively stable and ranges from 9 to 10. The defined interaction for all considered variants of RBD are characterized by the presence of a pocket between the subinterfaces, which is formed by the residues R403, Y453, Q493, S494, Y495, G496, F497, Q498, N501, and Y505 conditionally original variant. According to the results of the molecular dynamics simulation, the Y453F replacement has little effect on the overall topology of the cavity but sufficiently reduces the polarity of the pocket part of its localization, which leads to the impossibility of forming any polar interactions. In contrast, N501Y, due to a larger size of the tyrosine radical and the presence of parahydroxyl, forms two equivalent mutually exclusive hydrogen bonds with the carbonyls of the peptide groups G496 and Y495. Additional stabilization of Y501 is provided by interplanar stacking with Y505. In addition to the anchored position in ~ 25% of the trajectory, there is another “open” conformation Y501, at which the radical of this tyrosine does not interact with the rest of the protein. Conclusions. 1) The interface of the interaction of SARS-CoV-2 RBD with hACE2 is not continuous, and it can be conditionally divided into two subinterfaces: N-terminal and C-terminal. Either of them is characterized by its own pattern of connections and changes according to the RBD N501Y and Y453F replacements considered. However, despite the presence of significant molecular ear angements caused by N501Y and Y453F, the total number of hydrogen bonds is almost the same for all mutants. 2) Between the identified interaction subinterfaces, SARS-CoV-2 RBD contains a caveola, which due to its location may be potentially suitable for finding promising candidates for drugs aimed at inhib-iting the interaction of this protein with hACE2. In this case, the replacements of N501Y and Y453F have a significant impact on the topology of a particular pocket and can potentially modify the activity of inhibitors directed to this area. © Publisher PH «Akademperiodyka» of the NAS of Ukraine, 2022.

Molecular Basis of Mink ACE2 Binding to SARS-CoV-2 and Its Mink-Derived Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted between humans and minks, and some mutations in the spike (S) protein, especially in the receptor-binding domain (RBD), have been identified in mink-derived viruses. Here, we examined binding of the mink angiotensin-converting enzyme 2 (ACE2) receptor to mink-derived and important human-originating variants, and we demonstrated that most of the RBD variants increased the binding affinities to mink ACE2 (mkACE2). Cryo-electron microscopy structures of the mkACE2-RBD Y453F (with a Y-to-F change at position 453) and mkACE2-RBD F486L complexes helped identify the key residues that facilitate changes in mkACE2 binding affinity. Additionally, the data indicated that the Y453F and F486L mutations reduced the binding affinities to some human monoclonal antibodies, and human vaccinated sera efficiently prevented infection of human cells by pseudoviruses expressing Y453F, F486L, or N501T RBD. Our findings provide an important molecular mechanism for the rapid adaptation of SARS-CoV-2 in minks and highlight the potential influence of the main mink-originating variants for humans. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a broad range of hosts. Mink-derived SARS-CoV-2 can transmit back to humans. There is an urgent need to understand the binding mechanism of mink-derived SARS-CoV-2 variants to mink receptor. In this study, we identified all mutations in the receptor-binding domain (RBD) of spike (S) protein from mink-derived SARS-CoV-2, and we demonstrated the enhanced binding affinity of mink angiotensin-converting enzyme 2 (ACE2) to most of the mink-derived RBD variants as well as important human-originating RBD variants. Cryo-electron microscopy structures revealed that the Y453F and F486L mutations enhanced the binding forces in the interaction interface. In addition, Y453F and F486L mutations reduced the binding affinities to some human monoclonal antibodies, and the SARS-CoV-2 pseudoviruses with Y453F, F486L, or N501T mutations were neutralized by human vaccinated sera. Therefore, our results provide valuable information for understanding the cross-species transmission mechanism of SARS-CoV-2.

The emerging SARS-CoV-2 variants of concern.

Since emerging from Wuhan, China, in December of 2019, the coronavirus (SARS-CoV-2) has been causing devastating severe respiratory infections in humans worldwide. With the disease spreading faster than the medical community could contain it, death tolls increased at an alarming rate worldwide, causing the World Health Organization to officially sanction the SARS-CoV-2 outbreak as a pandemic, leading to a state of worldwide lockdown for the majority of the year 2020. There have been reports of new strains of the virus emerging in various parts of the world, with some strains displaying even greater infectivity and transmissibility. Areas of the emerging variant of concern arise from countries like the United Kingdom, South Africa, Brazil, and India. These mutations carry a lineage from N501Y, D614G, N439K, Y453F, and others, which are globally dominated by clades 20A, 20B, and 20C. This literature review intends to identify and report SARS-CoV-2 variants that are currently evolving and their disease implications.

SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity.

Many SARS-CoV-2 variants with naturally acquired mutations have emerged. These mutations can affect viral properties such as infectivity and immune resistance. Although the sensitivity of naturally occurring SARS-CoV-2 variants to humoral immunity has been investigated, sensitivity to human leukocyte antigen (HLA)-restricted cellular immunity remains largely unexplored. Here, we demonstrate that two recently emerging mutations in the receptor-binding domain of the SARS-CoV-2 spike protein, L452R (in B.1.427/429 and B.1.617) and Y453F (in B.1.1.298), confer escape from HLA-A24-restricted cellular immunity. These mutations reinforce affinity toward the host entry receptor ACE2. Notably, the L452R mutation increases spike stability, viral infectivity, viral fusogenicity, and thereby promotes viral replication. These data suggest that HLA-restricted cellular immunity potentially affects the evolution of viral phenotypes and that a further threat of the SARS-CoV-2 pandemic is escape from cellular immunity.

SARS-CoV-2 mutations acquired in mink reduce antibody-mediated neutralization.

Transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to farmed mink has been observed in Europe and the US. In the infected animals, viral variants arose that harbored mutations in the spike (S) protein, the target of neutralizing antibodies, and these variants were transmitted back to humans. This raised concerns that mink might become a constant source of human infection with SARS-CoV-2 variants associated with an increased threat to human health and resulted in mass culling of mink. Here, we report that mutations frequently found in the S proteins of SARS-CoV-2 from mink are mostly compatible with efficient entry into human cells and its inhibition by soluble angiotensin-converting enzyme 2 (ACE2). In contrast, mutation Y453F reduces neutralization by an antibody with emergency use authorization for coronavirus disease 2019 (COVID-19) therapy and sera/plasma from COVID-19 patients. These results suggest that antibody responses induced upon infection or certain antibodies used for treatment might offer insufficient protection against SARS-CoV-2 variants from mink.

Intranasal Infection of Ferrets with SARS-CoV-2 as a Model for Asymptomatic Human Infection.

Ferrets were experimentally inoculated with SARS-CoV-2 (severe acute respiratory syndrome (SARS)-related coronavirus 2) to assess infection dynamics and host response. During the resulting subclinical infection, viral RNA was monitored between 2 and 21 days post-inoculation (dpi), and reached a peak in the upper respiratory cavity between 4 and 6 dpi. Viral genomic sequence analysis in samples from three animals identified the Y453F nucleotide substitution relative to the inoculum. Viral RNA was also detected in environmental samples, specifically in swabs of ferret fur. Microscopy analysis revealed viral protein and RNA in upper respiratory tract tissues, notably in cells of the respiratory and olfactory mucosae of the nasal turbinates, including olfactory neuronal cells. Antibody responses to the spike and nucleoprotein were detected from 21 dpi, but virus-neutralizing activity was low. A second intranasal inoculation (re-exposure) of two ferrets after a 17-day interval did not produce re-initiation of viral RNA shedding, but did amplify the humoral response in one animal. Therefore, ferrets can be experimentally infected with SARS-CoV-2 to model human asymptomatic infection.