Omicron Spike Protein is Vulnerable to Reduction.

SARS-CoV-2 virus spike (S) protein is an envelope protein responsible for binding to the ACE2 receptor, driving subsequent entry into host cells. The existence of multiple disulfide bonds in the S protein makes it potentially susceptible to reductive cleavage. Using a tri-part split luciferase-based binding assay, we evaluated the impacts of chemical reduction on S proteins from different virus variants and found that those from the Omicron family are highly vulnerable to reduction. Through manipulation of different Omicron mutations, we found that alterations in the receptor binding module (RBM) are the major determinants of this vulnerability. Specifically we discovered that Omicron mutations facilitate the cleavage of C480-C488 and C379-C432 disulfides, which consequently impairs binding activity and protein stability. The vulnerability of Omicron S proteins suggests a mechanism that can be harnessed to treat specific SARS-CoV-2 strains.

In silico design of ACE2 mutants for competitive binding of SARS-CoV-2 receptor binding domain with hACE2

The receptor binding motif (RBM) within the S-protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been touted as one of the main targets for vaccine/therapeutic development due to its interaction with the human angiotensin II converting enzyme 2 (hACE2) to facilitate virus entry into the host cell. The mechanism of action is based on the disruption of binding between the RBM and the hACE2 to prevent virus uptake for replication. In this work, we applied in silico approaches to design specific competitive binders for SARS-CoV-2 S-protein receptor binding motif (RBM) by using hACE2 peptidase domain (PD) mutants. Online single point mutation servers were utilised to estimate the effect of PD mutation on the binding affinity with RBM. The PD mutants were then modelled and the binding free energy was calculated. Three PD variants were designed with an increased affinity and interaction with SARS-CoV-2-RBM. It is hope that these designs could serve as the initial work for vaccine/drug development and could eventually interfere the preliminary recognition between SARS-CoV-2 and the host cell. © 2022 Walter de Gruyter GmbH, Berlin/Boston. All rights reserved.

Receptor-Binding-Motif-Targeted Sanger Sequencing: a Quick and Cost-Effective Strategy for Molecular Surveillance of SARS-CoV-2 Variants.

Whole-genome sequencing (WGS) is the gold standard for characterizing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome and identification of new variants. However, the cost involved and time needed for WGS prevent routine, rapid clinical use. This study aimed to develop a quick and cost-effective surveillance strategy for SARS-CoV-2 variants in saliva and nasal swab samples by spike protein receptor-binding-motif (RBM)-targeted Sanger sequencing. Saliva and nasal swabs prescreened for the presence of the nucleocapsid (N) gene of SARS-CoV-2 were subjected to RBM-specific single-amplicon generation and Sanger sequencing. Sequences were aligned by CLC Sequence Viewer 8, and variants were identified based upon specific mutation signature. Based on this strategy, the present study identified Alpha, Beta/Gamma, Delta, and Omicron variants in a quick and cost-effective manner. IMPORTANCE The coronavirus disease 2019 (COVID-19) pandemic resulted in 427 million infections and 5.9 million deaths globally as of 21 February 2022. SARS-CoV-2, the causative agent of the COVID-19 pandemic, frequently mutates and has developed into variants of major public health concerns. Following the Alpha variant (B.1.1.7) infection wave, the Delta variant (B.1.617.2) became prevalent, and now the recently identified Omicron (B.1.1.529) variant is spreading rapidly and forming BA.1, BA.1.1, BA.2, BA.3, BA.4, and BA.5 lineages of concern. Prompt identification of mutational changes in SARS-CoV-2 variants is challenging but critical to managing the disease spread and vaccine/therapeutic modifications. Considering the cost involved and resource limitation of WGS globally, an RBM-targeted Sanger sequencing strategy is adopted in this study for quick molecular surveillance of SARS-CoV-2 variants.

Structural Basis for Human Receptor Recognition by SARS-CoV-2 Omicron Variant BA.1.

The highly contagious and fast-spreading omicron variant of SARS-CoV-2 infects the respiratory tracts efficiently. The receptor-binding domain (RBD) of the omicron spike protein recognizes human angiotensin-converting enzyme 2 (ACE2) as its receptor and plays a critical role in the tissue tropism of SARS-CoV-2. Here, we showed that the omicron RBD (strain BA.1) binds to ACE2 more strongly than does the prototypic RBD from the original Wuhan strain. We also measured how individual omicron mutations affect ACE2 binding. We further determined the crystal structure of the omicron RBD (engineered to facilitate crystallization) complexed with ACE2 at 2.6 Å. The structure shows that omicron mutations caused significant structural rearrangements of two mutational hot spots at the RBD/ACE2 interface, elucidating how each omicron mutation affects ACE2 binding. The enhanced ACE2 binding by the omicron RBD may facilitate the omicron variant's infection of the respiratory tracts where ACE2 expression level is low. Our study provides insights into the receptor recognition and tissue tropism of the omicron variant. IMPORTANCE Despite the scarcity of the SARS-CoV-2 receptor-human angiotensin-converting enzyme 2 (ACE2)-in the respiratory tract, the omicron variant efficiently infects the respiratory tract, causing rapid and widespread infections of COVID-19. The omicron variant contains extensive mutations in the receptor-binding domain (RBD) of its spike protein that recognizes human ACE2. Here, using a combination of biochemical and X-ray crystallographic approaches, we showed that the omicron RBD binds to ACE2 with enhanced affinity and also elucidated the role of each of the omicron mutations in ACE2 binding. The enhanced ACE2 binding by the omicron RBD may contribute to the omicron variant's new viral tropism in the respiratory tract despite the low level of ACE2 expression in the tissue. These findings help us to understand tissue tropism of the omicron variant and shed light on the molecular evolution of SARS-CoV-2.

Improved Binding Affinity of Omicron's Spike Protein for the Human Angiotensin-Converting Enzyme 2 Receptor Is the Key behind Its Increased Virulence.

The new variant of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), Omicron, has been quickly spreading in many countries worldwide. Compared to the original virus, Omicron is characterized by several mutations in its genomic region, including the spike protein's receptor-binding domain (RBD). We have computationally investigated the interaction between the RBD of both the wild type and Omicron variant of SARS-CoV-2 with the human angiotensin-converting enzyme 2 (hACE2) receptor using molecular dynamics and molecular mechanics-generalized Born surface area (MM-GBSA)-based binding free energy calculations. The mode of the interaction between Omicron's RBD with the hACE2 receptor is similar to the original SARS-CoV-2 RBD except for a few key differences. The binding free energy difference shows that the spike protein of Omicron has an increased affinity for the hACE2 receptor. The mutated residues in the RBD showed strong interactions with a few amino acid residues of hACE2. More specifically, strong electrostatic interactions (salt bridges) and hydrogen bonding were observed between R493 and R498 residues of the Omicron RBD with D30/E35 and D38 residues of the hACE2, respectively. Other mutated amino acids in the Omicron RBD, e.g., S496 and H505, also exhibited hydrogen bonding with the hACE2 receptor. A pi-stacking interaction was also observed between tyrosine residues (RBD-Tyr501: hACE2-Tyr41) in the complex, which contributes majorly to the binding free energies and suggests that this is one of the key interactions stabilizing the formation of the complex. The resulting structural insights into the RBD:hACE2 complex, the binding mode information within it, and residue-wise contributions to the free energy provide insight into the increased transmissibility of Omicron and pave the way to design and optimize novel antiviral agents.

Recombinant VLPs empower RBM peptides showing no immunogenicity in native SARS-COV-2 protein to elicit a robust neutralizing antibody response.

New SARS-COV-2 vaccine strategies are still urgently needed, especially for emerging virus mutations and variants. In this study, we focused on analyzing the antigenicity and vaccine potency of linear peptide epitopes located in receptor binding motif (RBM) of spike (S) protein. Nine 12 to 16-mer overlapping peptides (P1-P9) were synthesized chemically and coupled to carrier protein KLH for the immunization in mice. Four of identified peptides were further engineered to present on the surface of recombinant Hepatitis B core antigen (HBcAg) virus-like particles (VLPs) respectively. Antisera obtained from VLPs -immunized mice demonstrated strong reactivity and affinity to S1 protein or inactivated virus and neutralizing activity against virus infection in vitro. This study indicates that recombinant VLPs empower peptides which display underprivileged antigenicity in native protein to elicit high levels of neutralizing antibody, providing potential epitope candidates and an effective delivery strategy for the development of a multi-epitope vaccine.

SARS-CoV-2 Omicron Mutation Is Faster than the Chase: Multiple Mutations on Spike/ACE2 Interaction Residues.

Recently, a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (B.1.1.529) Omicron variant originated from South Africa in the middle of November 2021. SARS-CoV-2 is also called coronavirus disease 2019 (COVID-19) since SARS-CoV-2 is the causative agent of COVID-19. Several studies already suggested that the SARS-CoV-2 Omicron variant would be the fastest transmissible variant compared to the previous 10 SARS-CoV-2 variants of concern, interest, and alert. Few clinical studies reported the high transmissibility of the Omicron variant but there is insufficient time to perform actual experiments to prove it, since the spread is so fast. We analyzed the SARS-CoV-2 Omicron variant, which revealed a very high rate of mutation at amino acid residues that interact with angiostatin-converting enzyme 2. The mutation rate of COVID-19 is faster than what we prepared vaccine program, antibody therapy, lockdown, and quarantine against COVID-19 so far. Thus, it is necessary to find better strategies to overcome the current crisis of COVID-19 pandemic.

Modeling SARS-CoV-2 spike/ACE2 protein-protein interactions for predicting the binding affinity of new spike variants for ACE2, and novel ACE2 structurally related human protein targets, for COVID-19 handling in the 3PM context.

Aims: The rapid spread of new SARS-CoV-2 variants has highlighted the crucial role played in the infection by mutations occurring at the SARS-CoV-2 spike receptor binding domain (RBD) in the interactions with the human ACE2 receptor. In this context, it urgently needs to develop new rapid tools for quickly predicting the affinity of ACE2 for the SARS-CoV-2 spike RBD protein variants to be used with the ongoing SARS-CoV-2 genomic sequencing activities in the clinics, aiming to gain clues about the transmissibility and virulence of new variants, to prevent new outbreaks and to quickly estimate the severity of the disease in the context of the 3PM. Methods: In our study, we used a computational pipeline for calculating the interaction energies at the SARS-CoV-2 spike RBD/ACE2 protein-protein interface for a selected group of characterized infectious variants of concern/interest (VoC/VoI). By using our pipeline, we built 3D comparative models of the SARS-CoV-2 spike RBD/ACE2 protein complexes for the VoC B.1.1.7-United Kingdom (carrying the mutations of concern/interest N501Y, S494P, E484K at the RBD), P.1-Japan/Brazil (RBD mutations: K417T, E484K, N501Y), B.1.351-South Africa (RBD mutations: K417N, E484K, N501Y), B.1.427/B.1.429-California (RBD mutations: L452R), the B.1.141 (RBD mutations: N439K), and the recent B.1.617.1-India (RBD mutations: L452R; E484Q) and the B.1.620 (RBD mutations: S477N; E484K). Then, we used the obtained 3D comparative models of the SARS-CoV-2 spike RBD/ACE2 protein complexes for predicting the interaction energies at the protein-protein interface. Results: Along SARS-CoV-2 mutation database screening and mutation localization analysis, it was ascertained that the most dangerous mutations at VoC/VoI spike proteins are located mainly at three regions of the SARS-CoV-2 spike "boat-shaped" receptor binding motif, on the RBD domain. Notably, the P.1 Japan/Brazil variant present three mutations, K417T, E484K, N501Y, located along the entire receptor binding motif, which apparently determines the highest interaction energy at the SARS-CoV-2 spike RBD/ACE2 protein-protein interface, among those calculated. Conversely, it was also observed that the replacement of a single acidic/hydrophilic residue with a basic residue (E484K or N439K) at the "stern" or "bow" regions, of the boat-shaped receptor binding motif on the RBD, appears to determine an interaction energy with ACE2 receptor higher than that observed with single mutations occurring at the "hull" region or with other multiple mutants. In addition, our pipeline allowed searching for ACE2 structurally related proteins, i.e., THOP1 and NLN, which deserve to be investigated for their possible involvement in interactions with the SARS-CoV-2 spike protein, in those tissues showing a low expression of ACE2, or as a novel receptor for future spike variants. A freely available web-tool for the in silico calculation of the interaction energy at the SARS-CoV-2 spike RBD/ACE2 protein-protein interface, starting from the sequences of the investigated spike and/or ACE2 variants, was made available for the scientific community at: https://www.mitoairm.it/covid19affinities. Conclusion: In the context of the PPPM/3PM, the employment of the described pipeline through the provided webservice, together with the ongoing SARS-CoV-2 genomic sequencing, would help to predict the transmissibility of new variants sequenced from future patients, depending on SARS-CoV-2 genomic sequencing activities and on the specific amino acid replacement and/or on its location on the SARS-CoV-2 spike RBD, to put in play all the possible counteractions for preventing the most deleterious scenarios of new outbreaks, taking into consideration that a greater transmissibility has not to be necessarily related to a more severe manifestation of the disease. Supplementary Information: The online version contains supplementary material available at 10.1007/s13167-021-00267-w.

SARS-CoV-2 Delta (B.1.617.2) Variant: A Unique T478K Mutation in Receptor Binding Motif (RBM) of Spike Gene.

Over two hundred twenty-eight million cases of coronavirus disease 2019 (COVID-19) in the world have been reported until the 21st of September 2021 after the first rise in December 2019. The virus caused the disease called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Over 4 million deaths blame COVID-19 during the last one year and 8 months in the world. Currently, four SARS-CoV-2 variants of concern are mainly focused by pandemic studies with limited experiments to translate the infectivity and pathogenicity of each variant. The SARS-CoV-2 α, ß, γ, and δ variant of concern was originated from United Kingdom, South Africa, Brazil/Japan, and India, respectively. The classification of SARS-CoV-2 variant is based on the mutation in spike (S) gene on the envelop of SARS-CoV-2. This review describes four SARS-CoV-2 α, ß, γ, and δ variants of concern including SARS-CoV-2 ε, ζ, η, ι, κ, and B.1.617.3 variants of interest and alert. Recently, SARS-CoV-2 δ variant prevails over different countries that have 3 unique mutation sites: E156del/R158G in the N-terminal domain and T478K in a crucial receptor binding domain. A particular mutation in the functional domain of the S gene is probably associated with the infectivity and pathogenesis of the SARS-CoV-2 variant.