Binding affinity improvement analysis of multiple-mutant Omicron on 2019-nCov to human ACE2 by in silico predictions.

CONTEXT: Since the outbreak of COVID-19 in 2019, the 2019-nCov coronavirus has appeared diverse mutational characteristics due to its own flexible conformation. One multiple-mutant strain (Omicron) with surprisingly infective activity outburst, and affected the biological activities of current drugs and vaccines, making the epidemic significantly difficult to prevent and control, and seriously threaten health around the world. Importunately exploration of mutant characteristics for novel coronavirus Omicron can supply strong theoretical guidance for learning binding mechanism of mutant viruses. What's more, full acknowledgement of key mutated-residues on Omicron strain can provide new methodology of the novel pathogenic mechanism to human ACE2 receptor, as well as the subsequent vaccine development. METHODS: In this research, 3D structures of 32 single-point mutations of 2019-nCov were firstly constructed, and 32-sites multiple-mutant Omicron were finally obtained based one the wild-type virus by homology modeling method. One total number of 33 2019-nCov/ACE2 complex systems were acquired by protein-protein docking, and optimized by using preliminary molecular dynamic simulations. Binding free energies between each 2019-nCov mutation system and human ACE2 receptor were calculated, and corresponding binding patterns especially the regions adjacent to mutation site were analyzed. The results indicated that one total number of 6 mutated sites on the Omicron strain played crucial role in improving binding capacities from 2019-nCov to ACE2 protein. Subsequently, we performed long-term molecular dynamic simulations and protein-protein binding energy analysis for the selected 6 mutations. 3 infected individuals, the mutants T478K, Q493R and G496S with lower binding energies -66.36, -67.98 and -67.09 kcal/mol also presents the high infectivity. These findings indicated that the 3 mutations T478K, Q493R and G496S play the crucial roles in enhancing binding affinity of Omicron to human ACE2 protein. All these results illuminate important theoretical guidance for future virus detection of the Omicron epidemic, drug research and vaccine development.

SELP Asp603Asn and severe thrombosis in COVID-19 males.

Thromboembolism is a frequent cause of severity and mortality in COVID-19. However, the etiology of this phenomenon is not well understood. A cohort of 1186 subjects, from the GEN-COVID consortium, infected by SARS-CoV-2 with different severity was stratified by sex and adjusted by age. Then, common coding variants from whole exome sequencing were mined by LASSO logistic regression. The homozygosity of the cell adhesion molecule P-selectin gene (SELP) rs6127 (c.1807G > A; p.Asp603Asn) which has been already associated with thrombotic risk is found to be associated with severity in the male subcohort of 513 subjects (odds ratio = 2.27, 95% Confidence Interval 1.54-3.36). As the SELP gene is downregulated by testosterone, the odd ratio is increased in males older than 50 (OR 2.42, 95% CI 1.53-3.82). Asn/Asn homozygotes have increased D-dimers values especially when associated with poly Q ≥ 23 in the androgen receptor (OR 3.26, 95% CI 1.41-7.52). These results provide a rationale for the repurposing of antibodies against P-selectin as adjuvant therapy in rs6127 male homozygotes especially if older than 50 or with an impaired androgen receptor.

DGCddG: Deep Graph Convolution for Predicting Protein-Protein Binding Affinity Changes Upon Mutations.

Effectively and accurately predicting the effects of interactions between proteins after amino acid mutations is a key issue for understanding the mechanism of protein function and drug design. In this study, we present a deep graph convolution (DGC) network-based framework, DGCddG, to predict the changes of protein-protein binding affinity after mutation. DGCddG incorporates multi-layer graph convolution to extract a deep, contextualized representation for each residue of the protein complex structure. The mined channels of the mutation sites by DGC is then fitted to the binding affinity with a multi-layer perceptron. Experiments with results on multiple datasets show that our model can achieve relatively good performance for both single and multi-point mutations. For blind tests on datasets related to angiotensin-converting enzyme 2 binding with the SARS-CoV-2 virus, our method shows better results in predicting ACE2 changes, may help in finding favorable antibodies. Code and data availability: https://github.com/lennylv/DGCddG.

Retrospective in silico mutation profiling of SARS-CoV-2 structural proteins circulating in Uganda by July 2021: Towards refinement of COVID-19 disease vaccines, diagnostics, and therapeutics.

The SARS-CoV-2 virus, the agent of COVID-19, caused unprecedented loss of lives and economic decline worldwide. Although the introduction of public health measures, vaccines, diagnostics, and therapeutics disrupted the spread of the SARS-CoV-2, the emergence of variants poses substantial threat. This study traced SARS-CoV-2 variants circulating in Uganda by July 2021 to inform the necessity for refinement of the intervention medical products. A comprehensive in silico analysis of the SARS-CoV-2 genomes detected in clinical samples collected from COVID-19 patients in Uganda revealed occurrence of structural protein variants with potential of escaping detection, resisting antibody therapy, or increased infectivity. The genome sequence dataset was retrieved from the GISAID database and the open reading frame encoding the spike, envelope, membrane, or nucleocapsid proteins was translated. The obtained protein sequences were aligned and inspected for existence of variants. The variant positions on each of the four alignment sets were mapped on predicted epitopes as well as the 3D structures. Additionally, sequences within each of the sets were clustered by family. A phylogenetic tree was constructed to assess relationship between the encountered spike protein sequences and Wuhan-Hu-1 wild-type, or the Alpha, Beta, Delta and Gamma variants of concern. Strikingly, the frequency of each of the spike protein point mutations F157L/Del, D614G and P681H/R was over 50%. The furin and the transmembrane serine protease 2 cleavage sites were unaffected by mutation. Whereas the Delta dominated the spike sequences (16.5%, 91/550), Gamma was not detected. The envelope protein was the most conserved with 96.3% (525/545) sequences being wild-type followed by membrane at 68.4% (397/580). Although the nucleocapsid protein sequences varied, the variant residue positions were less concentrated at the RNA binding domains. The dominant nucleocapsid sequence variant was S202N (34.5%, 205/595). These findings offer baseline information required for refining the existing COVID-19 vaccines, diagnostics, and therapeutics.

SARS-CoV-2 Delta Variant: Interplay between Individual Mutations and Their Allosteric Synergy.

Since its first appearance in April 2021, B.1.617.2, also termed variant Delta, catalyzed one major worldwide wave dominating the second year of coronavirus disease 2019 (COVID-19) pandemic. Despite its quick disappearance worldwide, the strong virulence caused by a few point mutations remains an unsolved problem largely. Along with the other two sublineages, the Delta variant harbors an accumulation of Spike protein mutations, including the previously identified L452R, E484Q, and the newly emerged T478K on its receptor binding domain (RBD). We used molecular dynamics (MD) simulations, in combination with free energy perturbation (FEP) calculations, to examine the effects of two combinative mutation sets, L452R + E484Q and L452R + T478K. Our dynamic trajectories reveal an enhancement in binding affinity between mutated RBD and the common receptor protein angiotensin converting enzyme 2 (ACE2) through a net increase in the buried molecular surface area of the binary complex. This enhanced binding, mediated through Gln493, sets the same stage for all three sublineages due to the presence of L452R mutation. The other mutation component, E484Q or T478K, was found to impact the RBD-ACE2 binding and help the variant to evade several monoclonal antibodies (mAbs) in a distinct manner. Especially for L452R + T478K, synergies between mutations are mediated through a complex residual and water interaction network and further enhance its binding to ACE2. Taking together, this study demonstrates that new variants of SARS-CoV-2 accomplish both "attack" (infection) and "defense" (antibody neutralization escape) with the same "polished sword" (mutated Spike RBD).

Case report: BA.1 subvariant showing a BA.2-like pattern using a variant-specific PCR assay due to a single point mutation downstream the spike 69/70 deletion.

BACKGROUND: SARS-CoV-2 variant tracking is key to the genomic surveillance of the COVID-19 pandemic. While next-generation sequencing (NGS) is commonly used for variant determination, it is expensive and time-consuming. Variant-specific PCR (vsPCR) is a faster, cheaper method that detects specific mutations that are considered variant-defining. These tests usually rely on specific amplification when a mutation is present or a specific melting temperature peak after amplification. CASE PRESENTATION: A discrepant result between vsPCR and NGS was found in seventeen SARS-CoV-2 samples from Galicia, Spain. A cluster of BA.1 Omicron SARS-CoV-2 variant showed a BA.2-like melting temperature pattern due to a point mutation (C21772T) downstream the deletion of the spike amino acids 69/70. As the 69/70 deletion is widely used for differentiation between BA.1 and BA.2 by vsPCR, C21772T can cause BA.1 samples to be misinterpreted as BA.2. Over a thousand BA.1 sequences in the EpiCoV database contain this mutation. CONCLUSIONS: To our knowledge, this is the first case of a point mutation causing a vsPCR algorithm to misclassify BA.1 samples as BA.2. This is an example of how mutations in the probe target area of vsPCR tests based on melting curve analysis can lead to variant misclassification. NGS confirmation of vsPCR results is relevant for the accuracy of the epidemiological surveillance. In order to overcome the possible impact of novel mutations, diagnostic tools must be constantly updated.

Comparison of Insertion, Deletion, and Point Mutations in the Genomes of Human Adenovirus HAdvC-2 and SARS-CoV-2.

Virus genome mutation profiles with insertion, deletion, and point mutations have recently been revealed to differ remarkably between viruses. In RNA viruses like human coronaviruses or influenza viruses, genome samples collected over two to three decades usually show point mutations in 10-20% of the bases, while the rate of insertion and/or deletion mutations (indels) largely depends on the virus. This study evaluates the mutation profiles of DNA viruses by comparing a recently sampled genome of human adenovirus species C type 2 (isolate SG06/HAdvC2/2016) with a genome of the same species sampled in the 1970s. It was found insertions of 23 bases at seven sites and deletions of 22 bases at nine sites. The longest indels were 6-base insertions in E2B and L4. All indels in the coding regions were in-frame mutations with base lengths in multiples of three. In the non-coding regions, the lengths of the indels ranged from 1-4 consecutive bases. Long indels with more than 10 consecutive bases, which comprise nearly half of indels in the SARS-CoV-2 genome, were absent. In other sites, the point mutation rate was approximately 0.3%, which was significantly lower than in RNA viruses. In summary, the estimated point mutation rate in human adenovirus species C type 2 (HAdvC-2) was over 10 times lower than in RNA viruses. Unlike the relatively long indels in the SARS-CoV-2 genome, the indels in HAdvC-2 were short, with 6 or fewer consecutive bases.

How Do Point Mutations Enhancing the Basic Character of the RBDs of SARS-CoV-2 Variants Affect Their Transmissibility and Infectivity Capacities?

The spread of SARS-CoV-2 variants in the population depends on their ability to anchor the ACE2 receptor in the host cells. Differences in the electrostatic potentials of the spike protein RBD (electropositive/basic) and ACE2 receptor (electronegative/acidic) play a key role in both the rapprochement and the recognition of the coronavirus by the cell receptors. Accordingly, point mutations that result in an increase in electropositively charged residues, e.g., arginine and lysine, especially in the RBD of spike proteins in the SARS-CoV-2 variants, could contribute to their spreading capacity by favoring their recognition by the electronegatively charged ACE2 receptors. All SARS-CoV-2 variants that have been recognized as being highly transmissible, such as the kappa (κ), delta (δ) and omicron (o) variants, which display an enhanced electropositive character in their RBDs associated with a higher number of lysine- or arginine-generating point mutations. Lysine and arginine residues also participate in the enhanced RBD-ACE2 binding affinity of the omicron variant, by creating additional salt bridges with aspartic and glutamic acid residues from ACE2. However, the effects of lysine- and arginine-generating point mutations on infectivity is more contrasted, since the overall binding affinity of omicron RBD for ACE2 apparently results from some epistasis among the whole set of point mutations.

Origin of the tight binding mode to ACE2 triggered by multi-point mutations in the omicron variant: a dynamic insight.

The continuous spread of the newly emerged SARS-CoV-2 Omicron variant (B.1.1.529) has become an important reason for the surge in COVID-19 infections. Its numerous mutated residues containing key sites on the receptor-binding domain (RBD) undoubtedly pose new challenges for epidemic control. Although the preventive measures are becoming more sophisticated, the effects of mutations on the binding of the virus to the receptor protein remain to be elucidated. Here, we used molecular dynamics (MD) simulations to investigate the differences in the binding mode between the Omicron variant and the angiotensin-converting enzyme 2 (ACE2) compared to the wild-type strain (WT). Multi-point mutations in the Omicron variant RBD could cause the conformation shift in the large Loop (where T478K and E484A are located), which makes it easier to wrap the N-terminal helix of ACE2 and form tighter contacts. The stronger electrostatic interaction was the main reason for its enhanced binding affinity as compared to WT. This was due to the large number of positively charged patches (N440K, T478K, Q493R, Q498R, and Y505H) formed by the substitution of neutral amino acids at multiple sites. The appearance of these highly polar hydrophilic amino acids may cause local perturbations and affect the electrostatic complementarity of RBD with the ACE2, and further mediate conformational changes. Thus, a more extensive interaction network was found in the mutation system and the complex interaction cluster was formed near E37@ACE2, which was essential for the stable binding of the two. In addition, we speculated that these mutations may affect the electrostatic complementarity with the four potential antibodies to reduce the sensitivity of the virus to antibodies. This study reveals the key details of the Omicron variant binding to ACE2 and provides important theoretical views for the enhanced infectivity of this variant. We hope that these observations can provide timely molecular insights for responding to the Omicron variant pandemic.

SARS-CoV-2 Point Mutation and Deletion Spectra and Their Association with Different Disease Outcomes.

Mutant spectra of RNA viruses are important to understand viral pathogenesis and response to selective pressures. There is a need to characterize the complexity of mutant spectra in coronaviruses sampled from infected patients. In particular, the possible relationship between SARS-CoV-2 mutant spectrum complexity and disease associations has not been established. In the present study, we report an ultradeep sequencing (UDS) analysis of the mutant spectrum of amplicons from the nsp12 (polymerase)- and spike (S)-coding regions of 30 nasopharyngeal isolates (diagnostic samples) of SARS-CoV-2 of the first COVID-19 pandemic wave (Madrid, Spain, April 2020) classified according to the severity of ensuing COVID-19. Low-frequency mutations and deletions, counted relative to the consensus sequence of the corresponding isolate, were overwhelmingly abundant. We show that the average number of different point mutations, mutations per haplotype, and several diversity indices was significantly higher in SARS-CoV-2 isolated from patients who developed mild disease than in those associated with moderate or severe disease (exitus). No such bias was observed with RNA deletions. Location of amino acid substitutions in the three-dimensional structures of nsp12 (polymerase) and S suggest significant structural or functional effects. Thus, patients who develop mild symptoms may be a richer source of genetic variants of SARS-CoV-2 than patients with moderate or severe COVID-19. IMPORTANCE The study shows that mutant spectra of SARS-CoV-2 from diagnostic samples differ in point mutation abundance and complexity and that significantly larger values were observed in virus from patients who developed mild COVID-19 symptoms. Mutant spectrum complexity is not a uniform trait among isolates. The nature and location of low-frequency amino acid substitutions present in mutant spectra anticipate great potential for phenotypic diversification of SARS-CoV-2.

CovDif, a Tool to Visualize the Conservation between SARS-CoV-2 Genomes and Variants.

The spread of the newly emerged severe acute respiratory syndrome (SARS-CoV-2) virus has led to more than 430 million confirmed cases, including more than 5.9 million deaths, reported worldwide as of 24 February 2022. Conservation of viral genomes is important for pathogen identification and diagnosis, therapeutics development and epidemiological surveillance to detect the emergence of new viral variants. An intense surveillance of virus variants has led to the identification of Variants of Interest and Variants of Concern. Although these classifications dynamically change as the pandemic evolves, they have been useful to guide public health efforts on containment and mitigation. In this work, we present CovDif, a tool to detect conserved regions between groups of viral genomes. CovDif creates a conservation landscape for each group of genomes of interest and a differential landscape able to highlight differences in the conservation level between groups. CovDif is able to identify loss in conservation due to point mutations, deletions, inversions and chromosomal rearrangements. In this work, we applied CovDif to SARS-CoV-2 clades (G, GH, GR, GV, L, O, S and G) and variants. We identified all regions for any defining SNPs. We also applied CovDif to a group of population genomes and evaluated the conservation of primer regions for current SARS-CoV-2 detection and diagnostic protocols. We found that some of these protocols should be applied with caution as few of the primer-template regions are no longer conserved in some SARS-CoV-2 variants. We conclude that CovDif is a tool that could be widely applied to study the conservation of any group of viral genomes as long as whole genomes exist.

In-Flight Transmission of a SARS-CoV-2 Lineage B.1.617.2 Harbouring the Rare S:E484Q Immune Escape Mutation.

We describe a flight-associated infection scenario of seven individuals with a B.1.617.2 (Delta) lineage, harbouring an S:E484Q point mutation. In Sweden, at least 10% of all positive SARS-CoV-2 samples were sequenced in each county; the B.1.717.2 + S:E484Q combination was not detected in Sweden before and was imported within the scenario described in this report. The high transmission rate of the delta lineage combined with the S:E484Q mutation, associated with immune escape in other lineages, makes this specific genetic combination a possible threat to the global fight against the COVID-19 pandemic. Even within the Omicron wave, the B.1.617.2 + S:E484Q variant appeared in community samples in Sweden, as it seems that this combination has an evolutionary gain compared to other B.1.617.2 lineages. The here described genomic combination was not detectable with the common fasta file-based Pango-lineage analysis, hence increasing the probability of the true global prevalence to be higher.

Real-Time RT-PCR Allelic Discrimination Assay for Detection of N501Y Mutation in the Spike Protein of SARS-CoV-2 Associated with B.1.1.7 Variant of Concern.

The N501Y amino acid mutation caused by a single point substitution A23063T in the spike gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is possessed by three variants of concern (VOCs), B.1.1.7, B.1.351, and P.1. A rapid screening tool using this mutation is important for surveillance during the coronavirus disease 2019 (COVID-19) pandemic. We developed and validated a single nucleotide polymorphism real-time reverse transcription PCR assay using allelic discrimination of the spike gene N501Y mutation to screen for potential variants of concern and differentiate them from SARS-CoV-2 lineages without the N501Y mutation. A total of 160 clinical specimens positive for SARS-CoV-2 were characterized as mutant (N501Y) or N501 wild type by Sanger sequencing and were subsequently tested with the N501Y single nucleotide polymorphism real-time reverse transcriptase PCR assay. Our assay, compared to Sanger sequencing for single nucleotide polymorphism detection, demonstrated positive percent agreement of 100% for all 57 specimens displaying the N501Y mutation, which were confirmed by Sanger sequencing to be typed as A23063T, including one specimen with mixed signal for wild type and mutant. Negative percent agreement was 100% in all 103 specimens typed as N501 wild type, with A23063 identified as wild type by Sanger sequencing. The identification of circulating SARS-CoV-2 lineages carrying an N501Y mutation is critical for surveillance purposes. Current identification methods rely primarily on Sanger sequencing or whole-genome sequencing, which are time consuming, labor intensive, and costly. The assay described herein is an efficient tool for high-volume specimen screening for SARS-CoV-2 VOCs and for selecting specimens for confirmatory Sanger or whole-genome sequencing. IMPORTANCE During the coronavirus disease 2019 (COVID-19) pandemic, several variants of concern (VOCs) have been detected, for example, B.1.1.7, B.1.351, P.1, and B.1.617.2. The VOCs pose a threat to public health efforts to control the spread of the virus. As such, surveillance and monitoring of these VOCs is of the utmost importance. Our real-time RT-PCR assay helps with surveillance by providing an easy method to quickly survey SARS-CoV-2 specimens for VOCs carrying the N501Y single nucleotide polymorphism (SNP). Samples that test positive for the N501Y mutation in the spike gene with our assay can be sequenced to identify the lineage. Thus, our assay helps to focus surveillance efforts and decrease turnaround times.

Impact of ADAR-induced editing of minor viral RNA populations on replication and transmission of SARS-CoV-2.

Adenosine deaminases acting on RNA (ADAR) are RNA-editing enzymes that may restrict viral infection. We have utilized deep sequencing to determine adenosine to guanine (A→G) mutations, signifying ADAR activity, in clinical samples retrieved from 93 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients in the early phase of the COVID-19 pandemic. A→G mutations were detected in 0.035% (median) of RNA residues and were predominantly nonsynonymous. These mutations were rarely detected in the major viral population but were abundant in minor viral populations in which A→G was more prevalent than any other mutation (P < 0.001). The A→G substitutions accumulated in the spike protein gene at positions corresponding to amino acids 505 to 510 in the receptor binding motif and at amino acids 650 to 655. The frequency of A→G mutations in minor viral populations was significantly associated with low viral load (P < 0.001). We additionally analyzed A→G mutations in 288,247 SARS-CoV-2 major (consensus) sequences representing the dominant viral population. The A→G mutations observed in minor viral populations in the initial patient cohort were increasingly detected in European consensus sequences between March and June 2020 (P < 0.001) followed by a decline of these mutations in autumn and early winter (P < 0.001). We propose that ADAR-induced deamination of RNA is a significant source of mutated SARS-CoV-2 and hypothesize that the degree of RNA deamination may determine or reflect viral fitness and infectivity.

SARS-CoV-2 N Gene G29195T Point Mutation May Affect Diagnostic Reverse Transcription-PCR Detection.

Rapid onsite whole-genome sequencing of two suspected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N gene diagnostic escape samples revealed a previously unreported N gene point mutation at genome position 29195. Because the G29195T mutation occurs within a region probed by a commonly referenced U.S. CDC N gene reverse transcription (RT)-PCR assay, we hypothesize that the G29195T mutation rendered the N gene target of a proprietary commercial assay undetectable. The putative diagnostic escape G29195T mutation demonstrates the need for nearly real-time surveillance, as emergence of a novel SARS-CoV-2 variant with the potential to escape diagnostic tests continues to be a threat. IMPORTANCE Accurate diagnostic detection of SARS-CoV-2 currently depends on the large-scale deployment of RT-PCR assays. SARS-CoV-2 RT-PCR assays target predetermined regions in the viral genomes by complementary binding of primers and probes to nucleic acid sequences in the clinical samples. Potential diagnostic escapes, such as those of clinical samples harboring the G29195T mutation, may result in false-negative SARS-CoV-2 RT-PCR results. The rapid detection and sharing of potential diagnostic escapes are essential for diagnostic laboratories and manufacturers around the world, to optimize their assays as SARS-CoV-2 continues to evolve.

Single-Point Mutations in the N Gene of SARS-CoV-2 Adversely Impact Detection by a Commercial Dual Target Diagnostic Assay.

Accurate and rapid diagnostic tests are a critical component for the early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of the overall control strategy for the current pandemic. Nucleic acid amplification tests are the gold standard for diagnosis of acute SARS-CoV-2 infection, and many real-time PCR diagnostic assays have been developed. Mutations that occur within the primer/probe binding regions of the SARS-CoV-2 genome can negatively impact the performance of diagnostic assays. Here, we report two single-point mutations in the N gene of SARS-CoV-2 associated with N gene target detection failures in the Cepheid Xpert Xpress SARS-CoV-2 assay, the first a C to T mutation at position 29197, found in five patients, and the second a C to T mutation at position 29200, found in eight patients. By sequencing the Xpert amplicons, we showed both mutations to be located within the amplified region of the Xpert N gene target. This report highlights the necessity for multiple genetic targets and the continual monitoring and evaluation of diagnostic assay performance. IMPORTANCE This paper reports the identification of single-point mutations in the N gene of SARS-CoV-2 associated with a gene target failure by the Cepheid Xpert commercial system. In order to determine the mutation(s) responsible for the N gene detection failures, the genomic products from the Cepheid Xpert system were sequenced and compared to whole genomes of SARS-CoV-2 from clinical cases. This report is the first to our knowledge which characterizes the amplified PCR products of the Xpert system, confirming the mutations associated with the gene target failure. The mutations identified have previously been reported.

SARS-CoV-2 spike evolutionary behaviors; simulation of N501Y mutation outcomes in terms of immunogenicity and structural characteristic.

Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a large number of mutations in its genome have been reported. Some of the mutations occur in noncoding regions without affecting the pathobiology of the virus, while mutations in coding regions are significant. One of the regions where a mutation can occur, affecting the function of the virus is at the receptor-binding domain (RBD) of the spike protein. RBD interacts with angiotensin-converting enzyme 2 (ACE2) and facilitates the entry of the virus into the host cells. There is a lot of focus on RBD mutations, especially the displacement of N501Y which is observed in the UK/Kent, South Africa, and Brazilian lineages of SARS-CoV-2. Our group utilizes computational biology approaches such as immunoinformatics, protein-protein interaction analysis, molecular dynamics, free energy computation, and tertiary structure analysis to disclose the consequences of N501Y mutation at the molecular level. Surprisingly, we discovered that this mutation reduces the immunogenicity of the spike protein; also, displacement of Asn with Tyr reduces protein compactness and significantly increases the stability of the spike protein and its affinity to ACE2. Moreover, following the N501Y mutation secondary structure and folding of the spike protein changed dramatically.

SCoV2-MD: a database for the dynamics of the SARS-CoV-2 proteome and variant impact predictions.

SCoV2-MD (www.scov2-md.org) is a new online resource that systematically organizes atomistic simulations of the SARS-CoV-2 proteome. The database includes simulations produced by leading groups using molecular dynamics (MD) methods to investigate the structure-dynamics-function relationships of viral proteins. SCoV2-MD cross-references the molecular data with the pandemic evolution by tracking all available variants sequenced during the pandemic and deposited in the GISAID resource. SCoV2-MD enables the interactive analysis of the deposited trajectories through a web interface, which enables users to search by viral protein, isolate, phylogenetic attributes, or specific point mutation. Each mutation can then be analyzed interactively combining static (e.g. a variety of amino acid substitution penalties) and dynamic (time-dependent data derived from the dynamics of the local geometry) scores. Dynamic scores can be computed on the basis of nine non-covalent interaction types, including steric properties, solvent accessibility, hydrogen bonding, and other types of chemical interactions. Where available, experimental data such as antibody escape and change in binding affinities from deep mutational scanning experiments are also made available. All metrics can be combined to build predefined or custom scores to interrogate the impact of evolving variants on protein structure and function.

Activation of NF-κB and induction of proinflammatory cytokine expressions mediated by ORF7a protein of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for coronavirus disease 2019 (COVID-19) that emerged in human populations recently. Severely ill COVID-19 patients exhibit the elevation of proinflammatory cytokines, and such an unbalanced production of proinflammatory cytokines is linked to acute respiratory distress syndrome with high mortality in COVID-19 patients. Our study provides evidence that the ORF3a, M, ORF7a, and N proteins of SARS-CoV-2 were NF-κB activators. The viral sequence from infected zoo lions belonged to clade V, and a single mutation of G251V is found for ORF3a gene compared to all other clades. No significant functional difference was found for clade V ORF3a, indicating the NF-κB activation is conserved among COVID-19 variants. Of the four viral proteins, the ORF7a protein induced the NF-κB dictated proinflammatory cytokines including IL-1α, IL-1ß, IL-6, IL-8, IL-10, TNF-α, and IFNß. The ORF7a protein also induced IL-3, IL-4, IL-7, IL-23. Of 15 different chemokines examined in the study, CCL11, CCL17, CCL19, CCL20, CCL21, CCL22, CCL25, CCL26, CCL27, and CXCL9 were significantly upregulated by ORF7. These cytokines and chemokines were frequently elevated in severely ill COVID-19 patients. Our data provide an insight into how SARS-CoV-2 modulates NF-κB signaling and inflammatory cytokine expressions. The ORF7a protein may be a desirable target for strategic developments to minimize uncontrolled inflammation in COVID-19 patients.

SARS-CoV-2 beta variant substitutions alter spike glycoprotein receptor binding domain structure and stability.

The emergence of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and the subsequent COVID-19 pandemic have visited a terrible cost on the world in the forms of disease, death, and economic turmoil. The rapid development and deployment of extremely effective vaccines against SARS-CoV-2 have seemingly brought within reach the end of the pandemic. However, the virus has acquired mutations. and emerging variants of concern are more infectious and reduce the efficacy of existing vaccines. Although promising efforts to combat these variants are underway, the evolutionary pressures leading to these variants are poorly understood. To that end, here we have studied the effects on the structure and function of the SARS-CoV-2 spike glycoprotein receptor-binding domain of three amino-acid substitutions found in several variants of concern, including alpha (B.1.1.7), beta (B.1.351), and gamma (P.1). We found that these substitutions alter the receptor-binding domain structure, stability, and ability to bind to angiotensin converting enzyme 2, in such a way as to possibly have opposing and compensatory effects. These findings provide new insights into how these variants of concern may have been selected for infectivity while maintaining the structure and stability of the receptor binding domain.