Missense Variants of von Willebrand Factor in the Background of COVID-19 Associated Coagulopathy.

COVID-19 associated coagulopathy (CAC), characterized by endothelial dysfunction and hypercoagulability, evokes pulmonary immunothrombosis in advanced COVID-19 cases. Elevated von Willebrand factor (vWF) levels and reduced activities of the ADAMTS13 protease are common in CAC. Here, we aimed to determine whether common genetic variants of these proteins might be associated with COVID-19 severity and hemostatic parameters. A set of single nucleotide polymorphisms (SNPs) in the vWF (rs216311, rs216321, rs1063856, rs1800378, rs1800383) and ADAMTS13 genes (rs2301612, rs28729234, rs34024143) were genotyped in 72 COVID-19 patients. Cross-sectional cohort analysis revealed no association of any polymorphism with disease severity. On the other hand, analysis of variance (ANOVA) uncovered associations with the following clinical parameters: (1) the rs216311 T allele with enhanced INR (international normalized ratio); (2) the rs1800383 C allele with elevated fibrinogen levels; and (3) the rs1063856 C allele with increased red blood cell count, hemoglobin, and creatinine levels. No association could be observed between the phenotypic data and the polymorphisms in the ADAMTS13 gene. Importantly, in silico protein conformational analysis predicted that these missense variants would display global conformational alterations, which might affect the stability and plasma levels of vWF. Our results imply that missense vWF variants might modulate the thrombotic risk in COVID-19.

Better safe than sorry-Whole-genome sequencing indicates that missense variants are significant in susceptibility to COVID-19.

Undoubtedly, genetic factors play an important role in susceptibility and resistance to COVID-19. In this study, we conducted the GWAS analysis. Out of 15,489,173 SNPs, we identified 18,191 significant SNPs for severe and 11,799 SNPs for resistant phenotype, showing that a great number of loci were significant in different COVID-19 representations. The majority of variants were synonymous (60.56% for severe, 58.46% for resistant phenotype) or located in introns (55.77% for severe, 59.83% for resistant phenotype). We identified the most significant SNPs for a severe outcome (in AJAP1 intron) and for COVID resistance (in FIG4 intron). We found no missense variants with a potential causal function on resistance to COVID-19; however, two missense variants were determined as significant a severe phenotype (in PM20D1 and LRP4 exons). None of the aforementioned SNPs and missense variants found in this study have been previously associated with COVID-19.

Missense mutations in spike protein of SARS-CoV-2 delta variant contribute to the alteration in viral structure and interaction with hACE2 receptor.

INTRODUCTION: Many of the global pandemics threaten human existence over the decades among which coronavirus disease (COVID-19) is the newest exposure circulating worldwide. The RNA encoded severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is referred as the pivotal agent of this deadly disease that induces respiratory tract infection by interacting host ACE2 receptor with its spike glycoprotein. Rapidly evolving nature of this virus modified into new variants helps in perpetrating immune escape and protection against host defense mechanism. Consequently, a new isolate, delta variant originated from India is spreading perilously at a higher infection rate. METHODS: In this study, we focused to understand the conformational and functional significance of the missense mutations found in the spike glycoprotein of SARS-CoV-2 delta variant performing different computational analysis. RESULTS: From physiochemical analysis, we found that the acidic isoelectric point of the virus elevated to basic pH level due to the mutations. The targeted mutations were also found to change the interactive bonding pattern and conformational stability analyzed by the molecular dynamic's simulation. The molecular docking study also revealed that L452R and T478K mutations found in the RBD domain of delta variant spike protein contributed to alter interaction with the host ACE2 receptor. CONCLUSIONS: Overall, this study provided insightful evidence to understand the morphological and attributive impact of the mutations on SARS-CoV-2 delta variant.

The spike receptor-binding motif G496S substitution determines the replication fitness of SARS-CoV-2 Omicron sublineage.

The replication and pathogenicity of SARS-CoV-2 Omicron BA.2 are comparable to that of BA.1 in experimental animal models. However, BA.2 has rapidly emerged to overtake BA.1 to become the predominant circulating SARS-CoV-2 variant worldwide. Here, we compared the replication fitness of BA.1 and BA.2 in cell culture and in the Syrian hamster model of COVID-19. Using a reverse genetics approach, we found that the BA.1-specific spike mutation G496S compromises its replication fitness, which may contribute to BA.1 being outcompeted by BA.2 in the real world. Additionally, the BA.1-unique G496S substitution confers differentiated sensitivity to therapeutic monoclonal antibodies, which partially recapitulates the immunoevasive phenotype of BA.1 and BA.2. In summary, our study identified G496S as an important determinant during the evolutionary trajectory of SARS-CoV-2.

Pro108Ser mutation of SARS-CoV-2 3CLpro reduces the enzyme activity and ameliorates the clinical severity of COVID-19.

Recently, an international randomized controlled clinical trial showed that patients with SARS-CoV-2 infection treated orally with the 3-chymotrypsin-like protease (3CLpro) inhibitor PF-07321332 within three days of symptom onset showed an 89% lower risk of COVID-19-related hospital admission/ death from any cause as compared with the patients who received placebo. Lending support to this critically important result of the aforementioned trial, we demonstrated in our study that patients infected with a SARS-Cov-2 sub-lineage (B.1.1.284) carrying the Pro108Ser mutation in 3CLpro tended to have a comparatively milder clinical course (i.e., a smaller proportion of patients required oxygen supplementation during the clinical course) than patients infected with the same sub-lineage of virus not carrying the mutation. Characterization of the mutant 3CLpro revealed that the Kcat/Km of the 3CLpro enzyme containing Ser108 was 58% lower than that of Pro108 3CLpro. Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) revealed that the reduced activity was associated with structural perturbation surrounding the substrate-binding region of the enzyme, which is positioned behind and distant from the 108th amino acid residue. Our findings of the attenuated clinical course of COVID-19 in patients infected with SARS-CoV-2 strains with reduced 3CLpro enzymatic activity greatly endorses the promising result of the aforementioned clinical trial of the 3CLpro inhibitor.

Allostery and Missense Mutations as Intermittently Linked Promising Aspects of Modern Computational Drug Discovery.

Drug research and development is a multidisciplinary field with its own successes. Yet, given the complexity of the process, it also faces challenges over the long development stages and even includes those that develop once a drug is marketed, i.e. drug toxicity and drug resistance. Better success can be achieved via well designed criteria in the early drug development stages. Here, we introduce the concepts of allostery and missense mutations, and argue that incorporation of these two intermittently linked biological phenomena into the early computational drug discovery stages would help to reduce the attrition risk in later stages of the process. We discuss the individual or in concert mechanisms of actions of mutations in allostery. Design of allosteric drugs is challenging compared to orthosteric drugs, yet they have been gaining popularity in recent years as alternative systems for the therapeutic regulation of proteins with an action-at-a-distance mode and non-invasive mechanisms. We propose an easy-to-apply computational allosteric drug discovery protocol which considers the mutation effect, and detail it with three case studies focusing on (1) analysis of effect of an allosteric mutation related to isoniazid drug resistance in tuberculosis; (2) identification of a cryptic pocket in the presence of an allosteric mutation of falcipain-2 as a malarial drug target; and (3) deciphering the effects of SARS-CoV-2 evolutionary mutations on a potential allosteric modulator with changes to allosteric communication paths.

Statistical modeling of SARS-CoV-2 substitution processes: predicting the next variant.

We build statistical models to describe the substitution process in the SARS-CoV-2 as a function of explanatory factors describing the sequence, its function, and more. These models serve two different purposes: first, to gain knowledge about the evolutionary biology of the virus; and second, to predict future mutations in the virus, in particular, non-synonymous amino acid substitutions creating new variants. We use tens of thousands of publicly available SARS-CoV-2 sequences and consider tens of thousands of candidate models. Through a careful validation process, we confirm that our chosen models are indeed able to predict new amino acid substitutions: candidates ranked high by our model are eight times more likely to occur than random amino acid changes. We also show that named variants were highly ranked by our models before their appearance, emphasizing the value of our models for identifying likely variants and potentially utilizing this knowledge in vaccine design and other aspects of the ongoing battle against COVID-19.

Detection of SARS-CoV-2 and the L452R spike mutation using reverse transcription loop-mediated isothermal amplification plus bioluminescent assay in real-time (RT-LAMP-BART).

The new coronavirus infection (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be fatal, and several variants of SARS-CoV-2 with mutations of the receptor-binding domain (RBD) have increased avidity for human cell receptors. A single missense mutation of U to G at nucleotide position 1355 (U1355G) in the spike (S) gene changes leucine to arginine (L452R) in the spike protein. This mutation has been observed in the India and California strains (B.1.617 and B.1.427/B.1.429, respectively). Control of COVID-19 requires rapid and reliable detection of SARS-CoV-2. Therefore, we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay plus a bioluminescent assay in real-time (BART) to detect SARS-CoV-2 and the L452R spike mutation. The specificity and sensitivity of the RT-LAMP-BART assay was evaluated using synthetic RNAs including target sequences and RNA-spiked clinical nasopharyngeal and saliva specimens as well as reference strains representing five viral and four bacterial pathogens. The novel RT-LAMP-BART assay to detect SARS-CoV-2 was highly specific compared to the conventional real-time RT-PCR. Within 25 min, the RT-LAMP-BART assay detected 80 copies of the target gene in a sample, whereas the conventional real-time RT-PCR method detected 5 copies per reaction within 130 min. Using RNA-spiked specimens, the sensitivity of the RT-LAMP-BART assay was slightly attenuated compared to purified RNA as a template. The results were identical to those of the conventional real-time RT-PCR method. Furthermore, using a peptide nucleic acid (PNA) probe, the RT-LAMP-BART method correctly identified the L452R spike mutation. This is the first report describes RT-LAMP-BART as a simple, inexpensive, rapid, and useful assay for detection of SARS-CoV-2, its variants of concern, and for screening of COVID-19.

Evolution of the SARS-CoV-2 spike protein in the human host.

Recently emerged variants of SARS-CoV-2 contain in their surface spike glycoproteins multiple substitutions associated with increased transmission and resistance to neutralising antibodies. We have examined the structure and receptor binding properties of spike proteins from the B.1.1.7 (Alpha) and B.1.351 (Beta) variants to better understand the evolution of the virus in humans. Spikes of both variants have the same mutation, N501Y, in the receptor-binding domains. This substitution confers tighter ACE2 binding, dependent on the common earlier substitution, D614G. Each variant spike has acquired other key changes in structure that likely impact virus pathogenesis. The spike from the Alpha variant is more stable against disruption upon binding ACE2 receptor than all other spikes studied. This feature is linked to the acquisition of a more basic substitution at the S1-S2 furin site (also observed for the variants of concern Delta, Kappa, and Omicron) which allows for near-complete cleavage. In the Beta variant spike, the presence of a new substitution, K417N (also observed in the Omicron variant), in combination with the D614G, stabilises a more open spike trimer, a conformation required for receptor binding. Our observations suggest ways these viruses have evolved to achieve greater transmissibility in humans.

Missense variants in human ACE2 strongly affect binding to SARS-CoV-2 Spike providing a mechanism for ACE2 mediated genetic risk in Covid-19: A case study in affinity predictions of interface variants.

SARS-CoV-2 Spike (Spike) binds to human angiotensin-converting enzyme 2 (ACE2) and the strength of this interaction could influence parameters relating to virulence. To explore whether population variants in ACE2 influence Spike binding and hence infection, we selected 10 ACE2 variants based on affinity predictions and prevalence in gnomAD and measured their affinities and kinetics for Spike receptor binding domain through surface plasmon resonance (SPR) at 37°C. We discovered variants that reduce and enhance binding, including three ACE2 variants that strongly inhibited (p.Glu37Lys, ΔΔG = -1.33 ± 0.15 kcal mol-1 and p.Gly352Val, predicted ΔΔG = -1.17 kcal mol-1) or abolished (p.Asp355Asn) binding. We also identified two variants with distinct population distributions that enhanced affinity for Spike. ACE2 p.Ser19Pro (ΔΔG = 0.59 ± 0.08 kcal mol-1) is predominant in the gnomAD African cohort (AF = 0.003) whilst p.Lys26Arg (ΔΔG = 0.26 ± 0.09 kcal mol-1) is predominant in the Ashkenazi Jewish (AF = 0.01) and European non-Finnish (AF = 0.006) cohorts. We compared ACE2 variant affinities to published SARS-CoV-2 pseudotype infectivity data and confirmed that ACE2 variants with reduced affinity for Spike can protect cells from infection. The effect of variants with enhanced Spike affinity remains unclear, but we propose a mechanism whereby these alleles could cause greater viral spreading across tissues and cell types, as is consistent with emerging understanding regarding the interplay between receptor affinity and cell-surface abundance. Finally, we compared mCSM-PPI2 ΔΔG predictions against our SPR data to assess the utility of predictions in this system. We found that predictions of decreased binding were well-correlated with experiment and could be improved by calibration, but disappointingly, predictions of highly enhanced binding were unreliable. Recalibrated predictions for all possible ACE2 missense variants at the Spike interface were calculated and used to estimate the overall burden of ACE2 variants on Covid-19.

Analyses of Long-Term Epidemic Trends and Evolution Characteristics of Haplotype Subtypes Reveal the Dynamic Selection on SARS-CoV-2.

The scale of SARS-CoV-2 infection and death is so enormous that further study of the molecular and evolutionary characteristics of SARS-CoV-2 will help us better understand and respond to SARS-CoV-2 outbreaks. The present study analyzed the epidemic and evolutionary characteristics of haplotype subtypes or regions based on 1.8 million high-quality SARS-CoV-2 genomic data. The estimated ratio of the rates of non-synonymous to synonymous changes (Ka/Ks) in North America and the United States were always more than 1.0, while the Ka/Ks in other continents and countries showed a sharp decline, then a slow increase to 1.0, and a dramatic increase over time. H1 (B.1) with the highest substitution rate has become the most dominant haplotype subtype since March 2020 and has evolved into multiple haplotype subtypes with smaller substitution rates. Many evolutionary characteristics of early SARS-CoV-2, such as H3 being the only early haplotype subtype that existed for the shortest time, the global prevalence of H1 and H1-5 (B.1.1) within a month after being detected, and many high divergent genome sequences early in February 2020, indicate the missing of early SARS-CoV-2 genomic data. SARS-CoV-2 experienced dynamic selection from December 2019 to August 2021 and has been under strong positive selection since May 2021. Its transmissibility and the ability of immune escape may be greatly enhanced over time. This will bring greater challenges to the control of the pandemic.

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.

Omicron: A Heavily Mutated SARS-CoV-2 Variant Exhibits Stronger Binding to ACE2 and Potently Escapes Approved COVID-19 Therapeutic Antibodies.

The new SARS-CoV-2 variant of concern "Omicron" was recently spotted in South Africa and spread quickly around the world due to its enhanced transmissibility. The variant became conspicuous as it harbors more than 30 mutations in the Spike protein with 15 mutations in the receptor-binding domain (RBD) alone, potentially dampening the potency of therapeutic antibodies and enhancing the ACE2 binding. More worrying, Omicron infections have been reported in vaccinees in South Africa and Hong Kong, and that post-vaccination sera poorly neutralize the new variant. Here, we investigated the binding strength of Omicron with ACE2 and monoclonal antibodies that are either approved by the FDA for COVID-19 therapy or undergoing phase III clinical trials. Computational mutagenesis and free energy perturbation could confirm that Omicron RBD binds ACE2 ~2.5 times stronger than prototype SARS-CoV-2. Notably, three substitutions, i.e., T478K, Q493K, and Q498R, significantly contribute to the binding energies and almost doubled the electrostatic potential (ELE) of the RBDOmic-ACE2 complex. Omicron also harbors E484A substitution instead of the E484K that helped neutralization escape of Beta, Gamma, and Mu variants. Together, T478K, Q493K, Q498R, and E484A substitutions contribute to a significant drop in the ELE between RBDOmic-mAbs, particularly in etesevimab, bamlanivimab, and CT-p59. AZD1061 showed a slight drop in ELE and sotrovimab that binds a conserved epitope on the RBD; therefore, it could be used as a cocktail therapy in Omicron-driven COVID-19. In conclusion, we suggest that the Spike mutations prudently devised by the virus facilitate the receptor binding, weakening the mAbs binding to escape the immune response.

High diversity in Delta variant across countries revealed by genome-wide analysis of SARS-CoV-2 beyond the Spike protein.

The highly contagious Delta variant of SARS-CoV-2 has become a prevalent strain globally and poses a public health challenge around the world. While there has been extensive focus on understanding the amino acid mutations in the Delta variant's Spike protein, the mutational landscape of the rest of the SARS-CoV-2 proteome (25 proteins) remains poorly understood. To this end, we performed a systematic analysis of mutations in all the SARS-CoV-2 proteins from nearly 2 million SARS-CoV-2 genomes from 176 countries/territories. Six highly prevalent missense mutations in the viral life cycle-associated Membrane (I82T), Nucleocapsid (R203M, D377Y), NS3 (S26L), and NS7a (V82A, T120I) proteins are almost exclusive to the Delta variant compared to other variants of concern (mean prevalence across genomes: Delta = 99.74%, Alpha = 0.06%, Beta = 0.09%, and Gamma = 0.22%). Furthermore, we find that the Delta variant harbors a more diverse repertoire of mutations across countries compared to the previously dominant Alpha variant. Overall, our study underscores the high diversity of the Delta variant between countries and identifies a list of amino acid mutations in the Delta variant's proteome for probing the mechanistic basis of pathogenic features such as high viral loads, high transmissibility, and reduced susceptibility against neutralization by vaccines.

Structure and dynamics of SARS-CoV-2 proofreading exoribonuclease ExoN.

High-fidelity replication of the large RNA genome of coronaviruses (CoVs) is mediated by a 3'-to-5' exoribonuclease (ExoN) in nonstructural protein 14 (nsp14), which excises nucleotides including antiviral drugs misincorporated by the low-fidelity viral RNA-dependent RNA polymerase (RdRp) and has also been implicated in viral RNA recombination and resistance to innate immunity. Here, we determined a 1.6-Å resolution crystal structure of severe acute respiratory syndrome CoV 2 (SARS-CoV-2) ExoN in complex with its essential cofactor, nsp10. The structure shows a highly basic and concave surface flanking the active site, comprising several Lys residues of nsp14 and the N-terminal amino group of nsp10. Modeling suggests that this basic patch binds to the template strand of double-stranded RNA substrates to position the 3' end of the nascent strand in the ExoN active site, which is corroborated by mutational and computational analyses. We also show that the ExoN activity can rescue a stalled RNA primer poisoned with sofosbuvir and allow RdRp to continue its extension in the presence of the chain-terminating drug, biochemically recapitulating proofreading in SARS-CoV-2 replication. Molecular dynamics simulations further show remarkable flexibility of multidomain nsp14 and suggest that nsp10 stabilizes ExoN for substrate RNA binding to support its exonuclease activity. Our high-resolution structure of the SARS-CoV-2 ExoN-nsp10 complex serves as a platform for future development of anticoronaviral drugs or strategies to attenuate the viral virulence.

SARS-CoV-2 genomes from Saudi Arabia implicate nucleocapsid mutations in host response and increased viral load.

Monitoring SARS-CoV-2 spread and evolution through genome sequencing is essential in handling the COVID-19 pandemic. Here, we sequenced 892 SARS-CoV-2 genomes collected from patients in Saudi Arabia from March to August 2020. We show that two consecutive mutations (R203K/G204R) in the nucleocapsid (N) protein are associated with higher viral loads in COVID-19 patients. Our comparative biochemical analysis reveals that the mutant N protein displays enhanced viral RNA binding and differential interaction with key host proteins. We found increased interaction of GSK3A kinase simultaneously with hyper-phosphorylation of the adjacent serine site (S206) in the mutant N protein. Furthermore, the host cell transcriptome analysis suggests that the mutant N protein produces dysregulated interferon response genes. Here, we provide crucial information in linking the R203K/G204R mutations in the N protein to modulations of host-virus interactions and underline the potential of the nucleocapsid protein as a drug target during infection.

Two short low complexity regions (LCRs) are hallmark sequences of the Delta SARS-CoV-2 variant spike protein.

Low complexity regions (LCRs) are protein sequences formed by a set of compositionally biased residues. LCRs are extremely abundant in cellular proteins and have also been reported in viruses, where they may partake in evasion of the host immune system. Analyses of 28,231 SARS-CoV-2 whole proteomes and of 261,051 spike protein sequences revealed the presence of four extremely conserved LCRs in the spike protein of several SARS-CoV-2 variants. With the exception of Iota, where it is absent, the Spike LCR-1 is present in the signal peptide of 80.57% of the Delta variant sequences, and in other variants of concern and interest. The Spike LCR-2 is highly prevalent (79.87%) in Iota. Two distinctive LCRs are present in the Delta spike protein. The Delta Spike LCR-3 is present in 99.19% of the analyzed sequences, and the Delta Spike LCR-4 in 98.3% of the same set of proteins. These two LCRs are located in the furin cleavage site and HR1 domain, respectively, and may be considered hallmark traits of the Delta variant. The presence of the medically-important point mutations P681R and D950N in these LCRs, combined with the ubiquity of these regions in the highly contagious Delta variant opens the possibility that they may play a role in its rapid spread.

Molecular dynamic simulation analysis of SARS-CoV-2 spike mutations and evaluation of ACE2 from pets and wild animals for infection risk.

Coronavirus Disease 2019 (COVID-19) is an ongoing global health emergency that has caused tremendous stress and loss of life worldwide. The viral spike glycoprotein is a critical molecule mediating transmission of SARS-CoV-2 by interacting with human ACE2. However, through the course of the pandemics, there has not been a thorough analysis of the spike protein mutations, and on how these mutants influence the transmission of SARS-CoV-2. Besides, cases of SARS-CoV-2 infection among pets and wild animals have been reported, so the susceptibility of these animals requires great attention to investigate, as they may also link to the renewed question of a possible intermediate host for SARS-CoV-2 before it was transmitted to humans. With over 226,000 SARS-CoV-2 sequences obtained, we found 1573 missense mutations in the spike gene, and 226 of them were within the receptor-binding domain (RBD) region that directly interacts with human ACE2. Modeling the interactions between SARS-CoV-2 spike mutants and ACE2 molecules showed that most of the 74 missense mutations in the RBD region of the interaction interface had little impact on spike binding to ACE2, whereas several within the spike RBD increased the binding affinity toward human ACE2 thus making the virus likely more contagious. On the other hand, modeling the interactions between animal ACE2 molecules and SARS-CoV-2 spike revealed that many pets and wild animals' ACE2 had a variable binding ability. Particularly, ACE2 of bamboo rat had stronger binding to SARS-CoV-2 spike protein, whereas that of mole, vole, Mus pahari, palm civet, and pangolin had a weaker binding compared to human ACE2. Our results provide structural insights into the impact on interactions of the SARS-CoV-2 spike mutants to human ACE2, and shed light on SARS-CoV-2 transmission in pets and wild animals, and possible clues to the intermediate host(s) for SARS-CoV-2.

The alpha/B.1.1.7 SARS-CoV-2 variant exhibits significantly higher affinity for ACE-2 and requires lower inoculation doses to cause disease in K18-hACE2 mice.

The alpha/B.1.1.7 SARS-CoV-2 lineage emerged in autumn 2020 in the United Kingdom and transmitted rapidly until winter 2021 when it was responsible for most new COVID-19 cases in many European countries. The incidence domination was likely due to a fitness advantage that could be driven by the receptor-binding domain (RBD) residue change (N501Y), which also emerged independently in other variants of concern such as the beta/B.1.351 and gamma/P.1 strains. Here, we present a functional characterization of the alpha/B.1.1.7 variant and show an eightfold affinity increase towards human angiotensin-converting enzyme-2 (ACE-2). In accordance with this, transgenic hACE2 mice showed a faster disease progression and severity after infection with a low dose of B.1.1.7, compared to an early 2020 SARS-CoV-2 isolate. When challenged with sera from convalescent individuals or anti-RBD monoclonal antibodies, the N501Y variant showed a minor, but significant elevated evasion potential of ACE-2/RBD antibody neutralization. The data suggest that the single asparagine to tyrosine substitution remarkable rise in affinity may be responsible for the higher transmission rate and severity of the B.1.1.7 variant.

MDA5 disease variant M854K prevents ATP-dependent structural discrimination of viral and cellular RNA.

Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoinflammatory disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more stably to synthetic Alu:Alu dsRNA. CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 sidechain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle- RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via an allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides insights on how MDA5 recognizes dsRNA in health and disease.