Antibiotics daptomycin interacts with S protein of SARS-CoV-2 to promote cell invasion of Omicron (B1.1.529) pseudovirus.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has posed enormous challenges to global public health. The use of antibiotics has greatly increased during the SARS-CoV-2 epidemic owing to the presence of bacterial co-infection and secondary bacterial infections. The antibiotics daptomycin (DAP) is widely used in the treatment of infectious diseases caused by gram-positive bacteria owing to its highly efficient antibacterial activity. It is pivotal to study the antibiotics usage options for patients of coronavirus infectious disease (COVID-19) with pneumonia those need admission to receive antibiotics treatment for bacterial co-infection in managing COVID-19 disease. Herein, we have revealed the interactions of DAP with the S protein of SARS-CoV-2 and the variant Omicron (B1.1.529) using the molecular docking approach and Omicron (B1.1.529) pseudovirus (PsV) mimic invasion. Molecular docking analysis shows that DAP has a certain degree of binding ability to the S protein of SARS-CoV-2 and several derived virus variants, and co-incubation of 1-100 µM DAP with cells promotes the entry of the PsV into human angiotensin-converting enzyme 2 (hACE2)-expressing HEK-293T cells (HEK-293T-hACE2), and this effect is related to the concentration of extracellular calcium ions (Ca2+). The PsV invasion rate in the HEK-293T-hACE2 cells concurrently with DAP incubation was 1.7 times of PsV infection alone. In general, our findings demonstrate that DAP promotes the infection of PsV into cells, which provides certain reference of antibiotics selection and usage optimization for clinicians to treat bacterial coinfection or secondary infection during SARS-CoV-2 infection.

Cell-engineered virus-mimetic nanovesicles for vaccination against enveloped viruses.

Enveloped viruses pose a significant threat to human health, as evidenced by the recent COVID-19 pandemic. Although current vaccine strategies have proven effective in preventing viral infections, the development of innovative vaccine technologies is crucial to fortify our defences against future pandemics. In this study, we introduce a novel platform called cell-engineered virus-mimetic nanovesicles (VNVs) and demonstrate their potential as a vaccine for targeting enveloped viruses. VNVs are generated by extruding plasma membrane-derived blebs through nanoscale membrane filters. These VNVs closely resemble enveloped viruses and extracellular vesicles (EVs) in size and morphology, being densely packed with plasma membrane contents and devoid of materials from other membranous organelles. Due to these properties, VNVs express viral membrane antigens more extensively and homogeneously than EVs expressing the same antigen. In this study, we produced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) VNVs expressing the SARS-CoV-2 Spike glycoprotein (S) on their surfaces and assessed their preclinical efficacy as a COVID-19 vaccine in experimental animals. The administration of VNVs successfully stimulated the production of S-specific antibodies both systemically and locally, and immune cells isolated from vaccinated mice displayed cytokine responses to S stimulation.

Viral envelope proteins fused to multiple distinct fluorescent reporters to probe receptor binding.

Enveloped viruses carry one or multiple proteins with receptor-binding functionalities. Functional receptors can be glycans, proteinaceous, or both; therefore, recombinant protein approaches are instrumental in attaining new insights regarding viral envelope protein receptor-binding properties. Visualizing and measuring receptor binding typically entails antibody detection or direct labeling, whereas direct fluorescent fusions are attractive tools in molecular biology. Here, we report a suite of distinct fluorescent fusions, both N- and C-terminal, for influenza A virus hemagglutinins and SARS-CoV-2 spike RBD. The proteins contained three or six fluorescent protein barrels and were applied directly to cells to assess receptor binding properties.

RaptGen-Assisted Generation of an RNA/DNA Hybrid Aptamer against SARS-CoV-2 Spike Protein.

Optimization of aptamers in length and chemistry is crucial for industrial applications. Here, we developed aptamers against the SARS-CoV-2 spike protein and achieved optimization with a deep-learning-based algorithm, RaptGen. We conducted a primer-less SELEX against the receptor binding domain (RBD) of the spike with an RNA/DNA hybrid library, and the resulting sequences were subjected to RaptGen analysis. Based on the sequence profiling by RaptGen, a short truncation aptamer of 26 nucleotides was obtained and further optimized by a chemical modification of relevant nucleotides. The resulting aptamer is bound to RBD not only of SARS-CoV-2 wildtype but also of its variants, SARS-CoV-1, and Middle East respiratory syndrome coronavirus (MERS-CoV). We concluded that the RaptGen-assisted discovery is efficient for developing optimized aptamers.

Agonists or positive allosteric modulators of α7 nicotinic acetylcholine receptor prevent interaction of SARS-Cov-2 receptor-binding domain with astrocytoma cells.

SARS-Cov-2, the virus causing COVID-19, penetrates host target cells via the receptor of angiotensin-converting enzyme 2 (ACE2). Disrupting the virus interaction with ACE2 affords a plausible mechanism for prevention of cell penetration and inhibiting dissemination of the virus. Our studies demonstrate that ACE2 interaction with the receptor binding domain of SARS-Cov-2 spike protein (RBD) can be impaired by modulating the α7 nicotinic acetylcholine receptor (α7 nAChR) contiguous with ACE2. U373 cells of human astrocytoma origin were shown to bind both ACE2-specific antibody and recombinant RBD in Cell-ELISA. ACE2 was found to interact with α7 nAChR in U373 cell lysates studied by Sandwich ELISA. Our studies demonstrate that inhibition of RBD binding to ACE2-expressing U373 cells were defined with α7 nAChR agonists choline and PNU282987, but not a competitive antagonist methyllicaconitine (MLA). Additionally, the type 2 positive allosteric modulator (PAM2) PNU120596 and hydroxyurea (HU) also inhibited the binding. Our studies demonstrate that activation of α7 AChRs has efficacy in inhibiting the SARS-Cov-2 interaction with the ACE2 receptor and in such a way can prevent virus target cell penetration. These studies also help to clarify the consistent efficacy and positive outcomes for utilizing HU in treating COVID-19.

Testing the feasibility of targeting a conserved region on the S2 domain of the SARS-CoV-2 spike protein.

The efficacy of vaccines against the SARS-CoV-2 virus significantly declines with the emergence of mutant strains, prompting investigation into the feasibility of targeting highly conserved but often cryptic regions on the S2 domain of spike protein. Using tools from molecular dynamics, we find that exposure of a conserved S2 epitope located in the central helices below the receptor binding domains would require large-scale motion beyond receptor binding domain up-down motion, but, along the reaction coordinates we explored, it is unlikely to be exposed by such large-scale dynamic fluctuations of the S1 domain without any external facilitating factors, despite some previous computational evidence suggesting transient exposure of this region. Furthermore, glycans, particularly those on N165 and N234, hinder S2-exposing opening dynamics, and thus stabilize spike in addition to immunologically shielding the protein surface. Although the S2 epitope region examined here is central to large-scale conformational changes during viral entry, free energy landscape analysis obtained using the path coordinate formalism reveals no inherent "loaded spring" effect, suggesting that a vaccine immunogen would tend to present the epitope in a prefusion-like conformation and may be effective in neutralization. These findings contribute to a deeper understanding of the dynamic origins of the function of the spike protein, as well as further characterizing the feasibility of the S2 epitope as a therapeutic target.

Identification of mouse CD4+ T cell epitopes in SARS-CoV-2 BA.1 spike and nucleocapsid for use in peptide:MHCII tetramers.

Understanding adaptive immunity against SARS-CoV-2 is a major requisite for the development of effective vaccines and treatments for COVID-19. CD4+ T cells play an integral role in this process primarily by generating antiviral cytokines and providing help to antibody-producing B cells. To empower detailed studies of SARS-CoV-2-specific CD4+ T cell responses in mouse models, we comprehensively mapped I-Ab-restricted epitopes for the spike and nucleocapsid proteins of the BA.1 variant of concern via IFNγ ELISpot assay. This was followed by the generation of corresponding peptide:MHCII tetramer reagents to directly stain epitope-specific T cells. Using this rigorous validation strategy, we identified 6 immunogenic epitopes in spike and 3 in nucleocapsid, all of which are conserved in the ancestral Wuhan strain. We also validated a previously identified epitope from Wuhan that is absent in BA.1. These epitopes and tetramers will be invaluable tools for SARS-CoV-2 antigen-specific CD4+ T cell studies in mice.

Combinatorial Approach with Mass Spectrometry and Lectin Microarray Dissected Site-Specific Glycostem and Glycoleaf Features of the Virion-Derived Spike Protein of Ancestral and γ Variant SARS-CoV-2 Strains.

The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has impacted public health globally. As the glycosylation of viral envelope glycoproteins is strongly associated with their immunogenicity, intensive studies have been conducted on the glycans of the glycoprotein of SARS-CoV-2, the spike (S) protein. Here, we conducted intensive glycoproteomic analyses of the SARS-CoV-2 S protein of ancestral and γ-variant strains using a combinatorial approach with two different technologies: mass spectrometry (MS) and lectin microarrays (LMA). Our unique MS1-based glycoproteomic technique, Glyco-RIDGE, in addition to MS2-based Byonic search, identified 1448 (ancestral strain) and 1785 (γ-variant strain) site-specific glycan compositions, respectively. Asparagine at amino acid position 20 (N20) is mainly glycosylated within two successive potential glycosylation sites, N17 and N20, of the γ-variant S protein; however, we found low-frequency glycosylation at N17. Our novel approaches, glycostem mapping and glycoleaf scoring, also illustrate the moderately branched/extended, highly fucosylated, and less sialylated natures of the glycoforms of S proteins. Subsequent LMA analysis emphasized the intensive end-capping of glycans by Lewis fucoses, which complemented the glycoproteomic features. These results illustrate the high-resolution glycoproteomic features of the SARS-CoV-2 S protein, contributing to vaccine design and understanding of viral protein synthesis.

Mechanistic insights into ligand dissociation from the SARS-CoV-2 spike glycoprotein.

The COVID-19 pandemic, driven by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spurred an urgent need for effective therapeutic interventions. The spike glycoprotein of the SARS-CoV-2 is crucial for infiltrating host cells, rendering it a key candidate for drug development. By interacting with the human angiotensin-converting enzyme 2 (ACE2) receptor, the spike initiates the infection of SARS-CoV-2. Linoleate is known to bind the spike glycoprotein, subsequently reducing its interaction with ACE2. However, the detailed mechanisms underlying the protein-ligand interaction remain unclear. In this study, we characterized the pathways of ligand dissociation and the conformational changes associated with the spike glycoprotein by using ligand Gaussian accelerated molecular dynamics (LiGaMD). Our simulations resulted in eight complete ligand dissociation trajectories, unveiling two distinct ligand unbinding pathways. The preference between these two pathways depends on the gate distance between two α-helices in the receptor binding domain (RBD) and the position of the N-linked glycan at N343. Our study also highlights the essential contributions of K417, N121 glycan, and N165 glycan in ligand unbinding, which are equally crucial in enhancing spike-ACE2 binding. We suggest that the presence of the ligand influences the motions of these residues and glycans, consequently reducing accessibility for spike-ACE2 binding. These findings enhance our understanding of ligand dissociation from the spike glycoprotein and offer significant implications for drug design strategies in the battle against COVID-19.

Molecular Insights into the Variability in Infection and Immune Evasion Capabilities of SARS-CoV-2 Variants: A Sequence and Structural Investigation of the RBD Domain.

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continuously emerge, an increasing number of mutations are accumulating in the Spike protein receptor-binding domain (RBD) region. Through sequence analysis of various Variants of Concern (VOC), we identified that they predominantly fall within the ο lineage although recent variants introduce any novel mutations in the RBD. Molecular dynamics simulations were employed to compute the binding free energy of these variants with human Angiotensin-converting enzyme 2 (ACE2). Structurally, the binding interface of the ο RBD displays a strong positive charge, complementing the negatively charged binding interface of ACE2, resulting in a significant enhancement in the electrostatic potential energy for the ο variants. Although the increased potential energy is partially offset by the rise in polar solvation free energy, enhanced electrostatic potential contributes to the long-range recognition between the ο variant's RBD and ACE2. We also conducted simulations of glycosylated ACE2-RBD proteins. The newly emerged ο (JN.1) variant has added a glycosylation site at N-354@RBD, which significantly weakened its binding affinity with ACE2. Further, our interaction studies with three monoclonal antibodies across multiple SARS-CoV-2 strains revealed a diminished neutralization efficacy against the ο variants, primarily attributed to the electrostatic repulsion between the antibodies and RBD interface. Considering the characteristics of the ο variant and the trajectory of emerging strains, we propose that newly developed antibodies against SARS-CoV-2 RBD should have surfaces rich in negative potential and, postbinding, exhibit strong van der Waals interactions. These findings provide invaluable guidance for the formulation of future therapeutic strategies.

Structural basis and analysis of hamster ACE2 binding to different SARS-CoV-2 spike RBDs.

Pet golden hamsters were first identified being infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) delta variant of concern (VOC) and transmitted the virus back to humans in Hong Kong in January 2022. Here, we studied the binding of two hamster (golden hamster and Chinese hamster) angiotensin-converting enzyme 2 (ACE2) proteins to the spike protein receptor-binding domains (RBDs) of SARS-CoV-2 prototype and eight variants, including alpha, beta, gamma, delta, and four omicron sub-variants (BA.1, BA.2, BA.3, and BA.4/BA.5). We found that the two hamster ACE2s present slightly lower affinity for the RBDs of all nine SARS-CoV-2 viruses tested than human ACE2 (hACE2). Furthermore, the similar infectivity to host cells expressing hamster ACE2s and hACE2 was confirmed with the nine pseudotyped SARS-CoV-2 viruses. Additionally, we determined two cryo-electron microscopy (EM) complex structures of golden hamster ACE2 (ghACE2)/delta RBD and ghACE2/omicron BA.3 RBD. The residues Q34 and N82, which exist in many rodent ACE2s, are responsible for the lower binding affinity of ghACE2 compared to hACE2. These findings suggest that all SARS-CoV-2 VOCs may infect hamsters, highlighting the necessity of further surveillance of SARS-CoV-2 in these animals.IMPORTANCESARS-CoV-2 can infect many domestic animals, including hamsters. There is an urgent need to understand the binding mechanism of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants to hamster receptors. Herein, we showed that two hamster angiotensin-converting enzyme 2s (ACE2s) (golden hamster ACE2 and Chinese hamster ACE2) can bind to the spike protein receptor-binding domains (RBDs) of SARS-CoV-2 prototype and eight variants and that pseudotyped SARS-CoV-2 viruses can infect hamster ACE2-expressing cells. The binding pattern of golden hamster ACE2 to SARS-CoV-2 RBDs is similar to that of Chinese hamster ACE2. The two hamster ACE2s present slightly lower affinity for the RBDs of all nine SARS-CoV-2 viruses tested than human ACE2. We solved the cryo-electron microscopy (EM) structures of golden hamster ACE2 in complex with delta RBD and omicron BA.3 RBD and found that residues Q34 and N82 are responsible for the lower binding affinity of ghACE2 compared to hACE2. Our work provides valuable information for understanding the cross-species transmission mechanism of SARS-CoV-2.

Effective expression and characterization of the receptor binding domains in SARS-CoV-2 Spike proteins from original strain and variants of concern using Bombyx mori nucleopolyhedrovirus in silkworm.

A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the global pandemic of COVID-19 in 2020. Through structural analysis, it was found that several amino acid residues in the human angiotensin-converting enzyme-2 (hACE2) receptor directly interact with those in the receptor binding domain (RBD) of the spike glycoprotein (S-protein). Various cell lines, including HEK293, HeLa cells, and the baculovirus expression vector system (BEVS) with the insect cell line Sf9, have been utilized to produce the RBD. In this study, we investigated the use of Bombyx mori nucleopolyhedrovirus (BmNPV) and BEVS. For efficient production of a highly pure recombinant RBD protein, we designed it with two tags (His tag and STREP tag) at the C-terminus and a solubilizing tag (SUMO) at the N-terminus. After expressing the protein using BmNPV and silkworm and purifying it with a HisTrap excel column, the eluted protein was digested with SUMO protease and further purified using a Strep-Tactin Superflow column. As a result, we obtained the RBD as a monomer with a yield of 2.6 mg/10 mL serum (equivalent to 30 silkworms). The RBD showed an affinity for the hACE2 receptor. Additionally, the RBDs from the Alpha, Beta, Gamma, Delta, and Omicron variants were expressed and purified using the same protocol. It was found that the RBD from the Alpha, Beta, Gamma, and Delta variants could be obtained with yields of 1.4-2.6 mg/10 mL serum and had an affinity to the hACE2 receptor.

Crystal Structures of Fusion Cores from CCoV-HuPn-2018 and SADS-CoV.

Cross-species spillover to humans of coronaviruses (CoVs) from wildlife animal reservoirs poses marked and global threats to human and animal health. Recently, sporadic infection of canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018) in hospitalized patients with pneumonia genetically related to canine and feline coronavirus were identified. In addition, swine acute diarrhea syndrome coronavirus (SADS-CoV) had the capability of broad tropism to cultured cells including from humans. Together, the transmission of Alphacoronaviruses that originated in wildlife to humans via intermediate hosts was responsible for the high-impact emerging zoonosis. Entry of CoV is mainly mediated by Spike and formation of a typical six helix bundle (6-HB) structure in the postfusion state of Spike is pivotal. Here, we present the complete fusion core structures of CCoV-HuPn-2018 and SADS-CoV from Alphacoronavirus at 2.10 and 2.59 Å, respectively. The overall structure of the CCoV-HuPn-2018 fusion core is similar to Alphacoronavirus like HCoV-229E, while SADS-CoV is analogous to Betacoronavirus like SARS-CoV-2. Collectively, we provide a structural basis for the development of pan-CoV small molecules and polypeptides based on the HR1-HR2 complex, concerning CCoV-HuPn-2018 and SADS-CoV.

Phylogenetic and Molecular Analysis of the Porcine Epidemic Diarrhea Virus in Mexico during the First Reported Outbreaks (2013-2017).

The characteristics of the whole PEDV genome that has circulated in Mexico from the first outbreak to the present are unknown. We chose samples obtained from 2013 to 2017 and sequenced them, which enabled us to identify the genetic variation and phylogeny in the virus during the first four years that it circulated in Mexico. A 99% identity was found among the analyzed pandemic strains; however, the 1% difference affected the structure of the S glycoprotein, which is essential for the binding of the virus to the cellular receptor. The S protein induces the most efficacious antibodies; hence, these changes in structure could be implicated in the clinical antecedents of the outbreaks. Antigenic changes could also help PEDV avoid neutralization, even in the presence of previous immunity. The characterization of the complete genome enabled the identification of three circulating strains that have a deletion in ORF1a, which is present in attenuated Asian vaccine strains. The phylogenetic analysis of the complete genome indicates that the first PEDV outbreaks in Mexico were caused by INDEL strains and pandemic strains related to USA strains; however, the possibility of the entry of European strains exists, which may have caused the 2015 and 2016 outbreaks.

Novel neutralizing SARS-CoV-2-specific mAbs offer detection of RBD linear epitopes.

BACKGROUND: To stop the spread of the COVID-19 disease, it is crucial to create molecular tools to investigate and diagnose COVID-19. Current efforts focus on developing specific neutralizing monoclonal antibodies (NmAbs) elicited against the receptor-binding domain (RBD). METHODS: In the present study, recombinant RBD (rRBD) protein was produced in E. coli, followed by immunizing mice with purified rRBD. ELISA was applied to screen the hybridomas for positive reactivity with rRBD protein. The linear and conformational epitopes of the mAbs were subsequently identified using western blot. Finally, the reactivity, affinity, and neutralization activity of the purified mAbs were evaluated using ELISA. RESULTS: All mAbs exhibited similar reactivity trends towards both eukaryotic RBD and prokaryotic rRBD in ELISA. Among them, 2E7-D2 and 2B4-G8 mAbs demonstrated higher reactivity than other mAbs. Additionally, in western blot assays, these two mAbs could detect reducing and non-reducing rRBD, indicating recognition of linear epitopes. Notably, five mAbs effectively blocked rRBD- angiotensin-converting enzyme 2 (ACE2) interaction, while two high-affinity mAbs exhibited potent neutralizing activity against eukaryotic RBD. CONCLUSION: In the current study, we generated and characterized new RBD-specific mAbs using the hybridoma technique that recognized linear and conformational epitopes in RBD with neutralization potency. Our mAbs are novel candidates for diagnosing and treating SARS-CoV-2.

Harnessing preexisting influenza virus-specific immunity increases antibody responses against SARS-CoV-2.

In pandemic scenarios involving novel human pathogenic viruses, it is highly desirable that vaccines induce strong neutralizing antibodies as quickly as possible. However, current vaccine strategies require multiple immunization doses to produce high titers of neutralizing antibodies and are poorly protective after a single vaccination. We therefore wished to design a vaccine candidate that would induce increased protective immune responses following the first vaccine dose. We hypothesized that antibodies against the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein could be increased by drawing upon immunity to a previous infection. We generated a fusion protein containing the influenza H1N1 PR8 virus nucleoprotein (NP) and the SARS-CoV-2 spike RBD. Mice with or without preexisting immunity to PR8 were then vaccinated with NP/RBD. We observed significantly increased SARS-CoV-2 neutralizing antibodies in mice with PR8 immunity compared to mice without preexisting PR8 immunity. Vaccination with NP/RBD protected mice from SARS-CoV-2-induced morbidity and mortality after a single dose. Additionally, we compared SARS-CoV-2 virus titers in the lungs and nasal turbinates 4 days post-challenge of mice vaccinated with NP/RBD. SARS-CoV-2 virus was detectable in the lungs and nasal turbinate of mice without preexisting PR8 immunity, while SARS-CoV-2 virus was completely undetectable in mice with preexisting PR8 immunity. We also found that CD4-positive T cells in mice with preexisting immunity to PR8 play an essential role in producing the increased antibody response against RBD. This vaccine strategy potentially can be modified to target other pathogens of concern and offers extra value in future pandemic scenarios.IMPORTANCEIncreased globalization and changes in human interactions with wild animals has increased the likelihood of the emergence of novel viruses with pandemic potential. Vaccines can be effective in preventing severe disease caused by pandemic viruses. However, it takes time to develop protective immunity via prime-boost vaccination. More effective vaccine designs should quickly induce protective immunity. We propose leveraging preexisting immunity to a different pathogen to boost protection against emerging viruses. We targeted SARS-CoV-2 as a representative pandemic virus and generated a fusion protein vaccine that combines the nucleoprotein from influenza A virus and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Our vaccine design significantly increased the production of RBD-specific antibodies in mice that had previously been exposed to influenza virus, compared to those without previous exposure. This enhanced immunity reduced SARS-CoV-2 replication in mice. Our results offer a vaccine design that could be valuable in a future pandemic setting.

Using big sequencing data to identify chronic SARS-Coronavirus-2 infections.

The evolution of SARS-Coronavirus-2 (SARS-CoV-2) has been characterized by the periodic emergence of highly divergent variants. One leading hypothesis suggests these variants may have emerged during chronic infections of immunocompromised individuals, but limited data from these cases hinders comprehensive analyses. Here, we harnessed millions of SARS-CoV-2 genomes to identify potential chronic infections and used language models (LM) to infer chronic-associated mutations. First, we mined the SARS-CoV-2 phylogeny and identified chronic-like clades with identical metadata (location, age, and sex) spanning over 21 days, suggesting a prolonged infection. We inferred 271 chronic-like clades, which exhibited characteristics similar to confirmed chronic infections. Chronic-associated mutations were often high-fitness immune-evasive mutations located in the spike receptor-binding domain (RBD), yet a minority were unique to chronic infections and absent in global settings. The probability of observing high-fitness RBD mutations was 10-20 times higher in chronic infections than in global transmission chains. The majority of RBD mutations in BA.1/BA.2 chronic-like clades bore predictive value, i.e., went on to display global success. Finally, we used our LM to infer hundreds of additional chronic-like clades in the absence of metadata. Our approach allows mining extensive sequencing data and providing insights into future evolutionary patterns of SARS-CoV-2.

Adaptation of SARS-CoV-2 to ACE2H353K mice reveals new spike residues that drive mouse infection.

The Coronavirus Disease 2019 (COVID-19) pandemic continues to cause extraordinary loss of life and economic damage. Animal models of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection are needed to better understand disease pathogenesis and evaluate preventive measures and therapies. While mice are widely used to model human disease, mouse angiotensin converting enzyme 2 (ACE2) does not bind the ancestral SARS-CoV-2 spike protein to mediate viral entry. To overcome this limitation, we "humanized" mouse Ace2 using CRISPR gene editing to introduce a single amino acid substitution, H353K, predicted to facilitate S protein binding. While H353K knockin Ace2 (mACE2H353K) mice supported SARS-CoV-2 infection and replication, they exhibited minimal disease manifestations. Following 30 serial passages of ancestral SARS-CoV-2 in mACE2H353K mice, we generated and cloned a more virulent virus. A single isolate (SARS2MA-H353K) was prepared for detailed studies. In 7-11-month-old mACE2H353K mice, a 104 PFU inocula resulted in diffuse alveolar disease manifested as edema, hyaline membrane formation, and interstitial cellular infiltration/thickening. Unexpectedly, the mouse-adapted virus also infected standard BALB/c and C57BL/6 mice and caused severe disease. The mouse-adapted virus acquired five new missense mutations including two in spike (K417E, Q493K), one each in nsp4, nsp9, and M and a single nucleotide change in the 5' untranslated region. The Q493K spike mutation arose early in serial passage and is predicted to provide affinity-enhancing molecular interactions with mACE2 and further increase the stability and affinity to the receptor. This new model and mouse-adapted virus will be useful to evaluate COVID-19 disease and prophylactic and therapeutic interventions.IMPORTANCEWe developed a new mouse model with a humanized angiotensin converting enzyme 2 (ACE2) locus that preserves native regulatory elements. A single point mutation in mouse ACE2 (H353K) was sufficient to confer in vivo infection with ancestral severe acute respiratory syndrome-coronavirus-2 virus. Through in vivo serial passage, a virulent mouse-adapted strain was obtained. In aged mACE2H353K mice, the mouse-adapted strain caused diffuse alveolar disease. The mouse-adapted virus also infected standard BALB/c and C57BL/6 mice, causing severe disease. The mouse-adapted virus acquired five new missense mutations including two in spike (K417E, Q493K), one each in nsp4, nsp9, and M and a single nucleotide change in the 5' untranslated region. The Q493K spike mutation arose early in serial passage and is predicted to provide affinity-enhancing molecular interactions with mACE2 and further increase the stability and affinity to the receptor. This new model and mouse-adapted virus will be useful to evaluate COVID-19 disease and prophylactic and therapeutic interventions.

Enhanced portable detection for Sars-CoV-2 utilizing DNA tetrahedron-tethered aptamers and a pressure meter.

Tethering oligonucleotide aptamers to a DNA tetrahedron structure can enhance the recognition of SARS-CoV-2 spike protein to effectively overcome challenges with its detection in current diagnostic assays. Building on this framework, we have developed a unique portable detection method for COVID-19 that provides exceptional sensitivity and selectivity via pressure meter readout. This innovative assay streamlines the detection process, providing a rapid, sensitive, cost-effective, and user-friendly diagnostic tool. This point-of-care test exhibits high sensitivity and specificity, achieving an impressive detection limit of 0.1 pg mL-1 for the spike protein. The effectiveness of this method was validated using pseudoviruses and oropharyngeal swab samples, and its utility for environmental monitoring is demonstrated by testing sewage samples. With a wide linear range and strong potential for clinical or home application, our assay represents a major innovation in point-of-care diagnostics and provides a vital contribution to the current toolkit for controlling the impacts of COVID-19.

Rapid Profiling of the Glycosylation Effects on the Binding of SARS-CoV-2 Spike Protein to Angiotensin-Converting Enzyme 2 Using MALDI-MS with High Mass Detection.

The spike protein receptor-binding domain (RBD) of SARS-CoV-2 binds directly to angiotensin-converting enzyme 2 (ACE2), mediating the host cell entry of SARS-CoV-2. Both spike protein and ACE2 are highly glycosylated, which can regulate the binding. Here, we utilized high-mass MALDI-MS with chemical cross-linking for profiling the glycosylation effects on the binding between RBD and ACE2. Overall, it was found that ACE2 glycosylation affects the binding more strongly than does RBD glycosylation. The binding affinity was improved after desialylation or partial deglycosylation (N690) of ACE2, while it decreased after degalactosylation. ACE2 can form dimers in solution, which bind more tightly to the RBD than the ACE2 monomers. The ACE2 dimerization and the binding of RBD to dimeric ACE2 can also be improved by the desialylation or deglycosylation of ACE2. Partial deglycosylation of ACE2 increased the dimerization of ACE2 and the binding affinity of RBD and ACE2 by more than a factor of 2, suggesting its high potential for neutralizing SARS-CoV-2. The method described in the work provided a simple way to analyze the protein-protein interaction without sample purification. It can be widely used for rapid profiling of glycosylation effects on protein-protein interaction for glycosylation-related diseases and the study of multiple interactions between protein and protein aggregates in a single system.