Human coronavirus HKU1 recognition of the TMPRSS2 host receptor.

The human coronavirus HKU1 spike (S) glycoprotein engages host cell surface sialoglycans and transmembrane protease serine 2 (TMPRSS2) to initiate infection. The molecular basis of HKU1 binding to TMPRSS2 and determinants of host receptor tropism remain elusive. We designed an active human TMPRSS2 construct enabling high-yield recombinant production in human cells of this key therapeutic target. We determined a cryo-electron microscopy structure of the HKU1 RBD bound to human TMPRSS2, providing a blueprint of the interactions supporting viral entry and explaining the specificity for TMPRSS2 among orthologous proteases. We identified TMPRSS2 orthologs from five mammalian orders promoting HKU1 S-mediated entry into cells along with key residues governing host receptor usage. Our data show that the TMPRSS2 binding motif is a site of vulnerability to neutralizing antibodies and suggest that HKU1 uses S conformational masking and glycan shielding to balance immune evasion and receptor engagement.

Differential epitope prediction across diverse circulating variants of SARS-COV-2 in Brazil.

COVID-19, caused by the SARS-COV-2 virus, induces numerous immunological reactions linked to the severity of the clinical condition of those infected. The surface Spike protein (S protein) present in Sars-CoV-2 is responsible for the infection of host cells. This protein presents a high rate of mutations, which can increase virus transmissibility, infectivity, and immune evasion. Therefore, we propose to evaluate, using immunoinformatic techniques, the predicted epitopes for the S protein of seven variants of Sars-CoV-2. MHC class I and II epitopes were predicted and further assessed for their immunogenicity, interferon-gamma (IFN-γ) inducing capacity, and antigenicity. For B cells, linear and structural epitopes were predicted. For class I MHC epitopes, 40 epitopes were found for the clades of Wuhan, Clade 2, Clade 3, and 20AEU.1, Gamma, and Delta, in addition to 38 epitopes for Alpha and 44 for Omicron. For MHC II, there were differentially predicted epitopes for all variants and eight equally predicted epitopes. These were evaluated for differences in the MHC II alleles to which they would bind. Regarding B cell epitopes, 16 were found in the Wuhan variant, 14 in 22AEU.1 and in Clade 3, 15 in Clade 2, 11 in Alpha and Delta, 13 in Gamma, and 9 in Omicron. When compared, there was a reduction in the number of predicted epitopes concerning the Spike protein, mainly in the Delta and Omicron variants. These findings corroborate the need for updates seen today in bivalent mRNA vaccines against COVID-19 to promote a targeted immune response to the main circulating variant, Omicron, leading to more robust protection against this virus and avoiding cases of reinfection. When analyzing the specific epitopes for the RBD region of the spike protein, the Omicron variant did not present a B lymphocyte epitope from position 390, whereas the epitope at position 493 for MHC was predicted only for the Alpha, Gamma, and Omicron variants.

Structural determinants of spike infectivity in bat SARS-like coronaviruses RsSHC014 and WIV1.

The recurrent spillovers of coronaviruses (CoVs) have posed severe threats to public health and the global economy. Bat severe acute respiratory syndrome (SARS)-like CoVs RsSHC014 and WIV1, currently circulating in bat populations, are poised for human emergence. The trimeric spike (S) glycoprotein, responsible for receptor recognition and membrane fusion, plays a critical role in cross-species transmission and infection. Here, we determined the cryo-electron microscopy (EM) structures of the RsSHC014 S protein in the closed state at 2.9 Å, the WIV1 S protein in the closed state at 2.8 Å, and the intermediate state at 4.0 Å. In the intermediate state, one receptor-binding domain (RBD) is in the "down" position, while the other two RBDs exhibit poor density. We also resolved the complex structure of the WIV1 S protein bound to human ACE2 (hACE2) at 4.5 Å, which provides structural basis for the future emergence of WIV1 in humans. Through biochemical experiments, we found that despite strong binding affinities between the RBDs and both human and civet ACE2, the pseudoviruses of RsSHC014, but not WIV1, failed to infect 293T cells overexpressing either human or civet ACE2. Mutagenesis analysis revealed that the Y623H substitution, located in the SD2 region, significantly improved the cell entry efficiency of RsSHC014 pseudoviruses, which is likely accomplished by promoting the open conformation of spike glycoproteins. Our findings emphasize the necessity of both efficient RBD lifting and tight RBD-hACE2 binding for viral infection and underscore the significance of the 623 site of the spike glycoprotein for the infectivity of bat SARS-like CoVs. IMPORTANCE: The bat SARS-like CoVs RsSHC014 and WIV1 can use hACE2 for cell entry without further adaptation, indicating their potential risk of emergence in human populations. The S glycoprotein, responsible for receptor recognition and membrane fusion, plays a crucial role in cross-species transmission and infection. In this study, we determined the cryo-EM structures of the S glycoproteins of RsSHC014 and WIV1. Detailed comparisons revealed dynamic structural variations within spike proteins. We also elucidated the complex structure of WIV1 S-hACE2, providing structural evidence for the potential emergence of WIV1 in humans. Although RsSHC014 and WIV1 had similar hACE2-binding affinities, they exhibited distinct pseudovirus cell entry behavior. Through mutagenesis and cryo-EM analysis, we revealed that besides the structural variations, the 623 site in the SD2 region is another important structural determinant of spike infectivity.

High Transferability of Neutralizing Antibodies against SARS-CoV-2 to Umbilical Cord Blood in Pregnant Women Vaccinated with BNT162b2 XBB.1.5: A Retrospective Cohort Study.

BACKGROUND: Coronavirus disease 2019 (COVID-19) can lead to severe respiratory illness, rapid disease progression, and higher rates of intensive care unit admission in pregnant women. Infection during pregnancy is associated with an increased risk of preterm delivery, cesarean section, fetal dysfunction, preeclampsia, and perinatal death. Vertical transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from pregnant women to their fetuses has also been observed. Although severe infections in neonates and infants are rare, newborns can experience serious consequences from COVID-19 due to their suboptimal humoral immune system protection. The amino acids in the structural proteins of SARS-CoV-2 are constantly mutating. Since around January 2023, COVID-19, caused by omicron-type SARS-CoV-2 variants, has been prevalent globally. These variants can evade the immune response triggered by traditional mRNA-based COVID-19 vaccines, such as BNT162b2. Therefore, vaccination with BNT162b2 XBB.1.5, which provides protection against omicron-type SARS-CoV-2 variants, is recommended. METHODS: This retrospective cohort study included 148 pregnant women who received the BNT162b2 XBB.1.5 vaccine at 30 partner medical institutions from September 2023 to January 2024. We examined the titers of anti-spike glycoprotein SARS-CoV-2 immunoglobin G (IgG) and IgA in the blood and umbilical cord blood obtained from the participants using ELISA. FINDINGS: Anti-spike glycoprotein SARS-CoV-2 IgG and IgA titers were highest in the blood and cord blood at late gestational age (28-34 weeks). No serious side effects or adverse events were observed in either the pregnant women or their newborns. INTERPRETATION: Pregnant women who received the BNT162b2 XBB.1.5 vaccine during gestational weeks 28 to 34 had the highest titers of anti-omicron SARS-CoV-2 variant antibodies in their blood. Moreover, these antibodies were transferred to their umbilical cord blood. To validate our findings, large cohort clinical studies involving numerous pregnant women are warranted. FUNDING: This study was funded by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) and Grants-in-Aid for Medical Research from the Japan Agency for Medical Research and Development (AMED).

Molecules targeting a novel homotrimer cavity of Spike protein attenuate replication of SARS-CoV-2.

The SARS-CoV-2 Spike glycoprotein (S) utilizes a unique trimeric conformation to interact with the ACE2 receptor on host cells, making it a prime target for inhibitors that block viral entry. We have previously identified a novel proteinaceous cavity within the Spike protein homotrimer that could serve as a binding site for small molecules. However, it is not known whether these molecules would inhibit, stimulate, or have no effect on viral replication. To address this, we employed structural-based screening to identify small molecules that dock into the trimer cavity and assessed their impact on viral replication. Our findings show that a cohort of identified small molecules binding to the Spike trimer cavity effectively reduces the replication of various SARS-CoV-2 variants. These molecules exhibited inhibitory effects on B.1 (European original, D614G, EDB2) and B.1.617.2 (δ) variants, while showing moderate activity against the B.1.1.7 (α) variant. We further categorized these molecules into distinct groups based on their structural similarities. Our experiments demonstrated a dose-dependent viral replication inhibitory activity of these compounds, with some, like BCC0040453 exhibiting no adverse effects on cell viability even at high concentrations. Further investigation revealed that pre-incubating virions with compounds like BCC0031216 at different temperatures significantly inhibited viral replication, suggesting their specificity towards the S protein. Overall, our study highlights the inhibitory impact of a diverse set of chemical molecules on the biological activity of the Spike protein. These findings provide valuable insights into the role of the trimer cavity in the viral replication cycle and aid drug discovery programs aimed at targeting the coronavirus family.

TLR2/4 are novel activating receptors for SARS-CoV-2 spike protein on NK cells.

Background: In early infected or severe coronavirus disease 2019 (COVID-19) patients, circulating NK cells are consistently reduced, despite being highly activated or exhausted. The aim of this paper was to establish whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (SP) may directly trigger NK cells and through which receptor(s). Methods: SP-stimulated human NK cells have been evaluated for the expression of activation markers, cytokine release, and cytotoxic activity, as well as for gene expression profiles and NF-kB phosphorylation, and they have been silenced with specific small interfering RNAs. Results: SPs from the Wuhan strain and other variants of concern (VOCs) directly bind and stimulate purified NK cells by increasing activation marker expression, cytokine release, and cytolytic activity, prevalently in the CD56brightNK cell subset. VOC-SPs differ in their ability to activate NK cells, G614, and Delta-Plus strains providing the strongest activity in the majority of donors. While VOC-SPs do not trigger ACE2, which is not expressed on NK cells, or other activating receptors, they directly and variably bind to both Toll-like receptor 2 (TLR2) and TLR4. Moreover, SP-driven NK cell functions are inhibited upon masking such receptors or silencing the relative genes. Lastly, VOC-SPs upregulate CD56dimNK cell functions in COVID-19 recovered, but not in non-infected, individuals. Conclusions: TLR2 and TLR4 are novel activating receptors for SP in NK cells, suggesting a new role of these cells in orchestrating the pathophysiology of SARS-CoV-2 infection. The pathogenic relevance of this finding is highlighted by the fact that free SP providing NK cell activation is frequently detected in a SARS-CoV-2 inflamed environment and in plasma of infected and long-COVID-19 subjects.

Dipeptidyl peptidase 4 interacts with porcine coronavirus PHEV spikes and mediates host range expansion.

Porcine hemagglutinating encephalomyelitis virus (PHEV), a neurotropic betacoronavirus, is prevalent in natural reservoir pigs and infects mice. This raises concerns about host jumping or spillover, but little is known about the cause of occurrence. Here, we revealed that dipeptidyl peptidase 4 (DPP4) is a candidate binding target of PHEV spikes and works as a broad barrier to overcome. Investigations of the host breadth of PHEV confirmed that cells derived from pigs and mice are permissive to virus propagation. Both porcine DPP4 and murine DPP4 have high affinity for the viral spike receptor-binding domain (RBD), independent of their catalytic activity. Loss of DPP4 expression results in limited PHEV infection. Structurally, PHEV spike protein binds to the outer surface of blades IV and V of the DPP4 ß-propeller domain, and the DPP4 residues N229 and N321 (relative to human DPP4 numbering) participate in RBD binding via its linked carbohydrate entities. Removal of these N-glycosylations profoundly enhanced the RBD-DPP4 interaction and viral invasion, suggesting they act as shielding in PHEV infection. Furthermore, we found that glycosylation, rather than structural differences or surface charges, is more responsible for DPP4 recognition and species barrier formation. Overall, our findings shed light on virus-receptor interactions and highlight that PHEV tolerance to DPP4 orthologs is a putative determinant of its cross-species transmission or host range expansion.IMPORTANCEPHEV is a neurotropic betacoronavirus that is circulating worldwide and has raised veterinary and economic concerns. In addition to being a reservoir species of pigs, PHEV can also infect wild-type mice, suggesting a "host jump" event. Understanding cross-species transmission is crucial for disease prevention and control but remains to be addressed. Herein, we show that the multifunctional receptor DPP4 plays a pivotal role in the host tropism of PHEV and identifies the conserved glycosylation sites in DPP4 responsible for this restriction. These findings highlight that the ability of PHEV to utilize DPP4 orthologs potentially affects its natural host expansion.

Disappearance of Imported Cases of Omicron Lineage BA.2.40 in West Kalimantan, Indonesia.

Background: The World Health Organization has declared Omicron as the fifth variant of concern with more than 50 mutations, particularly in the spike protein. Given increased viral infectivity due to mutations, worldwide genomic surveillance and detection of severe acute respiratory syndrome 2 (SARS-CoV-2) is essential. The present study aimed to track Omicron lineage BA.2.40 in West Kalimantan, Indonesia. Methods: In May-August 2022, nasopharyngeal swab samples (n=3,642) were collected from international travelers to West Kalimantan (active surveillance), and patients hospitalized due to SARS-CoV-2 infection (baseline surveillance). The samples were tested for Omicron lineages based on ORF1ab, N, and HV69-70del genes, followed by whole-genome sequencing. The sequences were then identified using two genomic databases, aligned against the reference genome (Wuhan/Hu-1/2019), and then compared with BA.2.40 lineage detected across the world. Phylogenetic analysis between the samples and other SARS-CoV-2 isolates was performed using molecular evolutionary genetics analysis software. Results: Based on the genomic databases, 10 isolates were identified as BA.2.40. All samples tested positive for the ORF1ab and N genes, but negative for the HV69-70del gene, which is a marker to detect the Omicron variant. Phylogenetic analysis showed the isolates were closely related to an isolate from Malaysia, an area dominated by BA.2.40. Conclusion: Omicron lineage BA.2.40 has no HV69-70 deletion in the spike protein, a marker used to screen for the Omicron variant. BA.2.40 showed a high similarity to an isolate from Malaysia and was detected only during certain periods, indicating the effect of internationally imported cases.

Correlation of ABO Blood Group Susceptibility to Disease Severity of SARS-COV-2: An Original Research.

COVID-19, the Ecumenical Pandemic that hit Wuhan, Hubei Province, China, in 2019 has instigated an emergency situation all over the globe. Current scientific corroborations highlighted the role of zoonotic cross-over species transmission for the spread of the deadly virus SARSCoV2. The proposition of ABO blood grouping to susceptibility for various infectious diseases has been documented in the past since blood group antigens constitute polymorphic traits that are inherited among humans, therefore are frequent targets in epidemiological studies. Aim: To correlate the ABO blood group susceptibility to disease severity in COVID-19-positive cases among Indian populations. Objectives: Association of ABO blood group patterns to disease severity in COVID-19-positive cases. Materials and Methods: A cross-sectional, observational study design was conducted among 700 confirmed COVID-19-positive cases admitted to the tertiary health care center in Maharashtra, India. The data collected were subjected to statistical analysis. Results: Blood group 'A' positive was frequent (40%) in severe COVID-19 (E group) disease, and 'O' positive blood group was frequent in moderate COVID-19 disease (34.62%). Conclusion: ABO Blood grouping can be used as one of the efficient biomarker for COVID-19, thereby providing a new platform for therapeutic applications in the field of research.

Humoral Immune Response to SARS-CoV-2 Spike Protein Receptor-Binding Motif Linear Epitopes.

The worldwide spread of SARS-CoV-2 has led to a significant economic and social burden on a global scale. Even though the pandemic has concluded, apprehension remains regarding the emergence of highly transmissible variants capable of evading immunity induced by either vaccination or prior infection. The success of viral penetration is due to the specific amino acid residues of the receptor-binding motif (RBM) involved in viral attachment. This region interacts with the cellular receptor ACE2, triggering a neutralizing antibody (nAb) response. In this study, we evaluated serum immunogenicity from individuals who received either a single dose or a combination of different vaccines against the original SARS-CoV-2 strain and a mutated linear RBM. Despite a modest antibody response to wild-type SARS-CoV-2 RBM, the Omicron variants exhibit four mutations in the RBM (S477N, T478K, E484A, and F486V) that result in even lower antibody titers. The primary immune responses observed were directed toward IgA and IgG. While nAbs typically target the RBD, our investigation has unveiled reduced seroreactivity within the RBD's crucial subregion, the RBM. This deficiency may have implications for the generation of protective nAbs. An evaluation of S1WT and S2WT RBM peptides binding to nAbs using microscale thermophoresis revealed a higher affinity (35 nM) for the S2WT sequence (GSTPCNGVEGFNCYF), which includes the FNCY patch. Our findings suggest that the linear RBM of SARS-CoV-2 is not an immunodominant region in vaccinated individuals. Comprehending the intricate dynamics of the humoral response, its interplay with viral evolution, and host genetics is crucial for formulating effective vaccination strategies, targeting not only SARS-CoV-2 but also anticipating potential future coronaviruses.

Persistent immune imprinting occurs after vaccination with the COVID-19 XBB.1.5 mRNA booster in humans.

Immune imprinting describes how the first exposure to a virus shapes immunological outcomes of subsequent exposures to antigenically related strains. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Omicron breakthrough infections and bivalent COVID-19 vaccination primarily recall cross-reactive memory B cells induced by prior Wuhan-Hu-1 spike mRNA vaccination rather than priming Omicron-specific naive B cells. These findings indicate that immune imprinting occurs after repeated Wuhan-Hu-1 spike exposures, but whether it can be overcome remains unclear. To understand the persistence of immune imprinting, we investigated memory and plasma antibody responses after administration of the updated XBB.1.5 COVID-19 mRNA vaccine booster. We showed that the XBB.1.5 booster elicited neutralizing antibody responses against current variants that were dominated by recall of pre-existing memory B cells previously induced by the Wuhan-Hu-1 spike. Therefore, immune imprinting persists after multiple exposures to Omicron spikes through vaccination and infection, including post XBB.1.5 booster vaccination, which will need to be considered to guide future vaccination.

Steroidal lactones from Withania somnifera effectively target Beta, Gamma, Delta and Omicron variants of SARS-CoV-2 and reveal a decreased susceptibility to viral infection and perpetuation: a polypharmacology approach.

Prevention from disease is presently the cornerstone of the fight against COVID-19. With the rapid emergence of novel SARS-CoV-2 variants, there is an urgent need for novel or repurposed agents to strengthen and fortify the immune system. Existing vaccines induce several systemic and local side-effects that can lead to severe consequences. Moreover, elevated cytokines in COVID-19 patients with cancer as co-morbidity represent a significant bottleneck in disease prognosis and therapy. Withania somnifera (WS) and its phytoconstituent(s) have immense untapped immunomodulatory and therapeutic potential and the anticancer potential of WS is well documented. To this effect, WS methanolic extract (WSME) was characterized using HPLC. Withanolides were identified as the major phytoconstituents. In vitro cytotoxicity of WSME was determined against human breast MDA-MB-231 and normal Vero cells using MTT assay. WSME displayed potent cytotoxicity against MDA-MB-231 cells (IC50: 66 µg/mL) and no effect on Vero cells in the above range. MD simulations of Withanolide A with SARS-CoV-2 main protease and spike receptor-binding domain as well as Withanolide B with SARS-CoV spike glycoprotein and SARS-CoV-2 papain-like protease were performed using Schrödinger. Stability of complexes followed the order 6M0J-Withanolide A > 6W9C-Withnaolide B > 5WRG-Withanolide B > 6LU7-Withanolide A. Maximum stable interaction(s) were observed between Withanolides A and B with SARS-CoV-2 and SARS-CoV spike glycoproteins, respectively. Withanolides A and B also displayed potent binding to pro-inflammatory markers viz. serum ferritin and IL-6. Thus, WS phytoconstituents have the potential to be tested further in vitro and in vivo as novel antiviral agents against COVID-19 patients having cancer as a co-morbidity. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-023-00184-y.

Understanding SARS-CoV-2 spike glycoprotein clusters and their impact on immunity of the population from Rio Grande do Norte, Brazil.

SARS-CoV-2 genome underwent mutations since it started circulating within the human population. The aim of this study was to understand the fluctuation of the spike clusters concomitant to the population immunity either due to natural infection and/or vaccination in a state of Brazil that had both high rate of natural infection and vaccination coverage. A total of 1725 SARS-CoV-2 sequences from the state of Rio Grande do Norte, Brazil, were retrieved from GISAID and subjected to cluster analysis. Immunoinformatics were used to predict T- and B-cell epitopes, followed by simulation to estimate either pro- or anti-inflammatory responses and to correlate with circulating variants. From March 2020 to June 2022, the state of Rio Grande do Norte reported 579,931 COVID-19 cases with a 1.4% fatality rate across the three major waves: May-Sept 2020, Feb-Aug 2021, and Jan-Mar 2022. Cluster 0 variants (wild type strain, Zeta) were prevalent in the first wave and Delta (AY.*), which circulated in Brazil in the latter half of 2021, featuring fewer unique epitopes. Cluster 1 (Gamma (P.1 + P.1.*)) dominated the first half of 2021. Late 2021 had two new clusters, Cluster 2 (Omicron, (B.1.1.529 + BA.*)), and Cluster 3 (BA.*) with the most unique epitopes, in addition to Cluster 4 (Delta sub lineages) which emerged in the second half of 2021 with fewer unique epitopes. Cluster 1 epitopes showed a high pro-inflammatory propensity, while others exhibited a balanced cytokine induction. The clustering method effectively identified Spike groups that may contribute to immune evasion and clinical presentation, and explain in part the clinical outcome.

COVID-19 Antibody Levels among Various Vaccination Groups, One-Year Antibody Follow-Up in Two University Hospitals from Western and Central Turkey.

Various clinical outcomes, reinfections, vaccination programs, and antibody responses resulted from the COVID-19 pandemic. This study investigated the time-dependent changes in SARS-CoV-2 antibody responses in infected and/or vaccinated and unvaccinated individuals and to provide insights into spike and nucleocapsid antibodies, which fluctuate during infectious and non-infectious states. This cohort study was carried out at the Ege University Faculty of Medicine hospital in Izmir (western Turkey) and the Erciyes University Faculty of Medicine hospital in Kayseri (central Turkey) between December 2021 and January 2023, which coincided with the second half of COVID-19 pandemic. The study included 100 COVID-19 PCR-positive patients and 190 healthcare workers (HCWs). Antibody levels were followed up via quantitative anti-SARS-CoV-2 spike and qualitative anti-nucleocapsid immunoassays (Elecsys™). Antibody levels declined after infection but persisted for at least 6-8 months. Individuals who had received only CoronaVac had higher anti-nucleocapsid antibody levels in the early months than those who received mixed vaccination. However, anti-spike antibodies persisted longer and at higher levels in individuals who had received mixed vaccinations. This suggests that combining two different vaccine platforms may provide a synergistic effect, resulting in more durable and broad-spectrum immunity against SARS-CoV-2. The study provides information about the vaccination and antibody status of healthcare workers in the second half of the pandemic and provides valuable insights into the dynamics of antibody responses to COVID-19 infection and vaccination.

Genome-wide bioinformatics analysis of human protease capacity for proteolytic cleavage of the SARS-CoV-2 spike glycoprotein.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) primarily enters the cell by binding the virus's spike (S) glycoprotein to the angiotensin-converting enzyme 2 receptor on the cell surface, followed by proteolytic cleavage by host proteases. Studies have identified furin and transmembrane protease serine 2 proteases in priming and triggering cleavages of the S glycoprotein, converting it into a fusion-competent form and initiating membrane fusion, respectively. Alternatively, SARS-CoV-2 can enter the cell through the endocytic pathway, where activation is triggered by lysosomal cathepsin L. However, other proteases are also suspected to be involved in both entry routes. In this study, we conducted a genome-wide bioinformatics analysis to explore the capacity of human proteases in hydrolyzing peptide bonds of the S glycoprotein. Predictive models of sequence specificity for 169 human proteases were constructed and applied to the S glycoprotein together with the method for predicting structural susceptibility to proteolysis of protein regions. After validating our approach on extensively studied S2' and S1/S2 cleavage sites, we applied our method to each peptide bond of the S glycoprotein across all 169 proteases. Our results indicate that various members of the proprotein convertase subtilisin/kexin type, type II transmembrane family serine protease, and kallikrein families, as well as specific coagulation factors, are capable of cleaving S2' or S1/S2 sites. We have also identified a potential cleavage site of cathepsin L at the K790 position within the S2' loop. Structural analysis suggests that cleavage of this site induces conformational changes similar to the cleavage at the R815 (S2') position, leading to the exposure of the fusion peptide and subsequent fusion with the membrane. Other potential cleavage sites and the influence of mutations in common SARS-CoV-2 variants on proteolytic efficiency are discussed.IMPORTANCEThe entry of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) into the cell, activated by host proteases, is considerably more complex in coronaviruses than in most other viruses and is not fully understood. There is evidence that other proteases beyond the known furin and transmembrane protease serine 2 can activate the spike protein. Another example of uncertainty is the cleavage site for the alternative endocytic route of SARS-CoV-2 entrance, which is still unknown. Bioinformatics methods, modeling protease specificity and estimating the structural susceptibility of protein regions to proteolysis, can aid in studying this topic by predicting the involved proteases and their cleavage sites, thereby substantially reducing the amount of experimental work. Elucidating the mechanisms of spike protein activation is crucial for preventing possible future coronavirus pandemics and developing antiviral drugs.

In Silico Study on Natural Chemical Compounds from Citric Essential Oils as Potential Inhibitors of an Omicron (BA.1) SARS-CoV-2 Mutants' Spike Glycoprotein.

BACKGROUND: SARS-CoV-2's remarkable capacity for genetic mutation enables it to swiftly adapt to environmental changes, influencing critical attributes, such as antigenicity and transmissibility. Thus, multi-target inhibitors capable of effectively combating various viral mutants concurrently are of great interest. OBJECTIVE: This study aimed to investigate natural compounds that could unitedly inhibit spike glycoproteins of various Omicron mutants. Implementation of various in silico approaches allows us to scan a library of compounds against a variety of mutants in order to find the ones that would inhibit the viral entry disregard of occurred mutations. METHODS: An extensive analysis of relevant literature was conducted to compile a library of chemical compounds sourced from citrus essential oils. Ten homology models representing mutants of the Omicron variant were generated, including the latest 23F clade (EG.5.1), and the compound library was screened against them. Subsequently, employing comprehensive molecular docking and molecular dynamics simulations, we successfully identified promising compounds that exhibited sufficient binding efficacy towards the receptor binding domains (RBDs) of the mutant viral strains. The scoring of ligands was based on their average potency against all models generated herein, in addition to a reference Omicron RBD structure. Furthermore, the toxicity profile of the highest-scoring compounds was predicted. RESULTS: Out of ten built homology models, seven were successfully validated and showed to be reliable for In Silico studies. Three models of clades 22C, 22D, and 22E had major deviations in their secondary structure and needed further refinement. Notably, through a 100 nanosecond molecular dynamics simulation, terpinen-4-ol emerged as a potent inhibitor of the Omicron SARS-CoV-2 RBD from the 21K clade (BA.1); however, it did not show high stability in complexes with other mutants. This suggests the need for the utilization of a larger library of chemical compounds as potential inhibitors. CONCLUSION: The outcomes of this investigation hold significant potential for the utilization of a homology modeling approach for the prediction of RBD's secondary structure based on its sequence when the 3D structure of a mutated protein is not available. This opens the opportunities for further advancing the drug discovery process, offering novel avenues for the development of multifunctional, non-toxic natural medications.

Novel Spike-stabilized trimers with improved production protect K18-hACE2 mice and golden Syrian hamsters from the highly pathogenic SARS-CoV-2 Beta variant.

Most COVID-19 vaccines are based on the SARS-CoV-2 Spike glycoprotein (S) or their subunits. However, S shows some structural instability that limits its immunogenicity and production, hampering the development of recombinant S-based vaccines. The introduction of the K986P and V987P (S-2P) mutations increases the production and immunogenicity of the recombinant S trimer, suggesting that these two parameters are related. Nevertheless, S-2P still shows some molecular instability and it is produced with low yield. Here we described a novel set of mutations identified by molecular modeling and located in the S2 region of the S-2P that increase its production up to five-fold. Besides their immunogenicity, the efficacy of two representative S-2P-based mutants, S-29 and S-21, protecting from a heterologous SARS-CoV-2 Beta variant challenge was assayed in K18-hACE2 mice (an animal model of severe SARS-CoV-2 disease) and golden Syrian hamsters (GSH) (a moderate disease model). S-21 induced higher level of WH1 and Delta variants neutralizing antibodies than S-2P in K18-hACE2 mice three days after challenge. Viral load in nasal turbinate and oropharyngeal samples were reduced in S-21 and S-29 vaccinated mice. Despite that, only the S-29 protein protected 100% of K18-hACE2 mice from severe disease. When GSH were analyzed, all immunized animals were protected from disease development irrespectively of the immunogen they received. Therefore, the higher yield of S-29, as well as its improved immunogenicity and efficacy protecting from the highly pathogenic SARS-CoV-2 Beta variant, pinpoint the S-29 mutant as an alternative to the S-2P protein for future SARS-CoV-2 vaccine development.

Unraveling viral drug targets: a deep learning-based approach for the identification of potential binding sites.

The coronavirus disease 2019 (COVID-19) pandemic has spurred a wide range of approaches to control and combat the disease. However, selecting an effective antiviral drug target remains a time-consuming challenge. Computational methods offer a promising solution by efficiently reducing the number of candidates. In this study, we propose a structure- and deep learning-based approach that identifies vulnerable regions in viral proteins corresponding to drug binding sites. Our approach takes into account the protein dynamics, accessibility and mutability of the binding site and the putative mechanism of action of the drug. We applied this technique to validate drug targeting toward severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein S. Our findings reveal a conformation- and oligomer-specific glycan-free binding site proximal to the receptor binding domain. This site comprises topologically important amino acid residues. Molecular dynamics simulations of Spike in complex with candidate drug molecules bound to the potential binding sites indicate an equilibrium shifted toward the inactive conformation compared with drug-free simulations. Small molecules targeting this binding site have the potential to prevent the closed-to-open conformational transition of Spike, thereby allosterically inhibiting its interaction with human angiotensin-converting enzyme 2 receptor. Using a pseudotyped virus-based assay with a SARS-CoV-2 neutralizing antibody, we identified a set of hit compounds that exhibited inhibition at micromolar concentrations.

Broad receptor tropism and immunogenicity of a clade 3 sarbecovirus.

Although Rhinolophus bats harbor diverse clade 3 sarbecoviruses, the structural determinants of receptor tropism along with the antigenicity of their spike (S) glycoproteins remain uncharacterized. Here, we show that the African Rhinolophus bat clade 3 sarbecovirus PRD-0038 S has a broad angiotensin-converting enzyme 2 (ACE2) usage and that receptor-binding domain (RBD) mutations further expand receptor promiscuity and enable human ACE2 utilization. We determine a cryo-EM structure of the PRD-0038 RBD bound to Rhinolophus alcyone ACE2, explaining receptor tropism and highlighting differences with SARS-CoV-1 and SARS-CoV-2. Characterization of PRD-0038 S using cryo-EM and monoclonal antibody reactivity reveals its distinct antigenicity relative to SARS-CoV-2 and identifies PRD-0038 cross-neutralizing antibodies for pandemic preparedness. PRD-0038 S vaccination elicits greater titers of antibodies cross-reacting with vaccine-mismatched clade 2 and clade 1a sarbecoviruses compared with SARS-CoV-2 S due to broader antigenic targeting, motivating the inclusion of clade 3 antigens in next-generation vaccines for enhanced resilience to viral evolution.

Probable human origin of the SARS-CoV-2 polybasic furin cleavage motif.

BACKGROUND: The key evolutionary step leading to the pandemic virus was the acquisition of the PRRA furin cleavage motif at the spike glycoprotein S1/S2 junction by a progenitor of SARS-CoV-2. Two of its features draw attention: (i) it is absent in other known lineage B beta-coronaviruses, including the newly discovered coronaviruses in bats from Laos and Vietnam, which are the closest known relatives of the covid virus; and, (ii) it introduced the pair of arginine codons (CGG-CGG), whose usage is extremely rare in coronaviruses. With an occurrence rate of only 3%, the arginine CGG codon is considered a minority in SARS CoV-2. On the other hand, Laos and Vietnam bat coronaviruses contain receptor-binding domains that are almost identical to that of SARS-CoV-2 and can therefore infect human cells despite the absence of the furin cleavage motif. RESULTS: Based on these data, the aim of this work is to provide a detailed sequence analysis between the SARS-CoV-2 S gene insert encoding PRRA and the human mRNA transcripts. The result showed a 100% match to several mRNA transcripts. The set of human genes whose mRNAs match this S gene insert are ubiquitous and highly expressed, e.g., the ATPase F1 (ATP5F1) and the ubiquitin specific peptidase 21 (USP21) genes; or specific genes of target organs or tissues of the SARS-CoV-2 infection (e.g., MEMO1, SALL3, TRIM17, CWC15, CCDC187, FAM71E2, GAB4, PRDM13). Results suggest that a recombination between the genome of a SARS-CoV-2 progenitor and human mRNA transcripts could be the origin of the S gene 12-nucleotide insert encoding the S protein PRRA motif. CONCLUSIONS: The hypothesis of probable human origin of the SARS-CoV-2 polybasic furin cleavage motif is supported by: (i) the nature of human genes whose mRNA sequence 100% match the S gene insert; (ii) the synonymous base substitution in the arginine codons (CGG-CGG); and (iii) further spike glycoprotein PRRA-like insertions suggesting that the acquisition of PRRA may not have been a single recombination event.