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The prevalence of "long COVID" is just one of the conundrums highlighting how little we know about the lung's response to viral infection, particularly to syndromecoronavirus-2 (SARS-CoV-2), for which the lung is the point of entry. We used an in vitro human lung system to enable a prospective, unbiased, sequential single-cell level analysis of pulmonary cell responses to infection by multiple SARS-CoV-2 strains. Starting with human induced pluripotent stem cells and emulating lung organogenesis, we generated and infected three-dimensional, multi-cell-type-containing lung organoids (LOs) and gained several unexpected insights. First, SARS-CoV-2 tropism is much broader than previously believed: Many lung cell types are infectable, if not through a canonical receptor-mediated route (e.g., via Angiotensin-converting encyme 2(ACE2)) then via a noncanonical "backdoor" route (via macropinocytosis, a form of endocytosis). Food and Drug Administration (FDA)-approved endocytosis blockers can abrogate such entry, suggesting adjunctive therapies. Regardless of the route of entry, the virus triggers a lung-autonomous, pulmonary epithelial cell-intrinsic, innate immune response involving interferons and cytokine/chemokine production in the absence of hematopoietic derivatives. The virus can spread rapidly throughout human LOs resulting in mitochondrial apoptosis mediated by the prosurvival protein Bcl-xL. This host cytopathic response to the virus may help explain persistent inflammatory signatures in a dysfunctional pulmonary environment of long COVID. The host response to the virus is, in significant part, dependent on pulmonary Surfactant Protein-B, which plays an unanticipated role in signal transduction, viral resistance, dampening of systemic inflammatory cytokine production, and minimizing apoptosis. Exogenous surfactant, in fact, can be broadly therapeutic.
Assuntos
COVID-19 , Pulmão , Organoides , SARS-CoV-2 , Internalização do Vírus , Humanos , SARS-CoV-2/fisiologia , SARS-CoV-2/imunologia , COVID-19/imunologia , COVID-19/virologia , Pulmão/virologia , Pulmão/imunologia , Pulmão/patologia , Organoides/virologia , Tratamento Farmacológico da COVID-19 , Células-Tronco Pluripotentes Induzidas/virologia , Enzima de Conversão de Angiotensina 2/metabolismo , Inflamação , Citocinas/metabolismo , ApoptoseRESUMO
Diagnostic approaches that combine the high sensitivity and specificity of laboratory-based digital detection with the ease of use and affordability of point-of-care (POC) technologies could revolutionize disease diagnostics. This is especially true in infectious disease diagnostics, where rapid and accurate pathogen detection is critical to curbing the spread of disease. We have pioneered an innovative label-free digital detection platform that utilizes Interferometric Reflectance Imaging Sensor (IRIS) technology. IRIS leverages light interference from an optically transparent thin film, eliminating the need for complex optical resonances to enhance the signal by harnessing light interference and the power of signal averaging in shot-noise-limited operation to achieve virtually unlimited sensitivity. In our latest work, we have further improved our previous 'Single-Particle' IRIS (SP-IRIS) technology by allowing the construction of the optical signature of target nanoparticles (whole virus) from a single image. This new platform, 'Pixel-Diversity' IRIS (PD-IRIS), eliminated the need for z-scan acquisition, required in SP-IRIS, a time-consuming and expensive process, and made our technology more applicable to POC settings. Using PD-IRIS, we quantitatively detected the Monkeypox virus (MPXV), the etiological agent for Monkeypox (Mpox) infection. MPXV was captured by anti-A29 monoclonal antibody (mAb 69-126-3) on Protein G spots on the sensor chips and were detected at a limit-of-detection (LOD) - of 200 PFU/ml (~3.3 attomolar). PD-IRIS was superior to the laboratory-based ELISA (LOD - 1800 PFU/mL) used as a comparator. The specificity of PD-IRIS in MPXV detection was demonstrated using Herpes simplex virus, type 1 (HSV-1), and Cowpox virus (CPXV). This work establishes the effectiveness of PD-IRIS and opens possibilities for its advancement in clinical diagnostics of Mpox at POC. Moreover, PD-IRIS is a modular technology that can be adapted for the multiplex detection of pathogens for which high-affinity ligands are available that can bind their surface antigens to capture them on the sensor surface.
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Patterns of transcriptional activity are encoded in our genome through regulatory elements such as promoters or enhancers that, paradoxically, contain similar assortments of sequence-specific transcription factor (TF) binding sites1-3. Knowledge of how these sequence motifs encode multiple, often overlapping, gene expression programs is central to understanding gene regulation and how mutations in non-coding DNA manifest in disease4,5. Here, by studying gene regulation from the perspective of individual transcription start sites (TSSs), using natural genetic variation, perturbation of endogenous TF protein levels and massively parallel analysis of natural and synthetic regulatory elements, we show that the effect of TF binding on transcription initiation is position dependent. Analysing TF-binding-site occurrences relative to the TSS, we identified several motifs with highly preferential positioning. We show that these patterns are a combination of a TF's distinct functional profiles-many TFs, including canonical activators such as NRF1, NFY and Sp1, activate or repress transcription initiation depending on their precise position relative to the TSS. As such, TFs and their spacing collectively guide the site and frequency of transcription initiation. More broadly, these findings reveal how similar assortments of TF binding sites can generate distinct gene regulatory outcomes depending on their spatial configuration and how DNA sequence polymorphisms may contribute to transcription variation and disease and underscore a critical role for TSS data in decoding the regulatory information of our genome.
Assuntos
Regulação da Expressão Gênica , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , Fatores de Transcrição , Sítio de Iniciação de Transcrição , Iniciação da Transcrição Genética , Humanos , Sítios de Ligação , Regulação da Expressão Gênica/genética , Genoma Humano/genética , Motivos de Nucleotídeos/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Fatores de Transcrição/metabolismo , Variação GenéticaRESUMO
The continuing mutability of the SARS-CoV-2 virus can result in failures of diagnostic assays. To address this, we describe a generalizable bioinformatics-to-biology pipeline developed for the calibration and quality assurance of inactivated SARS-CoV-2 variant panels provided to Radical Acceleration of Diagnostics programs (RADx)-radical program awardees. A heuristic genetic analysis based on variant-defining mutations demonstrated the lowest genetic variance in the Nucleocapsid protein (Np)-C-terminal domain (CTD) across all SARS-CoV-2 variants. We then employed the Shannon entropy method on (Np) sequences collected from the major variants, verifying the CTD with lower entropy (less prone to mutations) than other Np regions. Polyclonal and monoclonal antibodies were raised against this target CTD antigen and used to develop an Enzyme-linked immunoassay (ELISA) test for SARS-CoV-2. Blinded Viral Quality Assurance (VQA) panels comprised of UV-inactivated SARS-CoV-2 variants (XBB.1.5, BF.7, BA.1, B.1.617.2, and WA1) and distractor respiratory viruses (CoV 229E, CoV OC43, RSV A2, RSV B, IAV H1N1, and IBV) were assembled by the RADx-rad Diagnostics core and tested using the ELISA described here. The assay tested positive for all variants with high sensitivity (limit of detection: 1.72-8.78 ng/mL) and negative for the distractor virus panel. Epitope mapping for the monoclonal antibodies identified a 20 amino acid antigenic peptide on the Np-CTD that an in-silico program also predicted for the highest antigenicity. This work provides a template for a bioinformatics pipeline to select genetic regions with a low propensity for mutation (low Shannon entropy) to develop robust 'pan-variant' antigen-based assays for viruses prone to high mutational rates.
Assuntos
Antígenos Virais , COVID-19 , Proteínas do Nucleocapsídeo de Coronavírus , Fosfoproteínas , SARS-CoV-2 , SARS-CoV-2/imunologia , SARS-CoV-2/genética , Humanos , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/genética , COVID-19/diagnóstico , COVID-19/imunologia , COVID-19/virologia , Antígenos Virais/imunologia , Antígenos Virais/genética , Fosfoproteínas/imunologia , Fosfoproteínas/genética , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Teste Sorológico para COVID-19/métodos , Teste Sorológico para COVID-19/normas , Anticorpos Antivirais/imunologia , Anticorpos Monoclonais/imunologia , Biologia Computacional/métodos , Mutação , AnimaisRESUMO
Viral infection induces production of type I interferons and expression of interferon-stimulated genes (ISGs) that play key roles in inhibiting viral infection. Here, we show that the ISG guanylate-binding protein 5 (GBP5) inhibits N-linked glycosylation of key proteins in multiple viruses, including SARS-CoV-2 spike protein. GBP5 binds to accessory subunits of the host oligosaccharyltransferase (OST) complex and blocks its interaction with the spike protein, which results in misfolding and retention of spike protein in the endoplasmic reticulum likely due to decreased N-glycan transfer, and reduces the assembly and release of infectious virions. Consistent with these observations, pharmacological inhibition of the OST complex with NGI-1 potently inhibits glycosylation of other viral proteins, including MERS-CoV spike protein, HIV-1 gp160, and IAV hemagglutinin, and prevents the production of infectious virions. Our results identify a novel strategy by which ISGs restrict virus infection and provide a rationale for targeting glycosylation as a broad antiviral therapeutic strategy.
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Broad spectrum oral antivirals are urgently needed for the early treatment of many RNA viruses of clinical concern. We previously described the synthesis of 1-O-octadecyl-2-O-benzyl-glycero-3-phospho-RVn (V2043), an orally bioavailable lipid prodrug of remdesivir nucleoside (RVn, GS-441524) with broad spectrum antiviral activity against viruses with pandemic potential. Here we compared the relative activity of V2043 with new RVn lipid prodrugs containing sn-1 alkyl ether or sn-2 glycerol modifications. We found that 3-F-4-MeO-Bn, 3-CN-Bn, and 4-CN-Bn sn-2 glycerol modifications improved antiviral activity compared to V2043 when tested in vitro against clinically important RNA viruses from 5 virus families. These results support the continued development of V2043 and sn-2 glycerol modified RVn lipid prodrugs for the treatment of a broad range of RNA viruses for which there are limited therapies.
Assuntos
Antivirais , Pró-Fármacos , Antivirais/farmacologia , Pró-Fármacos/farmacologia , Nucleosídeos/farmacologia , Glicerol , Lipídeos/farmacologiaRESUMO
Objectives: Breastfeeding and human milk (HM) improve maternal and infant morbidities and mortality. Therefore, monitoring the safety of breastfeeding and access to HM is of critical importance. In this study, we assessed tools to monitor the presence of monkeypox virus (MPXV) in HM and whether standard Holder pasteurization inactivates MPXV. Materials and Methods: Heat-inactivated MPXV was added to HM or viral transport media (VTM) and analyzed using both research and clinical MPXV quantitative polymerase chain reaction (qPCR) tests. Infectious MPXV was added to HM and was exposed to 1 cycle of freeze-thaw, incubation for 1 hour at room temperature, or conditions of Holder pasteurization (62.5°C for 30 minutes) followed by infectious unit quantification by plaque assay. Results: Research and clinical nucleic acid tests detect MPXV that was added to HM but with reduced sensitivity compared with equivalent samples in VTM at low virus inoculum. MPXV added to HM to achieve a starting concentration of 225,000 plaque forming units (pfu)/mL remains infectious after freeze-thaw or 1 hour storage at room temperature. However, Holder pasteurization reduced infectious virus below the limit of detection, >2,000-fold reduction in viral titer. Conclusion: MPXV can be detected when added to HM using a clinical laboratory-developed qPCR test without modification, but the detection limit is reduced compared with equivalent samples in VTM. MPXV remains viable in HM should the virus ever gain access to HM, but Holder pasteurization reduces infectious MPXV to below detection limits and can be used to reduce the risk of MPXV transmission to infants who receive pasteurized (donor) HM.
Assuntos
Leite Humano , Monkeypox virus , Feminino , Humanos , Aleitamento Materno , Pasteurização , Temperatura AltaRESUMO
The factors contributing to the rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) BA.4 and BA.5 subvariants in populations that experienced recent surges of BA.2 and BA.2.12.1 infections are not understood. Neutralizing antibodies (NAbs) are likely to protect against severe disease if present in sufficient quantity. We found that after BA.2 or BA.2.12.1 infection, NAb responses were largely cross-neutralizing but were much less effective against BA.5. In addition, individuals who were infected and treated early with nirmatrelvir/ritonavir (Paxlovid) had lower NAb levels than untreated individuals.
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Early antiviral treatments, including intravenous remdesivir (RDV), reduce hospitalization and severe disease caused by COVID-19. An orally bioavailable RDV analog may facilitate earlier treatment of non-hospitalized COVID-19 patients. Here we describe the synthesis and evaluation of alkyl glyceryl ether phosphodiesters of GS-441524 (RVn), lysophospholipid analogs which allow for oral bioavailability and stability in plasma. Oral treatment of SARS-CoV-2-infected BALB/c mice with 1-O-octadecyl-2-O-benzyl-sn-glyceryl-3-phospho-RVn (60 mg/kg orally, once daily for 5 days starting 12h after infection) reduced lung viral load by 1.5 log10 units versus vehicle at day 2 and to below the limit of detection at day 5. Structure/activity evaluation of additional analogs that have hydrophobic ethers at the sn-2 of glycerol revealed improved in vitro antiviral activity by introduction of a 3-fluoro-4-methoxy-substituted benzyl or a 3- or 4-cyano-substituted benzyl. Collectively, our data support the development of RVn phospholipid prodrugs as oral antiviral agents for prevention and treatment of SARS-CoV-2 infections.
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Antivirais , COVID-19 , Animais , Camundongos , SARS-CoV-2 , FosfolipídeosRESUMO
Microglia, the resident macrophage of the central nervous system, are increasingly recognized as contributing to diverse aspects of human development, health, and disease. In recent years, numerous studies in both mouse and human models have identified microglia as a "double edged sword" in the progression of neurotropic viral infections: protecting against viral replication and cell death in some contexts, while acting as viral reservoirs and promoting excess cellular stress and cytotoxicity in others. It is imperative to understand the diversity of human microglial responses in order to therapeutically modulate them; however, modeling human microglia has been historically challenging due to significant interspecies differences in innate immunity and rapid transformation upon in vitro culture. In this review, we discuss the contribution of microglia to the neuropathogenesis of key neurotropic viral infections: human immunodeficiency virus 1 (HIV-1), Zika virus (ZIKV), Japanese encephalitis virus (JEV), West Nile virus (WNV), Herpes simplex virus (HSV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We pay special attention to recent work with human stem cell-derived microglia and propose strategies to leverage these powerful models to further uncover species- and disease-specific microglial responses and novel therapeutic interventions for neurotropic viral infections.
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COVID-19 , Infecção por Zika virus , Zika virus , Humanos , Animais , Camundongos , Microglia/metabolismo , Interações entre Hospedeiro e Microrganismos , Infecção por Zika virus/metabolismo , COVID-19/metabolismo , SARS-CoV-2RESUMO
Small molecule inhibitors of glycosylation enzymes are valuable tools for dissecting glycan functions and potential drug candidates. Screening for inhibitors of glycosyltransferases are mainly performed by in vitro enzyme assays with difficulties moving candidates to cells and animals. Here, we circumvent this by employing a cell-based screening assay using glycoengineered cells expressing tailored reporter glycoproteins. We focused on GalNAc-type O-glycosylation and selected the GalNAc-T11 isoenzyme that selectively glycosylates endocytic low-density lipoprotein receptor (LDLR)-related proteins as targets. Our screen of a limited small molecule compound library did not identify selective inhibitors of GalNAc-T11, however, we identify two compounds that broadly inhibited Golgi-localized glycosylation processes. These compounds mediate the reversible fragmentation of the Golgi system without affecting secretion. We demonstrate how these inhibitors can be used to manipulate glycosylation in cells to induce expression of truncated O-glycans and augment binding of cancer-specific Tn-glycoprotein antibodies and to inhibit expression of heparan sulfate and binding and infection of SARS-CoV-2.
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COVID-19 , SARS-CoV-2 , Animais , Glicosilação , SARS-CoV-2/metabolismo , Glicoproteínas/metabolismo , Polissacarídeos/metabolismoRESUMO
Transmembrane protease serine 2 (TMPRSS2) is a plasma membrane protease that activates both spike protein of coronaviruses for cell entry and oncogenic signaling pathways for tumor progression. TMPRSS2 inhibition can reduce cancer invasion and metastasis and partially prevent the entry of SARS-CoV-2 into host cells. Thus, there is an urgent need for both TMPRSS2-selective imaging and precise screening of TMPRSS2 inhibitors. Here, we report a TMPRSS2-responsive surface-potential-tunable peptide-conjugated probe (EGTP) with aggregation-induced emission (AIE) features for TMPRSS2 selective imaging and accurate inhibitor screening. The amphiphilic EGTP was constructed with tunable surface potential and responsive efficiency with TMPRSS2 and its inhibitor. The rational construction of AIE luminogens (AIEgens) with modular peptides indicated that the cleavage of EGTP led to a gradual aggregation with bright fluorescence in high TMPRSS2-expressing cells. This strategy may have value for selective detection of cancer cells, SARS-CoV-2-target cells, and screening of protease inhibitors.
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COVID-19 , Peptídeo Hidrolases , Humanos , SARS-CoV-2 , Membrana Celular , Inibidores de Proteases , Internalização do Vírus , Serina EndopeptidasesRESUMO
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) causes an acute respiratory distress syndrome (ARDS) that resembles surfactant deficient RDS. Using a novel multi-cell type, human induced pluripotent stem cell (hiPSC)-derived lung organoid (LO) system, validated against primary lung cells, we found that inflammatory cytokine/chemokine production and interferon (IFN) responses are dynamically regulated autonomously within the lung following SARS-CoV-2 infection, an intrinsic defense mechanism mediated by surfactant proteins (SP). Single cell RNA sequencing revealed broad infectability of most lung cell types through canonical (ACE2) and non-canonical (endocytotic) viral entry routes. SARS-CoV-2 triggers rapid apoptosis, impairing viral dissemination. In the absence of surfactant protein B (SP-B), resistance to infection was impaired and cytokine/chemokine production and IFN responses were modulated. Exogenous surfactant, recombinant SP-B, or genomic correction of the SP-B deletion restored resistance to SARS-CoV-2 and improved viability.
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Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a major health problem worldwide. Due to the fast emergence of SARS-CoV-2 variants, understanding the molecular mechanisms of viral pathogenesis and developing novel inhibitors are essential and urgent. Here, we investigated the potential roles of N6,2'-O-dimethyladenosine (m6Am), one of the most abundant modifications of eukaryotic messenger ribonucleic acid (mRNAs), in SARS-CoV-2 infection of human cells. Using genome-wide m6Am-exo-seq, RNA sequencing analysis, and Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing, we demonstrate that phosphorylated C-terminal domain (CTD)-interacting factor 1 (PCIF1), a cap-specific adenine N6-methyltransferase, plays a major role in facilitating infection of primary human lung epithelial cells and cell lines by SARS-CoV-2, variants of concern, and other coronaviruses. We show that PCIF1 promotes infection by sustaining expression of the coronavirus receptors angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) via m6Am-dependent mRNA stabilization. In PCIF1-depleted cells, both ACE2/TMPRSS2 expression and viral infection are rescued by re-expression of wild-type, but not catalytically inactive, PCIF1. These findings suggest a role for PCIF1 and cap m6Am in regulating SARS-CoV-2 susceptibility and identify a potential therapeutic target for prevention of infection.
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COVID-19 , Humanos , SARS-CoV-2/genética , Enzima de Conversão de Angiotensina 2 , RNA Mensageiro/genética , Proteínas Nucleares/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Serina EndopeptidasesRESUMO
We isolated a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) BA.2 variant from a person with coronavirus disease 2019 recrudescence after nirmatrelvir/ritonavir treatment. Antiviral sensitivity and neutralizing antibody testing were performed with both parental SARS-CoV-2 and multiple variants of concern. We found that neither nirmatrelvir resistance nor absence of neutralizing immunity was a likely cause of the recrudescence.
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COVID-19 , Humanos , SARS-CoV-2 , Ritonavir/uso terapêutico , Tratamento Farmacológico da COVID-19RESUMO
The recent development and mass administration of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) vaccines allowed for disease control, reducing hospitalizations and mortality. Most of these vaccines target the SARS-CoV-2 Spike (S) protein antigens, culminating with the production of neutralizing antibodies (NAbs) that disrupt the attachment of the virus to ACE2 receptors on the host cells. However, several studies demonstrated that the NAbs typically rise within a few weeks after vaccination but quickly reduce months later. Thus, multiple booster administration is recommended, leading to vaccination hesitancy in many populations. Detecting serum anti-SARS-CoV-2 NAbs can instruct patients and healthcare providers on correct booster strategies. Several in vitro diagnostics kits are available; however, their high cost impairs the mass NAbs diagnostic testing. Recently, we engineered an ACE2 mimetic that interacts with the Receptor Binding Domain (RBD) of the SARS-2 S protein. Here we present the use of this engineered mini-protein (p-deface2 mut) to develop a detection assay to measure NAbs in patient sera using a competitive ELISA assay. Serum samples from twenty-one patients were tested. Nine samples (42.8%) tested positive, and twelve (57.1%) tested negative for neutralizing sera. The data correlated with the result from the standard commercial assay that uses human ACE2 protein. This confirmed that p-deface2 mut could replace human ACE2 in ELISA assays. Using bacterially expressed p-deface2 mut protein is cost-effective and may allow mass SARS-CoV-2 NAbs detection, especially in low-income countries where economical diagnostic testing is crucial. Such information will help providers decide when a booster is required, reducing risks of reinfection and preventing the administration before it is medically necessary.
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COVID-19 , SARS-CoV-2 , Humanos , Enzima de Conversão de Angiotensina 2 , COVID-19/diagnóstico , Anticorpos Antivirais , Anticorpos Neutralizantes , Glicoproteína da Espícula de CoronavírusRESUMO
The emergence of Zika virus (ZIKV) as a global health threat has highlighted the unmet need for ZIKV-specific vaccines and antiviral treatments. ZIKV infects dendritic cells (DC), which have pivotal functions in activating innate and adaptive antiviral responses; however, the mechanisms by which DC function is subverted to establish ZIKV infection are unclear. Here we develop a genomics profiling method that enables discrete analysis of ZIKV-infected versus neighboring, uninfected primary human DCs to increase the sensitivity and specificity with which ZIKV-modulated pathways can be identified. The results show that ZIKV infection specifically increases the expression of genes enriched for lipid metabolism-related functions. ZIKV infection also increases the recruitment of sterol regulatory element-binding protein (SREBP) transcription factors to lipid gene promoters, while pharmacologic inhibition or genetic silencing of SREBP2 suppresses ZIKV infection of DCs. Our data thus identify SREBP2-activated transcription as a mechanism for promoting ZIKV infection amenable to therapeutic targeting.
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Infecção por Zika virus , Zika virus , Antivirais/farmacologia , Células Dendríticas , Humanos , Lipídeos , Transcrição GênicaRESUMO
Existing tools to detect and visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suffer from low selectivity, poor cell permeability, and high cytotoxicity. Here we report a novel self-immolative fluorescent probe (MP590) for the highly selective and sensitive detection of the SARS-CoV-2 main protease (Mpro). This fluorescent probe was prepared by connecting a Mpro-cleavable peptide (N-acetyl-Abu-Tle-Leu-Gln) with a fluorophore (i.e., resorufin) via a self-immolative aromatic linker. Fluorescent titration results show that MP590 can detect Mpro with a limit of detection (LoD) of 35 nM and is selective over interferents such as hemoglobin, bovine serum albumin (BSA), thrombin, amylase, SARS-CoV-2 papain-like protease (PLpro), and trypsin. The cell imaging data indicate that this probe can report Mpro in HEK 293T cells transfected with a Mpro expression plasmid as well as in TMPRSS2-VeroE6 cells infected with SARS-CoV-2. Our results suggest that MP590 can both measure and monitor Mpro activity and quantitatively evaluate Mpro inhibition in infected cells, making it an important tool for diagnostic and therapeutic research on SARS-CoV-2.
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COVID-19 , Proteases 3C de Coronavírus , Corantes Fluorescentes , COVID-19/diagnóstico , Proteases 3C de Coronavírus/análise , Humanos , SARS-CoV-2/enzimologiaRESUMO
As new variants of SARS-CoV-2 continue to emerge, it is important to assess the cross-neutralizing capabilities of antibodies naturally elicited during wild type SARS-CoV-2 infection. In the present study, we evaluate the activity of nine anti-SARS-CoV-2 monoclonal antibodies (mAbs), previously isolated from convalescent donors infected with the Wuhan-Hu-1 strain, against the SARS-CoV-2 variants of concern (VOC) Alpha, Beta, Gamma, Delta and Omicron. By testing an array of mutated spike receptor binding domain (RBD) proteins, cell-expressed spike proteins from VOCs, and neutralization of SARS-CoV-2 VOCs as pseudoviruses, or as the authentic viruses in culture, we show that mAbs directed against the ACE2 binding site (ACE2bs) are more sensitive to viral evolution compared to anti-RBD non-ACE2bs mAbs, two of which retain their potency against all VOCs tested. At the second part of our study, we reveal the neutralization mechanisms at high molecular resolution of two anti-SARS-CoV-2 neutralizing mAbs by structural characterization. We solve the structures of the Delta-neutralizing ACE2bs mAb TAU-2303 with the SARS-CoV-2 spike trimer and RBD at 4.5 Å and 2.42 Å resolutions, respectively, revealing a similar mode of binding to that between the RBD and ACE2. Furthermore, we provide five additional structures (at resolutions of 4.7 Å, 7.3 Å, 6.4 Å, 3.3 Å, and 6.1 Å) of a second antibody, TAU-2212, complexed with the SARS-CoV-2 spike trimer. TAU-2212 binds an exclusively quaternary epitope, and exhibits a unique, flexible mode of neutralization that involves transitioning between five different conformations, with both arms of the antibody recruited for cross linking intra- and inter-spike RBD subunits. Our study provides additional mechanistic understanding about how antibodies neutralize SARS-CoV-2 and its emerging variants and provides insights on the likelihood of reinfections.
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COVID-19 , SARS-CoV-2 , Anticorpos Monoclonais/química , Anticorpos Antivirais , Humanos , Testes de Neutralização , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a serious threat to human health and lacks an effective treatment. There is an urgent need for both real-time tracking and precise treatment of the SARS-CoV-2-infected cells to mitigate and ultimately prevent viral transmission. However, selective triggering and tracking of the therapeutic process in the infected cells remains challenging. Here, we report a main protease (Mpro)-responsive, mitochondrial-targeting, and modular-peptide-conjugated probe (PSGMR) for selective imaging and inhibition of SARS-CoV-2-infected cells via enzyme-instructed self-assembly and aggregation-induced emission (AIE) effect. The amphiphilic PSGMR was constructed with tunable structure and responsive efficiency and validated with recombinant proteins, cells transfected with Mpro plasmid or infected by SARS-CoV-2, and a Mpro inhibitor. By rational construction of AIE luminogen (AIEgen) with modular peptides and Mpro, we verified that the cleavage of PSGMR yielded gradual aggregation with bright fluorescence and enhanced cytotoxicity to induce mitochondrial interference of the infected cells. This strategy may have value for selective detection and treatment of SARS-CoV-2-infected cells.