RESUMO
The entry of coronaviruses is initiated by spike recognition of host cellular receptors, involving proteinaceous and/or glycan receptors. Recently, TMPRSS2 was identified as the proteinaceous receptor for HCoV-HKU1 alongside sialoglycan as a glycan receptor. However, the underlying mechanisms for viral entry remain unknown. Here, we investigated the HCoV-HKU1C spike in the inactive, glycan-activated, and functionally anchored states, revealing that sialoglycan binding induces a conformational change of the NTD and promotes the neighboring RBD of the spike to open for TMPRSS2 recognition, exhibiting a synergistic mechanism for the entry of HCoV-HKU1. The RBD of HCoV-HKU1 features an insertion subdomain that recognizes TMPRSS2 through three previously undiscovered interfaces. Furthermore, structural investigation of HCoV-HKU1A in combination with mutagenesis and binding assays confirms a conserved receptor recognition pattern adopted by HCoV-HKU1. These studies advance our understanding of the complex viral-host interactions during entry, laying the groundwork for developing new therapeutics against coronavirus-associated diseases.
Assuntos
Serina Endopeptidases , Glicoproteína da Espícula de Coronavírus , Internalização do Vírus , Humanos , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Polissacarídeos/metabolismo , Polissacarídeos/química , Células HEK293 , Ligação Proteica , Receptores Virais/metabolismo , Receptores Virais/química , Coronavirus/metabolismo , Modelos MolecularesRESUMO
The mammalian SID-1 transmembrane family members, SIDT1 and SIDT2, are multipass transmembrane proteins that mediate the cellular uptake and intracellular trafficking of nucleic acids, playing important roles in the immune response and tumorigenesis. Previous work has suggested that human SIDT1 and SIDT2 are N-glycosylated, but the precise site-specific N-glycosylation information and its functional contribution remain unclear. In this study, we use high-resolution liquid chromatography tandem mass spectrometry to comprehensively map the N-glycosites and quantify the N-glycosylation profiles of SIDT1 and SIDT2. Further molecular mechanistic probing elucidates the essential role of N-linked glycans in regulating cell surface expression, RNA binding, protein stability, and RNA uptake of SIDT1. Our results provide crucial information about the potential functional impact of N-glycosylation in the regulation of SIDT1-mediated RNA uptake and provide insights into the molecular mechanisms of this promising nucleic acid delivery system with potential implications for therapeutic applications.
Assuntos
Proteínas de Transporte de Nucleotídeos , RNA , Humanos , Transporte Biológico , Glicosilação , Mamíferos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , RNA/metabolismoRESUMO
The COVID-19 pandemic, caused by SARS-CoV-2, has led to over 750 million infections and 6.8 million deaths worldwide since late 2019. Due to the continuous evolution of SARS-CoV-2, many significant variants have emerged, creating ongoing challenges to the prevention and treatment of the pandemic. Therefore, the study of antibody responses against SARS-CoV-2 is essential for the development of vaccines and therapeutics. Here we perform single particle cryo-electron microscopy (cryo-EM) structure determination of a rabbit monoclonal antibody (RmAb) 9H1 in complex with the SARS-CoV-2 wild-type (WT) spike trimer. Our structural analysis shows that 9H1 interacts with the receptor-binding motif (RBM) region of the receptor-binding domain (RBD) on the spike protein and by directly competing with angiotensin-converting enzyme 2 (ACE2), it blocks the binding of the virus to the receptor and achieves neutralization. Our findings suggest that utilizing rabbit-derived mAbs provides valuable insights into the molecular interactions between neutralizing antibodies and spike proteins and may also facilitate the development of therapeutic antibodies and expand the antibody library.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Anticorpos Monoclonais , Pandemias , Microscopia Crioeletrônica , Anticorpos Antivirais , Receptores Virais/metabolismo , Anticorpos Neutralizantes , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
RNA 2'-O-methylation is widely distributed and plays important roles in various cellular processes. Mycoplasma genitalium RNase R (MgR), a prokaryotic member of the RNase II/RNB family, is a 3'-5' exoribonuclease and is particularly sensitive to RNA 2'-O-methylation. However, how RNase R interacts with various RNA species and exhibits remarkable sensitivity to substrate 2'-O-methyl modifications remains elusive. Here we report high-resolution crystal structures of MgR in apo form and in complex with various RNA substrates. The structural data together with extensive biochemical analysis quantitively illustrate MgR's ribonuclease activity and significant sensitivity to RNA 2'-O-methylation. Comparison to its related homologs reveals an exquisite mechanism for the recognition and degradation of RNA substrates. Through structural and mutagenesis studies, we identified proline 277 to be responsible for the significant sensitivity of MgR to RNA 2'-O-methylation within the RNase II/RNB family. We also generated several MgR variants with modulated activities. Our work provides a mechanistic understanding of MgR activity that can be harnessed as a powerful RNA analytical tool that will open up a new venue for RNA 2'-O-methylations research in biological and clinical samples.
Assuntos
Exorribonucleases/química , Mycoplasma genitalium/química , RNA/química , Ribose/metabolismo , Catálise , Domínio Catalítico/genética , Cromatografia Líquida , Cristalografia por Raios X , Escherichia coli/metabolismo , Exorribonucleases/metabolismo , Metilação , Mutagênese , Mutação , Mycoplasma genitalium/enzimologia , Ligação Proteica , Domínios Proteicos , RNA/metabolismo , Estabilidade de RNA , Proteínas Recombinantes , Especificidade por Substrato , Espectrometria de Massas em TandemRESUMO
SARS-CoV-2 infection is initiated by Spike glycoprotein binding to the human angiotensin-converting enzyme 2 (ACE2) receptor via its receptor binding domain. Blocking this interaction has been proven to be an effective approach to inhibit virus infection. Here we report the discovery of a neutralizing nanobody named VHH60, which was directly produced from an engineering nanobody library based on a commercialized nanobody within a very short period. VHH60 competes with human ACE2 to bind the receptor binding domain of the Spike protein at S351, S470-471and S493-494 as determined by structural analysis, with an affinity of 2.56 nM. It inhibits infections of both ancestral SARS-CoV-2 strain and pseudotyped viruses harboring SARS-CoV-2 wildtype, key mutations or variants at the nanomolar level. Furthermore, VHH60 suppressed SARS-CoV-2 infection and propagation 50-fold better and protected mice from death for twice as long as the control group after SARS-CoV-2 nasal infections in vivo. Therefore, VHH60 is not only a powerful nanobody with a promising profile for disease control but also provides evidence for a highly effective and rapid approach to generating therapeutic nanobodies.
Assuntos
Enzima de Conversão de Angiotensina 2 , Anticorpos Neutralizantes , COVID-19 , SARS-CoV-2 , Anticorpos de Domínio Único , Glicoproteína da Espícula de Coronavírus , SARS-CoV-2/imunologia , SARS-CoV-2/efeitos dos fármacos , Anticorpos de Domínio Único/farmacologia , Anticorpos de Domínio Único/imunologia , Humanos , Animais , COVID-19/imunologia , COVID-19/virologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/química , Camundongos , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Neutralizantes/farmacologia , Tratamento Farmacológico da COVID-19 , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/uso terapêutico , Células HEK293 , Camundongos Endogâmicos BALB C , Ligação Proteica , FemininoRESUMO
BACKGROUNDAs Omicron is prompted to replicate in the upper airway, neutralizing antibodies (NAbs) delivered through inhalation might inhibit early-stage infection in the respiratory tract. Thus, elucidating the prophylactic efficacy of NAbs via nasal spray addresses an important clinical need.METHODSThe applicable potential of a nasal spray cocktail containing 2 NAbs was characterized by testing its neutralizing potency, synergetic neutralizing mechanism, emergency protective and therapeutic efficacy in a hamster model, and pharmacokinetics/pharmacodynamic (PK/PD) in human nasal cavity.RESULTSThe 2 NAbs displayed broad neutralizing efficacy against Omicron, and they could structurally compensate each other in blocking the Spike-ACE2 interaction. When administrated through the intranasal mucosal route, this cocktail demonstrated profound efficacy in the emergency prevention in hamsters challenged with authentic Omicron BA.1. The investigator-initiated trial in healthy volunteers confirmed the safety and the PK/PD of the NAb cocktail delivered via nasal spray. Nasal samples from the participants receiving 4 administrations over a course of 16 hours demonstrated potent neutralization against Omicron BA.5 in an ex vivo pseudovirus neutralization assay.CONCLUSIONThese results demonstrate that the NAb cocktail nasal spray provides a good basis for clinical prophylactic efficacy against Omicron infections.TRIAL REGISTRATIONwww.chictr.org.cn, ChiCTR2200066525.FUNDINGThe National Science and Technology Major Project (2017ZX10202203), the National Key Research and Development Program of China (2018YFA0507100), Guangzhou National Laboratory (SRPG22-015), Lingang Laboratory (LG202101-01-07), Science and Technology Commission of Shanghai Municipality (YDZX20213100001556), and the Emergency Project from the Science & Technology Commission of Chongqing (cstc2021jscx-fyzxX0001).
Assuntos
Anticorpos Neutralizantes , Sprays Nasais , Animais , Cricetinae , Humanos , China , Traqueia , Voluntários SaudáveisRESUMO
Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) has been identified as a nuclear DNA sensor. Upon viral infection, hnRNP A2/B1 recognizes pathogen-derived DNA as a homodimer, which is a prerequisite for its translocation to the cytoplasm to activate the interferon response. However, the DNA binding mechanism inducing hnRNP A2/B1 homodimerization is unknown. Here, we show the crystal structure of the RNA recognition motif (RRM) of hnRNP A2/B1 in complex with a U-shaped ssDNA, which mediates the formation of a newly observed protein dimer. Our biochemical assays and mutagenesis studies confirm that the hnRNP A2/B1 homodimer forms in solution by binding to pre-generated ssDNA or dsDNA with a U-shaped bulge. These results depict a potential functional state of hnRNP A2/B1 in antiviral immunity and other cellular processes.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , Multimerização Proteica , DNA/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismoRESUMO
Due to the continuous evolution of SARS-CoV-2, the Omicron variant has emerged and exhibits severe immune evasion. The high number of mutations at key antigenic sites on the spike protein has made a large number of existing antibodies and vaccines ineffective against this variant. Therefore, it is urgent to develop efficient broad-spectrum neutralizing therapeutic drugs. Here we characterize a rabbit monoclonal antibody (RmAb) 1H1 with broad-spectrum neutralizing potency against Omicron sublineages including BA.1, BA.1.1, BA.2, BA.2.12.1, BA.2.75, BA.3 and BA.4/5. Cryo-electron microscopy (cryo-EM) structure determination of the BA.1 spike-1H1 Fab complexes shows that 1H1 targets a highly conserved region of RBD and avoids most of the circulating Omicron mutations, explaining its broad-spectrum neutralization potency. Our findings indicate 1H1 as a promising RmAb model for designing broad-spectrum neutralizing antibodies and shed light on the development of therapeutic agents as well as effective vaccines against newly emerging variants in the future.
Assuntos
Anticorpos Monoclonais , COVID-19 , Humanos , Anticorpos Monoclonais/farmacologia , SARS-CoV-2/genética , Microscopia CrioeletrônicaRESUMO
The SARS-CoV-2 Omicron variant evades most currently approved neutralizing antibodies (nAbs) and caused drastic decrease of plasma neutralizing activity elicited by vaccination or prior infection, urging the need for the development of pan-variant antivirals. Breakthrough infection induces a hybrid immunological response with potentially broad, potent and durable protection against variants, therefore, convalescent plasma from breakthrough infection may provide a broadened repertoire for identifying elite nAbs. We performed single-cell RNA sequencing (scRNA-seq) and BCR sequencing (scBCR-seq) of B cells from BA.1 breakthrough-infected patients who received 2 or 3 previous doses of inactivated vaccine. Elite nAbs, mainly derived from the IGHV2-5 and IGHV3-66/53 germlines, showed potent neutralizing activity across Wuhan-Hu-1, Delta, Omicron sublineages BA.1 and BA.2 at picomolar NT50 values. Cryo-EM analysis revealed diverse modes of spike recognition and guides the design of cocktail therapy. A single injection of paired antibodies cocktail provided potent protection in the K18-hACE2 transgenic female mouse model of SARS-CoV-2 infection.
Assuntos
COVID-19 , SARS-CoV-2 , Feminino , Animais , Camundongos , SARS-CoV-2/genética , Infecções Irruptivas , Soroterapia para COVID-19 , Anticorpos Neutralizantes , Camundongos Transgênicos , Anticorpos AntiviraisRESUMO
The emergence of the SARS-CoV-2 Omicron variant is dominant in many countries worldwide. The high number of spike mutations is responsible for the broad immune evasion from existing vaccines and antibody drugs. To understand this, we first present the cryo-electron microscopy structure of ACE2-bound SARS-CoV-2 Omicron spike. Comparison to previous spike antibody structures explains how Omicron escapes these therapeutics. Secondly, we report structures of Omicron, Delta, and wild-type spikes bound to a patient-derived Fab antibody fragment (510A5), which provides direct evidence where antibody binding is greatly attenuated by the Omicron mutations, freeing spike to bind ACE2. Together with biochemical binding and 510A5 neutralization assays, our work establishes principles of binding required for neutralization and clearly illustrates how the mutations lead to antibody evasion yet retain strong ACE2 interactions. Structural information on spike with both bound and unbound antibodies collectively elucidates potential strategies for generation of therapeutic antibodies.
Assuntos
COVID-19 , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Anticorpos Neutralizantes , Anticorpos Antivirais , Microscopia Crioeletrônica , Humanos , Fragmentos Fab das Imunoglobulinas , Glicoproteína da Espícula de CoronavírusRESUMO
The accessory protein Orf6 is uniquely expressed in sarbecoviruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which is an ongoing pandemic. SARS-CoV-2 Orf6 antagonizes host interferon signaling by inhibition of mRNA nuclear export through its interactions with the ribonucleic acid export 1 (Rae1)-nucleoporin 98 (Nup98) complex. Here, we confirmed the direct tight binding of Orf6 to the Rae1-Nup98 complex, which competitively inhibits RNA binding. We determined the crystal structures of both SARS-CoV-2 and SARS-CoV-1 Orf6 C-termini in complex with the Rae1-Nup98 heterodimer. In each structure, SARS-CoV Orf6 occupies the same potential mRNA-binding groove of the Rae1-Nup98 complex, comparable to the previously reported structures of other viral proteins complexed with Rae1-Nup98, indicating that the Rae1-Nup98 complex is a common target for different viruses to impair the nuclear export pathway. Structural analysis and biochemical studies highlight the critical role of the highly conserved methionine (M58) of SARS-CoVs Orf6. Altogether our data unravel a mechanistic understanding of SARS-CoVs Orf6 targeting the mRNA-binding site of the Rae1-Nup98 complex to compete with the nuclear export of host mRNA, which further emphasizes that Orf6 is a critical virulence factor of SARS-CoVs.
RESUMO
A new coronavirus (SARS-CoV-2) has been identified as the etiologic agent for the COVID-19 outbreak. Currently, effective treatment options remain very limited for this disease; therefore, there is an urgent need to identify new anti-COVID-19 agents. In this study, we screened over 6,000 compounds that included approved drugs, drug candidates in clinical trials, and pharmacologically active compounds to identify leads that target the SARS-CoV-2 papain-like protease (PLpro). Together with main protease (Mpro), PLpro is responsible for processing the viral replicase polyprotein into functional units. Therefore, it is an attractive target for antiviral drug development. Here we discovered four compounds, YM155, cryptotanshinone, tanshinone I and GRL0617 that inhibit SARS-CoV-2 PLpro with IC50 values ranging from 1.39 to 5.63 µmol/L. These compounds also exhibit strong antiviral activities in cell-based assays. YM155, an anticancer drug candidate in clinical trials, has the most potent antiviral activity with an EC50 value of 170 nmol/L. In addition, we have determined the crystal structures of this enzyme and its complex with YM155, revealing a unique binding mode. YM155 simultaneously targets three "hot" spots on PLpro, including the substrate-binding pocket, the interferon stimulating gene product 15 (ISG15) binding site and zinc finger motif. Our results demonstrate the efficacy of this screening and repurposing strategy, which has led to the discovery of new drug leads with clinical potential for COVID-19 treatments.
Assuntos
Proteases Semelhantes à Papaína de Coronavírus/química , Ensaios de Triagem em Larga Escala/métodos , Inibidores de Proteases/química , Antivirais/química , Antivirais/metabolismo , Antivirais/uso terapêutico , Sítios de Ligação , COVID-19/virologia , Proteases Semelhantes à Papaína de Coronavírus/genética , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Humanos , Imidazóis/química , Imidazóis/metabolismo , Imidazóis/uso terapêutico , Concentração Inibidora 50 , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Naftoquinonas/química , Naftoquinonas/metabolismo , Naftoquinonas/uso terapêutico , Inibidores de Proteases/metabolismo , Inibidores de Proteases/uso terapêutico , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , SARS-CoV-2/isolamento & purificação , Tratamento Farmacológico da COVID-19RESUMO
Accumulating mutations in the SARS-CoV-2 Spike (S) protein can increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, 3 receptor binding domain (RBD) specific monoclonal antibodies (mAbs), 58G6, 510A5 and 13G9, with high neutralizing potency blocking authentic SARS-CoV-2 virus display remarkable efficacy against authentic B.1.351 virus. Surprisingly, structural analysis has revealed that 58G6 and 13G9 both recognize the steric region S470-495 on the RBD, overlapping the E484K mutation presented in B.1.351. Also, 58G6 directly binds to another region S450-458 in the RBD. Significantly, 58G6 and 510A5 both demonstrate prophylactic efficacy against authentic SARS-CoV-2 and B.1.351 viruses in the transgenic mice expressing human ACE2 (hACE2), protecting weight loss and reducing virus loads. Together, we have evidenced 2 potent neutralizing Abs with unique mechanism targeting authentic SARS-CoV-2 mutants, which can be promising candidates to fulfill the urgent needs for the prolonged COVID-19 pandemic.