Live attenuated vaccines might elicit mucosal and sterilizing immunity against SARS-CoV-2 that the existing mRNA,adenoviral vector and inactivated vaccines fail to induce.Here,we describe a candidate live attenuated v...Live attenuated vaccines might elicit mucosal and sterilizing immunity against SARS-CoV-2 that the existing mRNA,adenoviral vector and inactivated vaccines fail to induce.Here,we describe a candidate live attenuated vaccine strain of SARS-CoV-2 in which the NSP16 gene,which encodes 2′-O-methyltransferase,is catalytically disrupted by a point mutation.This virus,designated d16,was severely attenuated in hamsters and transgenic mice,causing only asymptomatic and nonpathogenic infection.A single dose of d16 administered intranasally resulted in sterilizing immunity in both the upper and lower respiratory tracts of hamsters,thus preventing viral spread in a contact-based transmission model.It also robustly stimulated humoral and cell-mediated immune responses,thus conferring full protection against lethal challenge with SARS-CoV-2 in a transgenic mouse model.The neutralizing antibodies elicited by d16 effectively cross-reacted with several SARS-CoV-2 variants.Secretory immunoglobulin A was detected in the blood and nasal wash of vaccinated mice.Our work provides proof-of-principle evidence for harnessing NSP16-deficient SARS-CoV-2 for the development of live attenuated vaccines and paves the way for further preclinical studies of d16 as a prototypic vaccine strain,to which new features might be introduced to improve safety,transmissibility,immunogenicity and efficacy.展开更多
A unique feature of coronaviruses is their utilization of self-encoded nonstructural protein 16(nsp16),20-Omethyltransferase(20-O-MTase),to cap their RNAs through ribose 20-O-methylation modification.This process is c...A unique feature of coronaviruses is their utilization of self-encoded nonstructural protein 16(nsp16),20-Omethyltransferase(20-O-MTase),to cap their RNAs through ribose 20-O-methylation modification.This process is crucial for maintaining viral genome stability,facilitating efficient translation,and enabling immune escape.Despite considerable advances in the ultrastructure of SARS-CoV-2 nsp16/nsp10,insights into its molecular mechanism have so far been limited.In this study,we systematically characterized the 20-O-MTase activity of nsp16 in SARS-CoV-2,focusing on its dependence on nsp10 stimulation.We observed cross-reactivity between nsp16 and nsp10 in various coronaviruses due to a conserved interaction interface.However,a single residue substitution(K58T)in SARS-CoV-2 nsp10 restricted the functional activation of MERS-CoV nsp16.Furthermore,the cofactor nsp10 effectively enhanced the binding of nsp16 to the substrate RNA and the methyl donor Sadenosyl-L-methionine(SAM).Mechanistically,His-80,Lys-93,and Gly-94 of nsp10 interacted with Asp-102,Ser-105,and Asp-106 of nsp16,respectively,thereby effectively stabilizing the SAM binding pocket.Lys-43 of nsp10 interacted with Lys-38 and Gly-39 of nsp16 to dynamically regulate the RNA binding pocket and facilitate precise binding of RNA to the nsp16/nsp10 complex.By assessing the conformational epitopes of nsp16/nsp10 complex,we further determined the critical residues involved in 20-O-MTase activity.Additionally,we utilized an in vitro biochemical platform to screen potential inhibitors targeting 20-O-MTase activity.Overall,our results significantly enhance the understanding of viral 20-O methylation process and mechanism,providing valuable targets for antiviral drug development.展开更多
基金supported by the Hong Kong Health and Medical Research Fund grants COVID190121 to JF-WC and COVID190114 to D-YJthe Hong Kong Research Grants Council grants C7142-20GF and T11-709/21-N to D-YJ.
文摘Live attenuated vaccines might elicit mucosal and sterilizing immunity against SARS-CoV-2 that the existing mRNA,adenoviral vector and inactivated vaccines fail to induce.Here,we describe a candidate live attenuated vaccine strain of SARS-CoV-2 in which the NSP16 gene,which encodes 2′-O-methyltransferase,is catalytically disrupted by a point mutation.This virus,designated d16,was severely attenuated in hamsters and transgenic mice,causing only asymptomatic and nonpathogenic infection.A single dose of d16 administered intranasally resulted in sterilizing immunity in both the upper and lower respiratory tracts of hamsters,thus preventing viral spread in a contact-based transmission model.It also robustly stimulated humoral and cell-mediated immune responses,thus conferring full protection against lethal challenge with SARS-CoV-2 in a transgenic mouse model.The neutralizing antibodies elicited by d16 effectively cross-reacted with several SARS-CoV-2 variants.Secretory immunoglobulin A was detected in the blood and nasal wash of vaccinated mice.Our work provides proof-of-principle evidence for harnessing NSP16-deficient SARS-CoV-2 for the development of live attenuated vaccines and paves the way for further preclinical studies of d16 as a prototypic vaccine strain,to which new features might be introduced to improve safety,transmissibility,immunogenicity and efficacy.
基金supported by grants from the National Natural Science Foundation of China(82372223 and 82172243)the National Key R&D Program of China(2021YFF0702004)the Fundamental Research Funds for the Central Universities of China(2042022dx0003).
文摘A unique feature of coronaviruses is their utilization of self-encoded nonstructural protein 16(nsp16),20-Omethyltransferase(20-O-MTase),to cap their RNAs through ribose 20-O-methylation modification.This process is crucial for maintaining viral genome stability,facilitating efficient translation,and enabling immune escape.Despite considerable advances in the ultrastructure of SARS-CoV-2 nsp16/nsp10,insights into its molecular mechanism have so far been limited.In this study,we systematically characterized the 20-O-MTase activity of nsp16 in SARS-CoV-2,focusing on its dependence on nsp10 stimulation.We observed cross-reactivity between nsp16 and nsp10 in various coronaviruses due to a conserved interaction interface.However,a single residue substitution(K58T)in SARS-CoV-2 nsp10 restricted the functional activation of MERS-CoV nsp16.Furthermore,the cofactor nsp10 effectively enhanced the binding of nsp16 to the substrate RNA and the methyl donor Sadenosyl-L-methionine(SAM).Mechanistically,His-80,Lys-93,and Gly-94 of nsp10 interacted with Asp-102,Ser-105,and Asp-106 of nsp16,respectively,thereby effectively stabilizing the SAM binding pocket.Lys-43 of nsp10 interacted with Lys-38 and Gly-39 of nsp16 to dynamically regulate the RNA binding pocket and facilitate precise binding of RNA to the nsp16/nsp10 complex.By assessing the conformational epitopes of nsp16/nsp10 complex,we further determined the critical residues involved in 20-O-MTase activity.Additionally,we utilized an in vitro biochemical platform to screen potential inhibitors targeting 20-O-MTase activity.Overall,our results significantly enhance the understanding of viral 20-O methylation process and mechanism,providing valuable targets for antiviral drug development.
