根据犬瘟热病毒(C an ine d istem per v irus,CDV)融合蛋白(F)的基因序列,利用L earnCo il-VM F与ExPA Sy软件预测出两个七肽重复区(heptad repeat,HR 1与HR 2),应用搭桥PCR拼接的方法获得HR 1与HR 2基因,将其直接克隆到pGEX-6p-I表达...根据犬瘟热病毒(C an ine d istem per v irus,CDV)融合蛋白(F)的基因序列,利用L earnCo il-VM F与ExPA Sy软件预测出两个七肽重复区(heptad repeat,HR 1与HR 2),应用搭桥PCR拼接的方法获得HR 1与HR 2基因,将其直接克隆到pGEX-6p-I表达载体构建重组质粒,用PCR及双酶切方法鉴定阳性重组质粒,并对其进行测序鉴定。并在大肠杆菌中进行融合表达。展开更多
The continuous emergence of new severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)variants meansthere is a need to explore additional strategies to develop broad-spectrum vaccines or therapeutics for individu...The continuous emergence of new severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)variants meansthere is a need to explore additional strategies to develop broad-spectrum vaccines or therapeutics for individuals remaining at risk of coronavirus disease 2019(COVID-19).Neutralizing monoclonal antibody(mAb)that binds to theconserved S2 subunit of the SARS-CoV-2 spike(S)protein alone,or in combination with mAb that binds to the receptor-binding domain(RBD)of S protein,might be effective in eliciting protection from infection by a variety of SARS-CoV2 variants.Using high-throughput single-cell immunoglobulin sequencing of B cells from COVID-19-convalescent donors,we identified a high-affinity S2-specific mAb-39,that could inhibit original SARS-CoV-2 strain,Omicron BA.1,BA.2.86,BA.4,BA.5,and EG.5.1 S protein-mediated membrane fusion,leading to the neutralization of these pseudoviralinfections.Moreover,mAb-39 could also improve the neutralizing activity of anti-RBD antibody against the highlyneutralization-resistant Omicron variants.Molecular docking and point mutation analyses revealed that mAb-39 recognized epitopes within the conserved upstream region of the heptad repeat 2(HR2)motif of the S2 subunit.Collectively,these findings demonstrate that targeting the conserved upstream region of the HR2 motif(e.g.,using mAbs)provides anovel strategy for preventing the infection of SARS-CoV-2 and its variants.展开更多
The continuously emerging SARS-CoV-2 variants pose a great challenge to the efficacy of current drugs,this necessitates the development of broad-spectrum antiviral drugs.In the previous study,we designed a recombinant...The continuously emerging SARS-CoV-2 variants pose a great challenge to the efficacy of current drugs,this necessitates the development of broad-spectrum antiviral drugs.In the previous study,we designed a recombinant protein,heptad repeat(HR)121,as a variant-proof vaccine.Here,we found it can act as a fusion inhibitor and demonstrated broadly neutralizing activities against SARS-CoV-2 and its main variants.Structure analysis suggested that HR121 targets the HR2 domain in SARS-CoV-2 spike(S)2 subunit to block virus-cell fusion.Functional experiments demonstrated that HR121 can bind HR2 at serological-pH and endosomal-pH,highlighting its inhibition capacity when SARS-CoV-2 enters via either cellular membrane fusion or endosomal route.Importantly,HR121 can effectively inhibit SARS-CoV-2 and Omicron variant pseudoviruses entering the cells,as well as block authentic SARSCoV-2 and Omicron BA.2 replications in human pulmonary alveolar epithelial cells.After intranasal administration to Syrian golden hamsters,it can protect hamsters from SARS-CoV-2 and Omicron BA.2 infection.Together,our results suggest that HR121 is a potent drug candidate with broadly neutralizing activities against SARS-CoV-2 and its variants.展开更多
Binding sialates to hemagglutinin-neuramini- dase (HN) activates (triggers) the fusion protein (F) to start the membrane fusion process of paramyxovirus, but the mechanism by which the HN and F associate with each oth...Binding sialates to hemagglutinin-neuramini- dase (HN) activates (triggers) the fusion protein (F) to start the membrane fusion process of paramyxovirus, but the mechanism by which the HN and F associate with each other to induce membrane fusion is still unclear. It is noteworthy to study the interaction domains of HN and F of paramyxovirus. To screen interacting domains of the HN and F proteins of Avian parainfluenza virus-2 (APIV-2) and identify the struc- ture of binding proteins, the GST pull-down assay and mass spectroscopy (MS) and circular dichroism (CD) experiments were performed in this study. The study revealed that the globular head region of HN protein tends to form a complex with either the heptad repeat 1 (HR1) or the heptad repeat 2 (HR2) of F protein respectively. This paper discusses the novel fusion mechanism induced by paramyxovirus HN and F proteins.展开更多
文摘根据犬瘟热病毒(C an ine d istem per v irus,CDV)融合蛋白(F)的基因序列,利用L earnCo il-VM F与ExPA Sy软件预测出两个七肽重复区(heptad repeat,HR 1与HR 2),应用搭桥PCR拼接的方法获得HR 1与HR 2基因,将其直接克隆到pGEX-6p-I表达载体构建重组质粒,用PCR及双酶切方法鉴定阳性重组质粒,并对其进行测序鉴定。并在大肠杆菌中进行融合表达。
基金funded bythe National Natural Science Foundation of China(81972753,32170712,and 32170937)R&D Program of Guangzhou National Laboratory(SRPG22-003)+6 种基金the China Postdoctoral Science Foundation(2019M663093)Prevention and Control ofCOVID-2019 Research Program in University of GuangdongProvince(2020KZDZX1176)the Science and TechnologyProgram of Guangdong Province,China(2020A1515110410and 2021A1515010917)the Guangdong Medical Scienceand Technology Research Foundation(A2021336)ShenzhenKey Laboratory Foundation(ZDSYS20200811143757022)theShenzhen Science and Technology Basic Research Program(JCYJ20180507182203049 and JCYJ20230807142815034)and the Shenzhen University(SZU)Top Ranking Project(86000000210).
文摘The continuous emergence of new severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)variants meansthere is a need to explore additional strategies to develop broad-spectrum vaccines or therapeutics for individuals remaining at risk of coronavirus disease 2019(COVID-19).Neutralizing monoclonal antibody(mAb)that binds to theconserved S2 subunit of the SARS-CoV-2 spike(S)protein alone,or in combination with mAb that binds to the receptor-binding domain(RBD)of S protein,might be effective in eliciting protection from infection by a variety of SARS-CoV2 variants.Using high-throughput single-cell immunoglobulin sequencing of B cells from COVID-19-convalescent donors,we identified a high-affinity S2-specific mAb-39,that could inhibit original SARS-CoV-2 strain,Omicron BA.1,BA.2.86,BA.4,BA.5,and EG.5.1 S protein-mediated membrane fusion,leading to the neutralization of these pseudoviralinfections.Moreover,mAb-39 could also improve the neutralizing activity of anti-RBD antibody against the highlyneutralization-resistant Omicron variants.Molecular docking and point mutation analyses revealed that mAb-39 recognized epitopes within the conserved upstream region of the heptad repeat 2(HR2)motif of the S2 subunit.Collectively,these findings demonstrate that targeting the conserved upstream region of the HR2 motif(e.g.,using mAbs)provides anovel strategy for preventing the infection of SARS-CoV-2 and its variants.
基金supported in part by grants from the National Natural Science Foundation of China(82151218,81971548)National Key Research and Development Program of China(2021YFC2301703,2021YFC2301303,2022YFC2303700)+1 种基金Yunnan Key Research and Development Program(202103AC100005,202103AQ100001,202102AA310055,China)CAS“Light of West China”。
文摘The continuously emerging SARS-CoV-2 variants pose a great challenge to the efficacy of current drugs,this necessitates the development of broad-spectrum antiviral drugs.In the previous study,we designed a recombinant protein,heptad repeat(HR)121,as a variant-proof vaccine.Here,we found it can act as a fusion inhibitor and demonstrated broadly neutralizing activities against SARS-CoV-2 and its main variants.Structure analysis suggested that HR121 targets the HR2 domain in SARS-CoV-2 spike(S)2 subunit to block virus-cell fusion.Functional experiments demonstrated that HR121 can bind HR2 at serological-pH and endosomal-pH,highlighting its inhibition capacity when SARS-CoV-2 enters via either cellular membrane fusion or endosomal route.Importantly,HR121 can effectively inhibit SARS-CoV-2 and Omicron variant pseudoviruses entering the cells,as well as block authentic SARSCoV-2 and Omicron BA.2 replications in human pulmonary alveolar epithelial cells.After intranasal administration to Syrian golden hamsters,it can protect hamsters from SARS-CoV-2 and Omicron BA.2 infection.Together,our results suggest that HR121 is a potent drug candidate with broadly neutralizing activities against SARS-CoV-2 and its variants.
基金This work was supported by the National Natural Sciences Foundation of China (Grant No. 30228025)The Founda-tion of China Agricultural University (Grant No. 2004009).
文摘Binding sialates to hemagglutinin-neuramini- dase (HN) activates (triggers) the fusion protein (F) to start the membrane fusion process of paramyxovirus, but the mechanism by which the HN and F associate with each other to induce membrane fusion is still unclear. It is noteworthy to study the interaction domains of HN and F of paramyxovirus. To screen interacting domains of the HN and F proteins of Avian parainfluenza virus-2 (APIV-2) and identify the struc- ture of binding proteins, the GST pull-down assay and mass spectroscopy (MS) and circular dichroism (CD) experiments were performed in this study. The study revealed that the globular head region of HN protein tends to form a complex with either the heptad repeat 1 (HR1) or the heptad repeat 2 (HR2) of F protein respectively. This paper discusses the novel fusion mechanism induced by paramyxovirus HN and F proteins.
