摘要
目的通过邻位标记-质谱联用与生物信息学分析结合筛选与人冠状病毒229E(HCoV-229E)核衣壳蛋白(NP)相互作用的潜在宿主因子,并利用免疫共沉淀等技术验证,寻找HCoV-229E NP的相互作用蛋白,为揭示病毒复制增殖的分子机制,及不同人冠状病毒致病力差异奠定基础。方法首先构建了表达HCoV-229E NP与生物素连接酶标签融合蛋白(NP-TurboID)的重组腺病毒Ad-V5-NP^(HCoV-229E)-TurboID(Ad-N),感染人非小细胞肺癌细胞A549,48 h后添加外源生物素,通过生物素连接酶标记NP相互作用蛋白,利用链霉亲和素交联的磁珠纯化生物素标记蛋白,而后进行无标记(label-free)蛋白质组学质谱分析,筛选出潜在的与NP互作的蛋白质,并通过免疫沉淀和免疫荧光等实验进行验证。结果无标记蛋白质组学质谱筛选出584个潜在互作蛋白,从中选取糖原合成酶激酶3(GSK3)A和GSK3B进行免疫共沉淀和免疫荧光验证,实验结果表明,二者与NP^(HCoV-229E)存在相互作用。结论邻位标记-质谱联用技术可以用于病毒-宿主相互作用因子的挖掘,为进一步研究冠状病毒复制、增殖及致病机制奠定了基础。
Objective Coronavirus is a class of long-standing pathogens,which are enveloped single-stranded positive-sense RNA viruses.The genome all encodes 4 structural proteins:spike protein(S),nucleocapsid protein(N),membrane protein(M),and envelope protein(E).The nucleocapsid protein(NP)serves as a key structural component of coronaviruses,playing a vital function in the viral life cycle.NP acts as an RNA-binding protein,with a critical role in identifying specific sequences within the viral genome RNA,facilitating the formation of ribonucleoprotein(RNP)complexes with viral RNA to stabilize the viral genome and contribute to viral particles assembly.The NP consists of two primary structural domains,the N-terminal domain(NTD)and the C-terminal domain(CTD).The NTD is primarily responsible for RNA binding,whereas the CTD is involved in polymerization.The N protein demonstrated to trigger the host immune response and to modulate the cell cycle of·3020·生物化学与生物物理进展Prog.Biochem.Biophys.2024;51(11)infected cells by interacting with host proteins.The NP,one of the most abundant protein in coronaviruses,is essential in understanding the pathogenic mechanism of coronaviruses through its interaction with host factors,which response for determining the virus pathogenicity.HCoV-229E is a widely distributed coronavirus that typically causes mild upper respiratory tract diseases,accounting for a significant portion of common cold cases.However,its pathogenicity is notably lower compared to other coronaviruses like MERS-CoV,SARS-CoV,and SARS-CoV-2.The exact molecular mechanism behind remains unexplained,and how HCoV-229E N protein influences virus replication,host antiviral immunity,and pathogenesis need to be further explored.Methods Proximity labeling-mass spectrometry technique and bioinformatics analysis were used to screen for potential host factors interacting with the NP of human coronavirus 229E(HCoV-229E).In this study,a recombinant adenovirus Ad-V5-NP^(HCoV-229E)-TurboID was constructed to express the fusion protein of HCoV-229E NP and biotin ligase(TurboID).A549 cells were infected with the Ad-V5-NP^(HCoV-229E)-TurboID.After 30 min biotin treatment,NP interacting proteins were labeled with biotin by biotin ligase,and subsequently isolated with streptavidin cross-linked magnetic beads.The potential interacting proteins were identified using label-free proteomic mass spectrometry and further validated through immunoprecipitation and immunofluorescence assays.Results We identified a total of 584 potential interacting proteins.Gene ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis highlighted the enrichment of glycogen synthase kinase(GSK)3A and GSK3B in the glycolysis/gluconeogenesis pathway,indicating HCoV-229E NP connection to diabetes through aberrant activity.Moreover,SARS-CoV-2 infection can exacerbate hyperglycemia and metabolic dysregulation in diabetic individuals by activating the ACE2 receptor.Moreover,SARS-CoV-2 was observed to cause potentially harm to pancreaticβ‑cells and leading to insulin deficiency,which not only worsens the condition of diabetic patients but also raises the possibility of new-onset diabetes in non-diabetic individuals.We demonstrated that GSK3A and GSK3B interacted with NP of HCoV-229E,suggesting that the NP may engage in various coronavirus pathogenic processes by interacting with GSK3.Conclusion These findings suggest that proximity labeling-mass spectrometry technique is a valuable tool for identifying virus-host interaction factors,and lay the foundation for future investigations into the mechanisms underlying coronavirus replication,proliferation,and pathogenesis.
作者
琚睿霞
汪浩勇
刘海楠
刘萱
曹诚
JU Rui-Xia;WANG Hao-Yong;LIU Hai-Nan;Liu Xuan;CAO Cheng(College of Life Sciences and Health Engineering,Hubei University of Technology,Wuhan 430068,China;Academy of Military Medicine,Academy of Military Sciences,Beijing 100850,China)
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2024年第11期3011-3020,共10页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金(82101865)资助项目。
