新型冠状病毒非结构蛋白1重要变异对其与病毒5’UTR结合能力的影响

Effect of significant variation in nonstructural protein 1 of 2019-nCoV on its binding ability to viral 5′UTR

目的探讨新型冠状病毒(2019 novel coronavirus,2019-nCoV)非结构蛋白1(NSP1)重要变异对其与病毒5’UTR结合能力的影响,为抗病毒药物和疫苗的研发提供线索。方法对2019-nCoV基因组数据库进行生物信息分析,选取可能影响NSP1结构及与5’UTR的结合能力的氨基酸变异位点(T12A、R124L、N128I、K141A、GHVMV82-86DEL及KSF141-143DEL),PSIPRED在线工具分析变异体的二级结构,mCSM-NA预测其与RNA的结合能力,DynaMut webserver分析变异对蛋白稳定性的影响;构建变异体质粒,转染HEK-293T细胞,利用双荧光素酶报告基因实验和RNA结合蛋白免疫沉淀(RNA binding protein immunoprecipitation,RIP)实验检测NSP1变异体与病毒5’UTR的结合能力。结果生物信息分析预测除了R124L突变外,其他5种变异体均可改变蛋白二级结构,T12A、R124L、N128I和K141A突变均可以降低与RNA的结合能力,而T12A、R124L、N128I突变降低了蛋白质的稳定性,实验表明R124L、N128I、GHVMV82-86DEL及KSF141-143DEL显著减弱了NSP1与5’UTR的结合能力。结论部分NSP1氨基酸突变或缺失可改变其二级结构,并能够显著减弱NSP1与5’UTR的结合能力,提示可能改变病毒的致病能力,为抗病毒药物和疫苗的研发提供了理论依据。

Objective To explore the effect of significant variation in non-structural protein 1(NSP1)of 2019 novel coronavirus(2019-nCoV)on binding to 5′UTR,and to provide clues for the development of antiviral drugs and vaccines.Methods The bioinformatics analysis of 2019-nCoV genome database was conducted to select the amino acid variation sites(T12A,R124L,N128I,K141A,GHVMV82-86DEL and KSF141-143DEL)that may affect the structure of NSP1 and the ability binding to 5′UTR.PSIPRED online tool was used to analyze the secondary structure of the variants,mCSM-NA was used to predict their binding ability to RNA,and DynaMut webserver was used to analyze the influence of variants on protein stability.The variant plasmids were constructed and transfected into HEK-293T cells.The dual luciferase reporter gene assay and RNA binding protein immunoprecipitation(RIP)assay were used to detect the binding ability of the NSP1 variant for viral 5′UTR.Results Bioinformatics analysis predicted that except for R124L mutation,the other five variants could change the secondary structure of protein,and the mutations of T12A,R124L,N128I and K141A could reduce the binding ability of RNA,while the mutations of T12A,R124L and N128I reduced the stability of protein.The experimental results showed that R124L,N128I,GHVMV82-86DEL and KSF141-143DEL significantly weakened the binding ability of NSP1 to 5′UTR.Conclusions Some mutations or deletion of NSP1 amino acids could change the secondary structure and significantly weaken the binding ability of NSP1 to the 5′UTR,suggesting that the pathogenicity of the virus may be changed,which could provide a theoretical basis for the development of antiviral drugs and vaccines.