新型冠状病毒非结构蛋白1重要变异对其与核糖体40S亚单位结合能力的影响

Impact of important mutations in SARS-CoV-2 non-structural protein 1 on the binding ability to 40S ribosomal subunit

目的 探讨新型冠状病毒(SARS-CoV-2)非结构蛋白1(NSP1)重要变异对其与核糖体40S亚单位结合能力的影响.方法 基于生物信息学方法,选取可能影响与核糖体40S亚单位结合能力的NSP1氨基酸突变位点(D152A、E155A、F157S、T170S和R171L),PSIPRED在线工具分析突变体的二级结构,mCSM-NA预测其与核糖体的结合能力,DynaMut webserver分析突变对蛋白稳定性的影响;构建NSP1突变体,转染HEK-293T细胞,利用免疫共沉淀检测NSP1突变体与核糖体40S亚单位的结合能力,SUnSET实验评价突变体对蛋白合成率的影响;双荧光素酶报告基因实验、ELISA和RT-PCR方法进一步评价NSP1蛋白关键位点突变对IFN-β表达的影响.结果 生物信息预测5种突变体均可改变蛋白二级结构,其中F157S和R171L可降低NSP1与核糖体40S的结合能力.实验表明F157S和R171L突变显著减弱了 NSP1与核糖体40S的结合能力,增加了细胞总蛋白合成、IFN-β荧光素酶活性、细胞内IFN-β mRNA的表达及上清中IFN-β表达量(P<0.05).结论 NSP1 F157S和R171L突变显著减弱了 NSP1与核糖体40S的结合能力,增强了 IFN-1的反应,提示NSP1蛋白在免疫调节中起重要作用.

Objective To explore the effects of important mutations in non-structural protein 1(NSP1)of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)on the binding ability to the 40S subunit of ribosome.Methods Based on bioinformatics method,NSP1 amino acid mutation sites(D152A,E155A,F157S,T170S and R171L)that may affect the binding ability to 40S ribosomal subunit were selected.PSIPRED online tool was used to analyze the secondary structure of the mutants,mCSM-NA was used to predict their binding ability to ribosome,and DynaMut webserver was used to analyze the impact of mutations on protein stability.The mutant plasmids were constructed and transfected into HEK-293T cells.The co-immunoprecipitation was used to detect the binding ability of NSP1 mutants to 40S ribosomal subunit.The SUnSET assay was used to evaluate the effect of mutants on protein synthesis rate while the dual luciferase reporter assay system,ELISA,and RT-PCR methods were used to assess the effect of the key point mutations on the expression of IFN-β.Results Bioinformatics predicted that all five mutants could alter the protein secondary structure and F157S and R171L could both reduce the binding ability of NSP1 to 40S ribosomal subunit.The experiment showed that both mutations significantly weakened the binding of NSP1 to ribosomal subunit,significantly increased cell total protein synthesis,IFN-βluciferase activity,intracellular IFN-βmRNA expression and IFN-βexpression level in the supernatant(P<0.05).Conclusions The mutation at F157S and R171L positions in NSP1 could significantly weaken the binding of NSP1 to ribosome,and enhanced the response of IFN-1.The results suggested that NSP1 protein plays an important role in immune regulation.