猪流行性腹泻病毒非结构蛋白Nsp14真核表达载体构建及蛋白表达鉴定

猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)Nsp14蛋白是PEDV的一种非结构蛋白,由于它同时具有核酸外切酶活性和N7-甲基转移酶活性,在病毒的生命周期中发挥重要作用。为了对PEDV Nsp14蛋白进行更加深入的研究,从病变猪小肠中提取总RNA,并通过PCR扩增获取PEDV Nsp14基因,经过双酶切与连接试验,最终将其连接到PRK5-Myc载体上。通过构建PEDV Nsp14真核重组表达载体,并通过Western Blot技术和免疫荧光技术在真核细胞中验证其蛋白表达情况和细胞内定位情况,为后续研究PEDV Nsp14蛋白的功能奠定研究基础。

SARS冠状病毒Nsp14基因的克隆与表达

对SARS冠状病毒的非结构蛋白Nsp14的基因全长进行了克隆表达。根据公布的SARS冠状病毒的非结构蛋白Nsp14的基因序列设计引物,用PCR的方法把该基因从SARS冠状病毒的cDNA片段中扩增出来,经限制性内切酶BamHI与XhoI双酶切后插入到原核表达载体pET30a(+)中,建成重组载体pET30a-Exo。重组载体转化大肠杆菌并进行诱导表达,通过原核表达的方法得到了该蛋白。这为该蛋白的进一步研究奠定了基础。

SARS-CoV-2非结构蛋白NSP14的生物信息学分析

目的:通过生物信息学手段预测新型冠状病毒(SARS-CoV-2)中非结构蛋白NSP14的性质特征。方法:以SARS-CoV-2的NSP14氨基酸序列为研究对象,通过Blastp与SARS-CoV的NSP14进行比对,并利用ProtParam、ProtScale、PredictProtein和SWISS-MODEL等生物信息学软件工具对NSP14的理化性质、结构和活性区域等进行预测。结果:SARS-CoV-2的NSP14由527个氨基酸残基构成,是一种可溶性亲水蛋白,与SARS-CoV中NSP14氨基酸序列的一致率达到95%。NSP14二级结构中无规则卷曲含量最高,该蛋白质包含了N-糖基化位点、cAMP或cGMP依赖蛋白激酶磷酸化位点、蛋白激酶C磷酸化位点、酪蛋白激酶Ⅱ磷酸化位点和N-豆蔻酰化位点等5个保守基序。NSP14同时具备N端核糖核酸外切酶和N7-甲基转移酶2个结构域,其活性区域的总面积和体积分别为1469.712?~2和1708.967?~3。结论:基于SARS-CoV-2中NSP14的氨基酸序列,运用生物信息学相关软件分析和预测了NSP14蛋白的性质和结构,为新型冠状病毒疫苗研制和药物筛选提供理论依据。

Potential treatment with Chinese and Western medicine targeting NSP14 of SARS-CoV-2

The outbreak of coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is a serious global health threat.This raises an urgent need for the development of effective drugs against the deadly disease.SARS-CoV-2 non-structural protein 14(NSP14)carrying RNA cap guanine N7-methyltransferase and 30-50 exoribonuclease activities could be a potential drug target for intervention.NSP14 of SARS-CoV-2 shares 98.7%of similarity with the one(PDB 5NFY)of acute respiratory syndrome(SARS)by ClustalW.Then,the SARS-CoV-2 NSP14 structures were modelled by Modeller 9.18 using SARS NSP14(PDB 5NFY)as template for virtual screening.Based on the docking score from AutoDock Vina1.1.2,18 small molecule drugs were selected for further evaluation.Based on the 5 ns MD simulation trajectory,binding free energy(DG)was calculated by MM/GBSA method.The calculated binding free energies of Saquinavir,Hypericin,Baicalein and Bromocriptine for the N-terminus of the homology model wereà37.2711±3.2160,à30.1746±3.1914,à23.8953±4.4800,andà34.1350±4.3683 kcal/mol,respectively,while the calculated binding free energies wereà60.2757±4.7708,à30.9955±2.9975,à46.3099±3.5689,andà59.8104±3.5389 kcal/mol,respectively,when binding to the C-terminus.Thus,the compounds including Saquinavir,Hypericin,Baicalein and Bromocriptine could bind to the N-terminus and C-terminus of the homology model of the SARS-CoV-2 NSP14,providing a candidate drug against SARS-CoV-2 for further study.

Porcine epidemic diarrhea virus nsp14 inhibits NF-κB pathway activation by targeting the IKK complex and p65

Coronaviruses(CoVs)are a group of related enveloped RNA viruses that have severe consequences in a wide variety of animals by causing respiratory,enteric or systemic diseases.Porcine epidemic diarrhea virus(PEDV)is an economically important CoV distributed worldwide that causes diarrhea in pigs.nsp14 is a nonstructural protein of PEDV that is involved in regulation of innate immunity and viral replication.However,the function and mechanism by which nsp14 modulates and manipulates host immune responses remain largely unknown.Here,we report that PEDV nsp14 is an NF-κB pathway antagonist.Overexpression PEDV nsp14 protein remarkably decreases SeV-,poly(I:C)-and TNF-α-induced NF-κB activation.Meanwhile,expression of proinflammatory cytokines is suppressed by nspl4.nsp14 inhibits the phosphorylation of IKKs by interacting with IKKs and p65.Furthermore,nsp14 suppresses TNF-α-induced phosphorylation and nuclear import of p65.Overexpression nsp14 considerably increases PEDV replication.These results suggest a novel mechanism employed by PEDV to suppress the host antiviral response,providing insights that can guide the development of antivirals against CoVs.

SARS-CoV-2 virus NSP14 Impairs NRF2/HMOX1 activation by targeting Sirtuin 1

Most deaths from the COVID-19 pandemic are due to acute respiratory distress syndrome(ARDS)-related respiratory failure.Cytokine storms and oxidative stress are the major players in ARDS development during respiratory virus infections.However,it is still unknown how oxidative stress is regulated by viral and host factors in response to SARS-CoV-2 infection.Here,we found that activation of NRF2/HMOX1 significantly suppressed SARS-CoV-2 replication in multiple cell types by producing the metabolite biliverdin,whereas SARS-CoV-2 impaired the NRF2/HMOX1 axis through the action of the nonstructural viral protein NSP14.Mechanistically,NSP14 interacts with the catalytic domain of the NAD-dependent deacetylase Sirtuin 1(SIRT1)and inhibits its ability to activate the NRF2/HMOX1 pathway.Furthermore,both genetic and pharmaceutical evidence corroborated the novel antiviral activity of SIRT1 against SARS-CoV-2.Therefore,our findings reveal a novel mechanism by which SARS-CoV-2 dysregulates the host antioxidant defense system and emphasize the vital role played by the SIRT1/NRF2 axis in host defense against SARS-CoV-2.

新型冠状病毒非结构蛋白14的表达及酶活性分析

目的获得纯度较高和有酶活性的新型冠状(新冠)病毒(SARS-CoV-2)非结构蛋白14(Nsp14)。方法根据大肠埃希氏菌(E.coli)的密码子偏好性,分析新冠病毒原始株基因nsp14的稀有密码子,并对其进行优化。将nsp14克隆至4个不同的表达载体并进行表达;通过对表达量和可溶性的比较分析,选择一个表达效果最好的重组表达质粒并优化表达条件;目的蛋白经谷胱甘肽亲和柱纯化后,用半胱氨酸家族蛋白酶(3C)切除融合标签,再分别利用谷胱甘肽亲和柱和分子筛柱纯化蛋白质。最后通过尿素聚丙烯酰胺凝胶电泳对其活性进行鉴定。结果Nsp14的基因在大肠杆菌中表达存在较多的稀有密码子,其中部分稀有密码子呈现近距离分布和串联分布。pGEX6P1-GST-OPTI-Nsp14重组质粒在大肠杆菌中表达的可溶性效果最好,其最佳表达温度为30℃。经纯化后获得了较纯的不含标签的Nsp14,该蛋白具有核酸酶活性。结论本研究成功地表达和纯化出具有核酸酶活性的新冠病毒Nsp14,为进一步推进对新冠病毒Nsp14的结构和功能研究奠定了基础,为筛选靶向Nsp14的抗病毒治疗药物提供了有利的条件。