猪δ冠状病毒(Porcine deltacoronavirus,PDCo V)是一种新发现的肠道冠状病毒,可引起仔猪的腹泻。冠状病毒编码的非结构蛋白Nsp5是一种3C样蛋白酶,在病毒多聚前体蛋白加工以及免疫调节中发挥重要作用。本研究以PDCo V CHN-HN-2014株为模...猪δ冠状病毒(Porcine deltacoronavirus,PDCo V)是一种新发现的肠道冠状病毒,可引起仔猪的腹泻。冠状病毒编码的非结构蛋白Nsp5是一种3C样蛋白酶,在病毒多聚前体蛋白加工以及免疫调节中发挥重要作用。本研究以PDCo V CHN-HN-2014株为模板,通过RT-PCR扩增Nsp5基因并将其克隆到原核表达载体p ET-28a(+)中,构建了重组表达质粒。将重组质粒转化到大肠杆菌BL21(DE3),经IPTG诱导后SDS-PAGE检测,结果表明Nsp5可在大肠杆菌中高效、可溶性表达。采用镍亲和层析对表达的Nsp5蛋白进行纯化,并通过荧光共振能量转移方法检测了其蛋白酶活性,结果表明纯化的Nsp5蛋白能够有效切割含有PDCo V Nsp5氨基端自切割位点的十二肽底物,证实体外表达的Nsp5重组蛋白具有良好的蛋白酶切割活性。猪δ冠状病毒Nsp5蛋白的克隆、表达及其蛋白酶活性检测,为深入研究该蛋白的功能奠定了基础。展开更多
As a highly pathogenic human coronavirus,SARS-CoV-2 has to counteract an intricate network of antiviral host responses to establish infection and spread.The nucleic acid-induced stress response is an essential compone...As a highly pathogenic human coronavirus,SARS-CoV-2 has to counteract an intricate network of antiviral host responses to establish infection and spread.The nucleic acid-induced stress response is an essential component of antiviral defense and is closely related to antiviral innate immunity.However,whether SARS-CoV-2 regulates the stress response pathway to achieve immune evasion remains elusive.In this study,SARS-CoV-2 NSP5 and N protein were found to attenuate antiviral stress granule(avSG)formation.Moreover,NSP5 and N suppressed IFN expression induced by infection of Sendai virus or transfection of a synthetic mimic of dsRNA,poly(I:C),inhibiting TBK1 and IRF3 phosphorylation,and restraining the nuclear translocalization of IRF3.Furthermore,HEK293T cells with ectopic expression of NSP5 or N protein were less resistant to vesicular stomatitis virus infection.Mechanistically,NSP5 suppressed avSG formation and disrupted RIG-I–MAVS complex to attenuate the RIG-I–mediated antiviral immunity.In contrast to the multiple targets of NSP5,the N protein specifically targeted cofactors upstream of RIG-I.The N protein interacted with G3BP1 to prevent avSG formation and to keep the cofactors G3BP1 and PACT from activating RIG-I.Additionally,the N protein also affected the recognition of dsRNA by RIG-I.This study revealed the intimate correlation between SARS-CoV-2,the stress response,and innate antiviral immunity,shedding light on the pathogenic mechanism of COVID-19.展开更多
文摘猪δ冠状病毒(Porcine deltacoronavirus,PDCo V)是一种新发现的肠道冠状病毒,可引起仔猪的腹泻。冠状病毒编码的非结构蛋白Nsp5是一种3C样蛋白酶,在病毒多聚前体蛋白加工以及免疫调节中发挥重要作用。本研究以PDCo V CHN-HN-2014株为模板,通过RT-PCR扩增Nsp5基因并将其克隆到原核表达载体p ET-28a(+)中,构建了重组表达质粒。将重组质粒转化到大肠杆菌BL21(DE3),经IPTG诱导后SDS-PAGE检测,结果表明Nsp5可在大肠杆菌中高效、可溶性表达。采用镍亲和层析对表达的Nsp5蛋白进行纯化,并通过荧光共振能量转移方法检测了其蛋白酶活性,结果表明纯化的Nsp5蛋白能够有效切割含有PDCo V Nsp5氨基端自切割位点的十二肽底物,证实体外表达的Nsp5重组蛋白具有良好的蛋白酶切割活性。猪δ冠状病毒Nsp5蛋白的克隆、表达及其蛋白酶活性检测,为深入研究该蛋白的功能奠定了基础。
文摘旨在对猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)Nsp5基因进行原核表达,利用生物信息学软件,预测其编码蛋白的结构和功能,为了解PEDV致病机理奠定基础。本研究克隆PEDV/LY/2014/04毒株的Nsp5基因,亚克隆进pET-28a(+)原核表达载体,PCR和酶切验证重组质粒的构建,重组质粒pET-28a(+)-Nsp5转入E.coli BL21(DE3)中,SDS-PAGE检测Nsp5的表达和His band Ni+纯化情况,Vector NTI Advance等软件对Nsp5蛋白氨基酸组成、抗原表位、二级和三级结构进行预测和分析。结果表明,成功克隆并构建了重组质粒pET-28a(+)-Nsp5,表达重组蛋白大小约22 ku,且主要以包涵体形式存在,His band Ni+纯化后获得高纯度重组蛋白。Nsp5蛋白是由196个氨基酸残基组成的多肽,其分子质量的理论值为21 820.07 u,理论等电点(pI)为8.734,略偏碱;二级结构骨架中α-螺旋(h)占55.61%,β-折叠(t)占7.65%,无规则卷曲(c)占20.92%,延伸链(e)占15.82%;Nsp5蛋白质的三级结构中,中间段以α-螺旋为主作为骨架,C端多个复杂二级结构构成该蛋白的酶活性中心;B细胞抗原表位的预测,表明有15个潜在的B细胞优势表位。本研究为猪流行性腹泻病毒Nsp5蛋白质的生物学功能相关研究提供数据支持。
基金This work was supported by grants from the Key Research and Development Program of Shandong Province(2020CXGC011305 to P.-H.W)grants from the Natural Science Foundation of Shandong Province(ZR2020QC085 to P.-H.W)+7 种基金grants from the Natural Science Foundation of Jiangsu Province(BK20200225 to P.-H.W)grants from the Natural Science Foundation of China(82101856 to P.-H.W)grants from the National Key R&D Program of China(2021YFC2701203 to P.-H.W)grants from the Natural Science Foundation of China(81930039,31730026,81525012)awarded to C.G,the Fundamental Research Funds of Shandong University(21510078614099)the Fundamental Research Funds of Cheeloo College of Medicine(21510089393109)China Postdoctoral Science Foundation(2018M642662)Future Scholar Program of Shandong University,and the Natural Science Foundation of China(81901604)awarded to Y.Zgrants from the Key Research and Development Project of Shandong Province(2020SFXGFY08).
文摘As a highly pathogenic human coronavirus,SARS-CoV-2 has to counteract an intricate network of antiviral host responses to establish infection and spread.The nucleic acid-induced stress response is an essential component of antiviral defense and is closely related to antiviral innate immunity.However,whether SARS-CoV-2 regulates the stress response pathway to achieve immune evasion remains elusive.In this study,SARS-CoV-2 NSP5 and N protein were found to attenuate antiviral stress granule(avSG)formation.Moreover,NSP5 and N suppressed IFN expression induced by infection of Sendai virus or transfection of a synthetic mimic of dsRNA,poly(I:C),inhibiting TBK1 and IRF3 phosphorylation,and restraining the nuclear translocalization of IRF3.Furthermore,HEK293T cells with ectopic expression of NSP5 or N protein were less resistant to vesicular stomatitis virus infection.Mechanistically,NSP5 suppressed avSG formation and disrupted RIG-I–MAVS complex to attenuate the RIG-I–mediated antiviral immunity.In contrast to the multiple targets of NSP5,the N protein specifically targeted cofactors upstream of RIG-I.The N protein interacted with G3BP1 to prevent avSG formation and to keep the cofactors G3BP1 and PACT from activating RIG-I.Additionally,the N protein also affected the recognition of dsRNA by RIG-I.This study revealed the intimate correlation between SARS-CoV-2,the stress response,and innate antiviral immunity,shedding light on the pathogenic mechanism of COVID-19.
