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nterferon alpha （IFNα）-induced TRIM22 interrupts HCV ＇eplication by ubiquitinating ns5a

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Cellular & Molecular Immunology 2016年第1期13卷 94-102页

作者： Chen Yang Xinhao Zhao Dakang Sun Leilei Yang Chang Chong Yu Pan Xiumei Chi Yanhang Gao Moli Wang Xiaodong Shi Haibo Sun Juan Lv Yuanda Gao Jin Zhong Junqi Niu Bing Sun State Key Laboratory of Cell Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China Key Laboratory of Molecular Virology and Immunology Institut Pasteur of Shanghai Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China Hepatology Section First Hospital University of Jilin Changchun China Infectious Diseases Department Fourth Hospital University of Jilin Changchun China Experiment Center of Clinical Medicine Affiliated Hospital of Binzhou Medical University Binzhou China

TRIM22, a tripartite-motif （TRIM） protein, is upregulated upon interferon alpha （IFNa） administration to hepatitis C virus （HCV）-infected patients. However, the physiological role of TRIM22 upregulation remains unclear. Here, we describe a potential antiviral function of TRI M22＇s targeting of the HCV nsSA protein. ns5a is important for HCV replication and for resistance to I FNa therapy. During the first 24 h following the initiation of I FNa treatment, upregulation of TRIM22 in the peripheral blood mononuclear cells （PBMCs） of HCV patients correlated with a decrease in viral titer. This phenomenon was confirmed in the hepatocyte-derived cell line Huh-7, which is highly permissive for HCV infection. TRIM22 over-expression inhibited HCV replication, and Small interfering RNA （siRNA）-mediated knockdown of TRIM22 diminished IFNα-induced anti-HCV function. Furthermore, we determined that TRIM22 ubiquitinates ns5a in a concentration-dependent manner. In summary, our results suggest that TRIM22 upregulation is associated with HCV decline during IFNα treatment and Dlavs an important role in controlling HCV replication in vitro.

关键词： HCV IFNα ns5a TRIM22 ubiquitin

牦牛源BVDV非结构蛋白ns5a的原核表达及抗原表位分析

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中国畜牧兽医 2022年第8期49卷 3180-3189页

作者：王慧慧冯茜莉蒲飞洋李易聪汪梦竹周小凯赵泽阳马忠仁李倬马晓霞西北民族大学生物医学研究中心兰州730030 西北民族大学生命科学与工程学院兰州730010

【目的】利用原核表达系统体外表达牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)ns5a基因,获得非结构蛋白ns5a,对其进行核苷酸、氨基酸序列分析,以解析BVDV非结构蛋白ns5a的功能。【方法】参考BVDV-1型毒株V006的ns5a基因序列(G... 详细信息

【目的】利用原核表达系统体外表达牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)ns5a基因,获得非结构蛋白ns5a,对其进行核苷酸、氨基酸序列分析,以解析BVDV非结构蛋白ns5a的功能。【方法】参考BVDV-1型毒株V006的ns5a基因序列(GenBank登录号:KX170647)设计并合成1对特异性引物,以分离到的牦牛BVDV GSTZ毒株cDNA为模板,PCR扩增ns5a基因片段,并克隆至表达载体pET-28a(+)中,构建重组原核表达载体pET28a-ns5a。经酶切初步鉴定及测序鉴定正确后,转化大肠杆菌BL21(DE3)感受态细胞,然后利用IPTG诱导表达。经10%SDS-PAGE电泳及Western blotting分析鉴定重组蛋白的表达,并根据ns5a基因的序列构建遗传发育进化树,利用DNAStar软件预测ns5a蛋白的亲水性、表面可塑性和抗原性等特性,并结合二级结构的预测对ns5a蛋白的B细胞抗原表位进行预测。【结果】PCR扩增ns5a目的基因片段为1488 bp,双酶切和测序鉴定结果证明,重组质粒pET28a-ns5a构建成功。经10%SDS-PAGE电泳及Western blotting鉴定重组蛋白,表达出了大小为55 ku的目的蛋白,大小与预期结果相符。通过对不同BVDV毒株ns5a基因序列构建遗传发育进化树,显示GSTZ毒株ns5a在遗传进化特征上属于BVDV-1型。ns5a蛋白的亲水性主要位于12—21、32—69、75—113、120—135、143—147、152—163、165—180、215—230、265—274、296—340、348—378、389—447、455—463、469—495位氨基酸处,表面可塑性主要位于14—18、37—42、76—81、86—109、154—160、169—178、218—228、297—309、348—358、365—373、414—442、430—437、454—460位氨基酸处,柔性区域较多,主要位于14—21、37—43、67—82、86—93、97—110、152—158、169—179、218—231、240—255、296—310、313—328、344—359、364—373、413—422和472—483位氨基酸处。ns5a蛋白的B细胞抗原表位主要位于15—18、76—81、154—158、169—178、218—228、297—309、348—358、365—373和414—422位氨基酸处。【结论】成功表达并鉴定了牦牛源BVDV的非结构蛋白ns5a,系统发育进化树表明BVDV GSTZ株基因型属于BVDV-1型,ns5a蛋白具有良好的抗原性,为深入解析BVDV非结构蛋白ns5a的自身结构功能、免疫学特性以及进一步研究非结构蛋白对病毒复制的影响提供参考。

关键词：牛病毒性腹泻病毒非结构蛋白 ns5a 原核表达

BVDV ns5a/E2蛋白纳米抗体的筛选及其对病毒复制的影响

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BVDV NS5A/E2蛋白纳米抗体的筛选及其对病毒复制的影响

作者：钱天皓石河子大学

学位级别：硕士

牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)可以引起腹泻、流产、发热、胎儿畸形等症状的病毒性传染病。由于疾病发病机制的复杂性,没有足够有效的手段来控制和治疗BVD。因此,必须要探索更有效的抗病毒策略来对抗BVDV的感染... 详细信息

牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)可以引起腹泻、流产、发热、胎儿畸形等症状的病毒性传染病。由于疾病发病机制的复杂性,没有足够有效的手段来控制和治疗BVD。因此,必须要探索更有效的抗病毒策略来对抗BVDV的感染。研究表明,BVDV-E2蛋白产生的中和抗体能够有效的抑制病毒感染,ns5a蛋白与ns5B蛋白共同构成病毒复制酶,且研究表明ns5B纳米抗体可以抑制BVDV复制。因此,这2个蛋白均有望成为BVDV防治药物的潜在靶标。目前,很多研究显示纳米抗体对于多种病毒,如HIV、HCV、SARS-Co V-2等有良好的抗病毒作用,但是对抗BVDV的效果还不十分清楚,研发BVDV纳米抗体为该病防控提供候选材料。目的:本研究旨在筛选针对BVDV-ns5a/E2蛋白的纳米抗体,得到高特异性的纳米抗体,探究其对病毒复制的影响,为研发抗BVDV生物制剂提供实验材料。方法:(1)将重组质粒p ET30a-E2/ns5a转化至大肠杆菌BL21(DE3)感受态细胞,经过诱导表达并纯化,获得重组蛋白,检测其反应原性。(2)分别用重组ns5a、E2蛋白免疫羊驼,分离淋巴细胞,使用巢式PCR扩增VHH基因,获得重组质粒p CANTAB-5E,并转化TG1感受态细胞,鉴定目的基因插入率以及文库容量;将噬菌体展示文库,通过三轮固相淘选,ELISA方法验证其结合力。(3)通过分析选择较好纳米抗体序列,构建重组表达载体,并转化,通过诱导表达并纯化,检测其反应原性。(4)将BVDV病毒分别与纳米抗体孵育后接种于MDBK细胞,通过q-PCR分析、评价不同浓度纳米抗体对BVDV病毒复制的阻断能力。结果:(1)获得重组蛋白ns5a、E2,分子量大小分别约为57、39 k Da,经WB验证反应原性良好。(2)获得1个插入率为88.5%,库容量为3.42×10CFU/m L的ns5a纳米抗体展示文库,1个插入率为83.3%,库容量为8.4×10CFU/m L的E2纳米抗体展示文库。(3)经过三轮淘选,ELISA验证纳米抗体粗提物反应,得到12条纳米抗体,其中5种与E2蛋白结合的纳米抗体,7种与ns5a蛋白结合的纳米抗体。(4)选择其中2条特异性纳米抗体Nb2、Nb8,并获得重组纳米抗体蛋白Nb2、Nb8,分子量大小约15 k Da,经ELISA检测反应原性良好。(5)经细胞试验验证,纳米抗体Nb2、Nb8均对病毒有一定中和能力,其中纳米抗体Nb2短时间内使BVDV拷贝数相比阳性对照组下降了10倍以上。结论:(1)成功原核表达重组蛋白ns5a、E2,构建ns5a/E2噬菌体展示文库,得到特异的纳米抗体。(2)利用原核系统得到Nb2、Nb8纳米抗体蛋白,为BVDV纳米抗体的生物学特性研究提供基础材料。(3)通过细胞试验证实纳米抗体可以阻断BVDV病毒复制。

关键词：纳米抗体牛病毒性腹泻病毒 ns5a E2

HCV ns5a abrogates p53 protein function by interfering with p53-DNA binding

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World Journal of Gastroenterology 2004年第15期10卷 2223-2227页

作者： Guo-ZhongGong Yong-FangJiang YanHe Li-YingLai Ying-HuaZhu Xian-ShiSu CenterforLiverDiseases SecondXiangyaHospitalCentralSouthUniversityChangsha410011HunanProvinceChina

AIM: To evaluate the inhibition effect of HCV ns5a on p53 transactivation on p21 promoter and explore its possible mechanism for influencing p53 ***: p53 function of transactivation on p21 promoter was studied with a luciferase reporter system in which the luciferase gene is driven by p21 promoter, and the p53-DNA binding ability was observed with the use of electrophoretic mobility-shift assay (EnsA). Lipofectin mediated p53 or HCV ns5a expression vectors were used to transfecthepatoma cell lines to observe whether HCV ns5a could abrogate the binding ability of p53 to its specific DNA sequence and p53 transactivation on p21 *** blot experiment was used for detection of HCVns5a and p53 proteins ***: Relative luciferase activity driven by p21 promoter increased significantly in the presence of endogenous p53 protein. Compared to the control group, exogenous p53 protein also stimulated p21 promoter driven luciferase gene expression in a dose-dependent way. HCV ns5aprotein gradually inhibited both endogenous andexogenous p53 transactivation on p21 promoter withincrease of the dose of HCV ns5a expression plasmid. By the experiment of ErVlSA, we could find p53 binding to its specific DNA sequence and, when co-transfected with increased dose of HCV ns5a expression vector, the p53binding affinity to its DNA gradually decreased and finally disappeared. Between the Huh 7 cells transfected with p53 expression vector alone or co-transfected with HCV ns5a expression vector, there was no difference in thep53 protein ***: HCV ns5a inhibits p53 transactivation onp21 promoter through abrogating p53 binding affinity toits specific DNA sequence. It does not affect p53 proteinexpression.

关键词： HCV ns5a p53蛋白质 p53-DNA粘合物基因表达 HCV 丙型肝炎病毒

Hepatitis C resistance to ns5a inhibitors:Is it going to be a problem?

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World Journal of Hepatology 2018年第9期10卷 543-548页

作者： Heidar Sharafi Seyed Moayed Alavian Baqiyatallah Research Center for Gastroenterology and Liver Diseases Baqiyatallah University of Medical SciencesTehran ***Iran Middle East Liver Diseases Center Tehran ***Iran

Treatment of hepatitis C virus(HCV) infection has evolved greatly through the recent decade. The availability of direct-acting antiviral agents(DAAs) targeting the functional proteins of HCV has resulted in the introduction of DAA-based combination therapies,providing an optimal rate of treatment success. Among the DAAs,ns5 A inhibitors are used in most of the introduced and approved HCV antiviral regimens. Resistance-associated substitutions(RASs) are amino acid substitutions in HCV protein sequences that result in decreased antiviral efficacy of the HCV DAAs. Among the HCV RASs,the ns5 A RASs were found to effectively modify and decrease treatment response to ns5 A inhibitor-containing regimens. As a baseline predictor of treatment response,ns5 A RAS draws attention for pretreatment testing in targeted patient groups. Given ns5 A RASs are either naturally-occurring or DAA-selected,the application of ns5 A RAS testing can be considered in two settings of ns5 A inhibitor-na?ve patients and ns5 A inhibitor-experienced patients. Less than 5% of ns5 A inhibitor-na?ve patients harbor naturally-occurring ns5 A RAS with high resistance level(> 100 X resistance foldchange). In ns5 A inhibitor-na?ve patients,ns5 A RAS testing accompanied by treatment optimization cannot increase treatment response more than 2%-3%,while in ns5 A inhibitor-experienced patients,> 75% are found to have ns5 A RASs > 100 X and ns5 A RAS testing in this group of patients seems to be reasonable. This editorial will address the debate on the application of ns5 A RAS testing and will discuss if the ns5 A RAS testing has any role in clinical management of hepatitis C.

关键词： Direct-acting antiviral agent Hepatitis C ns5a Resistance Treatment

HCV ns5a基因在hepG2细胞中的表达以及对PI3K的影响

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细胞与分子免疫学杂志 2007年第12期23卷 1133-1135页

作者：胡学芳邓小昭刁振宇张云周宗安刘玉南京军区军事医学研究所分子生物学研究室江苏南京210002

目的:探讨HCV-ns5a对PI3K表达的影响。方法:应用PCR技术从含有HCV全长开放阅读框的质粒中获得ns5a全长基因片段,利用基因重组技术将其克隆至真核表达载体pcDNA3.0(-)中。通过酶切、PCR及测序鉴定,ns5a基因已正确插入到pcDNA3.0(-)中,再... 详细信息

目的:探讨HCV-ns5a对PI3K表达的影响。方法:应用PCR技术从含有HCV全长开放阅读框的质粒中获得ns5a全长基因片段,利用基因重组技术将其克隆至真核表达载体pcDNA3.0(-)中。通过酶切、PCR及测序鉴定,ns5a基因已正确插入到pcDNA3.0(-)中,再利用脂质体转染HepG2细胞。结果:经RT-PCR及Western blot检测,HCV的ns5a基因在HepG2细胞中获得表达,而且在表达重组ns5a的转染HepG2细胞中,检测到PI3K蛋白的表达。结论:ns5a可在体外激活PI3K及其信号通路。

关键词： HCV ns5a HepG2细胞基因表达磷脂酰肌醇-3-激酶(PI3K)

Mutation in the ns5a Region of Chinese HCV-1b Isolates and Its Correlation with Efficacy of Interferon Alpha Therapy

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Journal of Nanjing Medical University 2000年第1期14卷 1-6页

作者： Zhang Yongxiang(张永祥) Sun Nanxiong(孙南雄)\ Huang Zuhu(黄祖瑚) Han Yaping(韩亚萍)\ Liu Ting(刘婷) Research Laboratory of Infectious Diseases, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, P. R. China

Objective\ To study the correlation between mutations in the ns5a region of hepatitis C virus genotype 1b and the efficacy of interferon alpha (IFN α) therapy. Methods\ Twenty patients with chronic HCV 1b infection, including 10 responders and 10 non responders to standard IFN α therapy, were investigated. HCV 1b ns5a 2209 2248 region was amplified by RT PCR and the second round product was directly sequenced before treatment. Results\ In IFN α response group, 4 out of 10 HCV isolates (40%) had mutations of amino acid in ns5a 2209 2248 region, which were all intermediate types; while in IFN α non response group, none of HCV isolates (0/10,0%) had mutations, which were all wild types (P5). There was no mutant type isolates in these two groups. All patients (4/4,100%) infected with intermediate type and 6 out of 16 patients(37.5%) infected with wild type of HCV 1b were responsive to IFN α therapy (P5). Conclusion\ The sequences in ns5a 2209 2248 region of Chinese HCV 1b isolates were relatively conservative. There may be a correlation between the efficacy of IFN α therapy and the mutations of amino acid in ns5a 2209 2248 region of Chinese HCV 1b isolates.

关键词： hepatitis C virus interferon alpha ns5a 2209 2248 genotypes

eEF1A Interacts with the ns5a Protein and Inhibits the Growth of Classical Swine Fever Virus

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eEF1A Interacts with the NS5A Protein and Inhibits the Growt...

丙型肝炎病毒ns5a高免疫原区人工合成多肽抗原性的研究

第十届全国免疫学学术大会

作者： Su Li Shuo Feng Jing-Han Wang Wen-Rui He Hua-yang Qin Hong Dong Lian-Feng Li Shao-Xiong Yu Yongfeng Li Hua-Ji Qiu eEF1A Interacts with the ns5a Protein and Inhibits the Growth of Classical Swine Fever Virus

Purpose:The ns5a protein of classical swine fever virus(CSFV)is involved in the RNA synthesis and viral ***,the ns5a-interacting cellular proteins engaged in the CSFV replication are poorly *** and Results:Using yeast two-hybrid screen,the eukaryotic elongation factor 1A(e EF1A)was identified to be an ns5a-binding *** ns5a–e EF1A interaction was confirmed by coimmunoprecipitation,glutathione S-transferase(GST)pulldown and laser confocal microscopy *** domain I of e EF1A was shown to be critical for the ns5a–e EF1A *** of e EF1A suppressed the CSFV growth markedly,and conversely,knockdown of e EF1A enhanced the CSFV replication ***,e EF1A,as well as ns5a,was found to reduce the translation efficiency of the internal ribosome entry site(IRES)of CSFV in a dose-dependent manner,as demonstrated by luciferase reporter *** pulldown assay revealed that e EF1A could bind to the CSFV ***:Our results suggest that eEF1A interacts with ns5a and negatively regulates the growth of CSFV.

关键词： classical swine fever virus virus–host interactions ns5a eukaryotic elongation factor 1A internal ribosome entry site

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中华实验和临床病毒学杂志 2004年第4期18卷 344-347页

作者：石理兰窦晓光冯国和 H.A.Fields Y.E.Khudyakov 中国医科大学附属二院感染病科沈阳110004 Centers for Diseases Control and Prevention

目的　检测不同基因型丙型肝炎病毒 (HCV)非结构 5a区 (ns5a)高免疫原区短链多肽的抗原性 ,确定该区的主要线性抗原表位并探讨基因异质性与免疫原性间的关系。方法　设计并合成特异性多肽 ;DNAStar软件对多肽氨基酸同源性进行比较 ;间接... 详细信息

目的　检测不同基因型丙型肝炎病毒 (HCV)非结构 5a区 (ns5a)高免疫原区短链多肽的抗原性 ,确定该区的主要线性抗原表位并探讨基因异质性与免疫原性间的关系。方法　设计并合成特异性多肽 ;DNAStar软件对多肽氨基酸同源性进行比较 ;间接ELISA法检测多肽的抗原性。结果多肽氨基酸的同源性在不同区及不同基因型间变化较大。氨基酸残基 2 2 12～ 2 2 4 1、2 2 72～ 2 30 1和2 30 2～ 2 331的多肽抗原性最强。 18个来自保守区的多肽可以与不同基因型抗 HCV阳性血清起反应 (阳性率高达 96 % ) ;而来自不同基因型间氨基酸序列保守性较低区的多肽抗原性与同基因血清的反应性较高。结论　HCVns5a高免疫原区主要的线性抗原位于氨基酸残基 2 2 12～ 2 2 4 1、2 2 72～2 30 1和 2 30 2～ 2 331的位置 ;人工合成的多肽可以有效地用于检测抗 HCV抗体 ;

关键词：多肽抗原 ns5a 丙型肝炎病毒免疫原抗原性基因型 HCV 氨基酸同源性人工合成特异性