HCV非结构蛋白5A对HIV长末端重复序列影响的初步探讨

目的本研究拟探讨HCV非结构蛋白5A(NS5A)对HIV长末端重复序列(long terminal repeat,LTR)影响,从而为HCV对HIV的影响提供实验依据。方法将构建的LTR启动子驱动的荧光素酶(Luc)报告基因表达质粒(pGL3-LTR-Luc)和含HCV NS5A基因的表达质粒pCNS5A共转染肝癌细胞株(Huh7细胞),采用免疫细胞化学技术、Western Blot及逆转录聚合酶链反应检测HCV NS5A蛋白及mRNA的表达;本实验分三组,将质粒pGL3-LTR-Luc(空白组)、质粒pRc/CMV+pGL3-LTR-Luc(对照组)、质粒pCNS5A+pGL3-LTR-Luc(实验组)分别转染Huh7细胞,48 h后收集细胞,采用Luc活性检测LTR的活性,以观察HCV NS5A对LTR的调控影响。所得荧光活性值以均数±标准差表示,采用Levene's方差齐性检验,多组间比较采用单因素方差分析,两两比较行LSD-t检验。结果转染pcNS5A质粒的Huh7细胞质经RT-PCR及Western Blot检测,HCV NS5A mRNA及蛋白在细胞中获得表达。方差分析结果提示LTR荧光活性在三组间有明显的差异(F=7.876,P=0.002),进一步比较各组间的差异,结果提示共转染质粒pcNS5A+pGL3-LTR-Luc组的Huh7细胞中Luc相对活性(22 476±4471)明显高于单转染pGL3-LTR-Luc组(15 887±3039,P=0.002)及共转染质粒pRc/CMV+pGL3-LTR-Luc组(16 321±4162,P=0.008),差异有统计学意义。结论表达HCV NS5A的质粒pCNS5A成功转染至Huh7细胞;HCV NS5A蛋白能激活HIV LTR,提示HCV NS5A可能为HCV促进HIV复制的分子机制之一。     

丙型肝炎病毒非结构蛋白5A对干扰素α-2b诱导的信号传导和转录激活因子1磷酸化及核转移的影响

目的探讨丙型肝炎病毒（HCV）非结构蛋白5A（NS5A）对干扰素α-2b诱导的Janus激酶-信号传导和转录激活子（JAK—STAT）信号传导途径中STAT1磷酸化及核转移的影响。方法用表达HCVNS5A的质粒（pCNS5A）转染Huh7细胞，应用免疫细胞化学技术检测HCVNS5A的表达，用免疫荧光和Western blot方法检测HCVNS5A对干扰素α-2b诱导的STAT1磷酸化和核转移的影响。结果转染了pCNS5A的Huh7细胞质可见HCVNS5A蛋白的表达；以干扰素α-2b诱导30min后，STAT1磷酸化及核转移在转染了表达HCVNS5A的质粒组比转染空白载体pRC／CMV组及未转染组减少，而未转染组及转染空白载体pRC／CMV组间无明显差别。结论表达HCVNS5A的质粒pCNS5A成功转染至Huh7细胞；HCV NS5A减弱干扰素α-2b诱导的STAT1的磷酸化及核转移，提示NS5A影响干扰素α-2b的JAKSTAT信号传导途径可能是HCV干扰素抵抗的机制之一。     

丙型肝炎病毒NS5A对p53活性调控机制研究

目的 研究HCV NS5A对抑癌蛋白p53活性的抑制作用及其作用机制。 方法 采用荧光素酶报告基因系统观察pwwp-luc,pwwp-mut-luc,pc53-NS3及pCNS5A分别转染或共同转染HepG2、Huh7细胞的荧光素酶活性。应用凝胶滞留试验观察HCV NS5A是否影响p53与其特异DNA序列的结合。 结果 内源性p53能激活p21启动子转录功能,使荧光素酶活性明显增加(3.49×10~5与0.60×10~5,t=5.92,P<0.01)。外源性p53也能激活p21启动子转录功能,荧光素酶活性为5.63×10~5,与对照组(0.47×10~5)比较差异具有显著性(t=10.12,P<0.01)。HCV NS5A能抑制内源性和外源性p53对p21启动子的激活作用,并呈剂量依赖性(F≥20.71,P<0.01)。凝胶滞留试验显示HCV NS5A能阻碍p53与其特异DNA序列的结合。 结论 HCV NS5A能抑制p53的反式激活功能使其目的基因p21启动子的转录功能下降,其机制是HCV NS5A能阻碍p53与其特异DNA序列的结合。     

丙型肝炎病毒非结构蛋白质5A激活Survivin基因的表达

目的探讨丙型肝炎病毒非结构蛋白质5A(HCV NS5A)对Survivin基因表达的影响。方法用含HCV NS5A基因的表达质粒(pCNS5A)转染HepG2细胞,采用免疫细胞化学技术检测HCV NS5A蛋白质的表达,采用逆转录聚合酶链反应和Western blot方法观察HCV NS5A对Survivin mRNA和蛋白质表达水平的影响。结果转染pCNS5A 的HepG2细胞胞浆可见HCV NS5A蛋白质的表达。转染pCNS5A的HepG2细胞内Survivin mRNA和蛋白质表达均比转染空白载体(pRc/CMV)的HepG2细胞及未转染质粒的HepG2细胞明显增强,而后两者Survivin mRNA和蛋白质表达相似。结论HCV NS5A可激活Survivin蛋白质表达,其途径可能通过调节Survivin基因的转录水平(mRNA转录增强)来实现。     

HCV NS2对细胞凋亡及DNA损伤应答信号的影响

目的构建HCV NS2的真核表达质粒,了解HCV NS2蛋白对细胞凋亡及DNA损伤应答信号的影响。方法以pCMV-tag2B为载体,采用双酶切法构建HCV NS2基因重组载体pCMV-tag2B-NS2,进行酶切分析和DNA测序鉴定;在Huh7细胞中转染相应质粒,分别采用Western blot和DAPI染色分析CPT处理或未处理时NS2对DNA损伤应答信号和凋亡的影响,采用流式细胞术检测NS2蛋白对细胞周期的影响。结果酶切和DNA测序证实pCMVtag2B-NS2重组质粒构建成功,HCV NS2蛋白可在Huh7细胞中正确表达并且活化DNA损伤应答信号,但不影响细胞周期进程;DAPI染色检查,NS2转染组和空载体转染组细胞凋亡率分别为(15.33±0.88)%和(4.66±0.88)%,差异有统计学意义(P0.05)。结论成功构建了真核表达载体pCMV-tag2B-NS2,其表达产物HCV NS2蛋白可诱导DNA损伤应答通路的活化并且促进Huh7细胞凋亡。     

丙型肝炎病毒非结构蛋白质5A抑制Hepcidin基因表达并促进肝细胞内铁储留

目的 探讨丙型肝炎病毒(HCV)非结构蛋白质5A (NS5A)对Hepcidin基因表达的影响. 方法 用含HCV NS5A基因的表达质粒pcNS5A瞬时转染QSG7701细胞,采用逆转录-聚合酶链反应及Western blot试验观察HCV NS5A和Hepcidin的mRNA转录水平及蛋白质表达水平,铁染色后观察细胞内铁储留情况.检测数据的多组间比较行单因素方差分析,两两比较行LSD-t检验.结果 转染pcNS5A质粒细胞内有HCV NS5A的mRNA和蛋白的表达,未转染质粒组和转染空白质粒pRc/CMV组细胞内无HCV NS5A的mRNA和蛋白的表达.未转染质粒组、转染pRc/CMV质粒组和转染pcNS5A质粒组细胞的Hepcidin mRNA相对表达量分别为0.711±0.049、0.718±0.052和0.264±0.030,转染pcNS5A组Hepcidin mRNA表达低于未转染质粒组和转染pRc/CMV质粒组(t值分别为- 13.523和- 13.045,P值均＜0.01).转染pcNS5A质粒组细胞Hepcidin蛋白表达较未转染质粒组及转染pRc/CMV质粒组下降,且随着转染pcNS5A质粒剂量的增加,Hepcidin蛋白表达下降更明显.铁染色结果显示,转染pcNS5A质粒细胞内铁较转染pRC/CMV质粒细胞和未转染质粒细胞高.结论 HCV NS5A蛋白能抑制Hepidin的mRNA和蛋白质表达,并引起细胞内铁的储留增加.     

丙型肝炎病毒核心蛋白对低氧诱导因子-1α和血管内皮生长因子表达的影响

目的 探讨HCV核心蛋白对低氧诱导因子1 α(HIF-1 α)和血管内皮生长因子(VEGF)表达的影响. 方法 将HCV核心蛋白基因的真核表达载体flag2B-core和HIF-1 αsiRNA转染Huh7.5.1细胞;采用RT-PCR和Western blot法分别检测HIF-1 α、VEGF在mRNA和蛋白水平的表达变化,采用酶联免疫吸附试验检测细胞上清液中VEGF的含量.对各组间数据进行t检验.结果 Huh7.5.1细胞转染flag2B-core后,HIF-1 α和VEGF的mRNA和蛋白表达水平升高,细胞上清液中VEGF含量明显高于对照组[(654.5±43.7) pg/ml与(365.9±26.8)pg/ml,t=653.1％,P＜ 0.01)];Huh7.5.1细胞共转染flag2B-core和HIF-1 α siRNA后,HIF-1 α和VEGF的mRNA和蛋白表达水平降低,细胞上清液VEGF含量明显低于对照组[(389.2±29.6) pg/ml与(768.8±47.3) pg/ml,t=1330.22,P＜0.01).结论 HCV核心蛋白能够上调HIF-1 α和VEGF的表达;HCV可能通过核心蛋白来调节HIF-1 α和VEGF的表达.     

丙型肝炎病毒非结构蛋白5A调节新生多肽复合物启动子转录活性的研究

目的探讨丙型肝炎病毒（HCV）非结构蛋白5A（NS5A）对新生多肽相关复合物（NACA）启动子转录活性的影响。方法以我室构建的能够表达NS5A蛋白的pcDNA3．1（-）-NS5A质粒和含有NACA基因启动子的PCAT3-NACA报告基因质粒共转染HepG2细胞系设为实验组，同时PCAT3-NACA单独转染HepG2细胞系作为对照组。用酶联免疫吸附法检测氯霉素乙酰转移酶（CAT）的表达活性。结果pCAT3-NACA单独转染HepG2细胞的CAT酶表达活性与pCAT3-NACA和pcDNA3．1（-）-NS5A共转染组HepG2细胞的CAT酶表达活性比较，共转染组A值下降了79．3％。结论HCV NS5A能够抑制NACA启动子的活性，对NACA的表达具有下调作用。     

丙型肝炎病毒非结构蛋白NS5A上调硫氧还蛋白还原酶1基因表达的研究

目的探讨丙型肝炎病毒(HCV)非结构蛋白NS5A对硫氧还蛋白还原酶1(TXNRD1)启动子转录的激活作用.方法以我室构建的丙型肝炎病毒非结构蛋白NS5A反式调节基因的cDNA文库抑制性消减杂交(SSH)筛选结果及基因表达谱芯片结果为基础,利用生物信息学技术确定TXNRD1的启动子区域(TXNRD1p),聚合酶链反应(PCR)扩增TXNRD1p,克隆至真核报告载体pCAT3-Basic中,构建pCAT3-TXNRD1p报告载体;以该质粒转染肝癌细胞系HepG2,用酶联免疫吸附法(ELISA)检测氯霉素乙酰转移酶(CAT)的表达活性;并与pcDNA3.1(-)-NS5A共转染HepG2细胞系,用ELISA法检测CAT的表达活性.结果pCAT3-TXNRD1p和pcDNA3.1(-)-NS5A瞬时转染的HepG2细胞的CAT表达活性是pCAT3-Basic空载体的9.6倍,pCAT3-TXNRD1p的2.1倍.本实验进一步验证了我室利用SSH技术及基因表达谱技术发现的HCV NS5A蛋白反式激活TXNRD1基因转录作用的结果.结论我室克隆的TXNRD1启动子有顺式激活下游基因的活性;HCV的NS5A蛋白具有对TXNRD1基因启动子的反式激活作用.     

丙型肝炎病毒NS5A在肝癌细胞对TLR4表达的影响

目的探讨丙型肝炎病毒非结构蛋白质5A（HCV NS5A）对Toll样受体4（TLR4）表达的影响。方法用含HCV NS5A基因的表达质粒pcNS5A瞬时转染QSG7701细胞,采用免疫细胞化学技术检测HCV NS5A及TLR4蛋白质的表达;采用逆转录聚合酶链反应观察HCV NS5A和TLR4的mRNA表达。结果转染pcNS5A质粒细胞内有HCV NS5A蛋白的表达。转染pcNS5A组TLR4 mRNA及蛋白质表达均较转染pRc/CMV和未转染质粒组明显增强,而后两组细胞TLR4 mRNA和蛋白质表达相似。结论 HCV NS5A可上调TLR4 mRNA和蛋白的表达。     

HCV NS5A abrogates p53 protein function by interfering with p53-DNA binding

AIM: To evaluate the inhibition effect of HCV NS5A on p53 transactivation on p21 promoter and explore its possible mechanism for influencing p53 function. METHODS: 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 (EMSA). Lipofectin mediated p53 or HCV NS5A expression vectors were used to transfect hepatoma cell lines to observe whether HCV NS5A could abrogate the binding ability of p53 to its specific DNA sequence and p53 transactivation on p21 promoter. Western blot experiment was used for detection of HCV NS5A and p53 proteins expression. RESULTS: 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 NS5A protein gradually inhibited both endogenous and exogenous p53 transactivation on p21 promoter with increase of the dose of HCV NS5A expression plasmid. By the experiment of EMSA, we could find p53 binding to its specific DNA sequence and, when co-transfected with increased dose of HCV NS5A expression vector, the p53 binding 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 the p53 protein expression. CONCLUSION: HCV NS5A inhibits p53 transactivation on p21 promoter through abrogating p53 binding affinity to its specific DNA sequence. It does not affect p53 protein expression.     

Hepatitis C virus NS5A promotes insulin resistance through IRS-1 serine phosphorylation and increased gluconeogenesis

AIM: To investigate the mechanisms of insulin resistance in human hepatoma cells expressing hepatitis C virus(HCV) nonstructural protein 5A(NS5A).METHODS: The human hepatoma cell lines,Huh7 and Huh7.5,were infected with HCV or transientlytransfected with a vector expressing HCV NS5 A. The effect of HCV NS5 A on the status of the critical players involved in insulin signaling was analyzed using realtime quantitative polymerase chain reaction and Western blot assays. Data were analyzed using Graph Pad Prism version 5.0.RESULTS: To investigate the effect of insulin treatment on the players involved in insulin signaling pathway,we analyzed the status of insulin receptor substrate-1(IRS-1) phosphorylation in HCV infected cells or Huh7.5 cells transfected with an HCV NS5 A expression vector. Our results indicated that there was an increased phosphorylation of IRS-1(Ser307) in HCV infected or NS5 A transfected Huh7.5 cells compared to their respective controls. Furthermore,an increased phosphorylation of Akt(Ser473) was observed in HCV infected and NS5 A transfected cells compared to their mock infected cells. In contrast,we observed decreased phosphorylation of Akt Thr308 phosphorylation in HCV NS5 A transfected cells. These results suggest that Huh7.5 cells either infected with HCV or ectopically expressing HCV NS5 A alone have the potential to induce insulin resistance by the phosphorylation of IRS-1 at serine residue(Ser307) followed by decreased phosphorylation of Akt Thr308,Fox01 Ser256 and GSK3β Ser9,the downstream players of the insulin signalingpathway. Furthermore,increased expression of PECK and glucose-6-phosphatase,the molecules involved in gluconeogenesis,in HCV NS5 A transfected cells was observed.CONCLUSION: Taken together,our results suggest the role of HCV NS5 A in the induction of insulin resistance by modulating various cellular targets involved in the insulin signaling pathway.     

Hepatitis C virus nonstructural protein 5A inhibits tumor necrosis factor-alpha-mediated apoptosis in Huh7 cells

To analyze the influence of hepatitis C virus nonstructural protein 5A (NS5A) on apoptosis, we established Huh7 cells that stably express NS5A, and induced apoptosis using tumor necrosis factor (TNF)-alpha. The viability of control Huh7 cells was reduced to 40%, compared with untreated cells, after TNF-alpha treatment, whereas that of Huh7-NS5A cells was reduced only to 80%. DNA fragmentation also decreased to <50% in Huh7-NS5A compared with control cells. Nuclear factor-kappaB activation was the same in both cell types, whereas caspase-8, -9, and -3 activity was decreased in Huh7-NS5A cells, compared with control cells, which indicates that the inhibition of caspase-8 activation is responsible for the antiapoptotic effect of the NS5A protein. The coexpression of NS5A did not inhibit apoptosis induced by caspase-8 or Fas-associating death domain protein expression. These findings suggest that the NS5A protein inhibits the apoptotic effect of TNF-alpha upstream of caspase-8 in the apoptosis cascade.