HCV非结构蛋白5A对HIV长末端重复序列影响的初步探讨  

Preliminary study on effect of hepatitis C virus nonstructural protein 5A on HIV LTR

作　　者：彭米林[1] 肖新强[1] 蒋永芳[1] 田沂[1] 许允[1] 张旻[1] 王文龙[1] 彭锋[1] 龚国忠[1] 

机构地区：[1]中南大学湘雅二医院感染科,长沙410011

出　　处：《临床肝胆病杂志》2014年第2期136-140,共5页Journal of Clinical Hepatology

基　　金：中南大学研究生创新课题(2010ssxt290);湖南省博士生科研创新项目资助(2502-71380100004)

摘　　要：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。ObjectiveTo investigate the effect of nonstructural protein 5A （NS5A） encoded by the human hepatitis C virus （HCV） RNA genome on human immunodeficiency virus （HIV） long terminal repeat （LTR） and to provide an experimental basis for the study on the effect of HCV on HIV. MethodsHepatocellular carcinoma Huh7 cells were divided into blank group, control group, and experimental group to be transfected with plasmid pGL3-LTR-Luc （containing luciferase reporter gene driven by the LTR promoter）, plasmid pRc/CMV plus plasmid pGL3-LTR-Luc, and plasmid pCNS5A （containing HCV NS5A gene） plus plasmid pGL3-LTR-Luc, respectively; Huh7 cells were collected 48 h later. The protein and mRNA expression levels of HCV NS5A were measured by immunocytochemistry, Western blot, and RT-PCR. The relative luciferase activity was measured to evaluate the HIV LTR activity and the effect of HCV NS5A on HIV LTR. The activity values were expressed as mean±SD, and Levene′s test of homogeneity of variance was used; comparison between all groups was made by one-way analysis of variance （ANOVA）, and comparison between two groups was made by least significant difference （LSD） test. ResultsThe mRNA and protein expression of HCV NS5A was detected in the cytoplasm of Huh7 cells in experimental group. The one-way ANOVA showed that there were significant differences in LTR luciferase activity between the three groups （F=7.876, P=0002）. The LSD test showed that the experimental group had a significantly higher relative luciferase activity than the blank group and control group （22476±4471 vs 15887±3039, P=0.002; 22476±4471 vs 16321±4162, P=0.008）. ConclusionHuh7 cells can be transfected with the HCV NS5A expression plasmid （pCNS5A）. HCV NS5A can activate HIV LTR, which suggests that HCV NS5A may be one of the molecular mechanisms of HCV promoting HIV replication.

关 键 词：肝炎病毒属 HIV 病毒非结构蛋白质类 HIV长末端重复序列 

分 类 号：R512.63[医药卫生—内科学]