丙型肝炎病毒5'端非编码区结构域Ⅰ、Ⅱ在其翻译启动活性中的作用具有细胞特异性  

Cell-specific roles of Domains Ⅰ and Ⅱ of HCV 5'untranslated region in the translation initiation activity

作　　者：黄小晔[1] 刘丽莎[1] 崔光晶 刘西霞[1] 刘美佟[1] 马琼山[1] 刘水平[1] 

机构地区：[1]中南大学湘雅医学院医学微生物学系,湖南长沙410078

出　　处：《南方医科大学学报》2014年第12期1826-1829,共4页Journal of Southern Medical University

基　　金：湖南省科技计划项目(2014SK3248);中南大学中央高校基本科研业务费专项资金

摘　　要：目的分析在不同宿主细胞的翻译体系下,缺失不同片段的HCV 5'UTR翻译启动活性的差异。方法以脂质体介导基因转染技术,将截短型HCV 5'UTR调控Fluc的真核表达质粒与Rluc真核表达质粒p RL-TK共转染至不同的细胞中,转染后36 h:6提取细胞RNA,半定量RT-PCR检测目的质粒的转录水平;6用双荧光素酶报告基因检测系统检测Fluc基因相对表达活性,分析HCV 5'UTR缺失不同结构域后在不同翻译体系中翻译启动活性的差异。结果 6缺失5'端44个碱基的HCV 5'UTR的翻译启动活性分别与缺失前相比:在He La细胞和C6细胞中无明显影响,在L-02细胞中活性下降为46%,而在293T细胞则为缺失前的146%;6缺失5'端118个碱基后,HCV 5'UTR的活性分别与缺失前相比:在He La细胞中,缺失后活性仅为缺失前的49%,而在L-02细胞、C6细胞和293T细胞中,活性分别为缺失前的140%、160%和235%。在本研究使用的四种细胞中,p CNl的翻译启动活性的差异无统计学意义,p CNl-d2在293T细胞中活性最高,在L-02细胞中活性最低。p CNl-d3在293T、C6和L-2细胞中活性相近,但在He La细胞中的活性明显比其他细胞低。结论 HCV 5'UTR的DomainⅠ和DomainⅡ对其翻译启动活性的影响与宿主细胞种类相关,具细胞特异性。Objective To investigate the roles of Domain I and Domain II of hepatitis C virus （HCV） 5＇ untranslated region （UTR） in the translation initiation activity of HCV 5＇UTR in different host cell lines. Methods The eukaryotic expression plasmid pCMVNCRLuc （pCN1）, in which full-length HCV 5＇UTR regulates firefly luciferase expression, was modified by deleting Domain I and the downstream single-stranded sequence （43 bp in total） from the UTR （pCNl-d2）, Domain I with the downstream single-stranded sequence and Domain II （118 bp in total） from the UTR （pCNl-d3）, or the total UTR （pCNl-d5）. The modified plasmids were transfected via liposome into different cell lines with pRL-TK plasmid co-transfected as the normalization control. At 36 h after the transfection, the total cellular RNA was harvested for semi-quantitative RT-PCR, and the relative expression activities of luciferase were assayed with a dual luciferase reporter gene assay system. The translation initiation activities of the truncated HCV 5＇UTRs in different translation systems were analyzed. Results Deletion of Domain I and the downstream single-stranded sequence caused no significant changes of the translational activity of HCV 5＇UTR in Hela or C6 cells, but decreased the translational activity by 46%in L-02 cells and increased the translational activity by 46%in 293T cells. Deletion of both Domain I and Domain II resulted in decreased translational activity of HCV 5＇UTR by 51%in HeLa cells, but increased the translational activity by 40%in L-02 cells, 60%in C6 cells and 135%in 293T cells. Conclusions Domain I and Domain II of HCV 5＇UTR perform cell type-specific roles in HCV IRES-driven translation initiation.

关 键 词：丙型肝炎病毒 5'端非编码区 翻译启动活性 细胞特异性 

分 类 号：R373.21[医药卫生—病原生物学]