结核分枝杆菌Rv2645刺激ISG15的表达上升促进体外丙型肝炎病毒的增殖

Rv2645 from Mycobacterium Tuberculosis RD13 Region Promotes ISG15 Expression and Overexpression of ISG15 Enhances HCV Replication in Vitro

为研究结核分枝杆菌(Mycobacterium tuberculosis,M.tb)与丙型肝炎病毒(Hepatitis C virus,HCV)感染之间的相关关系,本文研究毒性毒株M.tb H37Rv的RD13区中的Rv2645基因对免疫细胞中干扰素刺激基因(Interferonstimulated gene 15,ISG15)表达的影响,进而探讨ISG15的表达与HCV感染之间的相关性。利用携带Rv2645基因的无毒牛结核分枝杆菌卡介苗(Bacille calmette guerin,BCG)(即rBCG::Rv2645,rBCG)与亲本BCG分别感染小鼠或刺激小鼠巨噬细胞系RAW264.7,运用RT-qPCR以及蛋白印迹的方法检测,比较rBCG与BCG感染对小鼠CD4+T细胞及RAW264.7细胞中ISG15的mRNA及蛋白质表达的影响。同时克隆ISG15真核表达质粒和ISG15的沉默表达质粒-ISG15-shRNA,运用RT-qPCR及蛋白印迹分别检测ISG15过表达及沉默后,对HCV感染的人肝癌细胞系Huh7.5.1中HCV的mRNA、HCV非结构蛋白NS3及核心蛋白Core的表达。我们发现相对于BCG,携带Rv2645的BCG感染之后脾脏CD4+T细胞及体外RAW264.7中ISG15的mRNA及蛋白质的水平明显升高。此外,ISG15过表达导致Huh7.5.1中HCV的mRNA、NS3蛋白及Core蛋白的水平明显升高,而ISG15沉默后ISG15和HCV的mRNA的水平明显下调。本研究首次发现M.tb Rv2645能上调小鼠CD4+T细胞及巨噬细胞中ISG15的表达;而ISG15能促进Huh7.5.1中HCV增殖。本研究对阐明M.tb毒性菌株H37Rv的Rv2645基因的功能,以及为研究MTB感染与HCV感染的分子机制提供参考依据。

To study the relationship between Mycobacterium tuberculosis （M. tb） and hepatitis C virus （HCV） infection, we investigated the effect of the Rv2645 gene in the RD13 region of M. tb H37Rv on interferon-stimulated gene 15 （ISG15） expression in immune cells. Then, the relationship between ISG15 and HCV infection in Huh7.5.1 cell was investigated. Non-virulent bovine tuberculosis Bacillus Calmette Guerin（BCG）carrying the Rv2645 gene （BCG： .Rv2645, rBCG） and BCG （without Rv2645） were used to infect Balb/C mice and the mouse macrophage cell line RAW264.7, respectively. Expressions of ISG15 mRNA and protein in murine splenic CD4^＋T cell and RAW264.7 cells was detected by real-time quantitative polymerase chain reaction （RT-qPCR） and Western blotting （WB）. Furthermore, an ISG15 eukaryotic expression plasmid and ISG15-shRNA（short hairpin RNA） were constructed and transfected into HCV- infected Huh7.5.1 cells. Then the expressions of HCV mRNA, viral NS3 and Core protein were detected by RT-qPCR and WB, respectively. Results showed that the mRNA and protein levels of ISG15 in the mu- rine splenic CD4＋ T cells and RAW264. 7 were increased significantly after stimulated with rBCG.： Rv2645, compared with BCG. Furthermore, levels of HCV mRNA, NS3 and Core protein levels were increased remarkably in HCV-infected Huh7.5.1 cells in the context of ISG15 overexpression compared with the empty vector, ISG15 and HCV mRNA levels were down-regulated significantly after knock-down of ISG15. Rv2645 from M. tb H37Rv RDI3 region promotes the expression of ISG15, and overexpression of ISG15 enhances HCV replication in Huh7.5.1 cell. This study provides a basis for elucidating the function of Rv2645 in virulent M. tb H37Rv and provides an insight into the molecular mechanism of M. tb and HCV infections.