HIV Tat、Rev蛋白调节胞浆内DDX3水平对HCV复制影响的研究

Previous studies have shown that HIV co-infected with HCV could be observed in one hepatocyte. Lately, some groups reported that HIV Tat could upregulate the expression of DDX3 in lymphocyntes while HIV Rev could reduce the DDX3 expression in cytoplasm via combining with DDX3. As a working hypothesis we have assumed that HIV Tat -induced DDX3 will accelerate the replication of HCV while HIV Rev-DDX3 complex will reduce HCV replication in HCV/HIV co-infected cells. To test this hypothesis, we focused our research on the following aspects by using JFH-1-infected-Huh-7.5 cells: exploring whether HIV Tat protein could upregulate the expression of DDX3 in JFH-1-infected-Huh-7.5 cells; probing whether HIV Tat-induced DDX3 could accelerate the replication of HCV in JFH-1-infected-Huh-7.5 cells and finding out its potential mechanisms; investigating whether DDX3-Rev-CRM-1 complex could resist the acceleration effect of HIV Tat on HCV replication by inhibiting the formation of DDX3-Core complex competitively and finally providing its underlying mechanisms.

研究证实HCV和HIV可在同一肝细胞内形成共感染，HIV Tat蛋白可以在淋巴细胞内诱导DDX3高表达，DDX3可与HCV Core蛋白结合促进HCV复制，而HIV Rev蛋白可与DDX3结合，在核膜上形成复合体从而降低胞浆内DDX3的水平。可以假设，HCV和HIV共感染形成后，HIV Tat诱导的DDX3高表达可促进HCV复制，而HIV Rev-DDX3复合体形成可竞争性的抑制这种促进作用。我们拟以自主构建的JFH-1全基因组复制子感染Huh-7.5细胞模型为研究对象，验证这一假设，阐明：①HIV Tat蛋白在Huh-7.5细胞内对DDX3表达调控的影响；②HIV Tat诱导Huh-7.5细胞DDX3高表达后对HCV复制的促进作用及关键机制；③DDX3-Rev-CRM-1复合体是否通过竞争的方式抑制DDX3-Core复合体的形成，从而减弱Tat对HCV复制的促进作用及其机制。

已有研究表明HCV和HIV可在同一肝细胞内形成共感染，HIV Tat蛋白可以在淋巴细胞内诱导DDX3高表达,DDX3可与HCV Core蛋白结合促进HCV复制，HIV Rev蛋白可与DDX3结合，在核膜上形成复合体从而降低胞浆内DDX3的水平。本项目在国家自然科学基金的资助下，通过合成分别带有Tat、Rev基因的载体，并经PCR获取，将目的片段连接到真核表达载体pCDNA3.1上，并将带有目的基因Tat、Rev的真核表达载体转染至JFH-1-Huh-7.5细胞模型，并证实表达载体在细胞模型中能稳定表达。正在进行利用DDX3单克隆抗体对细胞胞浆内的DDX3进行捕获的实验，以及DDX3对HCV复制的影响。相关的论文正在撰写当中，目前本项目培养在读硕士研究生一名。本研究完成了相关细胞模型的建立，为接下来Tat、Rev蛋白对DDX3的调控，从而影响HCV复制的研究打下了基础。