Analysis of hepatitis C virus NS3 protein having transforming activity in mice

在日本，丙型肝炎病毒(丙型肝炎病毒)的持续感染与肝癌之间的密切关系是众所周知的。然而，其机制尚不清楚。已知的丙型肝炎病毒与黄病毒具有相同的结构。考虑到这一点，我们研究了丙型肝炎病毒NS3结构域的功能。我们还发现，丙型肝炎病毒NS3蛋白对小鼠细胞NIH3T3具有致瘤作用(J.Virol，69：3893,1995)。可以推测宿主蛋白如P53和NS3之间的相互作用。我们对此进行了测试，发现在NS3和P53载体导入的细胞中，裸鼠的细胞增殖能力和成瘤能力都显著降低。为了筛选出能与丙型肝炎病毒NS3相互作用的新宿主蛋白，我们利用HeLa文库进行了酵母双杂交筛选，最终筛选出两个NS3结合蛋白Sm-D1和SRCAP。SM-DI是小核核糖核蛋白复合体的组成部分，SRCAP是一种与细胞转录活性相关的蛋白质。分别用构建的Sm-D1和谷胱甘肽S-转移酶融合蛋白表达载体和标记标记的Sm-D1表达载体在体外和体内与Sm-D1和NS3结合，结果表明，含有GR重复序列的Sm-D1的C末端区域是NS3的结合区。此外，Sm-D1的表达特征受NS3共表达的影响。对KN73细胞的免疫染色分析表明，NS3蛋白和宿主蛋白均可在细胞核中检测到，NS3蛋白通常定位于细胞质中。Sm-D1与SRCAP共表达时，NS3蛋白的核移位明显大于SRCAP。这些结果表明，宿主蛋白，特别是核蛋白可能与丙型肝炎病毒NS3结合，并可能与丙型肝炎病毒NS3的细胞转化机制密切相关。

In Japan, a close relation between continued infection of hepatitis C virus (HCV) and liver cell cancer is wellknown. However, its mechanism is not clear. HCV (Hepacivirus) is known to show the structure same as Flavivirus. Paying an attention to this point, we have examined the function of a NS3 domain of HCV. And we discovered that HCV-NS3 protein showed a tumor formation ability for mouse cell NIH3T3 (J.Virol.,69:3893, 1995). An interaction of the host protein such as p53 and NS3 can be speculated. We tested this and found that in the NS3 and p53 vector introduced cells, both the cell-increase ability and the ability of the tumor formation in nude mouse were significantly decreased. To identify a new host protein that could interact with HCV-NS3, we performed a yeast two-hybrid screening using a HeLa cDNA library, and finally selected two NS3-binding proteins, Sm-D1 and SRCAP. Sm-DI is a component of small nuclear ribonucleoprotein complexes, and SRCAP is a protein relating to cell transcription activity. Both in vitro and in vivo bindings of Sm-D1 and NS3 were conducted using a constructed Sm-D1 and glutathione S-transferase fusion protein expression vector and a constructed FLAG-tagged Sm-D1 expression vector, respectively, and revealed that the C-terminal region of Sm-D1 containing the GR repeats was the binding region for NS3. In addition, the expression feature of Sm-D1 was affected by co-expression of NS3. Immunostaining assay using KN73 cells demonstrated that NS3 protein, which is usually localized in the cytoplasm of cells, was detectable in the nucleus when both NS3 and the host proteins were transfected into the cells. The nuclear shift of NS3 protein was greater in co-expression with Sm-D1 than that with SRCAP. These results suggest that the host proteins, especially nuclear proteins could connect with HCV-NS3 and may correlate closely with the cell transformation mechanism of HCV-NS3.