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

在日本，丙型肝炎病毒（HCV）的持续感染与肝细胞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HCV（肝炎病毒）具有与黄病毒相同的结构。注意到这一点，我们研究了HCV的NS3结构域的功能。我们发现HCV-NS3蛋白对小鼠细胞NIH3T3具有成瘤能力（J.Virol）。69:3893, 1995)。可以推测宿主蛋白如p53和NS3的相互作用。我们对此进行了测试，发现在NS3和p53载体引入的细胞中，裸鼠的细胞增殖能力和肿瘤形成能力都明显下降。为了寻找能与HCV-NS3相互作用的新宿主蛋白，我们利用HeLa cDNA文库进行酵母双杂交筛选，最终筛选出两个ns3结合蛋白Sm-D1和SRCAP。Sm-DI是小核核糖-核蛋白复合物的组成部分，而SRCAP是一种与细胞转录活性相关的蛋白质。利用构建的Sm-D1和谷胱甘肽s -转移酶融合蛋白表达载体和构建的flag标记Sm-D1表达载体分别对Sm-D1和NS3进行体外和体内结合，发现Sm-D1含有GR重复序列的c端区域是NS3的结合区。此外，NS3的共表达影响Sm-D1的表达特征。利用KN73细胞进行免疫染色实验，发现通常定位于细胞质中的NS3蛋白与宿主蛋白同时转染后，在细胞核中可检测到。NS3蛋白与Sm-D1共表达时核位移大于与SRCAP共表达时。这些结果表明，宿主蛋白，特别是核蛋白可以与HCV-NS3连接，并可能与HCV-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.