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

小鼠体内具有转化活性的丙型肝炎病毒NS3蛋白分析

• 批准号: 14570521
• 负责人: HASUMURA Yasushi
• 金额: $ 1.92万
• 依托单位: KANAZAWA MEDICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570521/
• 关键词: Hepatitis C virus; Nonstructural protein 3 (NS3); Cell transformation; 肝発癌

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.

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。但其作用机制尚不清楚。已知HCV（肝炎病毒）显示与黄病毒相同的结构。关注这一点，我们已经研究了HCV的NS 3结构域的功能。我们发现HCV-NS 3蛋白对小鼠细胞NIH 3 T3显示出肿瘤形成能力（J.Virol.，69：3893，1995）。可以推测宿主蛋白如p53和NS 3的相互作用。我们对此进行了测试，发现在NS 3和p53载体导入的细胞中，细胞增殖能力和裸鼠成瘤能力均显著降低。为了寻找一个与HCV-NS 3相互作用的新蛋白，我们利用HeLa cDNA文库进行酵母双杂交筛选，最终筛选出两个NS 3结合蛋白Sm-D1和SRCAP。Sm-DI是小核核糖核蛋白复合物的组分，SRCAP是与细胞转录活性相关的蛋白质。分别使用构建的Sm-D1和谷胱甘肽S-转移酶融合蛋白表达载体和构建的FLAG标记的Sm-D1表达载体进行Sm-D1和NS 3的体外和体内结合，并显示含有GR重复的Sm-D1的C-末端区域是NS 3的结合区域。此外，Sm-D1的表达特征受NS 3共表达的影响。使用KN 73细胞的免疫染色分析表明，通常位于细胞质中的NS 3蛋白，当NS 3和宿主蛋白都转染到细胞中时，在细胞核中可检测到。与Sm-D1共表达的NS 3蛋白核移位量大于与SRCAP共表达。这些结果表明，宿主蛋白，特别是核蛋白可能与HCV-NS 3蛋白结合，并可能与HCV-NS 3的细胞转化机制密切相关。

项目成果

Atsushi Iwai: "Hepatitis C Virus Nonstructural Protein NS3 Binds to Sm-D1, a Small Nuclear Ribonucleoprotein Associated with Autoimmune Disease"Microbiol.Immunol.. 47・8. 601-611 (2003)

Atsushi Iwai：“丙型肝炎病毒非结构蛋白 NS3 与与自身免疫性疾病相关的小核核糖核蛋白 Sm-D1 结合”Microbiol.Immunol.. 47・8 (2003)。

A.IWAI, Y.HASUMURA, T.NOJIMA, T.TAKEGAMI: "Hepatitis C virus nonstructural protein NS3 binds to SM-D1, a small nuclear ribonucleoprotein associated with autoimmune disease"Microbiol. Iuununol.. vol 47(8). 601-611 (2003)

A.IWAI、Y.HASUMURA​​、T.NOJIMA、T.TAKEGAMI：“丙型肝炎病毒非结构蛋白 NS3 与 SM-D1 结合，SM-D1 是一种与自身免疫性疾病相关的小核核糖核蛋白”Microbiol。

Atsushi Iwai: "Hepatitis C Virus Nonstructural Protein NS3 Binds to Sm-D1, a Small Nuclear Ribonucleoprotein Associated with Autoimmune Disease"Microbiol.Immunol.. 47. 601-611 (2003)

Atsushi Iwai：“丙型肝炎病毒非结构蛋白 NS3 与 Sm-D1（一种与自身免疫性疾病相关的小核核糖核蛋白）结合”Microbiol.Immunol.. 47. 601-611 (2003)