Development of anti-HCV therapy the regulation of NS5A function

抗HCV疗法的发展NS5A功能的调节

• 批准号: 12557053
• 负责人: ENOMOTO Nobuyuki
• 金额: $ 7.49万
• 依托单位: Tokyo Medical and Dental Uniersity
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12557053/
• 关键词: NS5A; HCV; Interferon; apoptosis; TNF alpha; signal transduction; chronic hepatitis; replicon; C型肝炎; Interferon; apoptosis

Hepatitis C virus (HCV) subgenomic replicon has been reported to replicate efficiently and continuously in human hepatoma Huh-7 cells. To extend the previous results to other isolated HCV clones, we have constructed another HCV replicon from HC-J4, one of the chimpanzee-infectious clones. An HCV-replicon derived from HC-J4 (RpJ4) consists of HCV-5'-UTR, neomycin phosphotransferase gene, the encephalomyocarditis virus IRES, HCV-nonstructural region, NS3 to NS5B, and HCV-3'-UTR. The adaptive mutations known to be required for HCV-Conl replicon were introduced in RpJ4 replicon, aa.(amino acids number according to HC-J4) 2197 serine to proline, deletion of serine at aa.2201, and aa.2204 serine to isoleucine (RpJ4-S2I97P, RpJ4-S22001del, and RpJ4-S2204I). RpJ4/ISDRmutant and RpJ4-S2201del/ISDRmutant were also constructed by introducing six amino acid mutations into the interferon sensitivity determining region (ISDR). Replicon RNA was transfected into Huh-7 cells, and stable replicon-expressing cell lines were established by G418 selection. After transfection to naive Huh-7 cells, RpJ4 and RpJ4/ISDRmutants did not produce any G418-resistant colonies. In contrast, G418-resistant cells were transduced efficiently by RpJ4-S2197P, RpJ4-S2204I, RpJ4-S2201del and RpJ4-S2201del/ISDRmutants, with the RpJ4-S2201del/ISDRmutant being most efficient. In conclusion, The HCV replicon derived from HC-J4 can replicate efficiently following the introduction of adaptive mutations into the upstream region of ISDR. Moreover, additional introduction of mutations into ISDR further enhances its replication. These findings demonstrate that the genetic structure of the NS5A domain is critical in HCV-1b replications.

丙型肝炎病毒（HCV）亚基因组复制子在人肝癌细胞Huh-7中能高效、持续复制。为了将以前的结果扩展到其他分离的HCV克隆，我们从黑猩猩感染性克隆之一HC-J 4构建了另一个HCV复制子。衍生自HC-J 4（RpJ 4）的HCV复制子由HCV-5 '-UTR、新霉素磷酸转移酶基因、脑心肌炎病毒IRES、HCV非结构区、NS 3至NS 5 B和HCV-3'-UTR组成。已知HCV-Conl复制子所需的适应性突变被引入RpJ 4复制子aa中。（根据HC-J 4的氨基酸编号）2197丝氨酸变为脯氨酸，aa. 2201处的丝氨酸缺失，和aa. 2204丝氨酸变为异亮氨酸（RpJ 4-S2 I97 P、RpJ 4-S22001 del和RpJ 4-S2204 I）。在干扰素敏感性决定区（ISDR）引入6个氨基酸突变，构建了RpJ 4/ISDR突变体和RpJ 4-S2201 del/ISDR突变体。将复制子RNA转染Huh-7细胞，通过G418筛选建立稳定表达复制子的细胞系。转染至初始Huh-7细胞后，RpJ 4和RpJ 4/ISDR突变体不产生任何G418抗性集落。相反，RpJ 4-S2197 P、RpJ 4-S2204 I、RpJ 4-S2201 del和RpJ 4-S2201 del/ISDR突变体可有效地转导G418抗性细胞，其中RpJ 4-S2201 del/ISDR突变体的转导效率最高。因此，HC-J 4基因的复制子在ISDR上游区域引入适应性突变后可以有效复制。此外，向ISDR中额外引入突变进一步增强其复制。这些发现表明NS 5A结构域的遗传结构在HCV-1b复制中至关重要。

项目成果

Nagayama K, Enomoto N, Izumi N, Kurosaki M, Miyasaka Y, Watanabe H, Itakura J, Chen C-H, Tazawa J, Ikeda T, Marumo F, Sato C.: "Sequences in the NS5A Protein of Genotype 1b Hepatitis C Virus and the Serum ALT Response to Interferon Therapy in Japanese Pat

Nagayama K、Enomoto N、Izumi N、Kurosaki M、Miyasaka Y、Watanabe H、Itakura J、Chen C-H、Tazawa J、Ikeda T、Marumo F、Sato C.：“基因型 1b 丙型肝炎病毒的 NS5A 蛋白序列和

Watanabe, H, Enomoto, N, et al.: "Number and Position of Mutations in the Interferon Sensitivity-Determining Region of the Gene for Nonstructural Protein 5A Correlates with Interferon Efficacy in Hepatitis C Virus Genotype 1b Infection"J Infect Dis. 183.

Watanabe, H, Enomoto, N 等人：“非结构蛋白 5A 基因干扰素敏感性决定区域中突变的数量和位置与丙型肝炎病毒基因型 1b 感染中的干扰素功效相关”J Infect Dis。

Nagayama, K, Enomoto, N, et al.: "Overexpression of interferon gamma-inducible protein 10 in the liver of patients with type I autoimmune hepatitis identified by suppression subtractive hybridization"Am J Gastroenterol. 96. 2211-2217 (2001)

Nagayama, K, Enomoto, N 等人：“通过抑制消减杂交鉴定出 I 型自身免疫性肝炎患者肝脏中干扰素 γ 诱导蛋白 10 的过度表达”Am J Gastroenterol。

Itakura, J, Enomoto, N, et al.: "CD81 Nucleotide Mutation in Hepatocellular Carcinoma and No CD81 Polymorphism in Patients with Various Stages of Hepatitis C Virus Infection"J Med Virol. 63. 22-28 (2001)

Itakura, J, Enomoto, N 等人：“肝细胞癌中的 CD81 核苷酸突变和丙型肝炎病毒感染各个阶段的患者中无 CD81 多态性”J Med Virol。

Yu S-H, Nagayama K, Enomoto N, Izumi N, Marumo F, Sato C.: "Intrahepatic mRNA expression of interferon-gamma inducible antiviral genes in liver diseases : PKR overexpression and RNase L inhibitor suppression in chronic hepatitis C"Hepatology. 32. 1089-109

Yu S-H、Nagayama K、Enomoto N、Izumi N、Marumo F、Sato C.：“肝病中干扰素-γ诱导型抗病毒基因的肝内 mRNA 表达：慢性丙型肝炎中 PKR 过度表达和 RNase L 抑制剂抑制”肝病学。