Analysis of anti-interferon mechanism by HCV NS5A protein using HCV replicon system

利用HCV复制子系统分析HCV NS5A蛋白抗干扰素机制

• 批准号: 14370175
• 负责人: ENOMOTO Nobuyuki
• 金额: $ 8.83万
• 依托单位: University of Yamanashi (2004)Tokyo Medical and Dental University (2002-2003)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370175/
• 关键词: Hepatitis C Virus; NS5A protein; IRF-1; innate immunity; replicon; interferon; レプリコン・システム; interferon regulatory factor 1; interferon sensitivity determining region; interferon regulatory factor 3; 分子標的治療; 抗ウイルス療法; NS5A領域; HCV; replicon; in

Hepatitis C virus (HCV) subgenomic replicon has been reported to replicate efficiently and continuously in human hepatoma Huh-7 cells. We have extended these results to other isolated HCV clones, and we have constructed another HCV replicon from HC-J4, one of the chimpanzee-infectious clones. An HCV replieon (RpJ4) was constructed from a chimpanzee-infectious clone, HC-J4, which consists of HCV-5'-UTR, neomycin phosphotransferase gene, the encephalomyocarditis virus IRES, HCV-non-structural region, NS3 to NS5B, and HCV-3'-UTR. The adaptive mutations known to be required for HCV-Con1 replicon were introduced in RpJ4 replicon, aa.(amino acids number according to HC-J4) 2197 serine to praline, aa.2201 serine to deletion, and aa.2204 serine to isoleucine (RpJ4-S2197P, 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). Replieon RNA was transfec … More ted 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 the introduction of RpJ4-S2197P, RpJ4-S2204I, RpJ4-S2201del and RpJ4-S2201del/ISDRmutants, with the RpJ4-S2201del/ISDR mutant being most efficient. The HCV replioon 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, for both the HCV-Con1 and the HC-J4 replicons.Cellular antiviral responses are mediated partly by the expression of interferon-stimulated genes, triggered by viral genomes, their transcripts and replicative intermediates. Persistent replication of a hepatitis C virus (HCV) replicon suggests that the replicon does not elicit cellular innate antiviral responses. In the present study, we investigated regulatory factors of the IFN-mediated antiviral system in cells expressing an HCV replieon. Luciferase reporter assays revealed that the baseline activity of the interferon-stimulated response element (ISRE) was significantly lower in cells harboring the replicon than in naive cells. Among the proteins involved in the IFN/Jak/STAT pathway and in ISRE activity, the expression level of interferon regulatory factor-1 (IRF-1) was found to be significantly lower in cells harboring the replicon. Transfection of an IRF-1 expression construct into cells harboring the replieon caused an increase of ISRE activity, accompanied by suppression of expression of the HCV replieon. Moreover in cured Huh7 cells, from which the HCV replicon had been eliminated, expression levels of IRF-1 and ISRE activity also were suppressed, demonstrating that the decrease of IRF-1 is attributable, not to active suppression by the viral proteins, but to adaptation of cells that enables replication of the HCV subgenome. The high permissiveness of the cured cells for the replicon was abolished by transgenic supplementation of IRF-1 expression. Taken together, IRF-1 is one of the key host factors that regulate intracellular HCV replication through modulation of ISG-mediated antiviral responses. Less

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。我们将这些结果扩展到其他分离的HCV克隆，并从黑猩猩感染性克隆之一HC-J4构建了另一个HCV复制子。从黑猩猩感染性克隆HC-J4中构建了HCV复制子（RpJ4），其由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、NS3至NS5B和HCV-3'-UTR组成。已知HCV-Con1复制子所需的适应性突变被引入RpJ4复制子，aa。（根据HC-J4的氨基酸编号）2197丝氨酸变为脯氨酸，aa.2201丝氨酸变为缺失，和aa.2204丝氨酸变为异亮氨酸（RpJ4-S2197 P、RpJ4-S22001 del和RpJ4-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RpJ4/ISDR突变体和RpJ4-S2201del/ISDR突变体。将外源RNA转移到 ...更多信息 转染Huh-7细胞，经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，RpJ4和RpJ4/ISDR突变体不产生任何G418抗性集落。相反，通过引入RpJ4-S2197P、RpJ4-S2204I、RpJ4-S2201del和RpJ4-S2201del/ISDR突变体，G418抗性细胞被有效地转导，其中RpJ4-S2201del/ISDR突变体是最有效的。在ISDR上游区域引入适应性突变后，HC-J4衍生的HCV复制子可以有效地复制。此外，在ISDR中额外引入突变进一步增强其复制。这些发现表明NS5A结构域的遗传结构在HCV-1b复制中是至关重要的，对于HCV-Con1和HC-J4复制子来说，细胞抗病毒反应部分由干扰素刺激的基因的表达介导，由病毒基因组、其转录物和复制中间体触发。丙型肝炎病毒（HCV）复制子的持续复制表明，复制子不引起细胞的先天性抗病毒反应。在本研究中，我们研究了表达HCV复制子的细胞中IFN介导的抗病毒系统的调节因子。荧光素酶报告基因分析显示，干扰素刺激的反应元件（ISRE）的基线活动是显着低于在幼稚细胞中的细胞窝藏的复制子。在参与IFN/Jak/STAT途径和ISRE活性的蛋白质中，发现干扰素调节因子-1（IRF-1）的表达水平在携带复制子的细胞中显著较低。将IRF-1表达构建体转染到含有复制子的细胞中引起ISRE活性的增加，伴随着HCV复制子表达的抑制。此外，在治愈的Huh7细胞中，HCV复制子已被消除，IRF-1的表达水平和ISRE活性也受到抑制，这表明IRF-1的减少不是由于病毒蛋白的主动抑制，而是由于能够复制HCV亚基因组的细胞的适应。通过转基因补充IRF-1表达，消除了治愈细胞对复制子的高容许性。总之，IRF-1是通过调节ISG介导的抗病毒应答来调节细胞内HCV复制的关键宿主因子之一。少

项目成果

Sakamoto N, Yokota T, et al.: "Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived siRNAs"EMBO Roports. 4. 602-608 (2003)

Sakamoto N、Yokota T 等人：“通过合成和载体衍生的 siRNA 抑制细胞内丙型肝炎病毒复制”EMBO Roports。

Maekawa S, Sakamoto N, et al.: "Introduction of NS5A Mutations Enables Subgenomic HCV-Replicon Derived from Chmpanzee-Infectious HC-J4 Isolate to Replicate Efficiently in Huh-7 Cells"J Viral Hepatitis. in press. (2004)

Maekawa S、Sakamoto N 等人：“NS5A 突变的引入使得源自黑猩猩感染性 HC-J4 分离株的亚基因组 HCV 复制子能够在 Huh-7 细胞中有效复制”J 病毒性肝炎。

Sakamoto N, et al.: "Synergistic inhibition of intracellular hepatitis C virus replication by combination of ribavirin and interferon-alpha"J Infect Dis. in press. (2004)

Sakamoto N 等人：“利巴韦林和干扰素-α 组合对细胞内丙型肝炎病毒复制的协同抑制”J Infect Dis。

Changes of HCV quasispecies during combination therapy with interferon and ribavirin

• DOI: 10.1016/j.hepres.2004.02.014
• 发表时间: 2004-06-01
• 期刊: HEPATOLOGY RESEARCH
• 影响因子: 4.2
• 作者: Ueda, E;Enomoto, N;Watanabe, M
• 通讯作者: Watanabe, M

Introduction of NS5A mutations enables subgenomic HCV replicon derived from chimpanzee‐infectious HC‐J4 isolate to replicate efficiently in Huh‐7 cells

• DOI: 10.1111/j.1365-2893.2004.00525.x
• 发表时间: 2004-09
• 期刊: Journal of Viral Hepatitis
• 影响因子: 2.5
• 作者: S. Maekawa;N. Enomoto;N. Sakamoto;M. Kurosaki;E. Ueda;T. Kohashi;H. Watanabe;C.‐H. Chen;T. Yamashiro;Y. Tanabe;N. Kanazawa;M. Nakagawa;C. Sato;M. Watanabe