Mechanism of interferon resistance of hepatitis C virus RNA

丙型肝炎病毒RNA干扰素抵抗机制

• 批准号: 09670585
• 负责人: DATE Takayasu
• 金额: $ 2.24万
• 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: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670585/
• 关键词: hepatitis C virus (HCV); interferon; core protein; NS5A; double-stranded RNA (dsRNA); dsRNA-activated protein kinase (PKR); 二本鎖RNA依存性プロテインキナーゼ(PKR); ゲルシフトアッセイ; two hybrid system; ISDR; GST融合タンパク質

Hepatitis C virus (HCV) core protein constitutes a viral nucleocapsid and possesses multiple functions. Some of functions may be mediated by the direct interaction with RNA.In order to clarify the interaction between the core protein and nucleic acids, we showed the property of glutathione S-transferase (GST)-core protein by activity gel in which protein is immobilized. It has been very difficult to electrophoretic mobility shift assay (EMSA) using the core protein due to extremely basic character. Therefore, the plasmid was constructed, encoding neutral fusion protein composed of GST, 47 amino acids-carboxyl terminal region of HCV nonstructural 5A protein (NS5Ac) which is acidic, and the core protein between amino acids 1 and 90 (C<@D21-90@>D2). The recombinant protein named GST-NS5Ac-C<@D21-90@>D2 was purified with homogeneity and used for biochemical analysis including EMSA.When 24-mer oligonucleotide corresponding to (CU<@D1n@>D1) repeat region of 3' terminal region of HCV was used … More as a probe, EMSA revealed that the GST NS5Ac-C<@D21-90@>D2 bound strongly to double-stranded RNA (dsRNA) but not to single-stranded RNA (ssRNA). The GST-NS5Ac-C<@D21-90@>D2 protein also strongly bound to dsDNA and DNA/RNA heteroduplex. Competition assays indicated that the order of affinity was dsRNA(]SY.di-substituted left.[) dsDNA (]SY.di-substituted left.[)(]SY.di-substituted left.[) ssRNA or ssDNA.The GST-NS5Ac-C<@D21-90@>D2 protein efficiently retarded some ssRNAs such as 5' non-coding region of HCV, and RNA including 5' portion of the HCV core gene although the affinity for ssRNA was lower than that for corresponding dsRNA, suggesting that the binding of the core protein to ssRNA is sequence-dependent manner, while the binding of the core to dsRNA is sequence-independent manner. The binding affinity of GST-NS5Ac-C<@D21-90@>D2 for dsRNA was comparable to that of interferon-inducible dsRNA dependent protein kinase (PKR) in vitro, and the equilibrium constant between GST-NS5Ac-C<@D21-90@>D2 and dsRNA was estimated about 1.5 * 10<@D1-8@>D1 M.When the PKR was incubated with the core fusion protein, PKR activity was inhibited depending on the concentration of GST-NS5Ac-C<@D21-90@>D2 protein but not GST-NS5Ac. These results suggest that the core protein potentiates to inhibit the PKR activity. Together with recent finding of the NS5A function, we propose the hypothesis that two HCV gene products, the core protein and NS5A protein, act in a synergistic manner and inhibit PKR activity to preclude the shutoff of protein synthesis. Less

丙型肝炎病毒（HCV）核心蛋白是病毒的核衣壳蛋白，具有多种功能。为了阐明核心蛋白与核酸之间的相互作用，我们采用固定化蛋白的活性凝胶法，研究了谷胱甘肽S-转移酶（GST）-核心蛋白的性质。由于核心蛋白的碱性很强，用其进行电泳迁移率变动分析（EMSA）非常困难。因此，构建了编码由GST、HCV非结构5A蛋白的47个氨基酸的羧基端区域（NS 5Ac）（其为酸性的）和氨基酸1至90之间的核心蛋白（C<@D21-90@>D2）组成的中性融合蛋白的质粒。将重组蛋白GST-NS 5Ac-C <@D21-90@>D2纯化后，用24-mer寡核苷酸（对应于HCV 3'端（CU<@D1n@>D1）重复区）进行电泳分析，结果表明，重组蛋白GST-NS 5Ac-C <@D21-90 @>D2具有良好的生物学活性 ...更多信息 以GST NS 5Ac-C <@D21-90@>D2为探针，EMSA分析表明GST NS 5Ac-C <@D21-90@>D2与双链RNA（dsRNA）结合较强，而与单链RNA（ssRNA）结合较弱。GST-NS 5Ac-C <@D21-90@>D2蛋白与dsDNA和DNA/RNA异源双链体也有较强的结合。竞争实验表明，双链RNA的亲和力大小顺序为：（1）SY. [）dsDNA（]SY. [）（]SY.di-substituted left. GST-NS 5Ac-C <@D21-90@>D2蛋白有效地阻滞了一些ssRNA，如HCV的5'非编码区，以及包括HCV核心基因5'部分的RNA，尽管对ssRNA的亲和力低于对相应dsRNA的亲和力，这表明核心蛋白与ssRNA的结合是序列依赖性的，而核心蛋白与dsRNA的结合是不依赖于序列的。GST-NS 5Ac-C <@D21-90@>D2与dsRNA的体外结合亲和力与干扰素诱导的dsRNA依赖性蛋白激酶（PKR）相当，并且GST-NS 5Ac-C <@D21-90@>D2与dsRNA之间的平衡常数估计为1.5 × 10<@D1-8@> D 1 M。GST-NS 5Ac-C <@D21-90@>D2蛋白对PKR活性有抑制作用，但对PKR活性无影响。这些结果表明，核心蛋白增强抑制PKR活性。结合最近发现的NS 5A功能，我们提出了两个HCV基因产物，核心蛋白和NS 5A蛋白，以协同的方式发挥作用，抑制PKR活性，以阻止蛋白质合成的关闭的假设。少