Mechanism of interferon resistance of hepatitis C virus RNA

丙型肝炎病毒核心蛋白是一种病毒核衣壳，具有多种功能。一些功能可能是通过与RNA的直接相互作用来实现的。为了阐明核心蛋白与核酸之间的相互作用，我们用固定蛋白的活性凝胶展示了谷胱甘肽S转移酶核心蛋白的性质。由于核心蛋白具有极其基本的性质，利用其进行凝胶迁移率改变分析(EMSA)一直是一项非常困难的工作。因此，构建了编码丙型肝炎病毒非结构5A蛋白(NS5Ac)的中性融合蛋白(C&lt；@D21-90@&gt；D2)。纯化的重组蛋白命名为GST-NS5Ac-C&lt；@D21-90@&gt；D2，用于包括EMSA在内的生化分析。当使用与丙型肝炎病毒3‘末端重复区(CU&lt；@D1n@&gt；d1)相对应的24聚体寡核苷酸进行…作为探针，EMSA显示GST NS5Ac-C&lt；@D21-90@&gt；D2与双链RNA(DsRNA)强烈结合，而不与单链RNA(SsRNA)结合。GST-NS5Ac-C&lt；@D21-90@&gt；D2蛋白也与dsDNA和DNA/RNA异源双链强烈结合。竞争分析表明，亲和力大小顺序为：dsRNA()SY.di-取代左.[)dsDNA(]SY.di-取代左.[)(]SY.di-取代左.[)ssRNA或ssDNA。D2蛋白有效地抑制了一些单链RNA，如丙型肝炎病毒5‘非编码区，以及包括丙型肝炎病毒核心基因5’端部分的RNA，尽管与单链RNA的亲和力低于与相应双链RNA的亲和力，表明核心蛋白与单链RNA的结合是序列依赖的，而核心与双链RNA的结合是非序列依赖的。GST-NS5Ac-C&lt；@D21-90@&gt；D2与dsRNA的结合亲和力与干扰素诱导的dsRNA依赖蛋白激酶(PKR)在体外的亲和力相当，GST-NS5Ac-C&lt；@D21-90@&gt；D2与dsRNA的平衡常数约为1.5×10&lt；@d1-8@&gt；D1M。当PKR与核心融合蛋白孵育时，PKR活性受到抑制，这取决于GST-NS5Ac-C&lt；@D21-90@&gt；2蛋白的浓度，而不是GST-NS5Ac。这些结果表明，核心蛋白增强了抑制PKR活性。结合最近对NS5A功能的发现，我们提出了两个丙型肝炎病毒基因产物，即核心蛋白和NS5A蛋白，以协同方式发挥作用，抑制PKR活性，从而阻止蛋白质合成的假说。较少

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