Analyzing the role of NS5A protein in regulation of interferon sensitivity using HCV replicon system.

丙型肝炎病毒非结构5A基因(NS5A)干扰素敏感性决定区(ISDR)的氨基酸替换数目与干扰素应答和病毒载量密切相关。一些基于丙型肝炎病毒复制子的研究已经报道了ISDR序列对病毒体外复制的影响。然而，目前还不清楚ISDR序列的多样性如何影响丙型肝炎病毒的复制。将临床上观察到的各种ISDR序列引入到丙型肝炎病毒复制子中，并通过克隆形成试验和/或瞬时复制试验来研究它们对病毒复制的贡献。对ISDR进行了定位研究，以确定对复制有重要影响的氨基酸位置。虽然在使用ISDR中有很少突变(0、1和3)的丙型肝炎病毒复制子的克隆形成试验中没有形成克隆，但使用具有6个突变的构建体出现了大量的克隆(&gt；200)。用多个…介绍各种不同的ISDR序列在瞬时检测中，e突变较多会导致复制增强。一项图谱研究确定了ISDR中几个严重改变复制的特定位点，包括2209密码子，它与对干扰素有强烈反应的患者密切相关。与临床干扰素应答和病毒载量相关的ISDR序列调节了丙型肝炎病毒复制子的复制，暗示了ISDR序列在丙型肝炎病毒感染中的重要性。接下来，我们研究了支持亚基因组丙型肝炎病毒复制的细胞(Huh7/Rep)中ISGs的表达谱，并筛选了它们抑制丙型肝炎病毒复制的活性。实时定量PCR分析显示，由于干扰素刺激反应元件(ISRE)的转录抑制，23个ISGs在Huh7/Rep细胞中的表达水平显著低于未感染的Huh7细胞。此外，在去除复制子的治愈Huh7细胞(CHuh7)中，ISGs的表达水平也降低，表明细胞通过下调ISGs来支持丙型肝炎病毒的复制。另一方面，包括PKR、MXA、IRF-9、GBP-1、IFI-6-16、IFI-27、25OAS和IRF-1在内的几个ISG的过表达显著抑制了丙型肝炎病毒复制子的表达。短发夹状RNA下调GBP-1、IFI-6-16和IFI-27基因，导致丙型肝炎病毒复制增加。较少

The number of amino acid substitutions in the interferon sensitivity-determining region (ISDR) in the nonstructural 5A (NS5A) gene of hepatitis C virus (HCV) is closely associated with the interferon response and viral load. Several HCV replicon-based studies have reported that ISDR sequences had an influence on viral replication in vitro. However, it is unclear how the variety of ISDR sequences affect HCV replication. Various clinically observed ISDR sequences were introduced into HCV replicons, and their contribution to viral replication was investigated using a colony formation assay and/or a transient replication assay. A mapping study of the ISDR was performed to identify the amino acid positions that critically affect replication. While no colonies were formed in colony formation assay using HCV replicons with few mutations (0, 1, and 3) in the ISDR, numerous colonies (>200) appeared using constructs with six mutations. Introduction of various distinct ISDR sequences with multipl … More e mutations resulted in replication enhancement in transient assays. A mapping study identified several specific sites in the ISDR that critically altered replication, including codon 2209 which was closely associated in patients with a strong response to IFN. ISDR sequences associated with a clinical interferon response and viral load modulated the replication of HCV replicons, suggesting the importance of the ISDR sequence in HCV infection.Next, we studied expression profiles of ISGs in cells supporting subgenomic HCV replication (Huh7/Rep), and screened their activities to suppress HCV replication. Real-time PCR analyses showed that the expression levels of 23 ISGs were significantly lower in Huh7/Rep than naive Huh7 cells due to transcriptional suppression of the interferon-stimulated response element (ISRE). Furthermore, the expression level of ISGs was also decreased in the cured Huh7 cells in which replicon had been eliminated (cHuh7), indicating adaptation of the cells to support HCV replication by downregulating ISGs. On the other hand, expression of HCV replicon was significantly suppressed by overexpression of several ISGs including PKR, MxA, IRF-9, GBP-1, IFI-6-16, IFI-27, 25OAS and IRF-1. Knock down of GBP-1, IFI-6-16 and IFI-27 by short hairpin RNA resulted in increase of HCV replication. Less