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

丙型肝炎病毒（HCV）NS 5A基因干扰素敏感性决定区（ISDR）的氨基酸替换数与干扰素应答和病毒载量密切相关。一些基于HCV复制子的研究已经报道ISDR序列对体外病毒复制有影响。然而，目前还不清楚ISDR序列的多样性如何影响HCV复制。将各种临床观察到的ISDR序列引入HCV复制子中，并使用集落形成测定和/或瞬时复制测定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。虽然在集落形成测定中使用在ISDR中具有很少突变（0、1和3）的HCV复制子没有形成集落，但是使用具有六个突变的构建体出现了大量集落（>200）。用多个引物引入各种不同的ISDR序列 ...更多信息 E突变导致瞬时测定中的复制增强。一项定位研究确定了ISDR中几个严重改变复制的特定位点，包括密码子2209，该位点与对IFN有强烈反应的患者密切相关。ISDR序列与临床干扰素应答和病毒载量相关，可调节HCV复制子的复制，表明ISDR序列在HCV感染中的重要性。接下来，我们研究了支持亚基因组HCV复制的细胞（Huh 7/Rep）中ISG的表达谱，并筛选了它们抑制HCV复制的活性。实时PCR分析表明，23 ISGs的表达水平显着低于Huh 7/Rep比幼稚Huh 7细胞由于干扰素刺激的反应元件（ISRE）的转录抑制。此外，在已消除复制子的治愈Huh 7细胞（cHuh 7）中，ISG的表达水平也降低，表明细胞通过下调ISG来支持HCV复制。另一方面，HCV复制子的表达被包括PKR、MxA、IRF-9、GBP-1、IFI-6-16、IFI-27、25 OAS和IRF-1的几种ISG的过表达显著抑制。通过短发夹RNA敲低GBP-1、IFI-6-16和IFI-27导致HCV复制增加。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