Site-specific mutation of the interferon sensitivity-determining region (ISDR) modulates hepatitis C virus replication

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 (IFN) 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 as to how different 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 the colony formation assay using HCV replicons with few mutations (0, 1 and 3) in the ISDR, numerous colonies (> 200) appeared when using constructs with six mutations. Introduction of various distinct ISDR sequences with multiple mutations resulted in replication enhancement in transient assays. A mapping study identified several specific sites in the ISDR that critically affected replication, including codon 2209 which, in patients, was closely associated with a strong response to IFN. ISDR sequences associated with a clinical IFN response and viral load modulated the replication of HCV replicons, suggesting the importance of the ISDR sequence in HCV infection.

丙型肝炎病毒（HCV）非结构蛋白5A（NS5A）基因中干扰素敏感性决定区（ISDR）的氨基酸替换数量与干扰素（IFN）应答和病毒载量密切相关。几项基于HCV复制子的研究报告称，ISDR序列在体外对病毒复制有影响。然而，不同的ISDR序列如何影响HCV复制尚不清楚。将各种临床观察到的ISDR序列引入HCV复制子中，并使用集落形成试验和/或瞬时复制试验研究它们对病毒复制的贡献。对ISDR进行了定位研究，以确定对复制有关键影响的氨基酸位置。在使用ISDR中突变较少（0、1和3个）的HCV复制子进行集落形成试验时，没有形成集落，而使用有6个突变的构建体时，出现了大量集落（>200个）。引入具有多个突变的各种不同的ISDR序列导致瞬时试验中的复制增强。一项定位研究确定了ISDR中几个对复制有关键影响的特定位点，包括密码子2209，在患者中，它与对干扰素的强烈应答密切相关。与临床干扰素应答和病毒载量相关的ISDR序列调节了HCV复制子的复制，表明ISDR序列在HCV感染中的重要性。