Elucidation of Interferon Sensitivity Determining Region in Hepatitis C Virus Genome

我们之前已经证明，在同一个体中同时检测到的丙型肝炎病毒（HCV）准种对干扰素的敏感性不同，并且在治疗期间选择干扰素耐药的HCV准种。为了确定其对干扰素耐药的遗传基础，从3例患者在干扰素治疗前和治疗期间的血清中提取HCV基因型1b，并测定其全长核苷酸和推断氨基酸序列。在不同个体中比较干扰素耐药和干扰素敏感HCV分离株，发现在NS5A区c端一半（密码子2154-2383）存在氨基酸簇差异，其中密码子2218处存在共同的独特氨基酸差异。从6例干扰素耐药型和9例干扰素敏感型丙型肝炎病毒中获得的NS5A c端一半序列数据证实，在干扰素敏感型丙型肝炎病毒中，在密码子2218附近（从密码子2209到密码子2248）的40个氨基酸序列中，存在更多的错义突变。另一方面，干扰素耐药HCV的这一区域与原型HCV基因型1b （HCV- j、HCV- jta或HC-J4）相同。我们将该区域指定为干扰素敏感性决定区。因此，具有原型干扰素敏感性决定区域的HCV基因型1b似乎是干扰素耐药菌株。这些突变的特殊性使得预测干扰素治疗的预后效果成为可能。随后，我们通过检测治疗前HCV的NS5A2209-2248来评估预测干扰素应答的可能性。84例感染HCV-1b的慢性丙型肝炎患者接受高剂量干扰素- α治疗。采用聚合酶链反应对从血清RNA中扩增的NS5A2209-2248进行直接测序，确定了NS5A2209-2248的氨基酸序列。30例野生型NS5A2209-2248（与原型HCV-1b相比没有氨基酸替代）患者中有0%的人在停止干扰素治疗后6个月内血清HCV-RNA呈阴性，38例中间型（1-3个氨基酸替代）患者中有13%，16例突变型（4个或更多氨基酸替代）患者中有100%的人完全缓解。表明突变型与完全缓解显著相关（P<0.001）。虽然突变型患者的血清HCV-RNA水平低于其他突变型患者（P<0.001），但多因素分析显示，NS5A2209-2248中氨基酸替换的数量是完全缓解的唯一独立预测因子（优势比5.4，P=0.007）。对干扰素的应答与NS5A2209-2248的结构密切相关，可预测慢性HCV-1b感染干扰素治疗的结果。少

We have previously demonstrated that sensitivity to interferon is different among hepatitis C virus (HCV) quasispecies simultaneously detected in same individuals and that interferon-resistant HCV quasispecies are selected during the treatment. To determine the genetic basis of their resistance to interferon, HCV genotype-1b was obtained from serum of three patients before and during interferon therapy, and their full-length nucleotide and deduced amino acid sequences were determined. Comparison of the paris of interferon-resistant and interferon-sensitive HCV isolates in respective individuals demonstrated clusters of amino acid differences in the C-terminal half of the NS5A region (codon 2154-2383), which contained a common unique amino acid difference at codon 2218. Additional sequence data of the C-terminal half of the NS5A region obtained from six interferon-resistant and nine interferon-sensitive HCV confirmed the exclusive existence of missense mutations in a 40 amino acid stret … More ch of the NS5A region around codon 2218 (from codon 2209 to codon 2248) in interferon-sensitive HCV.On the other hand, this region of interferon-resistant HCV was identical to that of prototype HCV genotype-1b (HCV-J,HCV-JTa or HC-J4). We designated this region as the interferon sensitivity determining region. Thus, HCV genotype-1b with the prototype-interferon sensitivity determining region appears to be interferon-resistant strains. The specific nature of these mutations would make it possible to predict prognostic effects of interferon treatment.Subsequently, we evaluated the possibility for predicting the response to interferon by examining the NS5A2209-2248 of pretherapy HCV.Eighty-four patients with chronic hepatitis C infected with HCV-1b were treated with high doses of interferon-alpha. The deduced amino acid sequence of the NS5A2209-2248 was determined by direct sequencing of the NS5A2209-2248 amplified from serum RNA by polymerase chain reaction. Complete responses with negative serum HCV-RNA for six months after the cessation of interferon were observed in 0 percent of 30 patients with the wild type NS5A2209-2248 (no amino acid substitution compared to the prototype HCV-1b), 13 percent of 38 patients with the intermediate type (1-3 amino acid substitutions), and 100percent of 16 patients with the mutant type (4 or more amino acid substitutions), indicating that the mutant type was significantly associated with the complete response (P<0.001). Although serum HCV-RNA level was lower in the mutant type than the others (P<0.001), multivariate analyzes revealed that the number of amino acid substitutions in the NS5A2209-2248 was the only independent predictor of the complete response (odds ratio 5.4, P=0.007). Responses to interferon are closely related to the structure of the NS5A2209-2248, which is predictive for the outcome of interferon therapy in chronic HCV-1b infection. Less