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(丙型肝炎病毒)准种对干扰素的敏感性不同，治疗过程中选择了具有干扰素抗性的丙型肝炎病毒准种。为探讨丙型肝炎患者干扰素耐药的遗传基础，从3例患者干扰素治疗前后的血清中提取丙型肝炎病毒1b亚型，测定其全长核苷酸序列和推导的氨基酸序列。比较耐干扰素和对干扰素敏感的丙型肝炎病毒分离株在各自个体中的巴黎序列，发现在NS5A区(密码子2154-2383)的C-末端半部分有一组氨基酸差异，其中在密码子2218处包含共同的唯一氨基酸差异。从6个干扰素耐药和9个干扰素敏感的丙型肝炎病毒中获得的NS5A区C末端一半的额外序列数据证实，在40个氨基酸的链…中唯一存在错义突变在干扰素敏感的丙型肝炎病毒中，2218密码子附近的NS5A区(密码子2209到2248)有更多的ch。另一方面，干扰素抵抗的丙型肝炎病毒与原型丙型肝炎病毒1b(丙型肝炎病毒J、丙型肝炎病毒JTA或丙型肝炎病毒J4)的NS5A区完全相同。我们将该区域命名为干扰素敏感性决定区域。因此，具有原型干扰素敏感性决定区域的丙型肝炎病毒基因-1b似乎是干扰素耐药株。随后，我们通过检测治疗前丙型肝炎病毒的NS5A2209-2248来评估预测干扰素疗效的可能性。84名感染丙型肝炎病毒的慢性丙型肝炎患者接受了大剂量的干扰素-α治疗。用聚合酶链式反应从血清RNA中扩增出NS5A2209-2248，通过直接测序确定NS5A2209-2248的氨基酸序列。在30例野生型NS5A2209-2248患者(与原型丙型肝炎病毒1b相比没有氨基酸替代)、38例中间型患者(1-3个氨基酸替代)和16例变异型(4个或4个以上氨基酸替代)患者中，观察到6个月血清丙型肝炎病毒RNA阴性的完全应答率为0%，表明突变型与完全应答显著相关(P&lt；0.001)。尽管变异型患者血清丙型肝炎病毒核糖核酸水平低于其他变异型患者(P&lt；0.001)，但多因素分析显示，NS5A2209-2248位氨基酸替换次数是完全应答的唯一独立预测因素(优势比为5.4P=0.007)。对干扰素的反应与NS5A2209-2248的结构密切相关，NS5A2209-2248的结构对慢性丙型肝炎病毒感染的干扰素治疗结果有预测作用。较少

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