Elucidation of Interferon Sensitivity Determining Region in Hepatitis C Virus Genome

我们以前已经证明，干扰素的敏感性是不同的丙型肝炎病毒（HCV）准种同时检测到在同一个人和干扰素耐药的HCV准种在治疗过程中选择。为了确定其对干扰素耐药的遗传基础，从3例患者干扰素治疗前和治疗期间的血清中获得HCV基因型-1b，并测定其全长核苷酸和推导的氨基酸序列。对干扰素耐药和干扰素敏感HCV分离株的巴黎序列进行比较，发现NS 5A区C-末端的一半（密码子2154-2383）存在一组氨基酸差异，在密码子2218处存在一个共同的独特氨基酸差异。从6个干扰素耐药和9个干扰素敏感的HCV中获得的NS 5A区C-末端的另外一半的序列数据证实了在一个40个氨基酸的序列中唯一存在错义突变。 ...更多信息 干扰素敏感型HCV的NS 5A区2218位密码子（2209 ~ 2248）的第一个氨基酸序列与干扰素耐药型HCV的NS 5A区2218位密码子（2209 ~ 2248）的第一个氨基酸序列相同，而干扰素耐药型HCV的NS 5A区2218位密码子（2209 ~ 2248）的第一个氨基酸序列与原型HCV基因型1b（HCV-J、HCV-JTa或HC-J 4）的NS 5A区相同。我们将该区域命名为干扰素敏感性决定区。因此，HCV基因型-1b与原型干扰素敏感性决定区似乎是干扰素耐药株。这些突变的特异性将使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NS 5A 2209 -2248来评估预测干扰素应答的可能性。NS 5A 2209 -2248的推导的氨基酸序列通过聚合酶链反应从血清RNA扩增的NS 5A 2209 - 2248的直接测序来确定。在30例野生型NS 5A 2209 -2248患者中，观察到干扰素停药后6个月血清HCV-RNA阴性的完全应答率为0（与原型HCV-1b相比没有氨基酸取代），38名中间型患者中有13% 16例突变型（4个或4个以上氨基酸替换）患者的阳性率为100%，表明突变型与完全缓解显著相关（P<0.001）。尽管变异型患者血清HCV-RNA水平低于其他变异型患者（P<0.001），但多因素分析显示NS 5A 2209 -2248的氨基酸替换数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NS 5A 2209 -2248的结构密切相关，NS 5A 2209 -2248的结构可预测干扰素治疗慢性HCV-1b感染的结果。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