Function of HCV NS5A protein and the mechanism of interferon resistance

将不同ISDR序列的全长或截短的NS5A cDNA克隆到酵母或哺乳动物表达载体上，形成GAL4 dna结合域与NS5A蛋白的融合蛋白。转染后，在GAL4结合位点的控制下，利用β-半乳糖苷酶（酵母）或氯霉素乙酰转移酶（哺乳动物细胞）报告基因表达来测定这些构建体的转录活性。结果表明，ISDR具有转录活性，并通过氨基酸突变增强，这也与病毒载量降低和干扰素敏感性增加有关。转录激活与干扰素敏感性或病毒复制之间可能存在的关联有待进一步研究。75例慢性HCV-2感染患者（HCV-2a型40例，HCV-2b型35例）接受干扰素- α治疗6个月，总剂量为468-860（平均±SD， 739±1.59）百万单位。预处理NS5A序列，RT-PCR产物直接测序确定更多的es。结果表明干扰素敏感性与HCV-2a感染中NS5A蛋白的氨基酸变化有关，与HCV-1b相似。比较22例HCV-1b型慢性肝炎患者干扰素治疗（总剂量2.4 - 8.8亿单位）前后ISDR准种的顺序变化。18例患者在停止干扰素治疗后血清HCV-RNA阳性无反应。其他4例患者在治疗后6个月内没有血清HCV-RNA的证据，完全缓解。利用聚合酶链式反应（PCR）对HCV基因组进行直接测序，确定优势准种的氨基酸序列，并与原型HCV-1b进行比较。通过PCR产物的克隆和测序，评价了ISDR准种的种群变化。干扰素治疗后血清中检测到的HCV在ISDR中的氨基酸取代比治疗前检测到的少。这种干扰素耐药HCV在治疗前已经作为次要准种存在于无应答者中，并被干扰素选择导致治疗失败。少

The full-length or truncated NS5A cDNA with different ISDR sequences was cloned into a yeast or mammalian expression vector to form a fusion protein consisting of the GAL4 DNA-binding domain and NS5A protein. Following transfection, the transcriptional activities of these constructs were determined using β-galactosidase (yeast) or chloramphenicol acetyltransferase (mammalian cell) reporter gene expression under the control of GAL4 binding sites. The results suggest that the ISDR has a transcriptional activity and it is enhanced by amino acid mutations, that are also related to decreased viral load and increased interferon sensitivity. The possible association between transcriptional activation and interferon sensitivity or viral replication should be studied further.Seventy-five patients with chronic HCV-2 infection (40 with HCV-2a and 35 with HCV-2b) were treated with interferon-alpha for 6 months with a total dose of 468-860 (mean±SD, 739±159) million units. Pretreatment NS5A sequenc … More es were determined by direct sequencing of RT-PCR products. The results indicate that interferon sensitivity is associated with amino acid variations in the NS5A protein in HCV-2a infection, like HCV-1b.Sequential changes in ISDR quasispecies were compared before and just after interferon therapy (total dose 240-880 million units) in 22 patients with chronic hepatitis caused by HCV-1b. Eighteen patients were non-responders with positive serum HCV-RNA after the cessation of interferon therapy. The other four patients were complete responders with no evidence of serum HCV-RNA for 6 months after therapy. Amino acid sequences of predominant quasispecies were determined by direct sequencing of the HCV genome amplified by polymerase chain reaction (PCR), and compared with that of the prototype HCV-1b. Populational changes of ISDR quasispecies were also evaluated by cloning and sequencing of PCR products. HCV detected in sera after interferon therapy have fewer amino acid substitutions in ISDR than those detected before therapy. Such interferon-resistant HCV are already present before therapy as minor quasispecies in non-responders, and are selected by interferon resulting in therapeutic failure. Less