BIOLOGY AND MOLECULAR BIOLOGY OF HEPATITIS C VIRUS

我们已经构建了一个假定的全长基因组cDNA克隆 在细胞培养和直接转染法中都得到了评价 将这个克隆的RNA转录到黑猩猩的肝脏中。而原来的 克隆不具传染性，大米最近准备了新的克隆 已经添加了哪个额外的序列。这些构造目前是 正在接受评估。 体外细胞培养实验继续主要使用肝细胞 线路，THLE5B。我们已经证明了丙型肝炎病毒的低水平复制 通过6代定量聚合酶链式反应，但没有显示任何 不断增加的复制水平，实际上已经失去了病毒 通过级别。这些实验正在重演。为了 在细胞培养中提高复制能力我们表达了多种丙型肝炎病毒 细胞培养中的非结构蛋白，如聚合酶 过度表达可能会改善复制的希望。 由于肝细胞癌是慢性前列腺癌最严重的并发症 丙型肝炎病毒感染，我们已经开始了实验，试图了解 病毒与致癌的关系。我们已经研究了 所有单个丙型肝炎病毒对P53和Rb肿瘤抑制因子的特异性蛋白 抗原。这些实验得到了很好的控制，但没有显示出特定的 我们也没有证明丙型肝炎病毒对P53和Rb的结合和切割 蛋白酶。我们还在寻找肝细胞癌(HCC) 特异性肿瘤抗原，因为我们发现，每四名患者中就有三名 肝癌细胞具有针对肝癌来源细胞表面的抗体 台词。由于我们对肝细胞癌的CTL反应也很感兴趣，所以我们对 7个肝细胞癌来源的细胞系，但发现6个细胞系的HL A类基因表达下调 并且只表达了HLAA基因座的一个等位基因(手稿 已呈交)。我们正在直接从肝癌组织中分离出CTL并进行测试 以它们在人类白细胞抗原相合的肝癌细胞系上的表达为靶点。已建立的CTL系 将被用来识别编码肝癌抗原的特定基因。

We have constructed a putative full length cDNA clone of the genome that has been evaluated in both cell culture and by direct transfection of RNA transcripts of this clone into chimpanzee liver. While the original clone was not infectious , Rice has recently prepared new clones in which additional sequence has been added. These constructs are presently being evaluated. In vitro cell culture experiments continue using primarily a liver cell line, THLE5b. We have demonstrated the low level replication of HCV by quantitative PCR through 6 passages but have failed to show any increasing levels of replication and in fact have lost the virus at that passage level. These experiments are being repeated. In order to improve the replication in cell culture we are expressing various HCV non-structural proteins such as the polymerase in the cell cultures in the hopes that over expression may improve replication. As hepatocellular carcinoma is the most serious complication of chronic HCV infection, we have initiated experiments to try to understand the relationship of the virus to carcinogenesis. We have studied binding of all the individual HCV specific proteins to p53 and RB tumor suppressor antigens. These experiments were well controlled but showed no specific binding nor have we demonstrated cleavage of p53 and RB by the HCV proteases. We are also looking for hepatocellular carcinoma (HCC) specific tumor antigens as we have found that 3 out of four patients with HCC have antibodies directed at the surface of hepatoma derived cell lines. As we are also interested in CTL responses to HCC, we HLA typed 7 HCC derived cell lines but found that 6 had down regulated HLA class I and only one allele of the HLA A-locus was expressed (manuscript submitted). We are isolating CTL directly from HCC tissue and testing them on HLA compatible HCC cell lines as targets. Established CTL lines will be used to identify specific genes coding for the HCC antigen.