Mechanism of Viral Liver Injury by analyzing escape mutant virus and HLA-binding viral peptide

用亲和层析法从稳定表达乙肝病毒的人肝癌细胞株(HepG2 2.2.15)中分离纯化了人类白细胞抗原结合肽。用LC/MS/MS测定人类白细胞抗原结合肽的氨基酸序列为NPLPOLPO，对从慢性乙型肝病患者血清中获得的全长HBVDNA进行测序，根据慢性肝病的进展在核心区和S前区积累氨基酸替换，通过反向聚合酶链式反应、克隆和测序来确定慢性活动性肝炎患者肝脏侵袭性细胞毒性T细胞的T细胞受体α链的谱系。最常见的是Valpha7.2基因与Jalpha33基因结合，我们建立了一步扩增3.2kbpHBVdna的方法。将克隆的全长HBVDNA导入人肝癌细胞(Huh7)，证实可复制。我们用Ile、Val、Ala、Leu、Lys、Arg和Thr替换YMDD基序的蛋氨酸，形成了7个突变体，以考察这些突变体的复制能力和拉米夫定的敏感性。4个变异体(Ile、Val、Ala和Leu)保持复制活性，而另外3个变异体(Lys、Arg和Thr)显示复制受损。丙型肝炎病毒(丙型肝炎病毒)、丙型肝炎病毒非结构区5A(NS5A)蛋白在GAL4的DNA结合区未融合146个氨基酸的情况下，在酵母和人肝癌细胞(HuH7)中强烈激活转录。丙型肝炎病毒NS5A蛋白的转录激活可能在病毒复制和肝癌发生中起作用。

Hepatitis B virus (HBV)We purified HLA-binding peptides from HBV stably-transfected human hepatoma cells (HepG2 2.2.15) by affinity chromatography using monoclonal Ab. Amino acid sequence of HLA-binding peptide was determined as NPLPOLPO by HPLC-mass spectrometry (LC/MS/MS).Full-length HBV DNAs obtained from sera of patients with type B-chronic liver diseases were sequenced Amino acid substitutions were accumulated in core-region and pre-S region according to the progression of chronic liver disease.The repertoire of T cell receptor alpha-chain of liver-invaded cytotoxic T cells in patiens with type B-chronic active hepatitis, was determined by inverse PCR,cloning and sequencing. The Valpha 7.2 gene was most frequently observed and found to bind Jalpha 33 gene.We established one-step amplification of 3.2 kbp of full-length HBV DNA by using long-PCR method. Cloned full-length HBV DNA was confirmed to replicate by transfection into human hepatoma cells (Huh7). We made 7 variants by substituting the methionine of the YMDD motif with Ile, Val, Ala, Leu, Lys, Arg and Thr, to explore replication ability and lamivudine sensitivity of these variants. Four variants (Ile, Val, Ala and Leu) remained replication competent, whereas 3 others (Lys, Arg and Thr) showed impaired replication. 2 variants (Ile and Val) were showed to be resistant to lamivudine.Hepatitis C virus (HCV)Hepatitis C virus nonstructural region 5A (NS5A) protein, without its 146 amino-terminal amino acids fused to the DNA-binding domain of GAL4 strongly activates transcription in yeast and human hepatoma cells (Huh7). Transcriptional activation by HCV NS5A protein may play a role in viral replication and hepatocarcinogenesis.