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

分析逃逸突变病毒和HLA结合病毒肽研究病毒性肝损伤机制

基本信息

批准号：
06404029
负责人：
OMATA Masao
金额：
$ 15.87万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1997
项目状态：
已结题

项目摘要

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.

B型肝炎病毒（HBV）我们用单克隆抗体通过亲和层析从HBV稳定转染的人肝癌细胞（HepG 2 2.2.15）中纯化HLA结合肽。HPLC-MS法测定其氨基酸序列为NPLPOLPO采用LC/MS/MS技术对乙型肝炎患者血清中HBV DNA进行了全序列测定，发现随着慢性肝病的进展，核心区和前S区的氨基酸替换逐渐增多，B型肝炎患者肝内侵袭性细胞毒性T细胞的T细胞受体α链库也逐渐增加。通过反向PCR、克隆和测序确定慢性活动性肝炎。我们建立了一步扩增3.2kbp全长HBV DNA的长链PCR方法。通过转染人肝癌细胞（Huh 7）证实克隆的全长HBV DNA复制。我们将YMDD基序中的蛋氨酸分别替换为Ile、瓦尔、Ala、Leu、Lys、Arg和Thr，制备了7个变异体，以探讨这些变异体的复制能力和拉米夫定敏感性。4个变异体（Ile、瓦尔、Ala和Leu）保持复制能力，而另外3个变异体（Lys、Arg和Thr）显示复制受损。丙型肝炎病毒（HCV）非结构区5A（NS 5A）蛋白（NS 5A）在酵母和人肝癌细胞（Huh 7）中强烈激活转录，但其氨基端146个氨基酸不与GAL 4的DNA结合域融合。HCV NS 5A蛋白的转录激活可能在病毒复制和肝癌发生中起作用。