Characterization of cell lines allowing tightly regulated expression of hepatitis C virus core protein
Characterization of cell lines allowing tightly regulated expression of hepatitis C virus core protein
复制标题
DOI：
10.1006/viro.1996.0397
复制
发表时间：
1996-08-01
期刊：
影响因子：
3.7
通讯作者：
Wands, JR
中科院分区：
文献类型：
--
作者：
Moradpour, D;Englert, C;Wands, JR
使用四环素调节系统产生细胞系，该细胞系允许包含5'非编码区、核心区和部分E1区的丙型肝炎病毒（HCV）cDNA的严格控制表达。21-kDa的加工核衣壳蛋白的生产可以在很宽的范围内调节存在于培养基中的四环素的浓度。使用HCV核心-荧光素酶融合构建体发现超过1000倍的诱导率。核心蛋白的细胞内半衰期为9小时，对应于HCV开放阅读框的173个氨基末端氨基酸的产物。连续免疫荧光显微术显示核心抗原首先以主要的核周细网状染色模式存在，随后也在细胞质颗粒和囊泡中存在。免疫电镜观察发现，核心蛋白位于内质网膜和细胞质脂滴表面。生长速率分析和集落形成效率测定显示HCV核心蛋白表达本身没有主要的细胞毒性作用。HCV基因表达可被靶向翻译起始密码子下游区域的反义寡核苷酸抑制。这些细胞系代表了研究HCV核心蛋白的结构和功能特性的重要工具，并且可用于评估细胞系统中针对HCV的基因治疗策略（C）1996 Academic Press，Inc.
A tetracycline-regulated system was used to generate cell lines allowing tightly controlled expression of a hepatitis C virus (HCV) cDNA comprising the 5' noncoding, the core, and part of the E1 regions. Production of 21-kDa processed nucleocapsid protein could be regulated over a broad range by the concentration of tetracycline present in the culture medium. Induction ratios of over 1000-fold were found using an HCV core-luciferase fusion construct. Core protein had an intracellular half-life of 9 hr and corresponded to the product of 173 amino-terminal amino acids of the HCV open reading frame. sequential immunofluorencence microscopy revealed the presence of core antigen first in a predominantly perinuclear fine-reticular staining pattern and subsequently also in cytoplasmic granules and vesicles. By immunoelectron microscopy core protein was found on the endoplasmic reticulum membrane and on the surface of cytoplasmic lipid droplets. Growth rate analyses and colony formation efficiency assays showed no major cytotoxic effect of HCV core protein expression per se. HCV gene expression could be inhibited by an antisense oligonucleotide targeting a region immediately downstream of the translation initiation codon. These cell lines represent important tools to investigate structural and functional properties of HCV core protein and may be useful to evaluate gene therapeutic strategies against HCV in a cellular system, (C) 1996 Academic Press, Inc.