Establishment of hepatitis C virus replicon cell lines possessing interferon-resistant phenotype
Establishment of hepatitis C virus replicon cell lines possessing interferon-resistant phenotype
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DOI：
10.1016/j.bbrc.2004.08.091
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发表时间：
2004-10-08
影响因子：
3.1
通讯作者：
Kato, N
中科院分区：
文献类型：
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作者：
Namba, K;Naka, K;Kato, N
为了阐明慢性肝炎患者中丙型肝炎病毒（HCV）对干扰素（IFN）耐药的机制，我们试图从先前建立的IFN敏感的50-1复制子中开发出IFN耐药的HCV复制子。通过用IFN-α的延长的低剂量处理来处理50-1复制子细胞，然后分离来自IFN-α处理的复制子细胞的总RNA，我们成功地获得了针对IFN-α（200 IU/ml）存活的HCV复制子细胞的四个克隆（命名为1、3、4和5）。这些克隆的细胞进一步用IFN-α或IFN-β（分别逐渐增加至2000或1000 IU/ml）处理。这导致四种具有IFN-α抗性表型的复制子细胞系（alphaR系列）和四种具有IFN-β抗性表型的复制子细胞系（PR系列）。此外，我们通过如上所述的IFN-α和RNA转染的两轮延长处理获得了具有IFN-α抗性表型的另外的复制子细胞系（alphaRmix）。这些获得的HCV复制子细胞系的表征揭示了PR系列对IFN-α和IFN-β两者都具有高度抗性，尽管含有alphaRmix的alphaR系列仅对IFN-α和IFN-β两者都具有部分抗性。对这些HCV复制子的遗传分析发现，PR系列的NS 4 B中有一个共同的氨基酸取代，NS 5A中有几个额外的氨基酸取代，这表明这些遗传改变与这些HCV复制子的IFN抗性有关。这些新建立的具有IFN抗性表型的HCV复制子细胞系是理解HCV在体内获得IFN抗性的机制的第一个有用的工具。(C)2004年爱思唯尔公司All rights reserved.
To clarify the mechanism underlying resistance to interferon (IFN) by the hepatitis C virus (HCV) in patients with chronic hepatitis, we attempted to develop an IFN-resistant HCV replicon from the IFN-sensitive 50-1 replicon established previously. By treating 50-1 replicon cells with a prolonged low-dose treatment of IFN-alpha and then transfecting the total RNA derived from the IFN-alpha-treated replicon cells, we successfully obtained four clones (named 1, 3, 4, and 5) of HCV replicon cells that survived against IFN-alpha (200 IU/ml). These cloned cells were further treated with IFN-alpha or IFN-beta (increased gradually to 2000 or 1000 IU/ml, respectively). This led to four replicon cell lines (alphaR series) possessing the IFN-alpha-resistant phenotype and four replicon cell lines (PR series) possessing the IFN-beta-resistant phenotype. Furthermore, we obtained an additional replicon cell line (alphaRmix) possessing the IFN-alpha-resistant phenotype by two rounds of prolonged treatment with IFN-alpha and RNA transfection as mentioned above. Characterization of these obtained HCV replicon cell lines revealed that the PR series were highly resistant to both IFN-alpha and IFN-beta, although the alphaR series containing alphaRmix were only partially resistant to both IFN-alpha and IFN-beta. Genetic analysis of these HCV replicons found one common amino acid substitution in the NS4B and several additional amino acid substitutions in the NS5A of the PR series, suggesting that these genetic alterations are involved in the IFN resistance of these HCV replicons. These newly established HCV replicon cell lines possessing IFN-resistant phenotypes are the first useful tools for understanding the mechanisms by which HCV acquires IFN resistance in vivo. (C) 2004 Elsevier Inc. All rights reserved.