Genetic characterization of hepatitis C virus in long-term RNA replication using Li23 cell culture systems.
Genetic characterization of hepatitis C virus in long-term RNA replication using Li23 cell culture systems.
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DOI：
10.1371/journal.pone.0091156
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Ikeda M
中科院分区：
文献类型：
--
作者：
Kato N;Sejima H;Ueda Y;Mori K;Satoh S;Dansako H;Ikeda M
丙型肝炎病毒（HCV）最显著的遗传特征是其显著的多样性和变异。为了理解这一特征，我们之前在人类肝癌huh -7衍生的HCV rna复制细胞系的长期培养中对HCV进行了遗传分析。另一方面，我们利用不同于HuH-7细胞的人肝癌Li23细胞建立了新的HCV rna复制细胞系。li23衍生的HCV rna复制细胞培养4年。我们对培养0年、2年和4年从这些细胞中回收的hcv进行了遗传分析。大多数分析分两部分进行：一部分覆盖从5 '端到NS2，这是RNA复制所必需的，另一部分覆盖从NS3到NS5B，这是RNA复制所必需的。这两个区域的基因突变均具有时间依赖性，5′端- ns2和NS3-NS5B区域的突变率分别为4.0-9.0×10−3和2.7-4.0×10−3个碱基替换/位点/年。这些结果表明NS3-NS5B区域的变异受到RNA复制压力的影响。从2年或4年培养的细胞中获得的HCV rna的结构区域检测到几个框架内缺失（3-105个核苷酸）。系统进化树分析清楚地表明，HCV的遗传多样性是以时间依赖的方式扩展的。正如之前在huh -7衍生的细胞系统中观察到的那样，HCV RNA的GC含量以时间依赖性的方式显着增加。这种现象的部分原因是人类细胞中密码子优化的密码子用法发生了变化。此外，我们证明这些长期培养的细胞可作为选择对抗HCV药物具有抗性的HCV克隆的有用来源。长期培养的HCV rna复制细胞可用于分析HCV的进化动力学和变异以及耐药性分析。
The most distinguishing genetic feature of hepatitis C virus (HCV) is its remarkable diversity and variation. To understand this feature, we previously performed genetic analysis of HCV in the long-term culture of human hepatoma HuH-7-derived HCV RNA-replicating cell lines. On the other hand, we newly established HCV RNA-replicating cell lines using human hepatoma Li23 cells, which were distinct from HuH-7 cells. Li23-derived HCV RNA-replicating cells were cultured for 4 years. We performed genetic analysis of HCVs recovered from these cells at 0, 2, and 4 years in culture. Most analysis was performed in two separate parts: one part covered from the 5′-terminus to NS2, which is mostly nonessential for RNA replication, and the other part covered from NS3 to NS5B, which is essential for RNA replication. Genetic mutations in both regions accumulated in a time-dependent manner, and the mutation rates in the 5′-terminus-NS2 and NS3-NS5B regions were 4.0–9.0×10−3 and 2.7–4.0×10−3 base substitutions/site/year, respectively. These results suggest that the variation in the NS3-NS5B regions is affected by the pressure of RNA replication. Several in-frame deletions (3–105 nucleotides) were detected in the structural regions of HCV RNAs obtained from 2-year or 4-year cultured cells. Phylogenetic tree analyses clearly showed that the genetic diversity of HCV was expanded in a time-dependent manner. The GC content of HCV RNA was significantly increased in a time-dependent manner, as previously observed in HuH-7-derived cell systems. This phenomenon was partially due to the alterations in codon usages for codon optimization in human cells. Furthermore, we demonstrated that these long-term cultured cells were useful as a source for the selection of HCV clones showing resistance to anti-HCV agents. Long-term cultured HCV RNA-replicating cells are useful for the analysis of evolutionary dynamics and variations of HCV and for drug-resistance analysis.
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影响因子：
13.5
作者：
Ghany, Marc G.;Nelson, David R.;Strader, Doris B.;Thomas, David L.;Seeff, Leonard B.
通讯作者：
Seeff, Leonard B.
影响因子：
3.7
作者：
Coelmont L;Hanoulle X;Chatterji U;Berger C;Snoeck J;Bobardt M;Lim P;Vliegen I;Paeshuyse J;Vuagniaux G;Vandamme AM;Bartenschlager R;Gallay P;Lippens G;Neyts J
通讯作者：
Neyts J
影响因子：
5.4
作者：
Ikeda, M;Yi, MK;Lemon, SA
通讯作者：
Lemon, SA
影响因子：
5
作者：
Kato, Nobuyuki;Mori, Kyoko;Ikeda, Masanori
通讯作者：
Ikeda, Masanori
影响因子：
4.9
作者：
Fridell, Robert A.;Qiu, Dike;Gao, Min
通讯作者：
Gao, Min