Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells
Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells
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DOI：
10.1128/jvi.76.6.2997-3006.2002
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发表时间：
2002-03-01
影响因子：
5.4
通讯作者：
Lemon, SA
中科院分区：
文献类型：
--
作者：
Ikeda, M;Yi, MK;Lemon, SA
来源于丙型肝炎病毒（HCV）的Con 1株的双顺反子、可选择的亚基因组复制子能够在培养的Huh 7细胞中自主复制（Lohmann等，Science 285：110-113，1999）。然而，NS 3、NS 5A和/或NS 5 B蛋白中的适应性突变对于这些RNA的有效复制是必需的，并且在Huh 7细胞转染后选择的G418抗性集落的数量级增加。在此，我们证明了衍生自第二种基因型1b病毒HCV-N的感染性分子克隆的亚基因组复制子（NNeo/3- 5 B）（Beard等，Hepatology 30：316-324，1999）也能够在Huh 7细胞中有效复制。用NNeo/3- 5 B RNA转染后选择的G418抗性细胞含有丰富的NS 5A抗原和可通过北方分析检测到的HCV RNA。三个克隆分离的细胞系之一中的复制子RNA在NS 3-NS 5 B多聚蛋白中不含突变，证实了在这些细胞中有效复制不需要适应性突变。然而，缺失野生型HCV-N中NS 5A蛋白的干扰素敏感性决定区（ISDR）内存在的独特的4-氨基酸插入显著降低了Huh 7细胞转染后获得的G418抗性集落的数量。这种效应可以通过包含先前描述的Con 1细胞培养适应性突变（S2005-->I）来逆转，证实了这种天然插入在决定野生型HCV-N RNA在Huh 7细胞中的复制能力方面具有控制作用。编码NS 2-NS 5 B、E1-NS 5 B或全长HCV多蛋白的另外的可选择的双顺反子RNA也能够复制，并且在转染Huh 7细胞后产生G418抗性细胞克隆。我们的结论是，来自这个记录的感染性分子克隆的RNA具有独特的能力，在没有额外的细胞培养适应性突变的情况下，在Huh 7细胞中复制。
Dicistronic, selectable subgenomic replicons derived from the Con1 strain of hepatitis C virus (HCV) are capable of autonomous replication in cultured Huh7 Cells (Lohmann et aL, Science 285:110-113, 1999). However, adaptive mutations in the NS3, NS5A, and/or NS5B proteins are required for efficient replication of these RNAs and increase by orders of magnitude the numbers of G418-resistant colonies selected following transfection of Huh7 cells. Here, we demonstrate that a subgenomic replicon (NNeo/3-5B) derived from an infectious molecular clone of a second genotype 1b virus, HCV-N (Beard et aL, Hepatology 30:316-324, 1999) is also capable of efficient replication in Huh7 cells. G418-resistant cells selected following transfection with NNeo/3-5B RNA contained abundant NS5A antigen and HCV RNA detectable by Northern analysis. Replicon RNA in one of three clonally isolated cell lines contained no mutations in the NS3-NS5B polyprotein, confirming that adaptive mutations are not required for efficient replication in these cells. However, the deletion of a unique 4-amino-acid insertion that is present within the interferon sensitivity- determining region (ISDR) of the NS5A protein in wild-type HCV-N drastically decreased the number of G418-resistant colonies obtained following transfection of Huh7 cells. This effect could be reversed by inclusion of a previously described Con1 cell culture-adaptive mutation (S2005-->I), confirming that this natural insertion has a controlling role in determining the replication capacity of wild-type HCV-N RNA in Huh7 cells. Additional selectable, dicistronic RNAs encoding NS2-NS5B, E1-NS5B, or the full-length HCV polyprotein were also capable of replication and gave rise to G418-resistant cell clones following transfection of Huh7 cells. We conclude that RNA derived from this documented infectious molecular clone has a unique capacity for replication in Huh7 cells in the absence of additional cell culture-adaptive mutations.