Tellinghuisen Timothy L, Lindenbach Brett D
Department of Infectology, The Scripps Research Institute, Jupiter, FL, USA.
Methods Mol Biol. 2009;510:165-75. doi: 10.1007/978-1-59745-394-3_12.
Since the advent of efficient cell-culture methods for HCV replication and, more recently, infection, there has been a need to efficiently sequence the viral RNA in these systems. This need is especially urgent in light of the error-prone nature of HCV RNA replication, which leads to a variety of interesting mutations. The adaptation of hepatitis C replicons to cell culture, which greatly increased their replication capacity, and the subsequent identification of viral point mutations responsible for this adaptation are prime examples of the type of phenotype-genotype connection that viral RNA sequencing methods can provide. More recently, researchers have used similar sequencing methods to identify changes in replicons that represent viral adaptation to engineered mutations, adaptation to a variety of host cells, and viral evasion of antiviral compound susceptibility. Here, we describe the cloning and isolation of HCV replicon-bearing cells, the extraction of total RNA, the generation of cDNA, and the amplification of specific HCV replicon sequences for sequence analysis. The methods we describe permit rapid and robust determination of HCV RNA sequences from cell culture.
自从出现了用于丙型肝炎病毒（HCV）复制以及最近用于感染的高效细胞培养方法以来，就需要在这些系统中对病毒RNA进行高效测序。鉴于HCV RNA复制具有易出错的特性，会导致各种有趣的突变，这种需求尤为迫切。丙型肝炎复制子适应细胞培养，这极大地提高了它们的复制能力，随后鉴定出负责这种适应的病毒点突变，是病毒RNA测序方法能够提供的表型-基因型联系类型的主要例子。最近，研究人员使用类似的测序方法来鉴定复制子中的变化，这些变化代表病毒对工程突变的适应、对多种宿主细胞的适应以及病毒对抗病毒化合物敏感性的逃避。在这里，我们描述了携带HCV复制子的细胞的克隆和分离、总RNA的提取、cDNA的生成以及用于序列分析的特定HCV复制子序列的扩增。我们描述的方法允许从细胞培养中快速且可靠地确定HCV RNA序列。
